Biology of Root Formation and Development (Basic Life Sciences)

Preface to the Third Edition

Evolution of roots was a fundamental development that enabled plants to migrate from aquatic to terrestrial habitats. Eventually it led to a division of functions between the carbohydrate-supplying shoot and the waterand mineral-supplying roots. Thus, it is not only the function of the individual shoots that constitutes the basis for the subsistence of global primary productivity and supports all animal and microbial life in terrestrial ecosystems, but also the concerted function of the shoots and roots. In spite of the great importance of roots for basic and applied scientific aspects and a long history of root research, there remains much to be learned about root development and functions. Roots are buried in the soil and any attempt to investigate their activity causes them irreversible damage. In addition, because roots live in very close contact with all other soil biota, the relationships may be so consolidated that it is hard to distinguish between a root as a plant organ and a root as a symbiont. Plant roots have remained an exciting and intriguing field of science. In the past five years, an exceptional proliferation of interest in root biology has developed, associated with intensive research activity in this field and contemporary developments in the understanding of root function and development. The new biological research involving the cloning and identification of genes and the control of their expression has deeply penetrated into root biology. More and more root-specific genes are being identified and the role of such genes in the control of root structure and function is being discovered. New methods and tools have been applied to root research, while old ideas and interpretations are being reexamined. As a result, it became necessary to update and expand our viewpoints. The chapters of this book are not intended to provide complete overviews. Our purpose in this expanded edition is to present a cross-section of the accomplishments of the past in and the future direction of root research. The third edition covers not only the numerous subjects of the previous editions but also the additional fields of genetics, molecular biology, growth-substance physiology, pH effects, biotechnology, and biomechanics. The book also tackles ecological problems and the multitude of interactions between roots and various types of soil organisms. We added some specific topics such as micropropagation, root signals, environmental sensing and direction finding, and expanded the scope of most of the other sections. Obviously, the chapters express the points of view of the writers. In some cases they are provocative. The presentation of a range of opinions introduces the reader to the current pressing questions in root studies, and points out new bearings for future research. The third edition serves as a major source of information for root scientists, botanists, plant physiologists, microbiologists, soil scientists, and those engaged in related professions. This book summarizes the previous information and designates the present frontiers of our knowledge in this field. The important questions that should be investigated in the future are pointed out. It presents a multidisciplinary view of the field of plant roots and its iii

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Preface to the Third Edition

state of the art. It covers various aspects of root origin, root structure, development and behavior, the interactions between roots and their environment, and the various uses of roots. The book contains 59 chapters, examining a wide range of critical topics and exploring potential applications and future directions. The following themes are covered: The origin and characteristics of roots Structure and development of root systems Root genetics Research techniques for root studies Regulation of root growth Physiological aspects of root systems Root growth under stress Root–rhizosphere interactions Roots of various ecological groups Root of economic value Some of the topics were given more attention than others, especially those in which the literature has proliferated recently or new techniques have been introduced. While we have made every effort to reach uniformity in terminology and style, the presented results, the expressed ideas, and the final shape of the chapters retain the personal imprint of the individual authors. We wish to express our sincere and warmest gratitude to all the eminent contributors, for their scholarly contribution and enthusiastic cooperation. Yoav Waisel Amram Eshel Uzi Kafkafi

Preface to the Second Edition

Plant roots have remained an exciting and intriguing field of science. During the years since the first edition of Plant Roots: The Hidden Half was published, an exceptional proliferation of interest in root biology has developed. This has been associated with the intensive research activity in this field and contemporary innovations in the understanding of root function and development. New methods have been applied, and old ideas and interpretations reexamined. Altogether, it became necessary to both update and expand the coverage of the first edition. We remain loyal to our original goals: to present the cardinal information about plant roots, to designate the frontiers of our knowledge in this field, and to point out the pressing questions that should be investigated. This book presents the state-of-the-art, multidisciplinary view of plant roots. It covers various aspects of root structure, development, and function; the interactions between roots and their environment; and the important benefits of roots to mankind. This is the story of the frontiers of the root sciences. It is a comprehensive volume, examining a wide range of critical topics, newly explored areas, potential applications, and future directions. The second edition covers nine aspects of the root sciences: Structure and development of the root system Methods of root studies Growth and metabolism Growth under stress Ion and water relations The rhizosphere Root pests Roots of various ecological groups Roots of economic value Because of the vast increase in information, this second edition is not only revised, but also expanded. Some of the topics were totally revised, especially those for which the literature has amply proliferated during the last five years or where new techniques of molecular genetics have been introduced. Notable among these are chapters related to root-pest interactions. Where necessary, some of the topics of the first edition were rewritten by new teams of root scientists. Thirty new contributors have joined those who participated in the first edition. Only one chapter was reproduced without change.

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Preface to the Second Edition

Three new themes are included in the second edition: 1. 2. 3.

Innovative methods of root studies Modeling and simulation Economic aspects of root biology

We have added some of the new topics to existing sections, whereas others are grouped into the following two new sections: Methods of Root Studies (Part II) includes chapters on modern techniques, fractal geometry, and simulation of root system architecture. Roots of Economic Value (Part IX) contains chapters addressing roots as a source of food, the biosynthetic potential of roots, and medicinal uses. Other new topics, including cell biology and development of root hairs, modeling of water uptake, simulation of ion uptake, drought rhizogenesis, and contractile roots, are distributed among the existing sections, according to their specific subject. The division of the 49 chapters into parts was rather difficult. In certain cases a chapter could fit equally well in more than one category. For example, Chapter 48, ‘‘Underground Plant Metabolism: The Biosynthetic Potential of Roots,’’ in Part IX, Roots of Economic Value, would have also been appropriate for Part III, Growth and Metabolism. Readers looking for a specific topic are therefore advised to browse through the entire volume. The numerous selected references, listed at the end of each chapter, direct the reader toward wider reading. These have been updated, many references cited in the first edition were replaced by new ones. Thus, the reader has the chance to unveil an entire epoch of root research, from 1672 to 1995. We wish to express our sincere gratitude to each of the eminent contributors for their scholarly contributions and for their enthusiastic and speedy cooperation. Yoav Waisel Amram Eshel Uzi Kafkafi

Preface to the First Edition

Roots, the ‘‘hidden half’’ of plants, serve a multitude of functions. They are responsible for anchorage, supply the plants with water and with nutrients, and exchange various growth substances with the shoots. Roots perform these functions in most ferns and in all seed plants, whereas additional traits (e.g., formation of storage organs, determination of the depth of the regenerating buds, or aeration of inundated organs) are characteristic of roots of exclusive groups of plants. The root–soil interface is the site where most interactions between the plants and their environment occur. Roots constitute a major source of organic material for the soil and thus affect its structure, aeration, and biological activities. While organic chemicals move out of the roots into the soil, inorganic ones move in: some of the entering materials are needed for normal metabolsim of the plants and are actively sought. Others are not required but are either neutral or toxic. Insufficient or excessive accumulation of most elements would damage plants, and therefore their uptake is controlled at the root surface. Our interest in the development and function of plant roots stems from the academic desire to understand their role in plant life, as well as from the important practical aspects they have. Most agricultural investment (i.e., plowing, seedbed preparation, irrigation, and fertilization) is spent to provide conducive conditions for the growth of roots of crop plants. Functional and healthy plant roots are essential for production of many of the resources on which human prosperity depends. The objectives of the present monograph are multiple: to review the recent contributions to the knowledge of the structure and function of roots, to outline the frontiers of root sciences, to point out the areas where gaps in knowledge exist, and to indicate the direction toward which basic and applied root research should proceed in the future. Plant Roots: The Hidden Half consists of 40 chapters that are grouped into the following seven sections. The first section deals with the structure and development of roots and with their assemblage into root systems. This section also tackles some of the genetic and physiological bases that control the development of individual roots and that eventually determine the structure, position, and function of roots systems. It also discusses in detail the individual root anatomy, with emphasis on root meristems, root caps, root hairs, and the cambium. The second section covers several aspects of the growth and metabolism of roots. It starts with the delicate hormonal relationships of roots and their effects on root growth and gravireaction, touches on various specific metabolic processes, and concludes with a discussion of root turnover and senescence. Studying roots, one can neither ignore the shoots nor disregard the constraints of the environment. The relationships between roots and shoots are, therefore, discussed here. The third section details with one of the very critical aspects of growth of plant roots—their encounter with stressing environments. This section covers the behavior of roots under temperature, low oxygen, heavy metal, and salinity stresses, as well as under the mechanical constraints of the soil. vii

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The fourth group of chapters discusses the uptake of minerals and water by roots and their subsequent transport into all other plant organs. Some chapters deal with nutrient availability, movement of ions across the soil–root interface, and the mechanisms of ion uptake. Three chapters discuss several aspects of the water relations of roots, and one chapter investigates an exceptional function of some roots: uptake of CO2. The various aspects of interactions that occur at the rhizosphere level are compiled in the fifth and sixth sections. Those include discussion of the interrelationship of roots with the biotic and abiotic components of their environment. In some cases the biotic interactions in the rhizosphere are obligatory and neither the roots nor the microorganisms can survive independently in nature. The application of these phenomena in agricultural practice is discussed in several of the chapters. In other cases the root–microorganism relationships are facultative. Still they may exert positive effects on the roots even if the soil organisms feed exclusively on root excretions and on their sloughed cells, without contacting live root cells. Other organisms exploit the roots as pathogens or as parasites and cause great damage to many crop plants. An understanding of the adaptation of plants to their respective habitats would be far from complete without a thorough knowledge of the role of their roots. Thus the last section discusses some of the aspects of plant adaptation at the ‘‘underground’’ level. Out of the great variety of ecological groups of plants that exist in nature, we have selected three unique ones, which present different trends of adaptation: desert plants, epiphytes, and submerged aquatic plants. An attempt to combine various topics of root science into one comprehensive treatise raises numerous difficulties, because of the complexity of integration of several aspects and because of the past division among different disciplines. Such a division has sometimes resulted in different and conflicting approaches and in a multiform terminology. For example, the term exudation is used by soil microbiologists to describe the release of organic materials by roots into their environment. The same term is used by plant physiologists for the completely different process of xylem sap flow out of the cut end of excised roots. The term growth is much less controversial. Still, some investigators use it to describe a general increase in root mass, whereas others employ it for description of the extension of a tap root or for initiation of primordia of lateral roots. These processes are intercorrelated but are certainly not interconvertible. Ion uptake by roots is a third example of the ambiguous use of terms. It was used by some scientists with the meaning of membrane transport, that is, a process that occurs between two solutions: one inside root cells and one outside them. These physiologists tended to disregard the roots’ environment, the existence of a boundary layer, and the contact with the charged surfaces of soil particles. Other investigators have referred to ion uptake in a much broader approach and included in this term all aspects of ion movement along and across root cells, including diffusion and mass flow through the free space in addition to the accumulation in the osmotic space. Soil scientists tend to look at the same process from their own point of view. To them, roots are organic cylinders of uniform quality that attract the soil solution. They describe ion uptake as a soil process that transports ions from the bulk of the soil into physiocochemical structures that by other people are named roots. In assembling this volume, our hope was to overcome the differences in approach to ‘‘roots’’ and to combine the intricate interrelationships of the complex quartet shoots–roots–soil–microorganisms into a single functional system. We have tried, as much as possible, to unify the vocabulary. Some of the contributors have agreed to use the more universal wording, whereas other adhered to the traditional sectorial terms. The wishes of those contributors were respected, and the battle for universal terms was left for future generations. We do hope that readers will not be annoyed by the multiform use of some terms, and we shall appreciate their patience and understanding. We have tried to keep each chapter complete and independent of the others. Therefore, some repetitions were unavoidable. To minimize overlapping without impairing the comprehensive nature of each chapter, such intersections were cross-referenced. The monograph is addressed to several groups of readers: professional scientists in plant sciences, agronomy, forestry, pathology, and soil science who seek to expand their knowledge to the related fields of root biology. The book is also directed to teachers at the university level and toward students of the rhizosphere sciences who intend to join the ‘‘avant garde’’ of root biology and would like to discover the pressing questions of this field. One of the unique features of this monograph is the attempt not only to present the objective state of the art in each domain, but also to emphasize the personal view of each contributor. How do the experts perceive the cardinal points in their own field? We felt that a treatise of this kind should form a rostrum for all manner of ideas, even for those that might seem a bit weird at present. After all, the unorthodox ideas are those that pave the way for progress in science. Although this monograph does not cover all aspects of the structure and function of roots, its backbone

Preface to the First Edition

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is composed of an interesting assortment of views combined with high-quality scientific information. We do hope that the result of our endeavors will ignite the imagination of readers and increase their interest in root biology. Yoav Waisel Amram Eshel Uzi Kafkafi

Contents

Preface to the Third Edition Preface to the Second Edition Preface to the First Edition Contributors I.

II.

iii v vii xv

The Origin and Characteristics of Roots 1.

The Origin of Roots ..............................................................................................................................1 Paul Kenrick

2.

Characteristics and Functions of Root Systems ................................................................................. 15 Alastair Fitter

The Root System: Structure and Development 3.

The Root Cap: Structure and Function.............................................................................................. 33 Andreas Sievers, Markus Braun, and Gabriele B. Monshausen

4.

Cellular Patterning in Root Meristems: Its Origins and Significance ................................................. 49 Peter W. Barlow

5.

Root Hairs: Hormones and Tip Molecules......................................................................................... 83 Robert W. Ridge and Masayuki Katsumi

6.

Secondary Growth of Roots: A Cell Biological Perspective............................................................... 93 Nigel Chaffey

7.

The Kinematics of Primary Growth ................................................................................................. 113 Wendy Kuhn Silk

8.

Lateral Root Initiation...................................................................................................................... 127 Pedro G. Lloret and Pedro J. Casero

9.

Functional Diversity of Various Constituents of a Single Root System .......................................... 157 Yoav Waisel and Amram Eshel xi

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Contents

III.

IV.

V.

10.

Biomechanics of Tree Root Anchorage ............................................................................................ 175 Alexia Stokes

11.

Root Systems of Arboreal Plants...................................................................................................... 187 Hans A˚. Persson

12.

Root–Shoot Relations: Optimality in Acclimation and Adaptation or the ‘‘Emperor’s New Clothes’’? ............................................................................................................... 205 Peter B. Reich

13.

Root Life Span, Efficiency, and Turnover ........................................................................................ 221 David M. Eissenstat and Ruth D. Yanai

Root Genetics 14.

Maize Root System and Genetic Analysis of Its Formation............................................................ 239 Gu¨nter Feix, Frank Hochholdinger, and Woong June Park

15.

Root Architecture—Wheat as a Model Plant................................................................................... 249 Gu¨nther G. B. Manske and Paul L. G. Vlek

16.

Banana Roots: Architecture and Genetics ........................................................................................ 261 Xavier Draye

17.

Molecular Root Bioengineering ........................................................................................................ 279 Marcel Bucher

Research Techniques for Root Studies 18.

Root Research Methods.................................................................................................................... 295 Janina Polomski and Nino Kuhn

19.

Aeroponics: A Tool for Root Research Under Minimal Environmental Restrictions..................... 323 Yoav Waisel

20.

Use of Microsensors for Studying the Physiological Activity of Plant Roots.................................. 333 D. Marshall Porterfield

21.

Rooting of Micropropagules ............................................................................................................. 349 Geert-Jan de Klerk

22.

Modeling Root System Architecture................................................................................................. 359 Loı¨c Page`s

The Regulation of Root Growth 23.

Auxins in the Biology of Roots ........................................................................................................ 383 Thomas Gaspar, Jean-Franc¸ois Hausman, Odile Faivre-Rampant, Claire Kevers, and Jacques Dommes

24.

Gibberellins ....................................................................................................................................... 405 Eiichi Tanimoto

25.

Roots and Cytokinins ....................................................................................................................... 417 R. J. Neil Emery and Craig A. Atkins

26.

Abscisic Acid in Roots—Biochemistry and Physiology.................................................................... 435 Eleonore Hose, Angela Sauter, and Wolfram Hartung

Contents

VI.

VII.

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27.

Role of Ethylene in Coordinating Root Growth and Development ................................................ 449 Ahmed Hussain and Jeremy A. Roberts

28.

Root Signals ...................................................................................................................................... 461 Mark A. Bacon, William J. Davies, Darren Mingo, and Sally Wilkinson

29.

Environmental Sensing and Directional Growth of Plant Roots ..................................................... 471 D. Marshall Porterfield

30.

Root Growth and Gravireaction: A Critical Study of Hormone and Regulator Implications ...................................................................................................................... 489 Paul-Emile Pilet

31.

Calcium and Gravitropism................................................................................................................ 505 B. W. Poovaiah, Tianbao Yang, and A. S. N. Reddy

Physiological Aspects of Root Systems 32.

Respiratory Patterns in Roots in Relation to Their Functioning..................................................... 521 Hans Lambers, Owen K. Atkin, and Frank F. Millenaar

33.

Root pH Regulation.......................................................................................................................... 553 Jo´ska Gerenda´s and R. George Ratcliffe

34.

Nutrient Absorption by Plant Roots: Regulation of Uptake to Match Plant Demand .................. 571 Anthony D. M. Glass

35.

Dynamics of Nutrient Movement at the Soil–Root Interface .......................................................... 587 Albrecht O. Jungk

36.

Root-Induced Changes in the Availability of Nutrients in the Rhizosphere.................................... 617 Gu¨nter Neumann and Volker Ro¨mheld

37.

Simulation of Ion Uptake from the Soil........................................................................................... 651 Moshe Silberbush

38.

Soil Water Uptake and Water Transport Through Root Systems ................................................... 663 John S. Sperry, Volker Stiller, and Uwe G. Hacke

39.

Ecological Aspects of Water Permeability of Roots ......................................................................... 683 Andrea Nardini, Sebastiano Salleo, and Melvin T. Tyree

40.

Inorganic Carbon Utilization by Root Systems................................................................................ 699 Michael D. Cramer

Root Growth Under Stress 41.

Temperature Effects on Root Growth .............................................................................................. 717 Bobbie L. McMichael and John J. Burke

42.

Root Growth and Metabolism Under Oxygen Deficiency ............................................................... 729 William Armstrong and Malcolm C. Drew

43.

Trace Element Stress in Roots .......................................................................................................... 763 Ju¨rgen Hagemeyer and Siegmar-W. Breckle

44.

Root Growth Under Salinity Stress .................................................................................................. 787 Nirit Bernstein and Uzi Kafkafi

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Contents

VIII.

IX.

X.

45.

High Soil Strength: Mechanical Forces at Play on Root Morphogenesis and in Root:Shoot Signaling ............................................................................................................. 807 Josette Masle

46.

Plant Roots Under Aluminum Stress: Toxicity and Tolerance ........................................................ 821 Hideaki Matsumoto

Root–Rhizosphere Interactions 47.

Root–Bacteria Interactions: Symbiotic N2 Fixation ......................................................................... 839 Carroll P. Vance

48.

Plant Growth Promotion by Rhizosphere Bacteria .......................................................................... 869 Yoram Kapulnik and Yaacov Okon

49.

Fungal Root Endophytes .................................................................................................................. 887 Thomas N. Sieber

50.

Mycorrhizae—Rhizosphere Determinants of Plant Communities .................................................... 919 Ingrid Kottke

51.

Root–Nematode Interactions: Recognition and Pathogenicity......................................................... 933 Hinanit Koltai, Edna Sharon, and Yitzhak Spiegel

52.

Interactions of Soilborne Pathogens with Roots and Aboveground Plant Organs .......................... 949 Jaacov Katan

Roots of Various Ecological Groups 53.

Ecophysiology of Roots of Desert Plants, with Special Emphasis on Agaves and Cacti ........................................................................................................................................... 961 Park S. Nobel

54.

Contractile Roots .............................................................................................................................. 975 Norbert Pu¨tz

55.

Roots of Banksia spp. (Proteaceae) with Special Reference to Functioning of Their Specialized Proteoid Root Clusters ......................................................................................... 989 John S. Pate and Michelle Watt

56.

Ecophysiology of Roots of Aquatic Plants..................................................................................... 1007 Craig Beyrouty

Roots of Economic Value 57.

Roots as a Source of Food ............................................................................................................. 1025 Daniel F. Austin

58.

Underground Plant Metabolism: The Biosynthetic Potential of Roots.......................................... 1045 Jorge M. Vivanco, Rejane L. Guimara˜es, and Hector E. Flores

59.

Roots as a Source of Metabolites with Medicinal Activity ............................................................ 1071 Zohara Yaniv and Uriel Bachrach

Index of Organism Names Subject Index

1093 1103

Contributors

William Armstrong, Ph.D., D.Sc., F.I. Biol., C. Biol. England Owen K. Atkin, Ph.D.

Department of Biology, University of York, York, England

Craig A. Atkins, Ph.D., D.Sc. Australia Daniel F. Austin, Ph.D.

Department of Biological Sciences, University of Hull, Hull,

Department of Botany, University of Western Australia, Perth, Western Australia,

Arizona-Sonora Desert Museum, Tucson, Arizona

Uriel Bachrach, Ph.D. Jerusalem, Israel

Department of Molecular Biology, The Hebrew University–Hadassah Medical School,

Mark A. Bacon, Ph.D.

Department of Biology, Lancaster University, Lancaster, Lancashire, England

Peter W. Barlow, D.Phil., D.Sc. Bristol, England

IACR-Long Ashton Research Station, University of Bristol, Long Ashton,

Nirit Bernstein, Ph.D. Institute of Soil, Water and Environmental Sciences, Agricultural Research Organization, The Volcani Center, Bet-Dagan, Israel Craig Beyrouty, Ph.D.

Department of Agronomy, Purdue University, West Lafayette, Indiana

Markus Braun, Dr. rer. nat. Siegmar-W. Breckle, Ph.D. Germany

Institute of Botany, University of Bonn, Bonn, Germany Department of Ecology, Faculty of Biology, University of Bielefeld, Bielefeld,

Marcel Bucher, Ph.D. Federal Institute of Technology, Institute of Plant Sciences, Plant Biochemistry and Physiology, Zurich, Switzerland

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Contributors

John J. Burke, Ph.D. Plant Stress and Germplasm Development Research Unit, U.S. Department of Agriculture– Agricultural Research Service, Lubbock, Texas Pedro J. Casero, Ph.D. Department of Morphological Science and Cellular and Animal Biology, Faculty of Science, Universidad de Extremadura, Badajoz, Spain IACR-Long Ashton Research Station, University of Bristol, Long Ashton, Bristol,

Nigel J. Chaffey, Ph.D. England

Department of Botany, University of Stellenbosch, Stellenbosch, South Africa

Michael D. Cramer, Ph.D.

Department of Biology, Lancaster University, Lancaster, Lancashire, England

William J. Davies, Ph.D.

Centre for Plant Tissue Culture Research, Lisse, The Netherlands

Geert-Jan de Klerk, Ph.D.

Department of Plant Biology, University of Lie`ge, Lie`ge, Belgium

Jacques Dommes, Ph.D. Xavier Draye, Ph.D. Belgium

Department of Applied Biology, Universite´ catholique de Louvain, Louvain-la-Neuve,

Department of Horticultural Sciences, Texas A & M University, College Station, Texas

Malcolm C. Drew, D.Phil. David M. Eissenstat, Ph.D. Pennsylvania

Department of Biology, Trent University, Peterborough, Ontario, Canada

R. J. Neil Emery, Ph.D. Amram Eshel, Ph.D.

Department of Horticulture, The Pennsylvania State University, University Park,

Department of Plant Sciences, Tel Aviv University, Tel Aviv, Israel

Odile Faivre-Rampant, Ph.D. Gu¨nter Feix, Dr. rer. nat. Alastair Fitter, Ph.D.

Department of Plant Biology, University of Lie`ge, Lie`ge, Belgium

Institute of Biology III, University of Freiburg, Freiburg, Germany

Department of Biology, University of York, York, England

Hector E. Flores, Ph.D. Pennsylvania

Department of Plant Pathology, The Pennsylvania State University, University Park,

Thomas Gaspar, Ph.D.

Department of Plant Biology, University of Lie`ge, Lie`ge, Belgium

Jo´ska Gerenda´s, Ph.D.

Institute for Plant Nutrition and Soil Science, University of Kiel, Kiel, Germany

Anthony D. M. Glass, Ph.D. Columbia, Canada

Department of Botany, University of British Columbia, Vancouver, British

Rejane L. Guimara˜es Pennsylvania

Department of Plant Pathology, The Pennsylvania State University, University Park,

Uwe G. Hacke, Ph.D.

Department of Biology, University of Utah, Salt Lake City, Utah

Ju¨rgen Hagemeyer, Priv-Doz.Dr. Germany

Department of Ecology, Faculty of Biology, University of Bielefeld, Bielefeld,

Contributors

xvii

Julius von Sachs Institut, University of Wu¨rzburg, Wu¨rzburg, Germany

Wolfram Hartung, Ph.D.

Jean-Franc¸ois Hausman, Ph.D.

Crebs Research Unit, CRP-Gabriel Lippman, Luxembourg

Frank Hochholdinger, Dr. rer. nat.

Julius von Sachs Institut, University of Wu¨rzburg, Wu¨rzburg, Germany

Eleonore Hose, Ph.D. Ahmed Hussain, Ph.D.

Division of Plant Science, School of Biosciences, University of Nottingham, England Institute for Agricultural Chemistry, Georg-August University of Go¨ttingen, Go¨ttingen,

Albrecht O. Jungk, Ph.D. Germany Uzi Kafkafi, Ph.D. Rehovot, Israel

Institute of Biology III, University of Freiburg, Freiburg, Germany

Department of Field Crops, Vegetables and Genetics, The Hebrew University of Jerusalem,

Yoram Kapulnik, Ph.D. Department of Agronomony and Natural Research Institute of Field and Garden Crops, Agricultural Research Organization, The Volcani Center, Bet-Dagan, Israel Masayuki Katsumi, Ph.D. Tokyo, Japan

Department of Plant Pathology and Microbiology, The Hebrew University of Jerusalem,

Jaacov Katan, Ph.D. Rehovot, Israel Paul Kenrick, Ph.D.

Division of Natural Sciences, Biology Department, International Christian University,

Department of Paleontology, The Natural History Museum, London, England

Claire Kevers, Ph.D.

Department of Plant Biology, University of Lie`ge, Lie`ge, Belgium

Hinanit Koltai, Ph.D. Dagan, Israel

Department of Nematology, Agricultural Research Organization, The Volcani Center, Bet-

Ingrid Kottke, Ph.D. Department of Systematic Botany, Mycology and Botanical Garden, Botanical Institute, University of Tu¨bingen, Tu¨bingen, Germany Nino Kuhn, Dr. sc. techn. Department of Forest and Environmental Protection, Swiss Federal Research Institute, Birmensdorf, Switzerland Hans Lambers, Ph.D. Australia, Australia

Department of Plant Sciences, University of Western Australia, Crawley, Western

Pedro G. Lloret, Dr.Sc. Department of Morphological Science and Cellular and Animal Biology, Faculty of Science, Universidad de Extremadura, Badajoz, Spain Gu¨nther G. B. Manske, Ph.D.

Center for Development Research, University of Bonn, Bonn, Germany

Josette Masle, Ph.D. Research School of Biological Sciences, Institute of Advanced Studies, Australian National University, Canberra, Australia Hideaki Matsumoto, Ph.D.

Research Institute for Bioresources, Okayama University, Okayama, Japan

xviii

Contributors

Bobbie L. McMichael, Ph.D. Plant Stress and Germplasm Development Research Unit, U.S. Department of Agriculture–Agricultural Research Service, Lubbock, Texas Frank F. Millenaar, Ph.D. Darren Mingo, B.Sc.

Department of Plant Ecophysiology, Utrecht University, Utrecht, The Netherlands

Department of Biology, Lancaster University, Lancaster, Lancashire, England

Gabriele B. Monshausen, Dr. rer. nat.

Department of Biology, University of Trieste, Trieste, Italy

Andrea Nardini, Ph.D.

Institut fu¨r Pflanzenerna¨hrung, University of Hohenheim, Stuttgart, Germany

Gu¨nter Neumann, Ph.D. Park S. Nobel, Ph.D. Angeles, California

Institute of Botany, University of Bonn, Bonn, Germany

Department of Organismic Biology, Ecology and Evolution, University of California, Los

Yaacov Okon, Ph.D. Department of Phytopathology and Microbiology, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot, Israel Loı¨ c Page`s, Ph.D.

Unite´ Plantes et Syste`mes de Culture Horticoles, INRA, Centre d’Avignon, Avignon, France Institute of Biology III, University of Freiburg, Freiburg, Germany

Woong June Park, Ph.D.

John S. Pate, D.Sc., F.R.S., F.A.A., F.L.S. Western Australia, Australia

Department of Botany, University of Western Australia, Nedlands,

Hans A˚. Persson, Ph.D. Department of Ecology and Environmental Research, Swedish University of Agricultural Sciences, Uppsala, Sweden Institute of Biology and Plant Physiology, University of Lausanne, Lausanne, Switzerland

Paul-Emile Pilet, Ph.D.

Janina Polomski, M.Agr. Department of Forest and Environmental Protection, Swiss Federal Research Institute, Birmensdorf, Switzerland B. W. Poovaiah, Ph.D. Department of Horticulture and Program in Plant Physiology, Washington State University, Pullman, Washington D. Marshall Porterfield, Ph.D. Norbert Pu¨tz, Dr. rer. nat

Department of Biological Sciences, University of Missouri-Rolla, Rolla, Missouri

Institute for Environmental Education, University of Vechta, Vechta, Germany

R. George Ratclifffe, D. Phil.

Department of Plant Sciences, University of Oxford, Oxford, England

A. S. N. Reddy, Ph.D. Department of Biology and Program in Cell and Molecular Biology, Colorado State University, Fort Collins, Colorado Peter B. Reich, Ph.D.

Department of Forest Resources, University of Minnesota, St. Paul, Minnesota

Robert W. Ridge, Ph.D. Tokyo, Japan Jeremy A. Roberts, Ph.D.

Division of Natural Sciences, Biology Department, International Christian University,

Division of Plant Science, School of Biosciences, University of Nottingham, England

Contributors

xix

Volker Ro¨mheld, Dr. agr. ing.

Department of Biology, University of Trieste, Trieste, Italy

Sebastiano Salleo, Ph.D. Angela Sauter

Institut fu¨r Pflanzenerna¨hrung, University of Hohenheim, Stuttgart, Germany

Julius von Sachs Institut, University of Wu¨rzburg, Wu¨rzburg, Germany Agricultural Research Organization, The Volcani Center, Bet-Dagan, Israel

Edna Sharon, Ph.D.

Department of Forest Sciences, Swiss Federal Institute of Technology, Zurich,

Thomas N. Sieber, Ph.D. Switzerland Andreas Sievers, Dr. rer. nat.

Institute of Botany, University of Bonn, Bonn, Germany

Moshe Silberbush, Ph.D. Wyler Department of Dryland Agriculture, Jacob Blaustein Institute for Desert Research, Ben-Gurion University of the Negev, Sede-Boker, Israel Wendy Kuhn Silk, Ph.D. California

Department of Land, Air, and Water Resources, University of California, Davis,

John S. Sperry, Ph.D.

Department of Biology, University of Utah, Salt Lake City, Utah

Yitzhak Spiegel, Ph.D. Dagan, Israel

Professor of Nematology, Agricultural Research Organization, The Volcani Center, Bet-

Volker Stiller, Ph.D.

Department of Biology, University of Utah, Salt Lake City, Utah

Alexia Stokes, Ph.D.

Laboratoire de Rhe´ologie du Bois de Bordeaux, Cestas Gazinet, France

Eiichi Tanimoto, Dr.Sci. Department of Information and Biological Sciences, Graduate School of Natural Sciences, Nagoya City University, Nagoya, Japan Melvin T. Tyree, Ph.D. Aiken Forestry Sciences Laboratory, U.S. Department of Agriculture Forest Service, South Burlington, Vermont U.S. Department of Agriculture–Agricultural Research Service, University of Minnesota,

Carroll P. Vance, Ph.D. St. Paul, Minnesota Jorge M. Vivanco, Ph.D. Fort Collins, Colorado Paul L. G. Vlek, Ph.D. Yoav Waisel, Ph.D.

Department of Horticulture and Landscape Architecture, Colorado State University,

Center for Development Research, University of Bonn, Bonn, Germany

Department of Plant Sciences, Tel Aviv University, Tel Aviv, Israel

Michelle Watt, Ph.D. Plant Cell Biology Group, Research School of Biological Science, Australian National University, Canberra, Australia Sally Wilkinson, Ph.D. Ruth D. Yanai, Ph.D. New York

Department of Biology, Lancaster University, Lancaster, Lancashire, England College of Environmental Science and Forestry, State University of New York, Syracuse,

xx

Tianbao Yang, Ph.D.

Contributors

Department of Horticulture, Washington State University, Pullman, Washington

Zohara Yaniv, Ph.D. Department of Genetic Resources and Seed Research, Agricultural Research Organization, The Volcani Center, Bet-Dagan, Israel

1 The Origin of Roots Paul Kenrick The Natural History Museum, London, England

I.

INTRODUCTION

the silent and unobtrusive cycling of minerals from soil to biosphere on a truly prodigious scale (Epstein, 1977), and root-mediated weathering of silicates is thought to have had worldwide consequences for the carbon cycle (Berner, 1998). This chapter reviews the origin and early evolution of roots in the major groups of vascular plants, focusing on new data from the fossil record, which is interpreted using modern phylogenetic methods. In addition to providing a narrative of what happened and when, the evolution of root systems and the homologies of roots are considered in detail. One aim is to provide the experimental biologist with a rational for choosing model organisms for research in root developmental biology. A second aim is to provoke the reader into testing some of the ideas of homology, which have consequences that could be examined at the molecular developmental level.

Roots originated during the early phase of the diversification of plants on land in the Devonian Period, some 363 million to 409 million years ago. This was a time of enormous changes in plant life that were to have far-reaching consequences for the evolution of land animals and for the chemical economy of life on earth (Epstein, 1977; DiMichele et al., 1992; Kenrick and Crane, 1997b; Algeo and Scheckler, 1998; Bateman et al., 1998; Berner, 1998). From small rootless organisms a few centimeters tall, plants evolved into large shrubs and trees with a range of specialized rooting structures. Roots combined with a fully integrated vascular system were essential to the evolution of large plants, enabling them to meet the requirements of anchorage, water, and nutrient acquisition. Large roots with secondary wood were widespread by the Late Devonian (378Ma) (Retallack, 1986; Algeo and Scheckler, 1998), possibly earlier (Elick et al., 1998), and this development coincided with the appearance of the earliest forest ecosystems. The impact of roots on the evolution of soils was enormous. Physical effects, such as the fracturing of rock, the binding of loose particles, and the introduction of large quantities of organic material, combined with the chemical consequences of actively pumping solutes through the system, led to the development of soils with modern profiles. Plants ploughed, tilled, and fertilized the land, and in so doing had a lasting influence on Earth geochemistry. The origin of roots set in motion

II.

ROOTS IN THE FOSSIL RECORD

Paleobotanists are continually hampered in their attempts to reconstruct plants from the past because much of the fossil record comprises broken or disarticulated fragments preserved in sediments deposited in rivers or lakes. Piecing together plants to form a conceptual whole is a difficult and painstaking task, and frequently one must be satisfied with an incomplete organism, simply because parts are missing. These missing parts are often the roots. Despite the difficul1

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ties inherent in reassembling whole plants, roots are occasionally preserved in fossils that have literally been uprooted and transported some distance. Also, it is possible to find environments where plants have been fossilized at their site of growth. Under circumstances such as these, preservation of roots can be exceptionally good. A.

Early Rooting Systems

1. The Rhynie Chert—An Early Terrestrial Biota The earliest examples of rooting structures preserved in growth position come from the 396-million-year-old Rhynie Chert from Scotland. This sequence of fossiliferous cherts—rocks composed of finely crystalline silica—provides a window onto an early terrestrial environment, capturing a period when plant life on land was at an early stage of development. Studies of the depositional environment of the chert show that plants grew on sandy substrates in and around the margins of pools on a plain that was receiving sediment from surrounding higher ground. Hydrothermal activity, driven by local volcanism, caused periodic flooding of the low-lying areas with hot water saturated in silicates, resulting in inundation and preservation of whole plants and the formation of the cherts (Trewin, 1994; Rice et al., 1995). Petrographic thin sections are the most widely used method of investigating and reconstructing the plants, and they reveal amazing details of cell structure. The first description of four vascular plants by Kidston and Lang (1921) revolutionized our understanding of the early land flora. Since then, the Rhynie Chert has continued to yield remarkable new information on the morphology and life cycles of the plants and arthropods of the early Devonian (Kevan et al., 1975; Rolfe, 1980; Edwards and Lyon, 1983; Lyon and Edwards, 1991; Taylor et al., 1992a,b; Remy et al., 1993; Kenrick, 1994; Remy et al., 1994; Taylor et al., 1995, 1997, 1999). 2. Stems Functioning as Roots The Rhynie Chert plants were all small and there is strong evidence that several species lacked roots. In these plants, absorptive and perhaps anchoring functions were performed by nonseptate rhizoids borne on prostrate stems (rhizomes) (Fig. 1). Rhizoids of this type have been documented in the extinct protracheophytes Horneophyton (Kidston and Lang, 1920a, 1921) and Aglaophyton (Kidston and Lang, 1920a). In Aglaophyton, rhizoid structure and development have

Figure 1 Unicellular rhizoids on the rooting stem of a fossil plant from the 396-million-year-old Rhynie Chert, Scotland. The Rhynie plants are silicified and have been fossilized in their growth position.

been studied in some detail (Edwards, 1986; Remy and Hass, 1996). Rhizoids developed from stomatal subsidiary cells. In rhizomes, these epidermal cells underwent division to produce a series of rectangular rhizoid mother cells. Simultaneously, cell division in the hypodermis produced a raised area on the stem. Mature rhizoids were therefore borne on small mounds on the lower surface of the rhizome and probably also on basal regions of erect stems. Stomates were interspersed among the rhizoids. Another specialization included the differentiation of some hypodermal cells into transfusion tissue that was connected with the vascular system. The evidence from Aglaophyton indicates that in protracheophytes (Fig. 2a) there was no clear developmental distinction between stem and rhizome. The rhizome was simply an older portion of the stem that underwent a limited amount of further differentiation to function as a rooting system. The development of rhizomes from prostrate stems resembling aerial branches was also a characteristic of many early vascular plants. The rhizome was much branched in groups such as zosterophylls—early relatives of living clubmosses (Lang, 1927; Lele and Walton, 1961; Gerrienne, 1988; Hao, 1989a) (Fig. 2b). In some species, multicellular trichomelike appendages were borne on both rhizomes and erect stems

Origin of Roots

3

(e.g., Hao, 1989b). The function of these appendages is unknown. Rhizoid development on rhizomes and at the basal regions of stems was probably widespread, but details such as these are seldom preserved in compression fossils formed in sandstone and shale. Exceptionally, preservation can occur where there has been substantial cutinization that extends to the rhizoids themselves, a phenomenon that has been observed in both bryophytes and vascular plants. One such example is the zosterophyll Serrulacaulis (Hueber and Banks, 1979). Preserved cuticles show that large deltoid spines were borne on stems of all sizes and that nonseptate heavily cutinized rhizoids developed on and among spines in the basal regions. 3.

Figure 2 Phylogenetic relationships among basal land plants. y ¼ extinct. A general summary is presented in part (a) and details of the two major groups within vascular plants in (b) (lycopsids) and (c) (euphyllophytes). (a) Relationships among land plants. Most phylogenetic analyses resolve bryophytes as paraphyletic to vascular plants, but relationships among the three major groups of bryophytes depicted are poorly resolved (for recent reviews and alternative hypotheses see Duff and Nickrent, 1999; Hedderson et al., 1998; Kenrick, 2000; Renzaglia et al., 2000). Note that nearly all known early fossil land plants are vascular plants or stem group vascular plants (Kenrick, 2000; Kenrick and Crane, 1997a,b). (b) Relationships among lycopsids with aspects of root evolution depicted (boxed text) (adapted from Kenrick and Crane, 1997a). For a detailed discussion of root homologies in lycopsids, see Rothwell and Erwin (1985). (c) Relationships among euphyllophytes from Rothwell (1999). For alternatives ideas see Kenrick and Crane (1997a), Pryer et al. (1995), and Stevenson and Loconte (1996).

Branches with Uniquely Rooting Function

An increased level of differentiation occurred in the early vascular plant Asteroxylon, also from the Rhynie Chert. Unlike the aerial stems, the rhizome was leafless and there were differences in the vascular system, which in stems was stellate but in the rhizoids was terete or elliptical. This difference is probably a consequence of the absence of vascular appendages (microphyllous leaves) in the rhizome. All branching appears to have been exogenous and there is no evidence of rhizoids or root hairs (Kidston and Lang, 1920b, 1921). Asteroxylon is related to lycopsids (Fig. 2b), and broadly similar roots have been documented in other less well preserved compression fossils of closely related plants such as Bathurstia (Kotyk and Basinger, 2000) and Drepanophycus (Rayner, 1984). These fossils document the early appearance of multicellular vascularized axes that are uniquely rooting in function. Dormant meristematic regions that have the potential to develop into new stems and perhaps roots were common to many early vascular plants (e.g., zosterophylls). These were most frequent on rhizomes but were also present on erect stems where they were usually confined to branch axils (Kenrick and Crane, 1997a). Sometimes they extended over a greater portion of the erect system (Edwards and Kenrick, 1986). These branches were exogenous, and they are known to have developed into new aerial branches in some species, whereas in others it is possible that they developed into roots (Edwards and Kenrick, 1986; Remy et al., 1986; Li, 1992). The pattern of distribution and the developmental plasticity of these enigmatic meristematic regions are notable similarities to the widely discussed rhizophore of living Selaginellaceae. Phylogenetic evidence indicates, however, that homol-

4

Kenrick

ogy is highly unlikely (Kenrick and Crane, 1997a) (Fig. 2b). The reason for this is that Selaginellaceae are phylogenetically remote from those zosterophylls with dormant meristems. Assuming homology of these structures would therefore be highly unparsimonious because it would imply multiple losses of dormant meristems/rhizophores in intervening groups. It has been suggested that the function of dormant meristems in the life cycle of the plant was to facilitate vegetative growth or reproduction or perhaps to enable regeneration following catastrophic burial by flood. 4. Bipolar Growth Plants with upright trunks and larger rooting systems are present in the fossil record by the Mid-Devonian (ca 380Ma). On the whole, these plants were still small and most had no secondary tissues. The Cladoxylopsida were a group of fernlike plants of uncertain systematic position. They are clearly euphyllophytes (i.e., members of the group that contains modern seed plants, ferns, and horsetails; Fig. 2c), but their precise relationships are poorly understood. This group has been implicated in the origins of both ferns and horsetails (Stein et al., 1984; Rothwell, 1999). One uprooted specimen, Lorophyton goense, provides an insight into the morphology of the whole plant. L. goense had a 2-cm-diameter trunk that bore tufts of leaflike branches from the apex and numerous bifurcating roots (maximum 1 cm diameter) from a slightly flared base. The whole plant is estimated to have been about 40 cm in height (Fairon-Demaret and Li, 1993). Schweitzer and Li (1996) have documented a similar growth form in the 50-cm-tall lycopsid Chamaedendron by the early part of the Late Devonian (Frasnian; 377Ma). The roots of Chamaedendron bifurcated at least four times and were at least 15 cm long (Fig. 3). These fossils mark an early departure from essentially unipolar to a form of bipolar growth. Roots were clearly differentiated as such, and they did not function as stems at any time during their ontogeny. B.

Roots of Trees

Trees first appear in the fossil record in the Late Devonian, and arborescence was widespread in progymnosperms—the free-sporing ancestors of living seed plants—and in clubmosses and horsetails, groups whose living relatives are predominantly herbaceous (Mosbrugger, 1990). This dramatic increase in plant

Figure 3 Uprooted fossil. A 377-million-year-old clubmoss (lycopsid) Chamaedendron multisporangiatum from China (Schweitzer and Li, 1996). The specimen illustrated is 35 cm long and shows a bifurcating root systems (R), a bifurcating stem (S) bearing numerous microphyllous leaves, and a slightly expanded root/stem junction (R/S).

Origin of Roots

size was a direct consequence of the evolution of the cambium, an innovation that also led to the development of much more extensive root systems. 1.

Horsetails

Paleozoic tree horsetails had roots that are very similar to those of their living relatives, which are all herbaceous (Eggert, 1962). The rhizome of living Equisetum is a modified stem that produces aerial branches and adventitious roots. During the Carboniferous the related tree horsetail Calamites grew to a height of 20 m. The rooting system was comparatively shallow and comprised an enormous, 40-cm-diameter horizontal rhizome that bore adventitious roots in whorls at nodes, much like branches on the erect stems. Roots also developed at the base of upright branches. Anatomically, the calamite rhizome was very similar to the stem, and it contained a large amount of secondary wood. The older and somewhat smaller horsetail Archaeocalamites (Early Carboniferous; Tournaisian; 363Ma) was of similar construction but had a greatly expanded woody rootstock at the base of a stem that bore numerous adventitious roots (Bateman, 1991). Scrambling or climbing forms such as Sphenophyllum produced adventitious roots at nodes, often with leaves. In this plant, it seems likely that as stem length increased basal portions assumed a horizontal position (Stewart and Rothwell, 1993). The rhizome-based rooting system of horsetails probably originated with plants such as the Mid-Devonian Metacladophyton, in which roots (8 cm long; 2.5 mm wide) were borne along one side of a horizontal rhizome (1 cm diameter) that developed into an upright stem (Wang and Geng, 1997). 2.

Clubmosses

Paleozoic tree clubmosses had highly distinctive roots that are termed rhizomorphs. The rhizomorph was a determinate structure that branched dichotomously to form a shallow but laterally extensive root system that would have exceeded 10 m in diameter in larger trees (Frankenberg and Eggert, 1969). Root apices terminated abruptly and bore a rimlike apical groove (Rothwell, 1984). Rootlets were cylindrical and helically arranged, developed near the apical meristem, and are thought to have been exogenous (Rothwell and Erwin, 1985). Each rootlet contained a large, airfilled cavity. No evidence of root caps or root hairs has been documented, but this may reflect poor preservation and lack of data from rootlet apices. On older portions, secondary growth of cortex and stele caused

5

rootlets to be abscised. Other Late Paleozoic arborescent clubmosses had a rather different rooting structure. Instead of the extended branched rhizomorph, plants such as Chaloneria and Paurodendron had unbranched rootstocks that were comparatively small and rounded, and that had a lobed cormose base (Pigg and Rothwell, 1983; Rothwell and Erwin, 1985). Rootlets of both the rhizomorphic and cormose club mosses had a unique type of stele located at the periphery of a large cavity. This is one of several striking anatomical similarities with the roots of their living relatives, Isoetes (Stewart, 1947). 3.

Tree Ferns

Tree ferns buttressed by extensive root mantles first appeared in the Early Carboniferous and became abundant during the Late Carboniferous and Permian. In general habit the earliest tree ferns resembled modern Dicksoniaceae and Cyatheaceae, but early fossils such as Psaronius are in fact most closely related to the Marattiales (Fig. 2c). Trunks of Psaronius belong to a plant that is estimated to have grown to 10 m in height. An extensive mantle of bifurcating roots that was widest at the base of the plant surrounded a comparatively narrow stem comprising exclusively primary tissues. Leaves were produced from a narrow crown. Roots arose endogenously from the peripheral vascular bundle of the stem and grew downward and outward to the periphery of the trunk. The roots of Psaronius are among the largest known for ferns, growing up to 2 cm in diameter (Ehret and Phillips, 1977). In some families of ferns and possibly also in some early seed plants, root mantles provided a ready alternative to cambial activity as a means of structural support in the evolution of the tree habit. 4.

Progymnosperms

Progymnosperms are a grade of extinct, free sporing plants that are closely related to seed plants (Fig. 2c). In many aspects of their morphology, progymnosperms are intermediate between pteridophytes and gymnosperms. Plants bore fernlike foliage and they ranged from small or medium-size homosporous shrubs (Aneurophytales) to large heterosporous trees (Archaeopteridales). Unlike ferns, progymnosperms produced gymnospermous wood from a bifacial cambium—one that produces both secondary xylem and phloem (Beck, 1971; Scheckler and Banks, 1971; Beck and Wight, 1988). Similarities with gymnosperms extended also to the roots (Scheckler, 1995).

6

Aneurophytales—the first progymnosperms to appear in the Mid-Devonian (Eifelian)—had bifurcating roots that were probably determinate. Most roots were endogenous, and some were adventitious. Cambial activity is evident in roots and rootlets of all sizes. Roots possessed cortical lacunae, periderm, persistent root hairs and possibly an exodermis. There is no evidence of mycorrhizae (Scheckler, 1995). Recent data from petrified trunks indicate that in addition to a well-developed root system with secondary wood, Archaeopteris had adventitious latent primordia similar to those produced by some living trees, which eventually develop into roots on stem cuttings (MeyerBerthaud et al., 1999). In the progymnosperm Eddya sullivanensis—possibly a juvenile Archaeopteris—there was a complex tap root system, comprising a strong main root that gave rise to a profusion of small, much branched, probably endogenous laterals (Beck, 1967).

Kenrick

(Delevoryas, 1955). Early seed plants occupied a broad range of environments including peat-forming swamps, flood plains, extra basin lowlands, and probably even higher ground (DiMichele et al., 1992). In some swamp-dwelling forms there is evidence for adaptation to growth under very wet conditions. Cordaites was a group of scrambling and upright shrubs or small trees that is related to conifers (Rothwell, 1988). Some Carboniferous swamp species had highly branched stilt roots that supported the stem (Cridland, 1964). Anatomically, these roots had an exarch actinostele with secondary xylem similar to that in the stem, well-developed periderm, lenticels that formed in the periderm, and a broad zone of aerenchymatous phelloderm that formed around the stele. The combination of aerenchyma, medullated protosteles, periderm, and lenticels has been likened to the stilt roots of plants growing in modern mangrove environments (Cridland, 1964).

5. Seed Plants The earliest seed plants were small to medium-size shrubs, many of which had a scrambling or semi-selfsupporting habit (Rowe et al., 1993). In this respect they were very similar to aneurophytalean progymnosperms. Adventitious roots borne along the stem are known to have been a feature of many species. In Callistophytaceae roots on aerial stems were axillary to buds or branches (Rothwell, 1975), they were also closely associated with leaves in Calamopityaceae (Meyer-Berthaud and Stein, 1995). However, in Lyginopteridaceae such roots were sometimes organized into vertical rows without apparent relation to other appendages (Taylor and Taylor, 1993). In all species there were many anatomical similarities between stems and roots. The stele was well defined and in some species possessed endodermis and pericycle. Secondary xylem and periderm were present in larger roots. The primary xylem was usually protostelic and diarch. Structures resembling secretory cells and resin canals have been documented in some species (Taylor and Taylor, 1993). Lateral roots were endogenous. Other early seed plants such as Medullosa were small trees (
4–5 m tall) with an upright trunk, and large, bifurcating, fernlike fronds (Stidd, 1981). The lower portion of the stem bore a periderm and numerous adventitious roots, and these roots are a common component of Carboniferous petrifactions (Rothwell and Whiteside, 1974). Roots turned downward between leaf bases and emerged near the base of the stem. Larger roots produced secondary xylem

III.

DISCUSSION

The early fossil record of land plants documents a continuum of variation in the evolution of rooting systems that began with simple rhizoid-bearing stems and developed, over a period of 40 million years, into a broad range of complex multicellular organs specialized in anchorage and nutrient acquisition. To define the term ‘‘root’’ one must draw an arbitrary line in this morphological continuum, and in applying the term to fossils further complications arise from a lack of anatomical information. Frequently, data on the presence/ absence of root cap and root hairs and on root development (endogenous or exogenous) are not available. Here, roots are defined as multicellular vascular organs without leaves or other multicellular appendages that function in anchorage and the acquisition of solutes and nutrients. A.

Root Origins

Fossil evidence demonstrates that roots evolved on land and that they were an early innovation of plant life. Roots, as defined here, are unique to vascular plants, but simpler functionally equivalent systems are more widespread. Rhizoid-bearing stems occur in living bryophytes and even some green algae. Evidence from complete plants fossilized in growth position in the Rhynie Chert confirms the presence of similar systems in the earliest vascular plants. In these plants, rhizoids probably developed in response to physical

Origin of Roots

contact of the stem with a substrate. The widespread occurrence of simple rhizoid-based systems in land plants indicates that this form of rooting system preceded the evolution of true roots and may well have been a legacy inherited from green algal ancestors. Shallow, rhizoid-bearing stems are adequate for small (30–50 cm tall) plants with low transpiration and a simple vascular system, but also present in the Rhynie Chert were plants with organs that were clearly and uniquely specialized for rooting. Asteroxylon and its cosmopolitan relative Drepanophycus possessed small roots that were probably positively geotropic. These plants are related to clubmosses, but other basal fossils in this clade are known to be rootless. Viewed in a phylogenetic context (Fig. 2b), the roots of lycopods are uniquely derived and are therefore not homologous with those of other vascular plants (Kenrick and Crane, 1997a).

B.

Root Diversity in the Late Devonian and Carboniferous

By the Late Devonian and Early Carboniferous an enormous variety of rooting structures had evolved and roots had been co-opted for an equally broad range of additional functions. The evolution of large erect plants and in particular trees placed increasing demands upon rooting systems, and these were solved in a variety of ways. From the outset, differentiation of tissues in the roots of arborescent plants followed closely patterns of tissue differentiation in the erect trunk. One of the major sources of divergence in root and stem anatomy resulted from the influence of lateral appendages in the stem. Arborescent horsetails maintained a rhizomatous rooting system that simply increased in girth through the addition of secondary tissues. Progymnosperms developed bipolar growth, where shoot and root were probably determined early in ontogeny as in their living relatives, the seed plants. Unlike horsetails, progymnosperms and seed plants had the ability to produce deeply penetrating roots. Bipolar growth also occurred in the extinct tree lycopods, but phylogenetic and ontogenetic studies clearly demonstrate that this growth form evolved independently of that in seed plants (Frankenberg and Eggert, 1969; Stubblefield and Rothwell, 1981; Rothwell and Erwin, 1985; Bateman et al., 1992). Roots were co-opted as buttresses or mantles in the formation of the false trunks of tree ferns. These provided a substrate for vascular plant epiphytes and root climbers, which are first observed in the fossil record

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on tree ferns in the Carboniferous Period (Rothwell, 1991; Krings and Kerp, 1997; Ro¨ßler, 2000). C.

Mycorrhizae—An Early Symbiosis

Root-mediated fungal symbioses are characteristic of most living land plants. Recent inferences from phylogenetic studies and direct observations from the fossil record demonstrate that this form of symbiosis is ancient. Most fungal symbioses of roots involve zygomycetes in the Glomales, which form arbuscular mycorrhizae in the rooting systems of bryophytes and most vascular plants (Smith and Read, 1997). The distribution of arbuscular mycorrhizae among living plants is consistent with an ancient origin of this plant/fungal relationship (Pirozynski and Malloch, 1975; chapter by Read in this volume). This hypothesis is borne out by two recent studies. A phylogenetic analysis of living members of the Glomales based on SSU rRNA reported an origin of endomycorrhizal associations in plants between 462 million and 353 million years ago (Simon et al., 1993). Ages were estimated based on the application of a molecular clock. These results were confirmed by direct observations of fossil endomycorrhizae in plants from the Rhynie Chert (Remy et al., 1994). Nonseptate hyphae and arbuscules have been observed in specialized meristematic regions of the cortex of Aglaophyton and Rhynia. In these plants, the fungal infection is widespread throughout the rhizome and the erect stems. The presence of arbuscular mycorrhizae in living bryophytes and in extinct plants such as Aglaophyton and Rhynia demonstrates that this fungal symbiosis preceded the evolution of roots. D.

Homologies of Roots in Clubmosses (Lycopods)—Observational Consequences in Living Plants

The origin of roots provides some remarkable examples of the co-option of organ systems from one role to a completely different one. Perhaps the most spectacular of these is the hypothesized origin of the root system in extinct tree lycopods. It has long been suspected that the unique rooting system of tree lycopods—the so-called rhizomorph—is in fact a shoot modified for rooting (Frankenberg and Eggert, 1969). The modified shoot hypothesis is based on similarities in anatomy, organization of appendages (rootlets/leaves), and embryology. Rhizomorphs, like stems, had a pith and an endarch xylem; rootlets were borne and abscised in a similar manner to leaves (Rothwell and

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Erwin, 1985); and evidence from fossil embryos shows that the rooting system developed from an early dichotomy of the shoot apex (Stubblefield and Rothwell, 1981). Under this interpretation ‘‘true’’ roots in tree lycopods have been lost and were substituted by a novel shoot system co-opted to a rooting function—developmental changes that may have accompanied the evolution of arborescence during the Devonian Period. The main trunk root is therefore interpreted as a transformed stem and the rootlets are modified leaves. This hypothesis is potentially testable because it has observational consequences in living plants. The closest living relatives of the tree lycopods are Isoetaceae (Bateman et al., 1992; Pigg, 1992; Kenrick and Crane, 1997a) (Fig. 2b). This ancient group comprises
130 species of terrestrial or aquatic plants with elongate linear leaves (sporophylls) borne, in most species, on a very short trunk (Jermy, 1990). Despite the short trunk length—which is interpreted as highly reduced—there are many similarities between Isoetaceae and their arborescent fossil relatives (Stewart, 1947). The interpretation of root origins in Isoetaceae, however, remains controversial. Noting some fundamental differences in embryogeny, Paolillo (1982) concluded that the root of Isoetaceae is homo-

Kenrick

logous with the embryonic roots of Lycopodiaceae and Selaginellaceae. In other words, these are true roots and not homologs of the rhizomorph. Rothwell and Erwin (1985) suggested an alternative hypothesis. Based on a reanalysis of the embryological data and on new information from fossils they concluded that the roots of Isoetaceae are in fact homologous with the rootlets in the rhizomorphic system. This conclusion is supported by the close anatomical similarities between Isoetes roots and those of extinct Stigmaria. If roots in these two plants are homologous, then by implication thoses of Isoetes also originated as rooting leaves. Modern molecular biology could provide a decisive test of the two hypotheses. If the root/leaf homology is correct, then the roots of Isoetaceae should share a greater similarity at the molecular developmental level with their own leaves than with the roots of close relatives in Lycopodiaceae or Selaginellaceae. E.

Roots, Soils, and the Devonian Environment

The Devonian Period witnessed a progressive increase in root biomass (Fig. 4) that had an enormous impact on various aspects of the environment, notably the development of soils and the evolution of the atmo-

Figure 4 Relative size, morphology, and penetration depth of root systems from selected Devonian plants. (From Algeo and Scheckler, 1998.)

Origin of Roots

sphere. Prior to the Devonian, soils, if developed at all, are thought to have been predominantly thin and of microbial origin (Retallack, 1990, 1992). The rhizomatous root systems of the earliest land plants were shallow and essentially superficial. They are unlikely to have contributed much to soil formation or to the weathering of the land surface. By the MidDevonian, the development of positively geotropic roots coupled with a progressive increase in plant size led to more extensive and penetrating rooting systems. These nurtured an environment suitable for the stabilization of shallow soils. A further significant step in the development of soils was the evolution of the cambium, which from the outset was expressed in both roots and stems. Cambial activity led rapidly to significant increase in plant size and the extent and depth of roots. Soil penetration depths were shallow (< 20 cm) during the Eifelian-Givetian (386–377Ma), but increased to 80–100 cm as archaeopterids spread during the Frasnian-Famenian (377–363Ma) (Algeo and Scheckler, 1998; Fig. 4). The diversity of soils also increased during the Devonian Period. This change was brought about by root-induced weathering and mixing. Roots increase rates of weathering by facilitating the pumping of solutes through soils and through the release of humic acids. Translocation of clays and iron was first recorded in wet lowland soils in the early Frasnian (Retallack, 1985). The effectiveness of soil mixing was boosted through anatomical innovations in roots (Algeo and Scheckler, 1998). The evolution of the cambium initiated continuous perennial root growth and long-term survival of roots in soils. The development of endogenous and adventitious roots, particularly in progymnosperms, enabled repeated penetration of a given soil volume. By the end of the Devonian there was an increase in soil clay content, structure, and profile maturity that correlates with increases in the depth of root penetration. Soils with modern profiles are recognizable at this time (Retallack, 1990, 1992). Deeper soils and larger plants go hand in hand, but the evolution of both may have been more intimately connected than previously suspected. One of the most remarkable phenomena of the Late Devonian is the independent but probably concurrent evolution of large trees in several plant groups. Mosbrugger (1990) argued that this phenomenon may in fact be caused by positive feedback between soil and plant, mediated by the roots. In essence, the idea is based on the observations that large plants generally require more stable and comparatively deeper soils, and that plants themselves contribute to soil formation. Under

9

these circumstances, increments in plant size are expected to lead to increments in the quality and depth of the soil profile, which in turn would provide soils suitable for even larger plants. Plant size and soil depth therefore increased in parallel until other limiting factors came into play. The broader relevance of the root/soil relationship to the early evolution of terrestrial and marine ecosystems is widely appreciated (Retallack, 1990, 1992; Algeo and Scheckler, 1998; Berner, 1998). One area of particular importance is the effect of root-mediated weathering on the atmosphere. In the long-term or geochemical carbon cycle, Ca and Mg silicate minerals in soils react with atmospheric CO2 in solution (HCO 3 ) to produce CaCO3 and MgCO3. These carbonates are transferred to the oceans by rivers, where they are precipitated as minerals. Carbonate formation combined with the burial of organic carbon are responsible for most of the draw down of CO2 from the atmosphere. Carbon dioxide is eventually returned to the atmosphere over long time scales by the weathering of organic carbon and by the thermal breakdown of carbonates at depth (Berner, 1998). The evolution of roots is thought to have had a major influence on this process by increasing the rate of weathering of Ca/Mg silicates. This phenomenon is a byproduct of the release of humic acids by roots, the channeling of solutes through the soil profile, and the physical effects of root penetration. Observations on modern root systems indicate that roots can increase weathering by a factor of 7 (Drever and Zobrist, 1992; Cochran and Berner, 1993; Berner, 1994; Cochran and Berner, 1997). By including a root effect factor, models of atmospheric gases through the Phanerozoic predict a massive decline in CO2 concentrations during the Paleozoic from 4–20PAL (pre-Devonian) to 1PAL (mid Carboniferous) (Berner, 1994), and this is broadly consistent with data from other sources (Yapp and Poths, 1992; Mora et al., 1996; McElwain, 1998).

IV.

LEADING TOPICS, GAPS, AND THE FUTURE

The investigation of root origins depends critically on information from the fossil record and on the development of a well-corroborated phylogenetic framework for interpreting the data. Here, I suggest three areas of special relevance to future progress in understanding root origins: 1. Fossils are important because they bridge the huge morphological gaps that separate the major

10

groups of living land plants. In this respect, the early fossil record of vascular plants is of enormous value and it is yielding data relevant to a raft of issues concerning root origins. The study of plants fossilized in their growth position is providing the most significant results, and the exceptional Rhynie Chert biota is a key source of data (e.g., Remy et al., 1994; Remy and Hass, 1996). More high-quality and detailed data on root morphology are needed from vascular plants in the euphyllophyte and lycopsid stem groups. 2. The integration of morphological data from living and fossil plants into phylogenetic research is having a major impact on our understanding of plant morphology. One consequence is that botanists are able to construct more rigorous hypotheses of homology. In discussing the origins of organs, this helps to sharpen the focus of an investigation by narrowing it down to a small number of plausible alternative hypotheses. Progress in our understanding of land plant phylogeny has been spectacular in the past 10 years, but further integration of molecular phylogenies with those that include fossils is necessary to fully exploit the paleobotanical data. 3. Recent advances in the field of molecular developmental biology provide a powerful new set of tools that could be applied to the investigation of root origins. Although the focus of this chapter has been on the fossil record, some of the hypotheses on root origins discussed here have observational consequences for living plants. REFERENCES Algeo TJ, Scheckler SE. 1998. Terrestrial-marine teleconnections in the Devonian: links between the evolution of land plants, weathering processes, and marine anoxic events. Phil Trans R Soc Lond B Biol Sci 353:113–130. Bateman RM. 1991. Palaeobiological and phylogenetic implications of anatomically-preserved Archaeocalamites from the Dinantian of Oxroad Bay and Loch Humphrey Burn, southern Scotland. Palaeontographica B223:1–59. Bateman RM, DiMichele WA, Willard DA. 1992. Experimental cladistic analysis of anatomically preserved lycoposids from the Carboniferous of Euramerica: an essay on paleobotanical phylogenetics. Ann Mo Bot Gdn 79:500–559. Bateman RM, Crane PR, DiMichele WA, Kenrick P, Speck T, Rowe NP, Stein WE. 1998. Early evolution of land plants: phylogeny, physiology, and ecology of the primary terrestrial radiation. Annu Rev Ecol Syst 29:263– 292.

Kenrick Beck CB. 1967. Eddya sullivanensis, gen. et sp. nov., a plant of gymnospermic morphology from the Upper Devonian of New York. Palaeontographica B121:1–21. Beck CB. 1971. On the anatomy and morphology of lateral branch systems of Archaeopteris. Am J Bot 58:758-784. Beck CB, Wight DC. 1988. Progymnosperms. In: Beck CB, ed. Origin and Evolution of Gymnosperms. New York; Columbia University Press, pp 1–84. Berner RA. 1994. GEOCARB II: a revised model of atmospheric CO2 over Phanerozoic time. Am J Sci 294:56– 91. Berner RA. 1998. The carbon cycle and CO2 over Phanerozoic time: the role of land plants. Phil Trans R Soc Lond B Biol Sci 353:75–82. Cochran MF, Berner RA. 1993. Enhancement of silicate weathering rates by vascular land plants: quantifying the effect. Chem Geol 107:213–215. Cochran MF, Berner RA. 1997. Promotion of chemical weathering by higher plants: field observations on Hawaiian basalts. In: Stillings LL, ed. Chemical and Biological Control on Mineral Growth and Dissolution Kinetics. Amsterdam; Elsevier, pp 71–77. Cridland AA. 1964. Amyelon in American coal balls. Palaeontology 7:186–209. Delevoryas T. 1955. The Medullosae—structure and relationships. Palaeontographica B97:114–167. DiMichele WA, Hook RW, Beerbower R, Boy JA, Gastaldo RA, Hotton III N, Phillips TM, Scheckler SE, Shear WA, Sues H-D. 1992. Paleozoic terrestrial ecosystems. In: Behrensmeyer AK, Damuth JD, DiMichele WA, Potts R, Sues H-D, Wing SL, eds. Terrestrial Ecosystems Through Time: Evolutionary Paleoecology of Terrestrial Plants and Animals. Chicago; University of Chicago Press, pp 205–325. Drever JI, Zobrist J. 1992. Chemical weathering of silicate rocks as a function of elevation in the southern Swiss Alps. Geo Cos Act 56:3209–3216. Duff JR, Nickrent DL. 1999. Phylogenetic relationships of land plants using mitochondrial small-subunit rDNA sequences. Am J Bot 86:372–386. Edwards DS. 1986. Aglaophyton major, a non-vascular landplant from the Devonian Rhynie Chert. Bot J Lin Soc 93:173–204. Edwards D, Kenrick P. 1986. A new zosterophyll from the Lower Devonian of Wales. Bot J Lin Soc 92:269–283. Edwards DS, Lyon AG. 1983. Algae from the Rhynie Chert. Bot J Lin Soc 86:37–55. Eggert DA. 1962. The ontogeny of Carboniferous arborescent sphenopsida. Palaeontographica B110:99–127. Ehret DL, Phillips TL. 1977. Psaronius root systems—morphology and development. Palaeontographica B161:147–164. Elick JM, Driese SG, Mora CI. 1998. Very large plant and root traces from the Early to Middle Devonian; implications for early terrestrial ecosystems and atmospheric pCO2. Geology 26:143–146.

Origin of Roots Epstein E. 1977. The role of roots in the chemical economy of life on earth. Bioscience 27:783–787. Fairon-Demaret M, Li C-S. 1993. Lorophyton goense gen. et sp. nov. from the Lower Givetian of Belgium and a discussion of the Middle Devonian Cladoxylopsida. Rev Pal Pal 77:1–22. Frankenberg JM, Eggert DA. 1969. Petrified Stigmaria from North America. I. Stigmaria ficoides, the underground portions of Lepidodendraceae. Palaeontographica B128:1–47. Gerrienne P. 1988. Early Devonian plant remains from Marchin (north of Dinant Synclinorium, Belgium). I. Zosterophyllum deciduum sp. nov. Rev Pal Pal 55:317335. Hao S-G. 1989a. Gumuia zyzzata—a new plant from the Lower Devonian of Yunnan, China. Act Bot Sin 31:954–961. Hao S-G. 1989b. A new zosterophyll from the Lower Devonian (Siegenian) of Yunnan, China. Rev Pal Pal 57:155–171. Hedderson TA, Chapman RL, Cox CJ. 1998. Bryophytes and the origins and diversification of land plants: new evidence from molecules. In: Bates JW, Ashton NW, Duckett JG, eds. Bryology for the Twenty-first Century. Leeds, UK: Maney & Son, pp 65–77. Hueber FM, Banks HP. 1979. Serrulacaulis furcatus gen. et sp. nov., a new zosterophyll from the lower Upper Devonian of New York State. Rev Pal Pal 28:169–189. Jermy AC. 1990. Isoetaceae. In: Kramer KU, Green PS, eds. Pteridophytes and Gymnosperms. Berlin; SpringerVerlag, pp 26–31. Kenrick P. 1994. Alternation of generations in land plants: new phylogenetic and palaeobotanical evidence. Biol Rev Camb Phil Soc 69:293–330. Kenrick P. 2000. The relationships of vascular plants. Phil Trans R Soc Lond B Biol Sci 355:847–855. Kenrick P, Crane PR. 1997a. The Origin and Early Diversification of Land Plants: A Cladistic Study. Washington; Smithsonian Institution Press. Kenrick P, Crane PR. 1997b. The origin and early evolution of plants on land. Nature 389:33–39. Kevan PG, Chaloner WG, Savile DBO. 1975. Interrelationships of early terrestrial arthropods and plants. Palaeontology 18:391–417. Kidston R, Lang WH. 1920a. On Old Red Sandstone plants showing structure, from the Rhynie Chert Bed, Aberdeenshire. Part II. Additional notes on Rhynia gwynne-vaughani, Kidston and Lang; with descriptions of Rhynia major, n. sp., and Hornea lignieri, n. g., n. sp. Trans R Soc Edin 52:603–627. Kidston R, Lang WH. 1920b. On Old Red Sandstone plants showing structure, from the Rhynie Chert Bed, Aberdeenshire. Part III. Asteroxylon mackiei, Kidston and Lang. Trans R Soc Edin 52:643–680. Kidston R, Lang WH. 1921. On Old Red Sandstone plants showing structure, from the Rhynie Chert Bed, Aberdeenshire. Part IV. Restorations of the vascular

11 cryptogams, and discussion on their bearing on the general morphology of the pteridophyta and the origin of the organization of land-plants. Trans R Soc Edin 52:831–854. Kotyk ME, Basinger JF. 2000. The early Devonian (Pragian) zosterophyll Bathurstia denticulata Hueber. Can J Bot 78:193–207. Krings M, Kerp H. 1997. Cuticles of Lescuropteris genuina from the Stephanian (Upper Carboniferous) of central France: evidence for a climbing growth habit. Bot J Lin Soc 123:73–89. Lang WH. 1927. Contributions to the study of the Old Red Sandstone flora of Scotland. VI. On Zosterophyllum myretonianum, Penh., and some other plant-remains from the Carmyllie Beds of the Lower Old Red Sandstone. VII. On a specimen of Pseudosporochnus from the Stromness beds. Trans R Soc Edin 55:443– 456. Lele KM, Walton J. 1961. Contributions to the knowledge of ‘‘Zosterophyllum myretonianum’’ Penhallow from the Lower Old Red Sandstone of Angus. Trans R Soc Edin 64:469–475. Li C-S. 1992. Hsua robusta, an early Devonian plant from Yunnan Province, China and its bearing on some structures of early land plants. Rev Pal Pal 71:121– 147. Lyon AG, Edwards D. 1991. The first zosterophyll from the Lower Devonian Rhynie Chert, Aberdeenshire. Trans R Soc Edin Earth Sci 82:323–332. McElwain JC. 1998. Do fossil plants signal palaeoatmospheric CO2 concentration in the geological past? Phil Trans R Soc Lond B Biol Sci 353:83–96. Meyer-Berthaud B, Stein WE. 1995. A reinvestigation of Stenomyelon from the Late Tournaisian of Scotland. Int J Plant Sci 156:863–895. Meyer-Berthaud B, Scheckler SE, Wendt J. 1999. Archaeopteris is the earliest known modern tree. Nature 398:700–701. Mora CI, Driese SG, Colarusso LA. 1996. Middle to late Paleozoic atmospheric CO2 levels from soil carbonate and organic matter. Science 271:1105–1107. Mosbrugger V. 1990. The tree habit in land plants. Lecture Notes in Earth Science 28:1–161. Paolillo DJ. 1982. Meristems and evolution: developmental correspondance among the rhizomorphs of the lycopsids. Am J Bot 69:1032–1042. Pigg KB. 1992. Evolution of Isoetalean lycopsids. Ann Mo Bot Gdn 79:589–612. Pigg KB, Rothwell GW. 1983. Chaloneria gen. nov.; heterosporous lycophytes from the Pennsylvanian of North America. Bot Gaz 144:132–147. Pirozynski KA, Malloch DW. 1975. The origin of land plants: a matter of mycotropism. Biosystems 6:153– 164. Pryer KM, Smith AR, Skog JE. 1995. Phylogenetic relationships of extant ferns based on evidence from morphology and rbcL sequences. Am Fern J 85:205–282.

12 Rayner RJ. 1984. New finds of Drepanophycus spinaeformis Go¨ppert from the Lower Devonian of Scotland. Trans R Soc Edin: Earth Sci 75:353–363. Remy W, Hass H. 1996. New information on gametophytes and sporophytes of Aglaophyton major and inferences about possible environmental adaptations. Rev Pal Pal 90:175–194. Remy W, Hass H, Schultka S. 1986. Anisophyton potoniei nov. spec. aus den Ku¨hlbacher Schichten (Emsian) vom Steinbruch Ufersmu¨hle, Wiehltalsperre. Argumenta Palaeobotanica 7:123–138. Remy W, Gensel PG, Hass H. 1993. The gametophyte generation of some early Devonian land plants. Int J Plant Sci 154:35–58. Remy W, Taylor TN, Hass H, Kerp H. 1994. Four hundredmillion-year-old vesicular arbuscular mycorrhizae. Proc Natl Acad Sci USA 91:11841–11843. Renzaglia KS, Duff RJ, Nickrent DL, Garbary DJ. 2000. Vegetative and reproductive innovations of early land plants: implications for a unified phylogeny. Phil Trans R Soc Lond B Biol Sci 355:769– 793. Retallack GJ. 1985. Fossil soils as grounds for interpreting the advent of large plants and animals on land. Phil Trans R Soc Lond B Biol Sci 309:105–142. Retallack GJ. 1986. The fossil record of soils. In: Wright VP, ed. Paleosols: Their Recognition and Interpretation. Oxford, UK: Blackwell, pp 1–57. Retallack GJ. 1990. Soils of the Past. London; UnwinHyman. Retallack GJ. 1992. Paleozoic paleosols. In: Martini IP, Chesworth W, eds. Weathering, Soils and Paleosols. Amsterdam; Elsevier, pp 543–564. Rice CM, Ashcroft WA, Batten DJ, Boyce AJ, Caulfield JBD, Fallick AE, Hole MJ, Jones E, Pearson MJ, Rogers G. 1995. A Devonian auriferous hot spring system, Rhynie, Scotland. J Geol Soc Lond 152:229– 250. Rolfe WDI. 1980. Early invertebrate terrestrial faunas. In: Panchen AL, ed. The Terrestrial Environment and the Origin of Land Vertebrates. London; Academic Press, pp 117–157. Ro¨ßler R. 2000. The late Palaeozoic tree fern Psaronius; an ecosystem unto itself. Rev Pal Pal 108:55–74. Rothwell GW. 1975. The Callistophytaceae (Pteridospermopsida): I. Vegetative structures. Palaeontographica B151:171–196. Rothwell GW. 1984. The apex of Stigmaria (Lycopsida), rooting organ of Lepidodendrales. Am J Bot 71:1031–1034. Rothwell GW. 1988. Cordaitales. In: Beck CB, ed. Origin and Evolution of Gymnosperms. New York; Columbia University Press, pp 273–297. Rothwell GW. 1991. Botryopteris forensis (Botryopteridaceae), a trunk epiphyte of the tree fern Psaronius. Am J Bot 78:782–788.

Kenrick Rothwell GW. 1999. Fossils and ferns in the resolution of land plant phylogeny. Bot Rev 65:188–218. Rothwell GW, Erwin DM. 1985. The rhizomorph apex of Paurodendron: implications for homologies among the rooting organs of Lycopsida. Am J Bot 72:86–98. Rothwell GW, Whiteside KL. 1974. Rooting structures of the Carboniferous medullosan pteridosperms. Can J Bot 52:97–102. Rowe NP, Speck T, Galtier J. 1993. Biomechanical analysis of a Palaeozoic gymnosperm stem. Proc R Soc Lond 252:19–28. Scheckler SE. 1995. Progymnosperms have gymnospermous roots. In: Hemsley AR, Kurmann MH, eds. Proceedings Royal Botanic Gardens, Kew London (abstract). Scheckler SE, Banks HP. 1971. Anatomy and relationships of some Devonian progymnosperms from New York. Am J Bot 58:737–751. Schweitzer H-J, Li C-S. 1996. Chamaedendron nov. gen., eine multisporangiate Lycophyte aus dem Frasnium su¨dchinas. Palaeontographica B238:45–69. Simon L, Bousquet J, Le´vesque C, Lalonde M. 1993. Origin and diversification of endomycorrhizal fungi with vascular plants. Nature 363:67–69. Smith SE, Read DJ. 1997. Mycorrhizal Symbiosis. London; Academic Press. Stein WE, Wight DC, Beck CB. 1984. Possible alternatives for the origin of Sphenopsida. Syst Bot 9:102–118. Stevenson DW, Loconte H. 1996. Ordinal and familial relationships of pteridophyte genera. In: Camus JM, Gibby M, Johns RJ, eds. Pteridology in Perspective. Kew, UK: Royal Botanic Gardens, pp 435–467. Stewart WN. 1947. A comparative study of stigmarian appendages and Isoetes roots. Am J Bot 34:315–324. Stewart WN, Rothwell GW. 1993. Paleobotany and the evolution of plants. Cambridge, UK: Cambridge University Press. Stidd BM. 1981. The current status of the medullosan seed ferns. Rev Pal Pal 32:63–101. Stubblefield SP, Rothwell GW. 1981. Embryogeny and reproductive biology of Bothrodendrostrobus mundus (Lycopsida). Am J Bot 68:625–634. Taylor TN, Taylor EL. 1993. The Biology and Evolution of Fossil Plants. Englewood Cliffs, NJ: Prentice Hall. Taylor TN, Hass H, Remy W. 1992a. Devonian fungi: interactions with the green alga Palaeonitella. Mycologia 84:901–910. Taylor TN, Remy W, Hass H. 1992b. Parasitism in a 400million-year-old green alga. Nature 357:493–494. Taylor TN, Hass H, Remy W, Kerp H. 1995. The oldest fossil lichen. Nature 378:244. Taylor TN, Hass H, Kerp H. 1997. A cyanolichen from the Lower Devonian Rhynie Chert. Am J Bot 84:992– 1004. Taylor TN, Hass H, Kerp H. 1999. The oldest fossil ascomycetes. Nature 399:648.

Origin of Roots Trewin NH. 1994. Depositional environment and preservation of biota in the Lower Devonain hot-springs of Rhynie, Aberdeenshire, Scotland. Trans R Soc Edin Earth Sci 84:433-442.

13 Wang Z, Geng B-Y. 1997. A new Middle Devonian plant: Metacladophyton tetraxylum gen. et sp. nov. Palaeontographica B243:85–102. Yapp CJ, Poths H. 1992. Ancient atmospheric CO2 pressures inferred from natural goethites. Nature 355:342–344.

2 Characteristics and Functions of Root Systems Alastair Fitter University of York, York, England

I.

FUNCTIONS OF ROOT SYSTEMS

Malloch, 1975; Lewis, 1987). Modern plants with similar underground parts, such as those with ‘‘magnolioid’’ roots, i.e., thick, little-branched root systems typified by the primitive family Magnoliaceae (Baylis, 1975), or achlorophyllous orchids (e.g., Neottia, Epipogium), are habitually or even obligately mycorrhizal. It is almost certain that Aglaophyton also was mycorrhizal, since arbuscules, the diagnostic structures of the most abundant group of mycorrhizal fungi, have been identified in its fossils (Remy et al., 1994). Mycorrhizal fungal spores have been found in Silurian deposits that certainly predate the evolution of root systems (Redecker et al., 2000). The origins of the diversity of the root systems of modern plants can be seen as achieving the more effective performance of these primary functions. In this chapter, I review the range of variation in the gross morphology and architecture of root systems and suggest that these can be related to the evolutionary optimization of at least the primary functions. I do not deal with metabolism or with the storage and propagation functions, important though these may be in particular cases (e.g., tubers, root buds, and suckers). The acquisition of resources is, in contrast, both well understood at a physiological level and clearly related to the overall behavior of root systems, and this will be the underlying concept throughout this chapter. It is important to bear in mind the distinction between roots (i.e., individual members of a root system) and their integration as a root system.

The root systems of terrestrial plants perform two primary functions: the acquisition of soil-based resources (principally water and ions), and anchorage. Other root system functions, such as storage, synthesis of growth regulators, propagation, and dispersal, can be seen as secondary. Little is known of the early evolution of the nonaerial parts of plants, although root traces have been found in fossil soils from Silurian deposits (Retallack, 1997), but it is certain that most of the first land plants, such as Cooksonia, Aglaophyton, and Rhynia, dating from the late Silurian and early Devonian periods, had poorly developed root systems (Collinson and Scott, 1987; see also Chapter 1 by Kenrick in this volume). Such fragments as preserved are of large diameter, and sparingly and often dichotomously branched. Early land plants were small and lived in very wet environments; evidence for plants with developed root systems growing in welldrained soils does not appear until the late Devonian (Driese et al., 1997). Neither anchorage nor acquisition of water is therefore likely to have been a serious problem: the tree habit, necessitating deep rooting for anchorage, did not develop until the mid-Devonian, when the evolution of the seed further freed plants from dependence on wet environments (Algeo and Scheckler, 1998; Bateman et al., 1998). It is likely that the most difficult function for early root systems to perform was the acquisition of poorly mobile resources, especially phosphate (Pirozynski and 15

16

II.

Fitter

CHARACTERISTICS OF ROOTS

Whereas plant species can be identified readily from flowers and, within local floras, usually without great difficulty from leaves, roots show few distinctive external features that would permit identification. Variation in leaf form can be described as adaptive to particular environmental influences such as radiation and herbivory. Leaf shape and size influence the transfer of heat and water vapor through the boundary layer (Grace, 1977), but there is no parallel phenomenon below ground. Herbivorous insects can be deterred from attacking leaves by spines and other modifications to the leaf margin (Merz, 1959; Gilbert, 1971), but there seem to be no similar structures on roots despite the fact that root-feeding insects are important and widespread in many communities (Brown and Gange, 1991; see also Chapter 51 by Koltai et al. and Chapter 52 by Katan in this volume). The scarce variation in external features of roots is presumably related to the limited range of variation of the root environment. Such variations as occur may be interpretable in terms of particular root functions. The main features of individual roots that show systematic variation are diameter, color, growth potential, surface texture, and various physiological traits such as transport capability, hormone content, and membrane composition (see Chapter 9 by Waisel and Eshel in this volume).

A.

Root Diameter

The diameter of individual roots varies widely both within and between species. In many taxonomic groups of plants, especially grasses, rushes, and sedges (Poaceae, Juncaceae, and Cyperaceae), root systems have very fine terminal branches (< 100
m diameter; Table 1). These species seem to have roots approaching an effective minimum diameter, determined by the need for a central stele and surrounding tissues (endodermis, cortex, epidermis), to provide transport to and from the root tip and the absorbing cells (Lyford, 1975; Fitter, 1987). At the other extreme, families such as Alliaceae and Magnoliaceae, although remote from each other both taxonomically and ecologically, have typically very coarse terminal roots, often
0:5–1.0 mm in diameter, and many trees approach such figures. Root diameter determines the length of root that the plant can produce for unit input of resources to the system. Many trees that normally grow in association with ectomycorrhizal fungi (especially Pinaceae) have thick roots and very low root length densities, that is, low total lengths of roots per unit soil volume. The fine-rooted species tend to have a lesser tendency to be mycorrhizal (Peat and Fitter, 1994; Fig. 1). Equally, many plants produce finer roots when grown at low nutrient supply rates. This can be demonstrated either by measuring root diameter directly (e.g., Christie and Moorby, 1975) or by determining specific

Table 1 Radius of the Finest Elements of the Root Systems of Selected Plant Species Radius (
m) > 500 250–500 150–250

100–150

35–100

Species

Family

Podocarpus totara Glycyrrhiza lepidota Lygodesmia juncea Sporobolus longifolius Histiopteris incisa Andropogon nutans Smilacina stellata Solanum nigrum Yucca glauca Triticum aestivum Andropogon scoparius Elymus canadensis Holcus lanatus Zea mays Carex coriacea Quercus rubra Senecio aureus

Podocarpaceae Fabaceae Asteraceae Poaceae Dennstaedtiaceae Poaceae Liliaceae Solanaceae Agavaceae Poaceae Poaceae Poaceae Poaceae Poaceae Cyperaceae Fagaceae Asteraceae

Source Baylis (1975) Weaver (1919) Weaver (1919) Weaver (1919) Baylis (1975) Weaver (1919) Weaver (1919) Baylis (1975) Weaver (1919) Hackett (1972) Weaver (1919) Weaver (1919) McGonigle (1987) Miller (1981) Baylis (1975) Lyford (1980) Weaver (1919)

Characteristics and Functions

Figure 1 Numbers of plant species in the British flora that are, respectively, never or rarely, occasionally, and normally mycorrhizal in each of four root diameter classes. (Data from Peat and Fitter, 1993.)

root length (length of root per unit root weight; Fitter, 1985), but it is not a universal response, particularly when parts of a single root system exposed to a heterogeneous nutrient supply are examined (Fransen et al., 1999; Hodge et al., 1998). The tendency to produce fine roots in such conditions can largely be explained in terms of ion mobility and the volume of soil exploited (Baldwin, 1975; Robinson and Rorison, 1988). Fine roots of some species (specifically roots with a thin cortex) also tend to have a relatively greater hydraulic conductivity, increasing transport efficiency (Rieger and Litvin, 1999). The countervailing benefits of coarse roots to plants in nutrient-rich soils or to mycorrhizal plants with an adequate supply of immobile ions are uncertain. Thicker roots may be able to exert greater forces on soil and might possibly have greater ability to penetrate compacted soil (Goss, 1977); it is conceivable, too, that they are more resistant to herbivores. Thick roots are more likely to persist, branch, and contribute to the long-term development of the root system. Fine roots turn over more rapidly than coarse roots (Eissenstat and Yanai, 1997) and also have higher nutrient concentrations (Gordon and Jackson, 2000). Rapid turnover of resource-rich roots may impose a large burden on the plant. B.

Texture

The surfaces of roots vary in terms of the number, size, and duration of root hairs; the persistence of the epidermis and cortex; and the nature of the bark that eventually covers woody roots. The last feature was

17

covered briefly by Cutler et al. (1987), who recorded, however, much greater variation in internal anatomy, leading one to wonder whether an equally detailed study of external features might be profitable. Root hairs are considered in depth by Ridge and Katsumi (Chapter 5, volume). The epidermis is often an ephemeral structure in field-grown roots covering < 30% of the root surface of Citrus roots (Eissenstat and Achor, 1999). Roots in soils are associated with an extensive rhizosphere microflora, and although this offers no visible features, rhizosphere soil frequently adheres to roots removed from soil (McCully, 1995), partly because of the production of mucigel by this association. The extreme case of rhizosphere development is the ectomycorrhizal sheath, an important visible feature of the terminal roots of many (but by no means all) woody plants. Ectomycorrhizae also alter root system characters, such as branching patterns, as described in the chapters by Kottke (50) and Sieber (49) in this volume.

C.

Color

Roots vary surprisingly in color. Old roots are usually colored one of various shades of brown, but young roots can be unpigmented (‘‘white’’) or variously tinged with pink or orange, possibly because pigmented zones contain feeding deterrents. Roots colonized by arbuscular mycorrhizal fungi are often yellow; the physiological significance of this deposition of pigments is unclear (Klinger et al., 1995). Roots of some plants (Lemna, Datura, Triticum) are capable of forming chlorophyll when exposed to light; aerial roots of epiphytic orchids may also be green. Although it is well established that roots can grow autotrophically when transformed in hairy root culture (Flores et al., 1993), the assimilatory potential of green roots in intact plants is unknown.

D.

Growth Potential and Longevity

A root system can be thought of as a population of meristems which vary greatly in the duration of their activity. Some survive for only a few days or weeks and give rise to very short roots; others may continue growing for months or years, and generally produce much longer roots. Oak seedlings (Quercus robur) produce some laterals that grow rapidly at first but soon cease growth, and others that grow slowly initially but continue for a longer time (Page´s, 1995); the difference is

18

apparently related to the diameter of the apex, with the short-lived roots having thinner apices. The growth potential of the meristem and the longevity of the root it gives rise to seem therefore to be related. Some roots have very short lives: Garwood (1967) estimated half-lives of grass roots to be as low as 10 days, and the use of video cameras and rhizotrons has ensured that longevity patterns are increasingly understood. In sugar maple (Acer saccharum) forests in Michigan, Hendrick and Pregitzer (1993) measured half-lives for summer cohorts of roots of <100 days, but roots produced in autumn had much lower mortality rates. Mortality was also greater at a more southerly and warmer site than at a northerly site. Similarly, Fitter et al. (1998) showed that halflives of cohorts of roots of Festuca ovina could be <10 days in summer at a lowland site in northern England, but were much longer at an upland site. However, patterns of root demography in that study were related to PAR flux more than to soil or air temperature, and when the soil was directly heated by 3 C (Fitter et al., 1999), the acceleration in root turnover was not caused directly by temperature but apparently by increased N mineralization. Nutrient supply is known to affect root longevity, although reported effects are conflicting, including greater longevity of Poplar (Populus) roots (Pregitzer et al., 1993) and increased mortality of ryegrass (Lolium) roots (Hodge et al., 1998). Other factors known to influence root longevity include grazing by soil animals, and colonization by both pathogenic (Kosola et al., 1995) and mycorrhizal (Hooker and Atkinson, 1996) fungi. Roots under Nardus stricta grassland from the same site had shorter half-lives when grown under elevated CO2 concentration (ambient þ 250
mol mol1 Þ as compared to those grown under ambient atmospheric CO2 (Fitter et al., 1997; Fig. 2). Demographic studies tend to emphasize the shortlivedness of the bulk of the root system. Some plants, however, seem to have very different patterns of root longevity. Bulbous plants such as the bluebell (Hyacinthoides non-scripta) often have annual root systems, in which all roots survive for somewhat less than a year and then die synchronously (Merryweather and Fitter, 1995); the same is true of some tundra species, such as the cotton grass (Eriophorum angustifolium) (Shaver and Billings, 1975). In some species, the bulk of the roots appear to be much longer-lived; club mosses (e.g., Diphasiastrum complanatum and Lycopodium annotinum) can have fully functional roots as much as 12 years old (Headley et al., 1988). Woody roots can, of course, be as old as the plant

Fitter

Figure 2 Demography of a single cohort of roots under a Nardus stricta grassland on peaty soil taken from Great Dun Fell (UK) and grown at ambient (open symbols) or elevated (solid symbols) CO2 in solardomes. Elevated CO2 represented ambient þ 250
mol mol1 . (From Fitter et al., 1997.)

itself. Fitter (1999) suggested a classification of root system demography with analogies to leaf demography (Table 2). E.

Specialized Roots

In many species, some roots perform special functions. Prop roots and stilt roots are adventitious roots that develop from the stem base above ground and increase the stability of the main stem. They are found in many plants of waterlogged soils, especially mangroves, and also in some herbaceous plants, such as maize (Zea mays). A similar function is performed by the buttress roots of many trees, particularly in rain forests (Ennos, 1993). Contractile roots develop from organs such as bulbs or tubers; where daughter bulbs develop above the parent or following exposure of the parent, these roots pull the daughters down into the original position (see also Chapter 54 by Putz in this volume). They are generally noticeably thicker than other roots. Other specialist functions include attachment, as in ivy (Hedera helix), and penetration of host tissue, as in parasitic plants (e.g., Lathraea, Loranthus, Rhinanthus, Viscum).

III.

FEATURES OF ROOT SYSTEMS

Except for several woody root systems in which anastomosis occurs, root systems are trivalent branching structures; in other words, each node or vertex has three links or edges emanating from it. ‘‘Node’’ is

Characteristics and Functions

19

Table 2 Possible Classification of Root System Demographya Production Mortality

Synchronous

Synchronous

Strict deciduous Geophytes (Merryweather and Fitter, 1995)

Continuous

Seasonal evergreen Temperate trees (Hendrick and Pregitzer, 1992; Berntson and Bazzaz, 1996)

Continuous Deciduous Annuals, herbaceous perennials, some trees (Gibbs and Reid, 1992) Productive evergreen Grasses, tropical trees (Fitter et al., 1998)

a

Based on the timing of events of root birth (production) and death (mortality). The examples are of plant groups possessing given combinations of demographic characteristics, and analogies to leaf demography are shown in italic type.

here used to describe the point of origin of a branch in a root system. Root systems grow by a simple branching process, with laterals emerging from main roots some distance behind the tip. Normally, only a single branch emerges at any point (see also the chapters by Barlow [Chapter 4] and by Lloret and Casero [8] in this volume), but adventitious roots can be produced from the stem base in such profusion that it is not possible to resolve the sequence of emergence; this happens in tomato (Lycopersicon), willows (Salix), and many other species. The branching structure of root systems is their most fundamental characteristic and can be shown to be central to their function. The geometry of such a structure can be represented mathematically as a tree and can be resolved into several components: 1. The number of links in the system, separated into those that terminate in a meristem (exterior links) and those that terminate in a node (interior links). The number of exterior links is always 1 greater than the number of interior links, and is referred to as magnitude (Fig. 3), a term that can refer either to the entire system or to any particular link within the system: the magnitude of any individual link is the number of exterior links it serves. 2. The lengths of the links (i.e., the distance between meristem and first node and between subsequent nodes). 3. The distribution of branches within the system, or its topology. 4. Branching angles, which are of two kinds: the radial angle at which a lateral emerges depends on the internal anatomy of the root (Charlton, 1975, 1983), and the branch angle, between a link and its parent when viewed in the plane of growth (Fitter, 1987).

5. Relative diameter, which is the rate of increase of link diameter with magnitude. This has been little studied, but it varies between species and has a profound influence on the construction cost of the root system (cf. Eshel et al., 2000). IV.

MODELS OF ROOT SYSTEMS

An essential step in understanding any system is description. Indeed, this initial step is necessary for any further investigation. Since geometry is the funda-

Figure 3 Diagrammatic representation of a root system described as a mathematical tree. Numerals represent link magnitudes. Exterior links have magnitude 1, and interior link magnitudes are the sum of the two distal links. The magnitude of this system is 12, the same as that of the basal link.

20

Fitter

mental feature of root systems, a sine qua non of root system study is the establishment of a taxonomy of root systems. Several models have been used for such classifications. A.

likely that the search for the grail of a comprehensive root system taxonomy will always be unfulfilled. A more important goal is to quantify different branching patterns so as to relate them to various functional attributes.

Types of Root Systems B.

Root systems vary widely both within and between species, and many attempts have been made to produce classifications of the various types (e.g., Weaver, 1958), usually based more or less closely on the developmental model. One of the earliest and greatest investigators of roots, William Cannon, attempted to distil 40 years’ experience of observing root systems into a ‘‘tentative classification,’’ but it is a measure of the elusiveness of this particular grail that his system has received little use since. As with most other classifications, Cannon (1949) made an initial distinction between root systems based on the primary root that emerges from the seed and those that are based on adventitious roots from the stem base, rhizome, or other organ. The illustration from Cannon’s paper is reproduced here (Fig. 4), and while the types shown are distinctive, it is certain that intermediates between all or most of the types could easily be found. The primary/adventitious distinction is less suitable for woody root systems, where the main elements are of course persistent and often extremely long-lived. Indeed, it seems that the longevity of woody roots may be comparable to that of the system as a whole (see Kozlowski, 1971, Chapter 5; Head, 1973; see also Chapter 11 by Persson in this volume). Trees typically have a number of major woody roots radiating from the stem base at shallow angles (e.g., Sitka spruce [Picea sitchensis]; Coutts, 1983; and northern red oak [Quercus rubra], Lyford, 1980) from which numerous vertical roots descend. In some species, notably silver fir (Abies alba) and Scots pine (Pinus sylvestris), there is often a dominant vertical root as large as or larger than the large radiating roots (Ko¨stler et al., 1968). In many deciduous trees, e.g. beech (Fagus sylvatica), the large woody roots rapidly dissipate into branches of lesser diameter. These different branching forms are not, however, well defined, and wide variation can be found within a species depending on age (Ko¨stler et al., 1968), site conditions (Pomerleau and Lotie, 1962), and genotype (see Chapter 11 by Persson in this volume). The root systems of smaller woody species have been little studied outside arid regions, where individual root systems can easily be traced. Just as no satisfactory classification of aboveground tree branching systems has proved possible, so it seems

Developmental System

A seedling typically has one or several seminal roots, and these then produce laterals. Subsequently, any of these initial laterals may produce further laterals, and so on, for few or many further orders of branching. The most usual classification is, therefore, to refer roots to such developmental orders, often known as axes and laterals, primary, secondary, tertiary, and so on. This has been termed a developmental classification (Rose, 1983), and it is well suited to the description of the development of many root systems. However, it is common for the meristem of the axis to die or cease to develop and for a lateral to take over its function, often changing its direction and rate of growth. The problems are most severe, however, when the classification is applied to the description of existing (as opposed to experimentally developing) root systems, when wholly arbitrary decisions must often be made as to which of two roots emanating from a junction is of higher order (Fig. 5). Further, the developmental system cannot be used to derive predictive hypotheses about root system function. Nevertheless, this approach can be used to provide a good understanding of the growth of a root system. Almost all models of root system growth have followed this line, and it is possible to reproduce rooting patterns with considerable accuracy as long as the necessary parameters are included. Typical constraints required in simulations so as to achieve realistic architectures are that lateral branches should diverge from parents at wide angles, which increases the chances of their entering previously unexploited regions, and that the primary roots originating from the stem base should be regularly distributed, again minimizing overlap. C.

Topological System

Topological models for describing root systems (Fitter, 1985, 1987; Fitter et al., 1991a), based on the concept of the mathematical tree described in Section III, represent an attempt to avoid some of the problems associated with the developmental model. These models reverse the direction of classification, so that terminal branches (exterior links) become order 1, and

Characteristics and Functions

21

Figure 4 Examples of root system types used as the basis for a tentative classification of root systems by Cannon (1949). (Reprinted with the permission of the editor of Ecology.)

22

Fitter

Figure 5 Application of developmental ordering to a growing root system. The initial system is shown at center. To the left, the main axis (A–A) continues to grow; the order of the laterals (1,2,3) is unambiguous. To the right, the main axis dies, and a lateral takes over its role; consequently the order of the laterals changes. New orders are shown with original orders in parentheses. Root system members can, therefore, effectively change order.

interior links are classified by magnitude, the number of exterior links that they serve. In this classification, therefore, links change their ‘‘order’’ (i.e., magnitude) as the system grows; the generation of a new link anywhere in the subsystem served by an interior link increases the magnitude of that interior link by one. This classification is therefore unsuited to the description of the growth of a root system. Trees of equal magnitude (i.e., those with the same number of exterior links) can differ widely in topology from the dichotomous at one extreme to the herringbone at the other (Fig. 6). These differences can be quantified using two parameters, altitude (a) and total exterior path length (pe). Altitude is the number of links in the longest path from any exterior link to the base link, and pe is the sum of the number of links in all such paths (see Fig. 3). In Fig. 6a, a is 4 and pe is 32, whereas in Fig. 6b, a is 8 and pe is 43. Both these parameters reach their minimum in dichotomous trees and their maximum in herringbone trees. For a given magnitude, a wide range of topologies is possible between the two extremes, and these represent the outcome of different growth patterns. If branching is confined to the main axis, a herringbone structure evolves (Fig. 6b); this is the most ordered pattern possible. The other extreme topology, the dichotomous system, results when branching generation is equiprobable on all exterior links (Fig. 6a). If branching is equiprobable on all links, the effect is of random growth. For a population of trees of equal magnitude,
¼ n, that have developed by random growth, expected values of the topological parameters a and pe can be

derived, as can minimum and maximum values (Knuth, 1968; Werner and Smart, 1973; Fitter, 1985; Berntson, 1995): maxðaÞ ¼ n

minðaÞ ¼
log2 ðn  1Þ
þ 2   maxðpe Þ ¼ 0:5 n2 þ 3n  2

ð1Þ

minðpe Þ ¼ n½minðaÞ þ 1  2minðaÞ1

ð4Þ

Eðpe ;nÞ

ð2Þ ð3Þ

¼ 22ðn1Þ NðnÞ

Figure 6 Diagrammatic representations of (a) dichotomous and (b) herringbone topologies. Numerals are exterior path lengths, the number of links in the path from each exterior link to the base link.

Characteristics and Functions

23

NðnÞ in equation (5) is the number of topologically distinct trees of magnitude n. Estimation of these parameters allows quantification of the branching pattern of a root system. Comparisons of systems of similar magnitude can be made directly, but where systems vary greatly in the number of links, the best approach is to calculate the regression of log a or log pe on log magnitude, or to use covariance analysis to eliminate scale effects where ratios such pe/E(pe) are used, since these are highly scale dependent (Fitter, 1993; see also Fig. 7).

from similar but more detailed observational architectural data in field crops (Ozier-Lafontaine et al., 1999). Fractal approaches offer great potential for the understanding of root systems across a wide range of spatial scales and have been used to derive general rules for the allometry of plant structures (West et al., 1999), but their predictions are sensitive to deviations from self-similarity caused by heterogeneity in the soil environment and the plasticity of plant response to that. V.

FUNCTIONAL IMPLICATIONS OF ROOT SYSTEM ARCHITECTURE

D.

A.

Cost

Fractal Analyses

Because their modular construction creates self-similarity at a range of spatial scales, root systems are fractal objects, and they can be described by a noninteger dimensionality. Early studies used 2D representations (Tatsumi et al., 1989; Fitter and Stickland, 1992); these have now been extended to three dimensions (Nielsen et al., 1997; Eshel, 1998) The 2D fractal dimension has been shown to differ among genotypes of a single species (Masi and Maranville, 1998) and to be sensitive to nutrient supply in a range of species, while displaying interpretable correlations with other architectural parameters (Fitter and Stickland, 1992). If a root system is strictly fractal, it should be possible to predict its characteristics at one spatial scale from measurements at another. This property has been used to predict the size of the total root system both from measurements of the basal link and the branching rules in model systems (Van Noordwijk et al., 1994) and

Figure 7 Maximum, expected, actual and minimum altitude of a root system plotted against magnitude.

Analysis of the functional characteristics of root systems requires the trade-offs inherent in any biological structure to be quantified. The first requirement therefore is to define the cost of root systems. This involves the identification of an appropriate currency, which is usually taken to be carbon. It has repeatedly been shown that carbon may not always be the best currency to use in studies of allocation (Abrahamson and Caswell, 1982; Reekie and Bazzaz, 1987; Fitter and Setters, 1988; see also Chapter 12 by Reich in this volume), and Hunt et al. (1987) used water as currency in a study of Agave deserti roots. They showed that the biomass of the constitutive root system appeared to be optimal in these terms, but that plants produced fewer roots in response to rain than the optimization model predicted, suggesting that an alternative currency might be appropriate for these ephemeral roots. However, for simplicity, carbon will be used as the currency here. The carbon cost of a root system has two components: construction and maintenance. The latter may be large but is covered in detail by Lambers et al. (Chapter 32 in this volume), and is related directly to construction cost (Amthor, 1984). It is an important component in detailed cost-benefit analyses of root systems, since benefits always accrue over a time period during which maintenance costs may increase. The dynamics of construction and metabolism of the root system are therefore also an essential component of the cost function (see also Chapters 12 and 32 in this volume). The carbon cost of construction has two components: the carbon used for structure and that used in growth respiration. Structural cost is simply approximated by the ash-free dry weight of the root system, whereas growth respiration is linearly related to growth rate (Lambers et al., 1983). The benefits deri-

24

vable from a given investment in root dry weight, however, depend fundamentally on root system architecture. At its simplest, this can be seen by calculating specific root length (SRL, cm mg1 ) for different plant root systems. Since many root functions, such as water and ion uptake, are more closely related to root length than to root volume (the simplest geometrical correlate of root weight), high values of SRL can imply a greater ability to obtain such resources. High SRL is found in young root systems (Fitter, 1985), in those grown in low-nutrient soils (Christie and Moorby, 1975; Fitter, 1985; see also Fig. 8), and in the parts of split-root systems supplied with highest nutrient levels (Robinson and Rorison, 1988; Farley and Fitter, 1999). This last finding appears to be due to the generation of young roots of high SRL in the high-nutrient zones. In all these cases, high SRL is the consequence of a reduction in mean root diameter, assuming constant specific mass. However, SRL is not a simple function of root diameter: it may be more closely determined by tissue density (Ryser, 1996) and even where diameter does vary, that may in some cases be due to changes in cell size rather than cell number (Eissenstat and Achor, 1999). Construction cost is proportional to biomass, which is in turn a function of tissue volume to which root diameter contributes as its square. The effect of root diameter on construction cost depends on topology and on the relationship between magnitude and diameter. In many dicots, but not in grasses, root diameter increases with magnitude. This relationship derives at least in part from the need for increased xylem and phloem capacity in roots that serve several growing points, in order to maintain flow at all exterior

Fitter

links (cf. Eshel et al., 2000). However, the rate of increase varies among species (Table 3). In consequence, root system construction costs increased markedly as high-magnitude links are added to the system, since these are of larger diameter. Topology is important because herringbone systems have more highmagnitude links than dichotomous systems (see Fig. 3). In a dichotomous system, the number of links of a given magnitude (
) is:
syst ð6Þ nð
Þ ¼
1 2 where (
syst ) is the system magnitude (number of exterior links). In a herringbone system, the number is: nð
¼ 1Þ ¼
syst

ð7Þ

nð
> 1Þ ¼ 1

ð8Þ

The cost of a root system is proportional to tissue volume (v) which, for a given magnitude, is: vð
Þ ¼ rð
Þ2 lð
Þ

ð9Þ

where rð
Þ and lð
Þ are the radius and length, respectively, of a link of magnitude
. The tissue volume of the entire system is, therefore: vsyst ¼


syst nð
Þ X X

ð10Þ

vð
,iÞ


¼1 i¼1

which can be resolved into separate formulas for extreme topologies, as follows. For a dichotomous root system: vsyst ¼


syst X
syst vð
Þ
¼1

ð11Þ

2
1

Table 3 Mean Radius (r, mm) of External Links (Magnitude 1) of Various Plant Species Grown in Sand Culture in a Growth Room, and the Rate at Which Radius Increases with Magnitude (
r) Species Figure 8 Specific root length (LR =WR , cm mg1 ) of the annual grass Poa annua over a period of 30 days at two levels of nutrient supply. Plants were grown individually in sand culture in a glasshouse at high (filled circle) or low (open circle) rates of fertilizer addition; the low-rate was oneninth of the high. Values are derived from polynomial regressions of ln WR (root dry mass) and ln LR (root length) against time, and represent fitted means and 95% confidence limits. (From Fitter, 1985.)

Poa annua Geranium lucidum Geranium robertianum Geranium dissectum Geum urbanum Euphorbia helioscopia Lactuca sativa

r


r

0.07 0.09 0.10 0.11 0.11 0.11 0.11

0.005 0.001 0.002 0.001 0.005 0.008 0.008

Source: Fitter and Nichols (unpublished results).

Characteristics and Functions

25

and for a herringbone system: vsyst ¼
syst vð1Þ þ


syst X vð
Þ
¼2

2

ð12Þ

Where root diameter increases as a simple function of magnitude, herringbone systems will be very much more expensive to construct (Fig. 9). B.

Transport

One of the functions of a root system is to transport materials obtained by absorbing cells to the rest of the plant. An optimization model would predict that a branching pattern would be favored if it offers the lowest resistance transport pathway, and that this would involve minimizing the length of the pathway unless the cost of less efficient patterns could be recouped in other ways. There are two costs to be considered here: the construction and maintenance costs considered above, and a specifically transport-related cost, which derives from the requirement for a driving force for transport (i.e., a water potential gradient), which increases with distance. Distinct considerations apply to xylem and phloem; for the latter, the costs are likely to be directly related to distance transported, but for xylem, the cost arises from the need to maintain a water potential gradient. This implies that cells at the

upper end of the pathway must expend energy in order to operate at a low water potential. Major determinants of transport efficiency could therefore be the lengths of the interior and exterior links (internodes and terminal branches) and the resistance they offer to transport. However, longer links imply a greater absorbing surface, the return from which will to some extent compensate for their increased costs. In contrast, different branching patterns or topologies can alter the nature of the pathway with no effect on the total length of links in the system. When link lengths and resistances are equal, the efficiency of the transport systems is a direct function of path length. Since dichotomous root systems minimize path length, they also minimize transport distances. For example, in a tree of magnitude 8 (see Fig. 3), all eight exterior links are four links from the base link (i.e., the stem base), giving a total exterior path length of 32; in a herringbone system, pe is 43, i.e., 34% greater. This differential increases markedly with magnitude, in the ratio of the maximum path lengths (see equations [3] and [4], Section IV.B). The greater the total exterior path length, the farther materials must travel from the absorbing parts of the root system to the shoot base. Transport efficiency may therefore be related directly to topology as measured by pe, with herringbone systems being least efficient. However, the role of root resistance in

Figure 9 Root system tissue volume in arbitrary units, an index of construction cost, as a function of system magnitude. Dashed lines represent dichotomous, solid lines, herringbone topology. In the example shown, exterior link radius ½rð1Þ is set at 0.2 mm radius with magnitude of 0:1 mm=
; rð50Þ indicates the radius (mm) of a link of magnitude 50 and here is 0.7.

26

Fitter

determining this efficiency is important, since diameter of links declines with path length and therefore resistance increases, depending on the conductivity of the xylem vessels. This is a problem that occurs wherever materials (or heat, momentum, or charge) is transferred through a branched system; Plawsky (1993) has generalized the constraints that operate in a wide range of such systems. Transport effectiveness and efficiency are increased by the first few generations of branching in a dichotomous system, but an asymptote is rapidly achieved, and in many situations, herringbone topologies are the most beneficial. The implications of such relationships for models of root system function are large (West et al., 1999). C.

Soil Exploration and Exploitation

This fundamental activity of root systems has proved too complex for quantitative investigation. The recognition by Bray (1954) of the importance of ion mobility in soil controlling ion uptake by plants led to a realization of the importance of root distribution (Barley, 1970; Nye and Tinker, 1977). To understand this fully, it is necessary to determine the branching pattern that will result in a distribution of roots in space and time that will in turn produce the greatest return in terms of resources taken up by the plant for the smallest commitment of resources to roots. Since there is great variation in soil in the spatial and temporal distribution of resources, and since resources vary in mobility, this problem is complex. Roots are generally more abundant in surface soil layers than in deeper horizons, and this corresponds to the availability of resources. In arid zones, there are often distinct rooting patterns. Many plants, especially CAM succulents, have superficial root systems, whereas other, generally deciduous, species may have deeply penetrating tap roots (Cannon, 1911; Chapter 53 by Nobel in this volume). Again, this parallels the distribution of water in space and time and reflects habitat effects (see also chapters by Glass [34], by Jungk [35], and by Sperry et al. [38] in this volume). The fact that plants differ in root distribution demonstrates differences in geometry, but since an analytical solution to the optimum geometry problem is not available, a simulation model can be used (Fitter et al., 1991a). The model grows artificial root systems following rules derived from the developmental model and then creates depletion zones around them depending on the diffusivity of the resource. Convective flux is not considered separately. Different topologies are achieved by changing branch-

ing rules, and the exploitation of soil determined by measuring the volume of depletion zones in relation to the tissue volume of the root system. The slope of the relationship between depleted volume and tissue volume represents the exploitation efficiency of the system. The model illustrates that for mobile resources such as water and nitrate ions with diffusivities > 107 cm2 s1 , herringbone topologies have the highest exploitation efficiency (Fig. 10). This result arises because such a topology produces fewer high-order laterals (secondary and tertiary); these typically have lower growth rates and fail to extend out of the depletion zone of the parent root. For less mobile resources (e.g., H2 PO4 , whose diffusion coefficient in soil is usually < 108 cm2 s1 ), topology is unimportant, because depletion zones are so narrow. Such ions may be largely acquired by mycorrhizal hyphae in many plants (see Chapter by 35 in this volume). This analysis therefore suggests that whereas root systems with dichotomous topology are both cheaper to construct and maintain and more efficient at transporting immobile materials, they are less efficient in the acquisition of mobile resources. This would lead one to

Figure 10 The exploitation efficiency of a root system (defined as the volume of soil exploited per unit root tissue volume) increases as the topological index increases, in a computer simulation. High values ( 1) of the topological index represent little-branched (herringbone) root systems; low values represent dichotomous branching patterns. In this simulation, exploitation was a resource with a diffusion coefficient in soil of 107 cm2 s1 , similar to that of nitrate ions. The simulation was performed at a range of values of other architectural characteristics, namely, branching density and the relationship between radius and magnitude, which explains the scatter of the points. (From Fitter et al., 1991.)

Characteristics and Functions

predict that ‘‘expensive’’ herringbone systems would be favored by slow-growing perennials found in resourcepoor soils where acquisition of such resources is likely to limit growth and where allocation of resources to root growth will be favored. Conversely, annuals and other plants for which soil-based resources are less likely to limit growth should produce more nearly dichotomous systems. This prediction is borne out by data available for the limited number of species that have already been studied (Fitter et al., 1988; Fitter and Stickland, 1991; Taub and Goldberg, 1996; Fig. 11). D.

Anchorage

Many features of root systems contribute to their role in providing anchorage for the plant. The part played by stilt, prop, and buttress roots has already been mentioned; only the role of subterranean roots is considered here. Two situations (or a combination) are likely to be important in natural situations: (1) where a simple upward force is exerted on the plant (e.g., by a grazing animal), and (2) where a horizontal force is exerted on the stem (usually by wind) (see Fig. 12). In practice, the first of these situations is most probable for small, mainly nonwoody plants, and the

Figure 11 The topological index of root systems produced by slow-growing species is greater than that of faster-growing species, in accordance with the predictions based on the relationship in Fig. 10. Topological index is calculated from the slope of log altitude on log magnitude (as in Fig. 10) and ranges from approaching zero (in very large, dichotomously branched systems) to 1 (in a herringbone system). (From Fitter and Strickland, 1991.)

27

upward force may be at almost any angle and may include twisting movements. There is certainly variation between species and stages of development in the ease with which they can be uprooted, in the relative strengths of stem base and root system. Stellaria media, for example, shears at the stem base if pulled, leaving the root system and stem base with buds intact. Other species may be uprooted more easily, and in some cases (e.g., Holcus lanatus), this appears to be because they are shallow rooted (Fitter, 1986). It is also likely that the quantity of roots may influence strength, but there seems to be no information on this for herbaceous plants. The nature of the bond between individual roots and soil (determined by root surface area, root hair density, extent of mucigel production, and nature of the rhizosphere among other things) has received little attention (see Chapter 5 by Ridge and Kutsumi in this volume). Nevertheless, Bailey (1998) showed that the root-hair-deficient mutant (rhd1) of Arabidopsis thaliana had the same anchorage characteristics as the wildtype, suggesting that root hairs play a limited role in anchorage. In addition to root distribution and quantity, it is certain that the branching pattern of root systems must influence the strength of anchorage (Coutts, 1983; see Chapter 10 by Stokes in this volume). Where roots are stressed under tension, an analogy can be made with a problem in material science involving the pulling of a reinforcing fibre out of a matrix (Kelly and Macmillan, 1986). Again, it is necessary to know something of the mechanical characteristics of both roots and soil. This has been studied in relation to the effects of roots on the shear resistance of soils (Waldron, 1977), and tension imposed on roots will be transferred by shear to the soil matrix. One important feature is that the

Figure 12 Diagrammatic representation of forces involved in resistance of a tree to windthrow. Windward roots are in tension, those at the hinge in compression. (From Stokes et al., 1995b.)

28

mechanical properties of roots (e.g., stiffness and strength) alter considerably as they develop secondary thickening, whereas those of the soil matrix are less variable at any one site. Woody and herbaceous plants may therefore behave very differently. Ennos (1989, 1990) and Fitter and Ennos (1989) have shown that tension is progressively transferred to soil as one moves distally along a root. In consequence, it is the proximal parts of main roots that are responsible for anchorage strength. If a force is applied that is too great to be dissipated in this way, the root will break long before the tension can be transmitted to the distal parts. Those finely branched regions therefore play little role in anchorage. This is an important result, since it means that the form of fine root systems can be interpreted in terms of resource acquisition ignoring anchorage. E. Plasticity of Root Systems Much of the discussion of root system form and function in this chapter has centered on the concept of optimization. Since the environment of root systems is highly heterogeneous both in time and space, it would appear essential that root systems would have the ability to react to that heterogeneity; in other words, they should possess phenotypic plasticity. In practice, the degree of plasticity is enormous and is largely responsible for the failure of attempts to classify root systems. All the important components of root system architecture are susceptible to environmental modification (see also Chapter 22 by Page´s in this volume). Nevertheless, there is some evidence that metric aspects of geometry (e.g., lengths and radii of links) are more plastic than topology. Root radius is certainly highly variable (Goss, 1977; Christie and Moorby, 1975), but link lengths are much more responsive to variations in nutrient supply than is topology (Fitter et al., 1988), although there is large interspecific variation in topology. One of the best-characterized plastic responses is that to local patches of high nutrient supply in soil. Almost 140 years ago, Nobbe (1862) drew attention to this phenomenon, which has been repeatedly investigated in natural soils (Sprague, 1933), in fertilizer bands in agricultural soils (Duncan and Ohirogge, 1958), and in water culture (Drew et al., 1973). As so often, however, most of this work has been performed on a limited set of species, mostly crop plants, which share certain ecological characteristics. These show rapid growth, a strong response to increased resource supply, and a low root/shoot ratio. Such plants

Fitter

evolved initially as opportunists, exploiting briefly available pulses and patches of high resource availability and were subsequently selected in agriculture for even greater responsiveness. One would expect them, therefore, to show a high degree of phenotypic and physiological plasticity. Many other species, however, normally experience lower and less variable levels of resources; it is likely that these would not have evolved such a high degree of plasticity. That plasticity of root development is itself an adaptive variable is suggested by an ingenious set of experiments by Campbell et al. (1991). By dripping nutrient solutions onto four spots on the surface of a bowl filled with sand, they were able to maintain four distinct quadrants, alternating high and low nutrient concentrations, but without any barriers to root growth. A single plant of each of a range of species was grown in each bowl and root growth in each quadrant measured. All species had greatest root growth in the nutrient-rich quadrants. However, in some species the difference between rich and poor quadrants was slight, whereas other species concentrated 80–90% of all root growth in the nutrient-rich quadrants. The species that showed greatest precision at placing roots in rich patches were also the smallest and least competitive (Fig. 13). Large, competitive species such as Urtica dioica were less selective, although because of their much faster growth rate, they achieved much higher root length densities in both rich and poor patches than the more precise species. Campbell et al. (1991) emphasized the contrast between scale and precision highlighted by these results, and that contrast needs to be viewed in relation to the pattern of heterogeneity that roots encounter in soil. In contrast, Einsmann et al. (1999) found no correlation between scale and precision of response. However, their set of species ranged from annuals to trees, making interpretation difficult. Patchiness may occur in both space and time, and patches may differ in the distribution (pattern in space, frequency in time), size of duration, and intensity. Farley and Fitter (1999) showed that each of seven coexisting herbaceous perennial had a unique set of responses, including changes in SRL, in topology (branching pattern), in mycorrhizal colonization, and in responsiveness to patch size and concentration. These studies demonstrate that plant species display a range of responses to heterogeneity. An apparent paradox is that several studies (Van Vuuren et al., 1996; Fransen et al., 1998; Hodge et al., 1998) using 15N as a label have shown that proliferation does not lead to increased N capture from N-

Characteristics and Functions

29

Figure 13 The precision with which roots are placed in patches of high nutrient concentration in the experiment of Campbell et al., (1991) is inversely related to plant size, as estimated from relative growth rate (R) and seed mass (Wsd) in a compound variable created by multiple regression. Large plants such as Urtica dioica grow roots at more or less random spatially. (From Fitter, A. H. [1994]. In Exploitation of Environmental Heterogeneity by Plants [M. M. Caldwell and R. W. Pearcy, Eds.]. Academic Press, New York, pp. 305–323.)

rich patches even though nitrate is a powerful trigger for localized proliferation (Zhang and Forde, 1998). This result is due to the mobility of nitrate in soil, which means that a low root length density can achieve a high N capture rate. The explanation for this paradox was provided by an experiment by Hodge et al. (1999) in which ryegrass (Lolium perenne) and smooth meadow grass (Poa pratensis) competed for N from an organic patch. In those circumstances the species with the highest root length density captured most N. Hence, proliferation must be seen as a response that promotes competitive ability, a result in striking contrast to the view of Campbell et al. (1991). Plasticity, as exemplified here by the proliferation response, is a fundamental characteristic of the growth of root systems. Their modular construction means that the fates of individual meristems in space and time can each be unique, giving rise to a large network of possible structures. Plasticity is perhaps the most important adaptive feature of roots to their environment, ensuring that they achieve their primary functions of resource acquisition and anchorage.

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Fitter Duncan WG, Ohlrogge A. 1958. Principles of nutrient uptake from fertiliser bands: II. Root development in the band. Agron J 50:605–608. Einsmann JC, Jones RH, Pu M, Mitchell RJ. 1999. Nutrient foraging traits in 10 co-occurring plant species of contrasting life forms. Ecology 87:609–619. Eissenstat DM, Achor DS. 1999. Anatomical characteristics of roots of citrus rootstocks that vary in specific root length. New Phytol 141:309–321. Eissenstat DM, Yanai R. 1997. The ecology of root life-span. Adv Ecol Res 27:1–60. Ennos AR. 1989. The mechanics of anchorage in seedlings of sunflower, Helianthus annuus L. New Phytol 113:185– 192. Ennos AR. 1990. The anchorage of leek seedlings: the effect of root length and soil strength. Ann Bot 65:409–416. Ennos AR. 1993. The function and formation of buttresses. Trends Ecol Evol 8:350–351. Eshel A. 1998. On the fractal dimensions of a root system. Plant Cell Environ 21:247–251. Eshel A, Schick I, Waisel Y. 2000. The efficiency of water conducting system of tomato roots. In: Stokes A, ed. The Supporting Roots of Trees and Woody Plants: Form, Function and Physiology. Dordrecht, Netherlands: Kluwer, pp 371–375. Farley RA, Fitter AH. 1999. The responses of seven cooccurring woodland herbaceous perennials to localized nutrient-rich patches. Ecology 87:849–859. Fitter AH. 1985. Functional significance of root morphology and root system architecture. In: Fitter AH, Atkinson D, Read DJ, Usher MB, eds. Ecological Interactions in Soil. Oxford, UK: Blackwell Scientific, pp 87–106. Fitter AH. 1986. Spatial and temporal patterns of root activity in a species rich alluvial grassland. Oecologia 69:594–599. Fitter AH. 1987. An architectural approach to the comparative ecology of plant root systems. New Phytol 106(suppl):61–77. Fitter AH. 1993. Architectural analysis of plant root systems. In: Hendry, GAF, Grime JP, eds. Methods in Comparative Plant Ecology: A Laboratory Manual. London; Chapman & Hall, pp 165–170. Fitter AH. 1994. Architecture and biomass allocation as components of the plastic response of root systems to soil heterogeneity. In: Caldwell, MM, Pearcy RM, eds. Exploitation of Environmental Heterogeneity by Plants. New York; Academic Press, pp 305–323. Fitter AH. 1999. Roots as dynamic systems: the developmental ecology of roots and root systems. In: Press MC, Scholes JD, Barker MG, eds. Plant Physiological Ecology. London; Blackwell Scientific, pp 115–131. Fitter AH, Ennos RA. 1989. Architectural constraints to root system function. In: Robinson D, ed. Roots and the Soil Environment. Aspects Appl Biol 2:15–22.

Characteristics and Functions Fitter AH, Setters NL. 1988. Vegetative and reproductive allocation of phosphorus and potassium in relation to biomass in six species of Viola. J Ecol 76:617–636. Fitter AH, Stickland TR. 1991. Architectural analysis of plant root systems II. Influence of nutrient supply on architecture in contrasting plant species. New Phytol 118:383–389. Fitter AH, Stickland TR. 1992. Fractal characterization of root-system architecture. Funct Ecol 6:632–635. Fitter AH, Nichols R, Harvey ML. 1988. Root system architecture in relation to life history and nutrient supply. Funct Ecol 2:345–351. Fitter AH, Stickland TR, Harvey ML, Wilson GW. 1991a Architectural analysis of plant root systems. I. Architectural correlates of exploitation efficiency. New Phytol 118:375–382. Fitter AH, Stickland TR, Harvey ML, Wilson GW. 1991b. Architectural analysis of plant root systems. II. Influence of nutrient supply on architecture in contrasting plant sciences. New Phytol 118:383–389. Fitter AH, Self GK, Wolfenden J, Van Vuuren M, Brown TK, Williamson L, Graves JD, Robinson D. 1997. Root production and mortality under elevated atmospheric carbon dioxide. Plant Soil 187:299–306. Fitter AH, Graves JD, Self GK, Brown TK, Bogie D, Taylor K. 1998. Root production, turnover and respiration under two grassland types along an altitudinal gradient: influence of temperature and solar radiation. Oecologia 114:20–30. Fitter AH, Self GK, Brown TK, Bogie D, Graves JD, Benham D, Ineson P. 1999. Root production and turnover in an upland grassland subjected to artificial soil warming respond to radiation flux and nutrients, not temperature. Oecologia 120:575–581. Flores HE, Dai YR, Cuello JL, Maldona-Mendoza IE, Loyola-Vargas VM. (1993). Green roots—photosynthesis and photoautotrophy in an underground plant organ. Plant Physiol 101:363–371. Fransen B, de Kroon H, Berendse F. 1998. Root morphological plasticity and nutrient acquisition of perennial grass species from habitats of different nutrient availability. Oecologia 115:351–358. Fransen,B, Blijenberg J, de Kroon H. 1999. Root morphological and physiological plasticity of perennial grass species and the exploitation of spatial and temporal heterogeneous nutrient patches. Plant Soil 211:179– 189. Garwood EA. 1967. Seasonal variation in appearance and growth of grass roots. J Br Grassland Soc 22:121–130. Gibbs RJ, Reid JB 1992. Comparison between net and gross root production by winter wheat and perennial ryegrass. NZ J Crop Hort Sci 20:483–487. Gilbert LE. 1971. Butterfly-plant coevolution: has Passiflora adenopoda won the selectional race with heliconid butterflies? Science 172:585–586.

31 Gordon WS, Jackson RB. 2000. Nutrient concentrations in fine roots. Ecology 81:275–280. Goss MJ. 1977. Effect of mechanical impedance on growth of seedlings. J Exp Bot 28:96–111. Grace J. 1977. Plant Response to Wind. London; Academic Press. Hackett C. 1972. A method of applying nutrients locally under controlled conditions and some morphological effects of locally applied nitrate on the branching of wheat roots. Aust J Biol Sci 25:1169–1180. Head GC. 1973. Shedding of roots. In: Kozlowski TT, ed. Shedding of Plant Parts. New York; Academic Press, pp 237–293. Headley AD, Callaghan TV, Lee JA. 1988. Water uptake and movements in the evergreen clonal plants Lycopodium annotinum L. and Diphasiastrum complantum (L.) Holub. New Phytol 110:487–495. Hendrick RL, Pregitzer KS. 1992. Patterns of the root mortality in two sugar maple forests. Nature 361:59–61. Hodge A, Stewart J, Robinson D, Griffiths BS, Fitter AH. 1998. Root proliferation, soil fauna and plant nitrogen capture from nutrient-rich patches in soil. New Phytol 139:479–494. Hooker JE, Atkinson D. 1996. Arbuscular mycorrhizal fungi-induced alteration to tree-root architecture and longevity. Z Pflanzenernhr Bodenk 159:229–234. Hunt ER, Zakir NJD, Nobel PS. 1987. Water costs and water revenues for established and rain-induced roots of Agave deserti. Funct Ecol 1:125–130. Kelly A, Macmillan NH. 1986. Strong Solids. Oxford, UK: Oxford University Press. Klinger A, Bothe H, Wray V, Marschner H. 1995. Identification of a yellow pigment found in maize roots upon mycorrhizal colonisation. Phytochemistry 38:53–55. Knuth DE. 1968. The Art of Computer Programming. Vol. 1. Reading MA; Addison-Wesley. Kosola KR, Eissenstat DM, Graham JH. 1995. Root demography of mature citrus trees: the influence of Phytophthora nicotianae. Plant Soil 171:283–288. Ko¨stler JN, Bruckner E, Bibelriether H. 1968. Die Wurzeln der Waldba¨ume. Hamburg, Germany: Parey. Kozlowski TT. 1971. Growth and Development of Trees. London; Academic Press. Lambers H, Szaniawski RK, de Visser R. 1983. Respiration of the growth, maintenance and ion uptake: an evaluation of concepts, methods, values and their significance. Physiol Plan 58:556–563. Lewis DH. 1987. Evolutionary aspects of mutualistic associations between fungi and photosynthetic organisms. In: Rayner ADM, Brasier CM, Moore D, eds. Evolutionary Biology of the Fungi. Cambridge, UK: Cambridge University Press, pp 161–178. Lyford WH. 1975. Rhizography of non-woody roots of trees in the forest floor. In: Torrey JG, Clarkson DT, eds.

32 The Development and Function of Roots. New York; Academic Press, pp 179–196. Lyford WH. 1980. Development of the Root System of Northern Red Oak (Quercus rubra L.). Harvard Forest Paper No. 21. Cambridge, MA: Harvard University Press. Masi CEA, Maranville JW. 1998. Evaluation of sorghum root branching using fractals. J Agric Sci 131:259–265. McCully ME. 1995. How do real roots work? Plant Physiol 109:1–6. McGonigle TP. 1987. Vesicular arbuscular mycorrhizas and plant performance in a semi-natural grassland. Ph.D. dissertation, University of York, York, UK. Merryweather JW, Fitter AH. 1995. Arbuscular mycorrhiza and phosphorus as controlling factors in the life history of Hyacinthoides non-scripta (L.) Chouard ex Rothm. New Phytol 129:629–636. Merz E. 1959. Pflanzen und Raupen: u¨ber einigen Prinzipien der Futterwahl bei Grossschmetterlingsraupen. Biol Zentralbl 78:152–158. Nielsen KL, Lynch JP, Weiss HN. 1997. Fractal geometry of bean root systems: correlations between spatial and fractal dimension. Am J Bot 84:26-33. Nobbe F. 1862. Uber die feinere Verastelung der Pflanzenwurzeln. Landwirtsch Versuchs 4:212–224. Nye PH, Tinker PB. 1977. Solute Movement in the Soil Root System. Oxford, UK: Blackwell Scientific. Ozier-Lafontaine H, Lecompte F, Sillon JF. 1999. Fractal analysis of the root architecture of Gliricidia sepium for the spatial prediction of root branching, size and mass: model development and evaluation in agroforestry. Plant Soil 209:167–180. Page´s L. 1995. Growth patterns of the lateral roots of young oak (Quercus robur L) tree seedlings: Relationship with apical diameter. New Phytol 130:503–509. Peat HJ, Fitter AH. 1994. The distribution of mycorrhizas in the British flora. New Phytol 125:845–854. Pirozynski KA, Malloch DW. 1975. The origin of land plants: a matter of mycotrophism. Biosystems 6:153– 164. Plawsky JL. 1993. Transport in branched systems 1: steadystate response. Chem Eng Comm 123:71–86. Pomerleau R, Lotie N. 1962. Relationships of dieback to the rooting depth of white birch. For Sci 8:219–224. Pregitzer KS, Hendrick RL, Fogel R. 1993. The demography of fine roots in response to patches of water and nitrogen. New Phytol 125:575–580. Redecker D, Kodner R, Graham LE. 2000. Glomalean fungi from the Ordovician. Science 289:1920–1921. Reekie EG, Bazzaz FA. 1987. Reproductive effect in plants: 2. Does carbon reflect the allocation of other resources: Am Nat 129:876–896. Remy W, Taylor TN, Hass H, Kerp H. 1994. Four hundred million year old vesicular arbuscular mycorrhizae. Proc Natl Acad Sci USA 91:11841–11843.

Fitter Retallack GJ. 1997. Early forest soils and their role in Devonian global change. Science 276:583–585. Rieger M, Litvin P. 1999. Root system hydraulic conductivity in species with contrasting root anatomy. J Exp Bot 50:201–209. Robinson D, Rorison IH. 1988. Plasticity in grass species in relation to nitrogen supply. Funct Ecol 2:249–257. Rose DA. 1983. The description of the growth of root systems. Plant Soil 75:405-415 Ryser P. 1996. The importance of tissue density for growth and life-span of leaves and roots: a comparison of five ecologically contrasting grasses. Funct Ecol 10:717– 723. Shaver GR, Billings WD. 1975. Root production and root turnover in a wet tundra ecosystem at Barrow, Alaska. Flora 165:247–267. Sprague HB. 1933. Root development of perennial grasses and its relation to soil condition. Soil Sci 36:189–209. Stokes A, Fitter AH. Coutts MP. 1995a. Responses of young trees to wind: effect on root growth. In: Coutts MP, Grace J, eds. Trees and Wind. Cambridge, UK: Cambridge University Press, pp 264–275. Stokes A, Fitter AH, Coutts MP. 1995b. Responses of young trees to wind and shading: effects on root architecture. J Exp Bot 46:1139–1146. Tatsumi J, Yamauchi A, Kono Y. 1989. Fractal analysis of plant root systems. Ann Bot 64:499–503. Taub DR, Goldberg D. 1996. Root system topology of plants from habitats differing in soil resource availability. Funct Ecol 10:258–264. Van Noordwijk M, Spek LY, de Willigen P. 1994. Proximal root diameter as predictor of total root size for fractal branching models. I. Theory. Plant Soil 164:107–117. Van Vuuren MMI, Robinson D, Griffiths BS. 1996. Nutrient inflow and root proliferation during the exploitation of a temporally and spatially discrete source of nitrogen in soil. Plant Soil 178:185–192. Waldron LJ. 1977. The shear resistance of root-permeated homogeneous and stratified soil. Soil Sci Soc Am J 41:843–849. Weaver JE. 1919. The ecological relations of roots. Carnegie Inst Wash Pub No. 286. Weaver JE. 1958. Classification of root systems of forms of grassland and a consideration of their significance. Ecology 39:393–401. Werner C, Smart JS. 1973. Some new methods of topologic classification of channel networks. Geogr Anal 5:271– 295. West GB, Brown JH Enquist BJ. 1999. A general model for the structure and allometry of plant vascular systems. Nature 400:664–667. Zhang H, Forde BG. 1998. An Arabidopsis MADS box gene that controls nutrient-induced changes in root architecture. Science 279:407–409.

3 The Root Cap: Structure and Function Andreas Sievers, Markus Braun, and Gabriele B. Monshausen University of Bonn, Bonn, Germany

I.

INTRODUCTION

II.

ROOT CAP IN EMBRYOGENESIS

In monocots, the different cell types of the root cap originate from the same cell during embryogenesis (Von Guttenberg, 1968), whereas in many dicots, the primary root cap is a composite of cells derived from two different origins. In the embryogenesis of Arabidopsis, the first asymmetric division of the zygote forms a terminal and a basal cell, both of which contribute to the generation of the root cap (Dolan et al., 1993; Scheres et al., 1994). Derivatives of the terminal cell give rise to the epidermal/lateral root cap initials. The initials of the central columella originate from the hypophysis, the uppermost derivative of the basal cell. In the triangular stage of embryogenesis the hypophysis divides to produce two daughter cells, one of which gives rise to the quiescent center, while the other—at the heart stage—forms the columella initials (Scheres et al., 1994). In garden cress (Lepidium sativum) amyloplast starch is accumulated within the prospective statocytes (Fig. 2A). These are the only root cells to do so. During embryogenesis columella cells are not structurally polarized, and the embryonic root, under in situ conditions, does not respond to gravity. In root cap cells of fully developed cress embryos, the transition to dormancy is accompanied by a loss of amyloplast starch (Fig. 2B) and by an accumulation of storage lipids and protein vacuoles (Friedrich and Sievers, 1985).

The root cap covers the outermost tip of roots, from the outgrowing primary root up to the outermost branches of the root system.Whatever the developmental stage is, the root cap protects the apical meristems, senses the direction of gravity and other environmental signals, and generates the rhizosphere. In contrast to most other plant tissues, the root cap (Fig. 1), as a persisting structural entity, consists of cells determined to eventually separate from the cap tissue. Renewed by meristematic divisions, statocytes (i.e., gravity-perceiving cells; Fig. 1C) develop and are transformed into mucilage producing secretion cells (Fig. 1D), which finally slough off from the cap, often as metabolically active border cells. The time for a single cell to proceed through the root cap depends on the cycle time, the size of the root cap, and the species. In general, only a few days are required to renew the cap completely, from 1 day in Zea mays to 6–9 days in Convolvulus (Barlow, 1975). In roots of open-type construction, as in most dicotyledonous species, there is no sharp boundary between the root cap and the root proper. In closed construction, as in grasses, there is a distinct separation. In spite of these anatomical features, the functions of the root caps of both types are the same. In the following sections we focus on the functional aspects of the root cap in the light of specific structural features.

33

34

Sievers et al.

Figure 1 (A) Median longitudinal section of cress root cap showing the regular pattern of cell arrangement. All cells shown here originate from the embryonic root cap, but during further development of the root, every cell generated by meristematic division will proceed through these stages. g— direction of gravity. Bar ¼ 10
m. Schematic representations of the different cell types indicated by solid lines are shown in panels (B–D). (B) Meristematic cell from the dermatocalyptrogen with a central nucleus (n) taking up much of the cell volume and proplastids (p) with some starch. (C) The nucleus (n) of a statocyte is placed at the proximal cell pole and a complex of ER at the distal cell pole. Amyloplasts (a; starch indicated in black) lie on this ER complex. Small vacuoles (v) and mitochondria (m) are seen in the cell center. (D) Secretion cell whose polarity was changed according to its differing function: at the distal (outer) cell pole dictyosomes (d) have accumulated, producing vesicles filled with mucilage. Fusion of these vesicles with the plasma membrane and the release of mucilage is shown by arrowheads. The nucleus (n) is found at the cell center and the vacuoles (v) have increased in size. (A, Original by Ulrike Wellenbrock.)

III.

ROOT STATOCYTES DURING GERMINATION

At the onset of imbibition of dry cress seeds, cells of the root cap become hydrated and starch is synthesized in the amyloplasts (Fig. 2C). At that phase, membranes of the endoplasmic reticulum (ER) are formed in the vicinity of the storage lipids. This is caused mainly by peripheral location of the lipid bodies that line up along the cell periphery (Sargent and Osborne, 1980; Hensel, 1986). Coincidentally, microtubules

appear near the distal cell edges of the statocytes, and an increasing amount of ER at the distal cell pole occurs about 10–15 h after the onset of imbibition. After 2 h of soaking, the plastids begin to synthesize starch, a process that increases during the next 8– 13 h. The increased weight and a reduction of the viscosity of the cytoplasm cause the amyloplasts to sediment into the physically lowermost part of the statocytes. Amyloplasts rest on the developing complex of distal ER in downward growing roots (cf. Fig. 1C, Fig. 3). The development of statocyte polarity is caused neither by the action of gravity per se nor by the gravity-dependent amyloplast sedimentation. This was shown by developing roots that were kept on the rotating horizontal axis of clinostats from the dry seed stage onward (Sievers et al., 1976; Hensel and Sievers, 1981) and by seed germination in a microgravity experiment on a Spacelab mission (Volkmann et al., 1986; Perbal et al., 1987). Instead, the polar differentiation is the result of a genetically prepatterned developmental program. The same argument holds true for the proximal position of the nucleus, which in embryonic cells and in very young (2–8 h after the onset of soaking) statocytes appears to be centrally located (Friedrich and Sievers, 1985). The distance between the nucleus and the proximal periclinal cell wall remains constant during the longitudinal growth of the statocytes, leading to the proximal nuclear position mentioned earlier (Hensel, 1986). Furthermore, microgravity influences starch metabolism and ER development, since less starch and more ER were found under microgravity (Moore et al., 1986, 1987; Volkmann et al., 1986) and under conditions of simulated weightlessness (Hoson et al., 1997). In addition, the diameter of lipid bodies increases under both conditions (Volkmann et al., 1986; Hoson et al., 1997). IV.

STATOCYTES IN MATURE ROOTS

A.

Nucleus

The nucleus occupies a position near the proximal periclinal cell wall of the statocytes of most plants. This has been shown for Arabidopsis thaliana Heynh. (Olsen et al., 1984), Hordeum vulgare (Moore, 1985), Lens culinaris (Perbal et al., 1987), Lepidium sativum (Fig. 3; see also Sievers and Volkmann, 1972), Pisum sativum (Olsen and Iversen, 1980), and Zea mays (e.g., Moore, 1983). Data for nuclei sedimenting in the direction of gravity have been reported (Hestnes and Iversen, 1978; Ransom and Moore, 1983; Lorenzi

Root Cap

35

Figure 2 Development of the root apex of cress. (A) During embryogenesis, the prospective statocytes of the embryonic root cap have accumulated starch (s, statenchyma filled with starch grains, i.e., amyloplasts). The embryo is still connected to the mother plant by the suspensor (su). (B) Starch is hydrolyzed before the seed enters dormancy, hence, between embryogenesis (panel A) and germination (panel C). m, meristem; I–VI denote the storeys of the root cap. (C) Starch grains (amyloplasts) are found during germination of the seed. This root cap was taken from a 10-h old seedling. Note that the root had been inverted; amyloplasts, which always sediment into the physically lowermost part of the cells, rest in the proximal area of the statocytes. Bars ¼ 10
m. (Originals by Ulrike Friedrich.)

and Perbal, 1990). The distance between the nucleus and proximal periclinal cell wall of cress statocytes remains constant irrespective of the increasing length of the statocytes. Since treatment with cytochalasin B (a drug known to destroy actin microfilaments) led to sedimentation of the nuclei, a role of actin microfila-

ments in anchoring the nucleus was suggested (Hensel, 1985; Lorenzi and Perbal, 1990). B.

Proplastids of the meristematic layer(s) accumulate starch and, because of this transformation into the amyloplast stage, sediment according to gravity (cf. Barlow et al., 1984). Amyloplasts from coleoptiles have negative zeta potentials of about 19:4 mV, as measured by rate of transport in an electric field, a feature that may also be valid for amyloplasts of root statocytes (Sack et al., 1983). C.

Figure 3 Statocyte of a cress root from a position near the median plane of the root. See Fig. 1C for explanation and abbreviations. Marker bar ¼ 1
m. (From Busch and Sievers, 1990.)

Amyloplasts

Endoplasmic Reticulum

Root statocytes of all plants investigated so far have only cortical ER (the distal ER complex included) but no ‘‘endoplasmic’’ ER. The location, amount, and even the internal organization of the ER in statocytes are reported to vary depending on the species being studied. Therefore, we shall introduce the well-studied cress statocytes (Fig. 3) as a model system and then describe some of the properties of other systems. In the first studies of cress root caps, Sievers and Volkmann (1972) and Volkmann (1974) noted that the number of ER layers in the distal ER complex increases with statocyte development. By means of inhibitor experi-

36

ments, it was shown that ER movement within statocytes and anchorage at the distal cell pole depend on the action of the cytoskeleton. The ER in mature statocytes is produced by an outgrowth of the outer membrane of the nuclear envelope (Hensel, 1985). An actin microfilament-dependent process translocates the newly formed ER membranes into the cortical cytoplasm of the cells (Hensel, 1988). Thereafter, coordinated action of plasma membrane-bridged microtubules and actin microfilaments translocates the ER into the distal cell pole (Hensel, 1987). The retranslocation of ER, displaced by centrifugation from the distal cell pole, is also driven by actin microfilament action (Wendt and Sievers, 1986). The distal ER complex is anchored in its position by microtubules arranged in an interlaced layer near the plasma membrane of the distal cell edges (Hensel, 1984). Actin microfilaments, however, also contribute to this anchorage, since only 7 min of treatment with cytochalasin D (an actin microfilament-destroying drug, more potent in action than cytochalasin B) led to disintegration of the complex (Hensel, 1987). It was suggested that cortical actin microfilaments may hold the ER complex under tension. This contributes to the regular appearance of the distal ER complex in control statocytes. Although the ER transported along the anticlinal cell walls may consist of smaller elements of the tubular type (Stephenson and Hawes, 1986; Hensel, 1987), the organization of the distal ER complex in cress was shown by freeze-etching to be cisternal (Sievers and Volkmann, 1977). Several species do not evolve an ER complex at the distal cell pole. Nevertheless, examples for distal ER complexes in plants other than cress were presented (Volkmann, 1974; Olsen and Iversen, 1980). An increase in ER during statocyte development appears to be a general feature for root caps (cf. Juniper and Clowes, 1965; Barlow and Sargent, 1978; Stephenson and Hawes, 1986; Hensel, 1987). Values that vary from 240
m2 ER area per statocyte up to > 9000
m2 for maize have been reported. The ER seems to be stable in its original location, possibly because of the anchoring action of the cytoskeleton. Only a few exceptions (e.g., Griffiths and Audus, 1964; Juniper and French, 1970, 1973) of an ER redistribution on gravitropic stimulation have been reported. Under certain experimental conditions, in particular inversion of the roots, formation of additional ER is induced beneath the amyloplasts at the proximal, then physically lowermost, part of the statocytes (Volkmann and Sievers, 1975). This indicates that gravity, obviously by an action of the sedimenting amyloplasts, may induce

Sievers et al.

the formation of ER at cell sites differing from those of normally grown roots. If roots grown in the normal vertical direction are rotated for 20 h on the horizontal axis of a slow (two rotations per minute) clinostat, the polar arrangement of the organelles is destroyed. The ER appears at different sites in the statocytes, often forming whorls or aggregates. As pointed out by Hensel and Sievers (1980), this overstimulation by continuous omnilateral gravistimulation leads to the selfdestruction of the statocytes. This is further indicated by a confluence of lipid bodies, the appearance of autophagosomes, the loss of amyloplast starch, and digestion of anticlinal cell walls. The effect of the self-destruction of statocytes was twofold: (1) the graviresponse of the overstimulated roots was drastically reduced, and (2) the root meristem responded to this damage by reducing the period of the cell cycle, thereby causing a faster repair of the statocytes (Hensel and Sievers, 1980). It should be mentioned that the unique lack of ‘‘endoplasmic’’ ER in mature statocytes—in conjunction with the lack of prominent endoplasmic cytoskeletal elements—facilitates the sedimentation of statoliths, thereby optimizing graviperception. The function of the remarkable amount of ER in mature statocytes can be interpreted in connection with the existence and the role of the Ca2þ -ATPase in plant ER membranes which was discovered in cress roots (Buckhout, 1983; see also the chapters of Pilet [30], Porterfield [20, 29], and of Poovaiah et al. [31] in this volume). D.

Other Organelles

Other organelles in statocytes, such as mitochondria, dictyosomes, vacuoles, and microbodies, are distributed randomly, although their distribution is limited to the area between the proximal nucleus and the distal ER complex with the sedimented amyloplasts. Lipid bodies remain, in general, in the vicinity of ER membranes. E.

Cytoskeleton

The cytoskeleton is a highly dynamic system of filamentous proteins with many functions in cell shaping, organization, motility, and signaling. Extensive tissuespecific arrangements of cortical and endoplasmic actin microfilaments and microtubules have been reported in the various cell types of roots and shoots. In statocytes, however, all labeling techniques applied so far have failed to visualize prominent endoplasmic actin microfilament bundles and microtubules

Root Cap

(Balusˇ ka et al., 1997). Soon after termination of the mitotic division in the prospective statocytes, distinct actin microfilament bundles can no longer be visualized and microtubules are limited to dense cortical arrays (Hensel, 1984; Balusˇ ka et al., 1997). This unique cytoskeletal arrangement is considered responsible for the cell polarity, the exclusion of larger organelles (e.g., nucleus, plastids, ER membranes) from the interior of the cells, and the absence of cytoplasmic streaming. In statocytes of other plant organs—coleoptiles and hypocotyls, where cytoplasmic streaming interferes with sedimentation of the amyloplast statoliths— actin cables have been observed (White and Sack, 1990). Gravity-directed sedimentation of the starchcontaining amyloplasts in root cap statocytes may also be related to the specific cytoskeletal properties which may be due to the specific calcium concentration in the cytosol. The diffuse actin labeling found at the cell periphery and close to the sedimented amyloplasts (Balus˘ ka et al., 1997; Blancaflor and Hasenstein, 1997) suggests that actin is organized in the form of delicate meshworks of oligomers, as is indicated by GFP-talin-transformed Arabidopsis seedlings (M. Jaideep, unpublished results). Interestingly, actin microfilament bundles were visualized in enzymatically extracted root cap tissues (Hensel, 1988; White and Sack, 1990), which indicates a potential ability of the actin cytoskeleton to rapidly change its organization. Inhibitor experiments indicate that the translocation and the polar distribution of the ER as well as the movements of the amyloplasts are based on the actomyosin system. The motor protein myosin was immunocytochemically detected in root tips of Allium cepa (Parke et al., 1986) and in statocytes of cress roots (Balusˇ ka and Hasenstein, 1997) by an antibody against animal myosin which cross-reacted on Western blots with a plant polypeptide of approximately 200,000 molecular weight. Myosin immunofluorescence was found in the vicinity of the sedimented amyloplasts in statocytes. Interconnections between actin microfilaments and plastids were described and are involved in the light-dependent movement of chloroplasts (Grolig and Wagner, 1988; Grolig, 1990). F.

Importance of Statocytes’ Structural Polarity

The structural polarity of statocytes appears to be a precondition for graviperception. This conclusion can be drawn from the fact that the polar differentiation is the result of an endogenous developmental program

37

and from the fact that the lack of endoplasmic structures like ER and prominent cytoskeletal elements facilitates sedimentation of statoliths. In addition, especially the results of two experiments support this conclusion: 1. The biogenic polarity was changed to a physical stratification by root-tip-directed centrifugation (20 min at 50–2000 g; Sievers and Heyder-Caspers, 1983). Within the following 8–10 min at 1 g, the structural polarity at the distal cell pole was reestablished in most statocytes, regardless of their orientation to the gravity vector. The lag phase of graviresponse was also increased by 8–10 min in centrifuged roots as compared to controls and independent of the applied centrifugation dose. The kinetics of the response were identical to controls. That means that some reorganization of the statocyte after stratification is necessary and sufficient for graviperception. 2. Treatment of roots with gibberellic acid and kinetin causes not only complete destarching of amyloplasts but also a total loss of structural polarity in statocytes and graviresponsiveness of roots (Busch and Sievers, 1990). Twenty-two hours after removal of the hormones, the polar arrangement of cell organelles was restored and starch was resynthesized so that the roots again responded gravitropically.

V.

MUCILAGE SECRETION BY ROOT CAPS

The outer layers of the root cap consist of mucilagesecreting cells (Fig. 4) which in maize may occupy as much as 40% of the cap volume (Moore, 1984). While in maize most of the mucilage is produced by lateral cap cells, one to two layers beneath the tissue surface, secretion also takes place in the most peripheral cap cells (Vermeer and McCully, 1982). The transition of statocytes to secretion cells is accompanied by a decrease in amyloplast starch and by an increase in the number of vacuoles and dictyosomes (Moore and McClelen, 1983). The trans-cisternae of the dictyosomes become hypertrophied and mucilage-containing Golgi vesicles are formed (Fig. 4D,E; Staehelin et al., 1990), which are transferred to the plasma membrane in an active, actin microfilament-dependent process for exocytosis (Mollenhauer and Morre´, 1976). In addition, accumulation of ER near the nucleus of secreting cells indicates a role of ER in the membrane flow (Volkmann and Czaja, 1981). Both membrane traffic from the ER to the dictyosomes and exocytosis of the mucilage-containing vesicles seem to be Ca2+-regulated processes. After treatment with cyclopiazonic

38

Figure 4 (A) Root tip of maize surrounded by a drop of mucilage. Bar ¼ 1 mm. (B) The mucilage contains numerous isolated cells which are sloughed off from the periphery of the root cap. Bar ¼ 0:1 mm. (C) The final fate of root cap cells of a Phleum root, shown in toto, the sloughing of the root cap cells is seen. Bar ¼ 10
m. (Original by Hanna Zieschang.) Corresponding images of secretion cells of cress root caps (D) after rapid freezing and freeze-etching and (E) after fixation and thin sectioning. Dictyosomes (d), Golgi vesicles (gv), ER, plasma membrane (pm), cell wall (cw), and the incorporation of mucilage-containing vesicles (asterisks) are shown by surface view or in thin section, respectively. At the arrow (panel D) a late fusion stage of a Golgi vesicle with the plasma membrane can be seen (note the various intramembranous particles). Bars ¼ 0:1
m. (Originals by Dieter Volkmann.)

acid, an inhibitor of the Ca2+-ATPase in the ER, dense aggregates of ER and inactive dictyosomes were formed in the secretory cells (Busch and Sievers, 1993). Exocytosis was stimulated even by small increases in cytosolic [Ca2+] above the normal resting [Ca2+] (Carroll et al., 1998). Dialysis of the protoplasts with annexins (which were shown to be expressed in root cap cells as well as differentiating vascular tissue and elongating cells) also stimulated exocytosis at resting cytosolic [Ca2+], though there was no additive effect at very high [Ca2+] (Carroll et al., 1998; see also Chapter 31 by Poovaiah et al. in this volume).

Sievers et al.

Few investigations have studied the transition from statocytes to secretion cells. However, since cutting root caps into halves has initiated mucilage secretion in the then outer cells, Barlow (1984) has suggested a positional determination of cell function. In intact roots, the transition may also be a consequence of the time/differentiation sequence caused by the meristematic cycle time. The products of mucilage secretion are not the same in the various species investigated, but the main components of the mucilage are carbohydrates (94% [w/w] in maize), with fucose being one of the most prominent sugars (Bacic et al., 1986). A glycine-rich protein may be the major component of the maize mucilage protein fraction (Matsuyama et al., 1999a), which makes up
6% of the mucilage dry weight (Bacic et al., 1986). Only 3% (w/w) uronic acids were found in maize mucilage, indicating that maize root mucilage contains a rather low proportion of acidic pectic polysaccharides and that its enormous capacity for gel formation may be due, at least in part, to interactions of various other polysaccharides (Bacic et al., 1986). Even when fully hydrated, a drop of maize root mucilage retains its integrity and the associated water (99.9% of wet weight) is entrapped by a polymer network of relatively minute dry weight (Guinel and McCully, 1986). The water potential of the fully expanded gel is very low (7:3 kPa), indicating that the mucilage would lose water to all but the wettest soils (Guinel and McCully, 1986). Interestingly, the physical properties of the root cap mucilage change considerably with changing degrees of hydration: upon loss of water, the surface tension of the mucilage is strongly reduced, whereas its viscosity increases (Read and Gregory, 1997). These findings support the idea that mucilage plays a major role in the maintenance of root–soil contact in drying soil: as the surface tension decreases, the ability of the mucilage to wet the surrounding soil particles becomes greater, and as the viscosity and elasticity increase, the rhizosphere structure is stabilized and hydraulic continuity is upheld (Read and Gregory, 1997). The mucilage secreted by the root cap cells forms sizable pockets in the periplasmic space and is exuded into the extracellular space even under conditions of water stress, though it is not known how the large polymer passes through the wall (Guinel and McCully, 1986). The pattern of ice crystal formation in freeze-substituted material suggests that the content of the secretory vesicles and the mucilage in the periplasmic space are much less hydrated than the extracellular mucilage, possibly because physical pressure (turgor, compres-

Root Cap

sion) prevents water uptake until the mucilage is free to expand (Guinel and McCully, 1986). The intercellular connection between peripheral secretory cells is maintained at primary pit fields where mucilage does not accumulate. The plasmodesmata are apparently occluded prior to separation of the cells from the root cap (Vermeer and McCully, 1982). The latter process is dependent on the expression of a pectinmethylesterase (Hawes et al., 1998). Though some of the detached cells (Fig. 4B,C) are destined to die quickly (Matsuyama et al., 1999b), many remain alive and metabolically active for extended periods (Vermeer and McCully, 1982). The differentiation of root cap cells into these border cells (which can be induced in culture to divide and differentiate into organized tissue) is accompanied by a dramatic switch in gene expression and many of the synthesized proteins are quickly exported into the extracellular environment (Hawes et al., 1998). Apart from affecting the rhizosphere, border cells also seem to regulate the turnover of the root cap. The mitotic activity of the root cap meristem is apparently continuous when roots are grown under conditions where border cells are constantly dispersed. However, root cap mitosis is inhibited if border cells do not separate but remain appressed to the root periphery in the absence of free water (Brigham et al., 1998, and references cited therein). Upon removal of the border cells, mitosis is quickly (within 5–15 min) induced in the meristem, and the expression of genes involved in physiological processes specific for different cap regions (starch synthesis in columella, cell wall degradation at cap periphery) is increased concomitantly. These findings support the notion that root cap differentiation is a coordinated and regulated continuous process (Barlow, 1984). Nevertheless, individual cells may remain for some time in intermediate stages, depending on the state of accumulation of the border cells (Brigham et al., 1998).

VI.

ROLE OF ROOT CAP IN SENSING ENVIRONMENTAL SIGNALS

The basic functions of the root system are anchorage of the plant in the soil, uptake of water and mineral nutrients, and delivery of growth substances. In order to perform these functions successfully, roots must be able to respond to their surroundings, e.g., by adjusting growth and actively modifying the rhizosphere. This necessitates the capacity to sense environmental signals. As the outermost tissue that covers the grow-

39

ing root apex, the root cap plays a central role in the perception of such signals. A.

Water Gradients

Near-surface soils (
upper 0.3 m) are subject to intensive drying by direct water evaporation as well as by root water extraction. At very low water contents, steep humidity gradients develop in the soil (Wraith and Wright, 1998). These are sufficient to induce hydrotropic growth in roots (0:5 MPa mm1 for the agravitropic pea mutant Pisum sativum ageotropum; Takano et al., 1995). Though the hydrosensing cells have not yet been identified, it has been shown that perception of a water gradient takes place in the root cap; decapped roots fail to curve hydrotropically (Takahashi and Scott, 1993), and applying a very localized water potential gradient directly to the root cap via sorbitol-containing agar blocks caused the root to grow toward the higher water potential (Takano et al., 1995). The reaction time required for the commencement of the hydrotropic response is
3–4 h in roots of the pea mutant ageotropum. Curvature is initiated in the apical region of the elongation zone (Takano et al., 1995) and is preceded by a decrease in cell wall extensibility on the prospective concave side of the root (Hirasawa et al., 1997). For additional details see Chapter 20 by Porterfield in this volume. B.

Mechanical Impedance

All roots growing in soil experience mechanical impedance to varying degrees. While most studies have concentrated on the long-term effects of strong mechanical stress on root growth and morphogenesis (Bengoun and Mullins, 1990; Chapter 45 by Masle, in this volume), the observation of mechanically induced membrane potential changes (Monshausen and Sievers, 1998) and increases in cytosolic [Ca2+] (Legue´ et al., 1997) has shown that roots are capable of perceiving weak mechanical stimuli of short duration, and that peripheral cells of the root cap are particularly sensitive (Legue´ et al., 1997). These results support earlier findings by Goss and Russell (1980) that a slight (persistent) mechanical stimulus has a remarkably large initial effect on root elongation. Upon encountering small, loosely packed glass beads, the elongation rate of an intact maize root was rapidly reduced by more than two-thirds for a period of 10 min, after which it quickly returned to the original, unimpeded value. No change in the rate of elongation was observed in maize roots which had been decapped

40

Sievers et al.

45 min prior to contact with the beads. Apparently, the root cap must have a central role in regulating the root response to very low mechanical resistance. The root cap may also reduce mechanical stress by sloughing off cells, thereby reducing the frictional resistance experienced by the growing root (Bengough and McKenzie, 1997). C.

Gravity

The requirement of the root cap for graviperception by roots is well established; experimental evidence for this goes back to the pioneering root tip-removal experiments of Charles Darwin (1880). The unequivocal proof was provided by the careful decapping experiments of Juniper et al. (1966). Decapped roots did not respond to gravitropic stimulation, whereas their growth was unaltered. More recent evidence for the statocyte function of the root cap columella cells comes from laser ablation experiments (Blancaflor et al., 1998) specifying the innermost columella cells as the most important sites for graviperception. The mechanism of graviperception is far from being understood. However, since gravity can only work on (deform or displace) masses, biological gravisensors must be equipped with receptors which are able to perceive the information resulting from the physical process of deformation or displacement known as susception. For graviperception by higher plants the starch–statolith theory published by Ne˘mec (1900) and Haberlandt (1900) is widely accepted. In gravisensitive tissues of shoots and roots, starch-containing sedimentable amyloplasts were observed in specialized cells, the statocytes, and are believed to act as susceptors or statoliths (cf. Volkmann and Sievers, 1979; Wilkins, 1984; Bjo¨rkman, 1988; Sack, 1991, 1997; Chen et al., 1999; Chapters by Pilet [30] and by Porterfield [20] in this volume). A similar phenomenon was found in rhizoids and protonemata of the green alga Chara, where vesicles filled with barium-sulfate crystals were identified as statoliths whose gravitydirected sedimentation promotes gravitropic curvature (Sievers et al., 1996; Braun, 1997). In higher plants, a good correlation was found between gravisensitivity and amyloplasts of different starch content (Iversen, 1969; Perbal and Rivie`re, 1976; Busch and Sievers, 1990; Sack, 1991; Kiss et al., 1996, 1997; Weise and Kiss, 1999). Even starchless amyloplasts produced high signal-to-noise ratios to activate the hypothetical receptor molecules. Furthermore, Arabidopsis mutants lacking the endodermal parenchyma in shoots and roots were reported to show no shoot but root gravi-

tropism (Fukaki et al., 1998). Since amyloplast-containing statocytes are present in the shoot endodermis but not in the root endodermis, these mutants strongly support the proposed hypothesis. Nonstatolith theories have suggested the possibility that amyloplast sedimentation might not be the sole mechanism of gravity sensing (Sack, 1997). However, there is evidence from high-gradient magnetic field experiments that amyloplast sedimentation in statocytes of roots and shoots is sufficient to initiate gravitropic curvature. Root curvature occurred in the direction in which the amyloplasts were displaced by the magnetic field (Kuznetsov and Hasenstein, 1996). Molecules involved in the mechanism of graviperception, the transduction of the physical process of sedimentation into a physiological signal, are still to be characterized. The presentation time is very short; for cress roots it is 12 s (Iversen and Larsen, 1973). By intermittent stimulation, the minimum time to be perceived (perception time) by Avena coleoptiles and cress roots was determined to be 0.5 s (cf. Hejnowicz et al., 1998). During the perception time, the amyloplasts are displaced
8 nm. Thus, attention should be paid to small displacements of statoliths in order to understand how gravity effects on statoliths are transferred to competent cellular structures (Sievers et al., 1991b). Cytoskeletal elements of the statocytes are the most likely candidates to be involved in the transduction of the statolith sedimentation into a physiological signal which is transmitted to the responding target cells in the root elongation zone (Sievers et al., 1991b). Circumstantial evidence for this model is provided by the finding that treatment of Phleum roots with the actin inhibitor cytochalasin D inhibited gravityinduced pH changes at the root surface (Monshausen et al., 1996). Eight minutes after tilting, the surface pH of gravistimulated control roots starts to decrease at the meristem and apical elongation zone of the physically upper root side (Zieschang et al., 1993; Monshausen et al., 1996) On the other hand, cytochalasin D had no effect on the graviresponse in roots of some other species (Staves et al., 1997). The unique lack of prominent actin bundles and microtubules in the statocyte interior probably facilitates unconstrained sedimentation of their statoliths (Balus˘ ka and Hasenstein, 1997; Volkmann and Balus˘ ka, 1999). A proposed delicate meshwork of oligomeric actin surrounding the statoliths and myosins localized in the vicinity of the sedimented statoliths (Balus˘ ka and Hasenstein, 1997) are the most likely basis for statolith movements and the displacement of statoliths under microgravity conditions (Fig. 5; Volkmann et al.,

Root Cap

1991; Driss-Ecole et al., 2000). This was also shown in single-cell systems (Braun, 1996, 1997). However, inhibitors affecting the polymerization of actin (cytochalasin, phalloidin) and tubulin (colchicine, taxol) cause significant differences in the sedimentation rate of amyloplasts (Sievers et al., 1989). These results also support the idea that actin microfilaments or microtubules are involved in statolith-mediated graviperception. Cytoskeletal filaments could be interconnected with mechanosensitive ion channels either in the cortical ER or other membranes (Fig. 6; Falke et al., 1988; Schroeder and Hedrich, 1989; Pickard and Ding, 1992; Hwang et al., 1997). Even the energy of small displacements of statoliths amplified by the cytoskeleton would allow dramatic changes in ion fluxes including that of calcium which appears to play a crucial role in signal transduction (Bjo¨rkman, 1988; Sievers et al., 1991a; Sievers and Busch, 1992; Chapter 31 by Poovaiah et al. in this volume). This is in full agreement with the tensegrity model of cytoskeletonmediated mechanotransduction (Ingber, 1993). In the characean rhizoids and protonemata a crucial but different role of actin in gravitropism was demonstrated (Braun and Wasteneys, 2000). Further support for a participation of the cytoskeleton in graviperception comes from a genetic approach. A gene has been identified that affects the graviperception phase in Arabidopsis mutants. The ARG1 locus encodes for a 45-kDa DnaJ-like protein

41

Figure 5 Light microscope photographs of median longitudinal sections through the statenchyma of cress roots. (A) Control cress root which was fixed with potassium permanganate at 1 g. (B) Cress root which was fixed at the end of the 6-min microgravity phase of the parabolic flight of a rocket (TEXUS). Microgravity conditions resulted in a considerable displacement of the statolith amyloplasts in the direction opposite to that of the gravity vector. Bar ¼ 10
m. (From Volkmann et al., 1991.)

Figure 6 Scheme of a central root statocyte of cress illustrating the difference in the tension of the proposed oligomeric actinnetwork and the asymmetrical activities of ion channels, both induced by gravistimulation. (A) Normal vertical orientation with most actin microfilaments in tension. (B) Horizontal orientation with asymmetrically stretched and relaxed actin microfilaments due to the gravity-induced displacement of the amyloplasts. (a) amyloplast (statolith); (ER) endoplasmic reticulum; (g) direction of gravity; (mf) actin microfilament; (mt) microtubulus; (n) nucleus; (PD) plasmodesma; (PM), plasma membrane. Open and closed stars symbolize postulated channels in two different activity states. (From Sievers et al., 1991b.)

42

containing a coiled-coil domain which is typical for cytoskeleton-interacting proteins (Sedbrook et al., 1999). A cytoskeleton membrane anchoring function seems possible because of the presence of several hydrophobic amino acids in the middle, and a putative transmembrane region. ARG1 may represent a component of the early cytoskeleton-mediated gravity signal transduction chain. However, because ARG1-protein is expressed in all plant tissues and is related to a conserved signal transduction molecule, a more general function of ARG1 in signal transduction, protein folding, or protein trafficking can not be ruled out. Recently, rapid changes in cytosolic pH have been demonstrated within Arabidopsis columella cells upon gravistimulation, suggesting that pH gradients might be involved at a very early stage of the gravitropic signal transduction pathway (Scott and Stro¨mgren Allen, 1999). Calcium and phosphoinositides possibly act as second messengers in the signal transduction pathway (Perera et al., 1999). High concentrations of calcium were detected in statocyte amyloplasts and membranes (Chandra et al., 1982; Busch et al., 1993), and calmodulin concentrations in statocytes are higher than in all other cell types (Allan and Trewavas, 1985), though the cytoplasmic [Ca2+] in statocytes is the same as in other root tissues (Legue´ et al., 1997). Furthermore, the degree of the polar arrangement of organelles in statocytes and the gravisensitivity of roots was reduced or eliminated after application of blockers of stretch-activated calcium channels and inhibitors of calmodulin or Ca2+ATPase activities (Biro et al., 1982; Bjo¨rkmann and Leopold, 1987; Stinemetz et al., 1987; Wendt and Sievers, 1989; Sievers and Busch, 1992). Lu and Feldman (1997) discussed the involvement of a Ca2+/calmodulin-dependent protein kinase in the light-dependent gravitropism of maize roots. These data indicate the involvement of calcium–calmodulin activity in the gravitropic signal-transduction pathway, but only of touch-induced changes (Legue´ et al., 1997). In this context it is noteworthy that a root is not only gravistimulated by tilting (dynamic gravistimulation; Sievers et al., 1991b). Vertically oriented roots commonly used as controls are also permanently (statically) stimulated. Statocytes of roots should be investigated in a stimulus-free microgravity environment— such as in space (Sievers, 1999). Nevertheless, calcium seems to act as a second messenger interfering with the polar transport of auxin within the root cap, and could mediate the redirection of auxin to the physically lower root flank (Lee et al., 1984; Lee and Evans, 1985;

Sievers et al.

Evans and Hasenstein, 1987). Additional information on gravitropic response mechanisms involving calcium and auxin is presented in this volume by Pilet (Chapter 31), by Porterfield (20), and by Poovaiah et al. (31). D.

Aluminum

Root exposure to toxic concentrations of aluminum causes a rapid decrease in the rate of root elongation. While this decrease is chiefly due to the inhibition of extension in the main elongation zone, the apical millimeter of the maize root (comprising the meristem and the apical part of the distal elongation zone) is the most Al-sensitive region of the root (Kollmeier et al., 2000). As aluminum treatment of this region inhibits extension in the more proximal part of the elongation zone, Kollmeier et al. (2000) suggest that a signaling pathway in the root apex mediates the Al signal between the meristem/distal elongation zone and the elongation zone proper (possibly via alterations in basipetal auxin transport). Aluminum binding by root cap mucilage may be one way of protecting the sensitive root apex from the toxic effects of Al (see also Chapter 46 by Matsumoto in this volume). Aluminum accumulates in the mucilage of maize roots, where it is tightly bound and thereby rendered nonphytotoxic (Li et al., 2000). However, Al binding by the mucilage does not confer effective protection to maize roots growing in hydroponic culture. This may reflect the relative amount of mucilage-bound Al to total Al in the bulk solution. In the soil, where Al movement is more restricted and where a root can modify its immediate environment via the activity of border cells (Hawes et al., 1998), binding of Al by mucilage might be more effective. Another explanation for this difference might be that in hydroponics the secreted mucilage is continuously washed away, leaving the apex exposed to the Al containing solution. Incubation of individual border cells with Al induces the production of a thick mucilage layer around each cell, which is correlated with the cessation of Al-induced border cell death (Hawes et al., 2000). E.

Pathogens

In the soil, the root is constantly exposed to a variety of pathogenic organisms. Protection of the root apex is especially vital, as the continued exploitation of the soil for water and nutrients depends on the root’s ability to move into new surroundings. Root cap border cells seem to play a major role in such protective

Root Cap

measures, ranging from temporarily immobilizing nematodes and the production of bacteria-repelling mucilage to acting as decoys for pathogenic fungi (Hawes et al., 2000).

VII.

REGENERATION OF THE ROOT CAP

Removal of the entire root cap without damaging cells of the root body, which is possible in roots of the closed type, provides an artificial system to use in studying the development of statocytes (Barlow, 1974a,b; Barlow and Grundwag, 1974; Hillman and Wilkins, 1982). After decapping, the cells of the quiescent center start to divide, initiating a new root cap. Prior to this, proplastids within the outermost apical cell layer of the quiescent center are transformed into amyloplasts and sediment in the direction of gravity. The onset of gravisensitivity is dependent on the existence of those sedimenting amyloplasts (Hillman and Wilkins, 1982). Twelve hours after decapping, amyloplasts are not displacable even by centrifugation (25 g), whereas after 24 h displacement of amyloplasts by centrifugation occurred. Hillman and Wilkins (1982) suggested a change in the characteristics of the cytoplasm (i.e., rearrangement of the actin cytoskeleton). By using a root cap-specific promoter to express a diphtheria toxin gene in Arabidopsis, several layers of the root caps were genetically ablated continuously throughout the plant’s life cycle (Tsugeki and Fedoroff, 1999). This had severe effects on root development. In these transgenic root caps two layers of columella cells and some of the lateral root cap cells were missing, and the remaining layers were increasingly disorganized. The transgenic roots were agravitropic and showed severely inhibited growth. Whereas the overall mitotic activity of the root meristem seemed decreased in the transgenic plants, cell division was observed in the normally mitotically inactive quiescent center. Epidermal root hair development, vascularization, and vacuolization commenced much closer to the apex than in wild-type roots and the transgenic roots were more branched (Tsugeki and Fedoroff, 1999). The authors conclude that essential components of a signaling system (specifically an auxin redistribution system) that determines root architecture reside in the root cap and disruption of this system by genetic ablation of the cap causes the observed changes in root development.

43

VIII.

CONCLUSIONS

As a consequence of its multitude of functions, such as protection of the meristem, production of mucilage, sensing gravity and other environmental signals, the root cap has attracted the attention of cell biologists and physiologists for over a century. Some of the aspects most thoroughly studied were the structure and function of the Golgi apparatus, mechanisms of membrane flow, exocytosis, development of cytoskeleton-dependent cell polarity, and cytoskeleton-mediated transduction of the gravistimulus. Future research will focus mainly on molecular characterization of elements involved in the manifold signal transduction pathways and on the generation and transmission of signals from the root cap directed at the apical elongation zone of the root. REFERENCES Allan E, Trewavas AJ. 1985. Quantitative changes in calmodulin and NAD kinase during early cell development in the root apex od Pisum sativum. Planta 165:493–501. Bacic A, Moody SF, Clarke AE. 1986. Structural analysis of secreted root slime from maize (Zea mays L.). Plant Physiol 80:771–777. Balus˘ ka F, Hasenstein KH. 1997. Root cytoskeleton: its role in perception of and response to gravity. Planta 203:S69–S78. Balus˘ ka F, Kreibaum A, Vitha S, Parker JS, Barlow PW, Sievers A. 1997. Central root cap cells are depleted of endoplasmic microtubules and actin filament bundles: implications for their role as gravity-sensing statocytes. Protoplasma 196:212–223. Barlow PW. 1974a. Regeneration of the cap of primary roots of Zea mays. New Phytol 73:937–954. Barlow PW. 1974b. Recovery of geotropism after removal of the root cap. J Exp Bot 25:1137–1146. Barlow PW. 1975. The root cap. In: Torrey JG, Clarkson DT, eds. The Development and Function of Roots. London; Academic Press, pp 21–54. Barlow PW. 1984. Positional controls in root development. In: Barlow PW, Carr DJ, eds. Positional Controls in Plant Development. Cambridge, UK: Cambridge University Press, pp 281–318. Barlow PW, Grundwag M. 1974. The development of amyloplasts in cells of the quiescent centre of Zea roots in response to removal of the root cap. Z Pflanzenphysiol 73:56–64. Barlow PW, Sargent JA. 1978. The ultrastructure of the regenerating root cap of Zea mays L. Ann Bot 42:791–799. Barlow PW, Hawes CR, Horne JC. 1984. Structure of amyloplasts and endoplasmic reticulum in the root caps of

44 Lepidium sativum and Zea mays observed after selective membrane staining and by high-voltage electron microscopy. Planta 160:363–371. Bengough AG, Mullins CE. 1990. Mechanical impedance to root growth: a review of experimental techniques and root growth responses. J Soil Sci 41:341–358. Bengough AG, McKenzie BM. 1997. Sloughing of root cap cells decreases the frictional resistance to maize (Zea mays L.) root growth. J Exp Bot 48:885–893. Biro RL, Hale CC II, Wiegand OF, Roux SJ. 1982. Effects of chlorpromazine on gravitropism in Avena coleoptiles. Ann Bot 50:735–747. Bjo¨rkman T. 1988. Perception of gravity by plants. Adv Bot Res 15:1–41. Bjo¨rkman T, Leopold AC. 1987. Effect of inhibitors of auxin transport and of calmodulin on a gravisensing-dependent current in maize roots. Plant Physiol 84:847–850. Blancaflor EB, Hasenstein K-H. 1997. The organization of the actin cytoskeleton in vertical and graviresponding primary roots of maize. Plant Physiol 113:1447–1455. Blancaflor EB, Fasano JM, Gilroy S. 1998. Mapping the functional roles of cap cells in the response of Arabidopsis primary roots to gravity. Plant Physiol 116:213–222. Braun M. 1996. Immunolocalization of myosin in rhizoids of Chara globularis Thuill. Protoplasma 191:1–8. Braun M. 1997. Gravitropism in tip-growing cells. Planta 203:S11–S19. Braun M, Wasteneys GO. 2000. Actin in characean rhizoids and protonemata. Tip growth, gravity sensing and photomorphogenesis. In: Staiger CJ, Balusˇ ka F, Volkmann P, Barlow P, eds. Actin: a Dynamic Framework for Multiple Plant Cell Functions. Dordrecht, Netherlands: Kluwer Academic Publishers, pp 237–258. Brigham LA, Woo H-H, Wen F, Hawes MC. 1998. Meristem-specific suppression of mitosis and a global switch in gene expression in the root cap of pea by endogenous signals. Plant Physiol 118:1223–1231. Buckhout TJ. 1983. ATP-dependent Ca2+-transport in endoplasmic reticulum isolated from the roots of Lepidium sativum L. Planta 159:84–90. Busch MB, Sievers A. 1990. Hormone treatment of roots causes not only a reversible loss of starch but also of structural polarity in statocytes. Planta 181:358–364. Busch MB, Sievers A. 1993. Membrane traffic from the endoplasmic reticulum to the Golgi apparatus is disturbed by an inhibitor of the Ca2+-ATPase in the ER. Protoplasma 177:23–31. Busch MB, Ko¨rtje KH, Rahmann H, Sievers A. 1993. Characteristic and differential calcium signals from cell structures of the root cap detected by energy-filtering electron microscopy (EELS/ESI). Eur J Cell Biol 60:88–100. Carroll AD, Moyen C, Van Kesteren P, Tooke F, Battey NH, Brownlee C. 1998. Ca2+, annexins, and GTP

Sievers et al. modulate exocytosis from maize root cap protoplasts. Plant Cell 10:1267–1276. Chandra S, Chabot JF, Morrison GH, Leopold AC. 1982. Localization of Ca2þ in amyloplasts of root cap cells using ion microscopy. Science 216:1221–1223. Chen R, Rosen E, Masson PH. 1999. Gravitropism in higher plants. Plant Physiol 120:343–350. Darwin C. 1880. The Power of Movement in Plants. London; John Murray. Dolan L, Janmaat K, Willemsen V, Linstead P, Poethig S, Roberts K, Scheres B. 1993. Cellular organisation of the Arabidopsis thaliana root. Development 119:71–84. Driss-Ecole D, Jeune B, Prouteau M, Julianus P, Perbal G. 2000. Lentil root statoliths reach a stable state in microgravity. Planta 211:396–405. Evans ML, Hasenstein K-H. 1987. Stimulus-response coupling in the action of auxin and gravity on roots. In: Cosgrove DJ, Knievel DP, eds. Physiology of Cell Expansion During Plant Growth. Bethesda, MD: American Society of Plant Physiologists, pp 202–214. Falke LC, Edwards KL, Pickard BG, Misler S. 1988. A stretch-activated anion channel in tobacco protoplasts. FEBS Lett 237:141–144. Friedrich U, Sievers A. 1985. Ontogeny of cell polarity in root statocytes of Lepidium sativum L. in the developing embryo and during germination. Eur J Cell Biol 36(suppl 7):18. Fukaki H, Wysocka-Diller J, Kato T, Fujisawa H, Benfey PN, Tasaka M. 1998. Genetic evidence that the endodermis is essential for shoot gravitropism in Arabidopsis thaliana. Plant J 14:425–430. Goss MJ, Russell RS. 1980. Effects of mechanical impedance on root growth in barley (Hordeum vulgare L.). J Exp Bot 31:577–588. Griffiths HJ, Audus LJ. 1964. Organelle distribution in the statocyte cells of the root-tip of Vicia faba in relation to geotropic stimulation. New Phytol 63:319–332. Grolig F. 1990. Actin-based organelle movements in interphase Spirogyra. Protoplasma 155:29–42. Grolig F, Wagner G. 1988. Light dependent chloroplast reorientation in Mougeotia and Mesotaenium:Biased by pigment-regulated plasmalemma anchorage sites to actin filaments. Bot Acta 101:2–6. Guinel FC, McCully ME. 1986. Some water-related physical properties of maize root-cap mucilage. Plant Cell Environ 9:657–665. Haberlandt G. 1900. U¨ber die Perzeption des geotropischen Reizes. Ber Dtsch Bot Ges 18:261–272. Hawes MC, Brigham LA, Wen F, Woo HH, Zhu Y. 1998. Function of root border cells in plant health: pioneers in the rhizosphere. Annu Rev Phytopathol 36:311327. Hawes MC, Gunawardena U, Miyasaka S, Zhao X. 2000. The role of root border cells in plant defense. Trends Plant Sci 5:128–133.

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45 Juniper BE, French A. 1973. The distribution and redistribution of endoplasmic reticulum (ER) in geoperceptive cells. Planta 109:211–224. Juniper BE, Groves S, Landau-Schachar B, Audus LJ. 1966. Root cap and the perception of gravity. Nature 209:93–94. Kiss JZ, Wright JB, Caspar T. 1996. Gavitropism in the roots of intermediate-starch mutants of Arabidopsis. Physiol Plant 97: 237–244. Kiss JZ, Katembe WJ, Edelmann RE. 1997. Gravitropism and development of wild-type and starch-deficient mutans of Arabidopsis during spaceflight. Physiol Plant 102:493–502. Kollmeier M, Felle HH, Horst WJ. 2000. Genotypical differences in aluminum resistance of maize are expressed in the distal part of the transition zone. Is reduced basipetal auxin flow involved in inhibition of root elongation by aluminum? Plant Physiol 122:945–956. Kuznetsov O, Hasenstein KH. 1996. Intracellular magnetophoresis of amyloplasts and induction of root curvature. Planta 198:87–94. Kuznetsov O, Hasenstein KH. 1997. Magnetophoretic induction of curvature in coleoptiles and hypocotyls. J Exp Bot 48:1951–1957. Lee JS, Evans ML. 1985. Polar transport of auxin across gravistimulated roots of maize and its enhancement by calcium. Plant Physiol 77:824. Lee JS, Mulkey TJ, Evans ML. 1984. Inhibition of polar calcium movement and gravitropism in roots treated with auxin-transport inhibitors. Planta 160:536–543. Legue´ V, Blancaflor E, Wymer C, Perbal G, Fantin D, Gilroy S. 1997. Cytoplasmic free Ca2+ in Arabidopsis roots changes in response to touch but not gravity. Plant Physiol 114:789–800 Li XF, Ma JF, Hiradate S, Matsumoto H. 2000. Mucilage strongly binds aluminum but does not prevent roots from aluminum injury in Zea mays. Physiol Plant 108:152–160. Lorenzi G, Perbal G. 1990. Actin filaments responsible for the location of the nucleus in the lentil statocyte are sensitive to gravity. Biol Cell 68:259–263. Lu Y-T, Feldman LJ. 1997. Light-regulated root gravitropism: a role for, and characterization of, a calcium/calmodulin-dependent protein kinase homolog. Planta 203:S91–S97. Matsuyama T, Satoh H, Yamada Y, Hashimoto T. 1999a. A maize glycine-rich protein is synthesized in the lateral root cap and accumulates in the mucilage. Plant Physiol 120:665–674. Matsuyama T, Yasumura N, Funakoshi M, Yamada Y, Hashimoto T. 1999b. Maize genes specifically expressed in the outermost cells of root cap. Plant Cell Physiol 40:469–476. Mollenhauer HH, Morre´ DJ. 1976. Cytochalasin B, but not colchicine, inhibits migration of secretory vesicles in root tips of maize. Protoplasma 87:39–45.

46 Monshausen GB, Sievers A. 1998. Weak mechanical stimulation causes hyperpolarisation in root cells of Lepidium. Bot Acta 111:303–306. Monshausen GB, Zieschang HE, Sievers A. 1996. Differential proton secretion in the apical elongation zone caused by gravistimulation is induced by a signal from the root cap. Plant Cell Environ 19:1408–1414. Moore R. 1983. A morphometric analysis of the ultrastructure of columella statocytes in primary roots of Zea mays L. Ann Bot 51:771–778. Moore R. 1984. Cellular volume and tissue partitioning in caps of primary roots of Zea mays. Am J Bot 71:1452– 1454. Moore R. 1985. A morphometric analysis of the redistribution of organelles in columella cells in primary roots of normal seedlings and agravitropic mutants of Hordeum vulgare. J Exp Bot 36:1275–1286. Moore R, McClelen CE. 1983. A morphometric analysis of cellular differentiation in the root cap of Zea mays. Am J Bot 70:611–617. Moore R, Fondren WM, Koon EC, Wang C-L. 1986. The influence of gravity on the formation of amyloplasts in columella cells of Zea mays L. Plant Physiol 82:867– 868. Moore R, Fondren WM, McClelen CE, Wang C-L. 1987. Influence of microgravity on cellular differentiation in root caps of Zea mays. Am J Bot 74:1006–1012. Ne˘mec B. 1900. U¨ber die Art der Wahrnehmung des Schwerkraftreizes bei den Pflanzen. Ber Dtsch Bot Ges 18:241–245. Olsen GM, Iversen T-H. 1980. Ultrastructure and movements of cell structures in normal pea and an ageotropic mutant. Physiol Plant 50:275–284. Olsen GM, Mirza JI, Maher EP, Iversen T-H. 1984. Ultrastructure and movements of cell organelles in the root cap of agravitropic mutants and normal seedlings of Arabidopsis thaliana. Physiol Plant 60:523–531. Parke J, Miller C, Anderton BH. 1986. Higher plant myosin heavy-chain identified using a monoclonal antibody. Eur J Cell Biol 41:9–13. Perbal G, Rivie`re S. 1976. Relation entre reaction ge´otropique et e´volution due statenchyme dans la racine d’asperge. Physiol Plant 38:39–47. Perbal G, Driss-Ecole D, Rutin J, Salle G. 1987. Graviperception of lentil seedling roots grown in space (Spacelab Dl mission). Physiol Plant 70:119-126. Perera IY, Heilmann I, Boss WF. 1999. Transient and sustained increases in inositol-1,2,5-trisphosphate precede the differential growth response in gravistimulated maize pulvini. Proc Natl Acad Sci USA 96:5838–5843. Pickard BG, Ding JP. 1992. Gravity sensing by higher plants. Adv Comp Environ Physiol 10:81–110. Ransom JS, Moore R. 1983. Geoperception in primary and lateral roots of Phaseolus vulgaris (Fabaceae): 1. Structure of columella cells. Am J Bot 70:1048–056.

Sievers et al. Read DP, Gregory PJ. 1997. Surface tension and viscosity of axenic maize and lupin root mucilages. New Phytol 137:623–628. Sack FD. 1991. Plant gravity sensing. Int Rev Cytol 127:193– 252. Sack FD. 1997. Plastids and gravitropic sensing. Planta 203:S63–S68. Sack FD, Priestley DA, Leopold AC. 1983. Surface charge on isolated maize-coleoptile amyloplasts. Planta 157:511–517. Sargent JA, Osborne DJ. 1980. A comparative study of the fine structure of coleorhiza and root cells during the early hours of germination of rye embryos. Protoplasma 104:91–103. Scheres B, Wolkenfelt H, Willemsen V, Terlouw M, Lawson E, Dean C, Weisbeek P. 1994. Embryonic origin of the Arabidopsis primary root and root meristem initials. Development 120:2475–2487. Schroeder JI, Hedrich R. 1989. Involvement of ion channels and active transport in osmoregulation and signaling of higher plant cells. TIBS 14:187–192. Scott AC, Stro¨mgren Allen N. 1999. Changes in cytosolic pH within Arabidopsis root columella cells play a key role in the early signaling pathway for root gravitropism. Plant Physiol 121:1291–1298. Sedbrook J, Chen R, Masson P. 1999. ARG1 (Altered Response to Gravity) encodes for a novel DnaJ-like protein which potentially interacts with the cytoskeleton. Proc Natl Acad Sci USA 96:1140–1145. Sievers A. 1999. Gravitational biology in Bonn. Am Soc Gravit Space Biol Newslett 15(3):15–22. Sievers A, Busch MB. 1992. An inhibitor of the Ca2+ATPases in the sarcoplasmic and endoplasmic reticula inhibits transduction of the gravity stimulus in cress roots. Planta 188:619–622. Sievers A, Heyder-Caspers L. 1983. The effect of centrifugal acceleration on the polarity of statocytes and on the graviperception of cress roots. Planta 157:64–70. Sievers A, Volkmann D. 1972. Verursacht differentieller Druck der Amyloplasten auf ein komplexes Endomembransystem die Geoperzeption in Wurzeln? Planta 102:160–172. Sievers A, Volkmann D. 1977. Ultrastructure of gravity-perceiving cells in plant roots. Proc R Soc Lond B 199:525–536. Sievers A, Volkmann D, Hensel W, Sobick V, Briegleb W. 1976. Cell polarity in root statocytes in spite of simulated weightlessness. Naturwissenschaften 63:343. Sievers A, Kruse S, Kuo-Huang L-L, Wendt M. 1989. Statoliths and microfilaments in plant cells. Planta 179:275–278. Sievers A, Kramer-Fischer M, Braun M, Buchen B. 1991a. The polar organization of the growing Chara rhizoid and the transport of statoliths are actin-dependent. Bot Acta 104:103–109.

Root Cap Sievers A, Buchen B, Volkmann D, Hejnowicz Z. 199lb. Role of the cytoskeleton in gravity perception. In: Lloyd CW, ed. The Cytoskeletal Basis of Plant Growth and Form. London; Academic Press, pp 169–182. Sievers A, Buchen B, Hodick D. 1996. Gravity sensing in tipgrowing cells. Trends Plant Sci 1:273–279. Sinclair W, Trewavas AL. 1997. Calcium in gravitropism. A re-examination. Planta 203:S85–S90. Staehelin LA, Giddings TH Jr, Kiss JZ, Sack FD. 1990. Macromolecular differentiation of Golgi stacks in root tips of Arabidopsis and Nicotiana seedlings as visualized in high pressure frozen and freeze-substituted samples. Protoplasma 157:75–91. Staves MP, Wayne R, Leopold AC. 1997. Cytochalasin D does not inhibit gravitropism in roots. Am J Bot 84:1530–1535. Stephenson JLM, Hawes CR. 1986. Stereology and stereometry of endoplasmic reticulum during differentiation in the maize root cap. Protoplasma 131:32–46. Stinemetz CL, Kuzmanoff KM, Evans ML, Jarrett HW. 1987. Correlation between calmodulin activity and gravitropic sensitivity in primary roots of maize. Plant Physiol 84:1337–1342. Takahashi H, Scott TK. 1993. Intensity of hydrostimulation for the induction of root hydrotropism and its sensing by the root cap. Plant Cell Environ 16:99–103. Takano M, Takahashi H, Hirasawa T, Suge H. 1995. Hydrotropism in roots: sensing of a gradient in water potential by the root cap. Planta 197:410–413. Tsugeki R, Fedoroff NV. 1999. Genetic ablation of root cap cells in Arabidopsis. Proc Natl Acad Sci USA 96:12941–12946. Vermeer J, McCully ME. 1982. The rhizosphere in Zea: new insight into its structure and development. Planta 156:45–61. Volkmann D. 1974. Amyloplasten und Endomembranen: das Geoperzeptionssystem der Prima¨rwurzel. Protoplasma 79:159–183. Volkmann D, Balusˇ ka F. 1999. Actin cytoskeleton in plants: from transport networks to signalling networks. Microsc Res Technique 47:135–154. Volkmann D, Czaja AWP. 1981. Reversible inhibition of secretion in root cap cells of cress after treatment with cytochalasin B. Exp Cell Res 135:229–236.

47 Volkmann D, Sievers A. 1975. Wirkung der Inversion auf die Anordnung des Endoplasmatischen Reticulums und die Polarita¨t von Statocyten in Wurzeln von Lepidium sativum. Planta 127:11–19. Volkmann D, Sievers A. 1979. Graviperception in multicellular organs. In: Haupt W, Feinleib ME, eds. Encyclopedia of Plant Physiology, New Series. Vol. 7, Physiology of Movements. Berlin; Springer-Verlag, pp 573–600. Volkmann D, Behrens HM, Sievers A. 1986. Development and gravity sensing of cress roots under microgravity. Naturwissenschaften 73:438–441. Volkmann D, Buchen B, Hejnowicz Z, Tewinkel M, Sievers A. 1991. Oriented movement of statoliths studied in a reduced gravitational field during parabolic flights of rockets. Planta 185:153–161. Von Guttenberg H. 1968. Der Prima¨re Bau der Angiospermenwurzel. In: Linsbauer K, ed. Handbuch der Pflanzenanatomie, 2nd ed., Vol. VIII, Part 5. Berlin; Borntraeger. Weise S, Kiss JZ. 1999. Gravitropism of inflorescence stems in starch-deficient mutants of Arabidopsis. Int J Plant Sci 160:521–527. Wendt M, Sievers A. 1986. Restitution of polarity in statocytes from centrifuged roots. Plant Cell Environ 9:17– 23. Wendt M, Sievers A. 1989. The polarity of statocytes and the gravisensitivity of roots are dependent on the concentration of calcium in statocytes. Plant Cell Physiol 30:929–932. White RG, Sack FD. 1990. Actin microfilaments in presumptive statocytes of root caps and coleoptiles. Am J Bot 77:17–26. Wilkins MB. 1984. Gravitropism. In: Wilkins MB, ed. Advanced Plant Physiology. London: Pitman, pp 163–185. Wraith JM, Wright CK. 1998. Soil water and root growth. Hort Sci 33:951–959. Zieschang HE, Koehler K, Sievers A. 1993. Changing proton concentrations at the surfaces of gravistimulated Phleum roots. Planta 190:546–554.

4 Cellular Patterning in Root Meristems: Its Origins and Significance Peter W. Barlow IACR–Long Ashton Research Station, University of Bristol, Long Ashton, Bristol, England

I.

INTRODUCTION

directly on root systems (e.g., Fitter, 1987), whereas others argue that the unit of selection is the whole plant (e.g., Peterson, 1992) on the grounds that it is the whole organism, rather than any particular organ, whose fitness is tested by selective pressures. However, it can also be argued that, on the one hand, populations are the subject of selection or, on the other hand, that it is the hereditary material (genes) which is selected. All arguments have some validity, particularly within the context of a hierarchically organized plant kingdom in which the levels of organization range from the molecular to the individual and the population (Barlow, 1999). Nevertheless, it cannot be denied that the root, like any other organ, contributes to fitness and, thus, is one of the elements of the plant which is offered for natural selection. Likewise, the cellular construction of roots also features somewhere in this general problem of fitness and plant evolution. Plant forms evolve as a result of an interaction between the energy levels inherent to the biotic and abiotic components of the total environment. Roots and their cells have undergone selection not only to resist potentially fatal encounters with the environment (e.g., heavy metals, drought), but also to serve the needs of the whole organism, or organ, of which they are, respectively, a part. Thus, they have characteristics that are relevant to both the evolutionary and functional aspects of plant biology. Each plant form is a

Roots are an integral component of nearly all plants. Where they are absent, as in certain water plants (Ceratophyllum, Salvinia, Utricularia, and Wolffia spp., for example), this is probably the result of evolutionary loss with the usual root function being performed by other organs. This last statement points to two important areas of biological enquiry which have been differentiated as evolutionary and functional biology (Mayr, 1961). In plants, evolutionary biology deals with problems of population genetics, ecology, and palaeontology, whereas functional biology includes the disciplines of anatomy, biochemistry, and embryology. In the present discussion of cellular patterning within root tissues, it is useful to keep these two avenues of enquiry in mind, for they should lead to an understanding not only of the advantages that cellular patterns confer in enhancing the fitness of the individual and hence of the population of which the individual is a member, but also the means by which such patterns are achieved. However, in considering the evolutionary significance of the cellular structure of roots, the question arises as to how such a property relates to natural selection. The units of living matter on which selection acts are topics of debate in the context of the neo-Darwinian interpretation of evolution. Some scholars suggest that natural selection acts 49

50

Barlow

member of a set of archetypes, these being particular energetically favourable biological constructions (cf. Hill, 1990). Certain archetypes might be favoured by natural selection on account of their positive contribution to fitness in a given environment. Examples, at the organism level, are the architectural ‘‘models’’ proposed for the shoot systems of tropical trees by Halle´ et al. (1978) and the rather similar models for root systems adopted by Jenı´ k (1978). Likewise, at the level of individual organs, the forms of shoots and roots represent another set of archetypes. Comparison of the forms of terrestrial roots with, for example, those of the river-dwelling members of the Podostemaceae, the cellular patterns of whose curious thalloid roots were described by Schnell (1967), illustrates the range of rhizogenetic possibilities that have been selected by the environment (Barlow, 1986, 1994a). By the same token, the characteristic pattern of cells that construct organs have also been determined by particular sets of conditions which reside at the various levels of organization whose processes impinge upon the level of the cell (Barlow, 1993, Fig. 1). These conditions include those which are inherent to (1) the cells themselves, (2) the organ to which the cells belong, and (3) the plant and its external environment. In this respect, it would not be surprising if the cellular patterns of roots and other organs also comprised a set of archetypes (Lu¨ck and Lu¨ck, 1986, 1993).

II.

SIGNIFICANCE OF CELLULAR PATTERNS

From time to time the role of cells in the development of plant organs and plant form is reappraised (Sinnott, 1960; Kaplan and Hagemann, 1991; Sitte, 1992; Barlow, 1994b). Some of the persistent concern surrounding this topic is a response to observations on the morphogenetic processes of animals. Here, cell migrations not only establish the sites of stem cells, which are responsible for tissue renewal, but also help generate the form of the growing embryo. Plant development, by contrast, is accompanied by much less movement of cells relative to one another: examples where this regularly occurs are in the processes of fertilization and intrusive growth of fibers in secondary tissues. However, the location of stem cells is a crucial feature of plant morphogenesis (Barlow, 1995a). Observations on the germination and growth of -irradiated seedlings, where cell divisions are largely abolished, are often mentioned as supporting the idea that the form of a plant organ (e.g., a root) develops with

only the minimal involvement of cell division (Haber, 1968). However, this evidence is relatively weak because both the cells comprising the organ and the polarity of cell and organ growth were formed prior to the experimental irradiation event. Since few or no cells are formed postirradiation, this system reveals only that the growth and polarity of preformed cells are not disturbed by irradiation. Nevertheless, in primary roots of -irradiated maize plantlets, it is possible to find that the cells of the quiescent center are provoked into division by the -rays (Barlow, unpublished), just as in the case of young, growing root apices irradiated with x-rays (Clowes, 1964). Other arguments against the necessity of cells for morphogenesis rely on examples from marine plants (Sinnott, 1960). Here, complex forms can be developed by organisms which are coenocytes, where one giant cell may contain thousands of nuclei (Jacobs, 1994). The aqueous environment is one in which plants are free-floating and buoyant. As a result, tissues and organs experience little of their own mass. On land, however, plant tissues and organs do experience their mass and also the forces exerted on them by adjoining organs. Furthermore, once plants grow away from the relatively calm conditions of the soil–air interface in which their seeds germinated, their shoots are subject to buffetting by the aerial environment and their roots experience the impedance of the medium in which they grow. In both these environments, the interpolation of internal mechanical struts and baffles helps counteract these externally applied physical forces which might otherwise compromise the integrity of the organism (Niklas, 1989). Cell walls provide some of the requisite resistance to these forces. The protoplasts also develop a force (osmotic pressure) which augments the support of the enclosing walls and contributes some rigidity to the organ (this is significant only when wall thickness is < 20% the radius of the cell). The processes of mitosis and cytokinesis which take place within a root meristem may therefore be viewed as a means of establishing a network of walls whose pattern has been optimized for its mechanical, force-resisting properties. Moreover, walls differentiate in specialized ways, especially in cells located deep within the interior of the



Sometimes, multinucleate cells of plant tissues are incorrectly described as ‘‘syncytia’’ (e.g., Olsen et al., 1995) when in fact they are coenocytes. There are two types of multinucleate cell. Multinucleate syncytial cells arise from cell fusion events, whereas coenocytic cells, such as those of algae and higher-plant endosperm, arise from a series of nuclear divisions within one cytoplasm.

Cellular Patterning in Root Meristems

organ whereby they contribute to the construction of conductive elements. The significance of the wall network is highlighted in dicots by the derivatives of the cambium, where secondary xylem and phloem simultaneously provide support as well as solute conduction. That is, the wall network confers both structure and function on the tissues. External boundary walls sometimes facilitate organ fusions. A further advantage of walls is that they can neutralize the pressures that might otherwise be exerted on the protoplast and which, unless attenuated, could lead to undesirable physiological disturbances of a thigmomorphogenic nature (Jaffe, 1985). An optimal wall network provides this attenuation. It also permits processes with low mass-sensing thresholds, originating within the protoplast, to initiate responses such as gravitropism (Barlow, 1995b). Thigmomorphogenesis and gravimorphogenesis require higher thresholds for their initiation and use the walls to transduce their respective stimuli (touch and mass perception) to the interior of the cell. As mentioned, initiation of gravitropism requires a low mass-sensing threshold. Although the walls are not directly used for gravity

51

perception, the walls in the zone of bending may, nevertheless, limit the intensity of the graviresponse. Division walls which limit contact between daughter cytoplasms may enable the differentiation of domains, which then function autonomously as tissues. Within these domains, particular metabolic processes can be elaborated without interference from processes in neighboring domains. Histological studies indicate that tissue differentiation begins within the meristematic region of the root and that tissue maturation continues to completion in the older zones behind the meristem. In the youngest zones of a meristem, the boundaries which demarcate tissues of stele, cortex, epidermis, and so on, are often already prefigured in the arrangement of the cell walls (Fig. 1). Presumably, the autonomy of each tissue becomes increasingly evident as (1) the number of cytoplasmic contacts (via plasmodesmata) across the longitudinal walls of the cell files diminishes (Juniper and Barlow, 1969; Cooke et al., 1996), and (2) the specific chemistry of the cell walls and the organization of the cytoplasm, by which tissues are recognized in histological studies, develop to their final states (Barlow, 1982). It is even

Figure 1 Three types of root apex construction based on cellular and histological patterns. (A) Closed type of meristem (Zea mays), where the tissues of cap, stele, and cortex/epidermis are discrete (410). (B) Open type of meristem (Vicia faba), where the tissues of cap, stele, and cortex appear to be in continuity (240). (C) Gymnosperm (Pinus sylvestris), where the stele is separate from the other tissues of the apex. Cap initials are sited upon its distal border (compare this with A, where the cap initials are sited upon the border of the epidermis) (320). (Modified from Clowes, 1959; with permission of Cambridge University Press.)

52

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possible that the cells themselves can regulate their plasmodesmatal permeability and hence construct physiological domains (Mezitt and Lucas, 1996). Thus, dilution by wall extension need not be the only way to modulate the degree of intercellular communication via plasmodesmata; plasmodesmata can perhaps be sealed and rendered inoperative and thus contribute to the establishment of positional information (PI). In contrast, it seems that secondary plasmodesmata can be inserted into walls (Seagull, 1983), at sites where the demand for rapid fluxes of solutes is likely to be great. III.

CELLULAR PATTERNS THAT ESTABLISH THE ROOT APEX

A.

The Embryo

An embryo develops within a polarized embryo sac and, in time, develops its own polarity. This then enables a root to form at the basal (micropylar) end of the proembryo. On the basis of recent research, it seems probable that not only does the internal organization and polarization of the zygotic cell’s cytoplasm have a profound influence on the initiation of embryonic organs, but so, too, do its boundary wall and membrane (Mordhorst et al., 1997; Vroemen et al., 1999). In angiosperms, the zygotic cell initially has a thicker wall at its micropylar end, whereas its opposite, chalazal end is usually wall-less and bounded only by a plasma membrane (Natesh and Rau, 1984). A particular cytoplasmic domain, together with specific wall or membrane properties at the micropylar region of the zygotic cell, determine that the cells which form here develop as suspensor. Conversely, conditions at the chalazal end may be conducive to embryonic development only, and the eventual development of a wall here, which finally encloses the embryo, may also be needed to maintain embryonic polarity. Indeed, Pennell et al. (1991) have shown that, in embryos of rape (Brassica napus), the plasma membrane of cells derived from the basal cell (cb) of the two-celled zygote have a positive reaction to the monoclonal antibody, JIM8, which recognizes an arabinogalactan protein epitope, whereas derivatives of the apical cell (ca) are unreactive. When a carrot cell with embryogenic potential divides, the daughter cell which becomes the embryonic initial can no longer be marked by JIM8, whereas its sister cell continues to display this protein (McCabe et al., 1997). A characteristic cell wall composition and cytoplasmic status are thus important for initiating the orderly development of both zygotic

embryos (Lu et al., 1996) as well as adventive embryoids (Williams and Maheswaran, 1986). That certain types of wall constituents actively promote cell division (Binns et al., 1987; Kreuger and van Holst, 1995; Toonen et al., 1997) may also be relevant in this respect. Patterns of division associated with early embryogenesis vary according to the species. Six basic embryogenic patterns (or types) have been described (Johansen, 1945; Wardlaw, 1955; Natesh and Rau, 1984). Correlated with these patterns is variation in the origin of root tissues (Fig. 2). One of the major differences is whether or not descendents of the cb, which lies at the micropylar end of the two-celled embryo, contribute to the root. In the onagrad (also called cruciferad), asterad, and solanad types of embryogeny they do so, whereas in the caryophyllad type they do not, and all root tissues arise from descendents of the ca of the two-celled embryo. In many cases, the patterns of early embryonic divisions appear to be regular, and so it is possible to deduce the genealogies of the cells and examine how these comply with the fates of the cells as they differentiate to form the major tissues of the embryonic root. In a number of cases enough of the requisite detail is available for such an analysis, but descriptions in the literature are often incomplete, especially in their three-dimensional detail and in the recording of the frequencies of alternative division sequences. Fortunately, the current interest in the development and genome of Arabidopsis thaliana has resulted in a complete description of the origin of the embryonic root (Ju¨rgens and Mayer, 1994). Arabidopsis conforms to the onagrad type of embryogeny. Numerous mutations have been discovered which affect various aspects of its embryogenesis (Mayer et al., 1991; Ju¨rgens et al., 1997), and many of these suggest that the orientation of new division walls at successive generations within the genealogical descendance are important for the establishment of the correct patterns of embryonic development. From descriptions and illustrations presented by Mayer et al. (1993) and Ju¨rgens and Mayer (1994) for A. thaliana, by Tykarska (1976, 1979) for Brassica napus, and by Swamy and Padmanabhan (1961) for Sphenoclea zeylanica, the genealogy and fate map for roots of three species with an onagrad embryogeny can be proposed. That for Arabidopsis is shown in Fig. 3; that for S. zeylanica was published earlier (Barlow, 1994b). The genealogies deriving from cells ca and cb are similar in all three species except for the number of cells that become suspensor (two cells

Cellular Patterning in Root Meristems

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Figure 2 Four types of angiosperm embryogeny. (A) Asterad. (B) Onagrad (or cruciferad). (C) Solanad. (D) Caryophyllad. In all four cases, the first division of the zygote is transverse (this division may be unequal [A] or equal [B, C, D]) and divides the cell into an apical cell (stippled) and a basal cell (clear). The root (indicated by r against the relevant tier of cells) may have a different origin in each embryogenic type. A thicker boundary wall encloses the embryo, the remaining group of cells is assigned to the suspensor. (From Sporne, 1974; with permission of Chapman and Hall.)

comprise the suspensor in Sphenoclea, eight or nine in Arabidopsis, up to 14 in Brassica). An important feature to notice in Arabidopsis is that among the descendants of cb, there are four cells descended from cell d1 which initiate the calyptrogen, and four cells which descend from cell h2 constitute a quiescent center (QC), the cells of which become proliferatively inert (Dolan et al., 1993). However, the QC of Petunia hybrida, with solanad embryogeny, has a different origin (Fig. 4). It is descended from ca (Vallade, 1970) via cell l00 6:1. The cap tissue of the root, along with the suspensor, are both descended from cb (Vallade, 1972).

It is possible that, in Petunia, additional cells also become quiescent later in embryogeny and thus increase the size of the QC. The ‘‘adding’’ of cells to the QC was also proposed by Clowes (1958, 1978). These results suggest that the QC could be composed of two parts. One is a ‘‘constitutive’’ group of cells directly formed as a consequence of cell genealogy (Q cells in Figs. 3 and 4), the other is a ‘‘facultative’’ group which forms later in root development as a response to PI. The facultative QC cells in Petunia, which may also descend from cell l00 6:1, serve as the most distal cells of the histogenetic

54

Barlow Figure 3 Genealogies of cells, and an indication of their eventual fates as tissues, during early embryogenesis of Arabidopsis thaliana (onagrad embryogeny). The zygote, z, divides transversely to give equally sized apical and basal cells, ca and cb, respectively. The embryo proper descends from ca which then divides longitudinally to give ql.l and q1.2; descendents of one of these two daughter cells (ql.l) are shown. Each cell continues to divide (although cells contributing to the suspensor undergo a limited number of divisions); their descendants are designated by various letters and numbers. The cell types and future tissues to which the cells will belong are as follows: su1-7, suspensor (a file of 7 cells); c, cap; Q, quiescent center; E, epidermis: Co, cortex; St, stele. These last three tissues become located within either root (R), hypocotyl (H), or shoot (S). Divisions may be of three types with reference to the long axis or to the surface of the embryo: transversal ( ), radial ( ), or periclinal ( ). A radial division may be at right angles to the direction of the previous one; this rotation of the division plane is indicated by the arrow ( ). The genealogy is based on results of investigators mentioned in the text (see also Ju¨rgens and Mayer, 1994).

plerome and periblem. They would be equivalent to the functional initials proposed by Barlow (1994c). Direct evidence for two types of cells in the QC is given by the reponse of root apices to feeding with 0.1 mM ascorbic acid solution for up to 64 h (Innocenti et al., 1990). Within 28 h, the outer, facultative cells of the QC had reentered the DNA-synthetic (S) phase of the mitotic cycle; the innermost, constitutive QC group remained held in the G1 phase of the cycle. In adventitious roots of Allium cepa, the number of these constitutive QC cells comprises

about 10% of the whole QC. However, using primary roots of Zea mays, Kerk and Feldman (1995) found that a 48-h exposure to 0.1 mM ascorbic acid induced all cells of the QC to enter S phase. As indicated in Figs. 3 and 4, the patterns of division and the lineages created in the onagrad and solanad embryogenies relate to the subsequent fates of the cells. The caryophyllad embryogeny may present yet another pathway for the origin of cap and QC. This pathway seems to be evident during embryogeny of apple (Malus pumilla cv. McIntosh) studied by Meyer (1958) (although he classified the embryogeny as solanad). Here, the cap and QC develop in the heart of the embryo and are derived from the fourth tier of embryonic cells (Fig. 5). This tier of cells descends from the ca. The supensor of apple embryos is particularly well developed. Its cells are derived mainly from cb but there is also a small contribution to it by cells descended from ca. A contrasting pattern of cell division vis-a`-vis tissue differentiation is found in embryos of graminaceous species—in Poa annua, for example, whose embryogeny conforms to the asterad type. Although less obvious than the examples mentioned above, the cellular pattern in embryos of P. annua also anticipates the pattern of tissue differentiation (Fig. 6). Root and cap tissues become evident when a thicker cell wall appears between these two areas (Fig. 6D,E). This wall was formed at the second division of the embryo. The base of the root seems to be formed from walls

Cellular Patterning in Root Meristems

55 Figure 4 Genealogies of cells and an indication of their eventual fates as tissues during early embryogenesis of Petunia hybrida (solanad embryogeny). The conventions used to depict this genealogy and the cell fates are similar to those shown in Fig. 3, although some of the letters used are different. The division of the zygote, z, results in a smaller ca cell and a larger cb cell (cf. Fig. 2). In the present scheme, however, the basal daughter cell (l 0 1:1) of the transverse division of ca gives rise to the root and hypocotyl and the apical daughter (l l.l) gives rise to the shoot (these descendants of l1.1 are not shown). Only one of the four clones of cells arising from cells l 00 1:1 and l 0 2:1 are followed in detail; the other three ðl 0 3:2–l 0 3:4) develop similarly. One of the descendents, l00 6:1, gives rise to a quiescent centre (Q) cell. There will be a total of four Q cells by the ninth division. The basal cell, cb, gives rise to suspensor (via cells cl, n, n 0 , m and f) and root cap (via cell d2). The cap can be further subdivided into central cap (cc) and cap flank (cf). (Based on observations of Vallade, 1970, 1972.)

laid down at the fifth division of the proembryo. Careful analysis was also given to embryos of Triticum aestivum by Batygina (1969). When the division sequence depicted by this author is closely scrutinized, it seems that, as is evident in P. annua, certain division walls consistently partition the embryo into regions which later correspond to various organs (coleorhiza, root, shoot) and tissues (epiblast, scutellum). Although Guignard (1961) assembled much useful comparative information for embryogenesis within

the Gramineae, the embryonic division patterns have not been studied with the attention they merit. Because of the ‘‘difficult’’ nature of their embryonic cell patterns, the Gramineae would be a useful system in which to apply molecular techniques for defining the extent of embryonic root tissue, such as Scheres et al. (1994) were able to do for germinating Arabidopsis embryos. Transgenic plants were used in which a marker gene was expressed upon the excision of a transposon. Excision occured at random and the gene product, highlighted by a coloured histochemical reaction for glucuronidase, distinguished clones of cells in which the gene had been activated. In this way, the proximal limit of hypocotylar tissue was identified. It coincided with the anatomical boundary between the shoot and the hypocotyl and, following the embryogenic scheme in Fig. 3, would have coincided with the wall which divided cell l1.1 from cell l 0 1:1 following the division of cell q2.1. Shorter stained segments of tissue corresponded to clones of cells derived from later divisions and these helped to pinpoint the divisions that delimited hypocotyl and root; two of these divisions were in the transversely dividing descendants of cells l 0 2:1 and l0 2:1 The longest segments, resulting from early excision events, ran from root tip (excluding cap and QC) into the cotyledons and marked a clone of cells whose distal boundary was probably the first division wall, between ca and cb. In certain species there is a free nuclear stage during early embryogenesis. Most gymnosperms (Roy Chowdhury, 1962; Singh, 1978), as well as Paeonia spp. (Cave et al., 1961) among the angiosperms, provide examples of this coenocytic state. Even embryoids

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Figure 5 Embryogenesis of apple (Malus pumila cv. McIntosh). (A, B) Early division of the zygote creates two unequally sized cells, ca and cb, and then four tiers of embryonic cells (1, 2, 3, and 4) are formed; they surmount the suspensor (su). (C–H) The four tiers can be recognized throughout embryogeny and are delimited by walls which have been more heavily inked. The root cap, quiescent center, and some of the root develop from tier 4. (From Meyer, 1958; with permission of American Journal of Botany.)

of carrot seem occasionally to be initiated following a multinucleate stage (Steward et al., 1958). Nuclear divisions occur without new walls being inserted between the daughter nuclei, as usually occurs at cytokinesis. In the gymnosperms, the walls may be inserted following the first two or three nuclear divisions (Cupressaceae) or after hundreds of nuclei have been produced (cycads), as is also the case for Paeonia. However, a coenocytic zygote need not be unstructured (Owens et al., 1995). Plant cell bodies, each one of which is composed of nuclear chromatin enclosed within a membrane-plus-cytoskeleton-organizing apparatus (Balus˘ ka et al., 1998), may well direct themselves to specific locations within the coenocytic chamber. From there, each cell body governs a certain cytoplasmic domain. In such circumstances, therefore, nuclear genealogies, or cytoplasmic-domain genealogies, could substitute for cellular genealogies in the

formalization of embryogeny. In gymnosperm and Paeonia embryos, once cellularization has occurred, tissue differentiation begins. Again, PI within the embryo, coupled with an interpretation of embryonic polarity, are probably important for this differentiation. Moreover, if nuclei have been associated with particular domains within the coenocytic cytoplasm which can impart histogenetic imprinting (Lyko and Para, 1999), the effects on differentiation might become manifest during the subsequent embryonic development. The simplest view of embryonic tissue differentiation with respect to cellular patterns is that, in all embryogenic types, the patterns of gene activity which establish tissue identity are specified relatively late, perhaps at the late globular stage, but certainly after many tens of cells have been generated. In all cases, differentiation is the result of the specifications

Cellular Patterning in Root Meristems

57

Figure 6 Meristem differentiation within embryos of Poa annua. (A-C) Third, fourth, and fifth division walls (as judged from a median longitudinal section in each case) are in place. Successively formed walls are labelled 1, 2, 3, etc. (D, E) At later stages of development, it becomes clear that the root is delimited by walls 1 and 5. Wall 2 separates root proper from root cap. Wall 3 approximately bisects the root longitudinally. The internal wall 5 similarly bisects the root cap. The root cap initials (identified by dots) are now evident. Another set of division walls (internal walls from the fourth division of the basal end of the embryo; not labelled, but drawn thicker in E) defines the outer wall of the root. (Modified from Guignard, 1961, who had reproduced drawings prepared by R Soue`ges, 1924.)

of PI in three planes, parallel to the embryonic axis (axial plane), and in two orthogonal directions at right angles to it (radial and tangential planes) (see Liu et al., 1993). In onagrad embryogeny, say, a specific pattern of differentiation appears to be superimposed upon the pattern of cell files, and there may be a

similar colocalization of these features in asterad embryogeny. However, separation of the division and differentiation processes could have adaptive significance because chance variations in the development of the cell lineage would have less serious consequences for tissue differentiation. Such a modus operandi

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would be characteristic of a regulative type of embryogenesis. But tending to argue against this are findings from Hypericum spp. where defects in the cell division program lead to embryo abortion (Bugnicourt, 1983) and thus suggest some type of lineage-dependent cell differentiation (mosaic, or determinate, type of embryogenesis). In these Hypericum spp., many of the aberrant divisions were found in the hypophysis, signifying an important regulatory role for this group of cells in normal embryogenesis. A comparative study of root meristem formation in zygotic and microspore-derived embryos of Brassica napus revealed that when the hypophysis failed to form correctly (as was the case in the microsporic embryos), the root then failed to grow (Yeung et al., 1996). This failure was associated with the absence of structural initials at the pole of the root. The systematic search for root meristem mutants in Arabidopsis has clarified not only these earlier observations of Bugnicourt (1983) but also sheds light on the conditions that initiate the hypophysis and all cells and tissues which are derived from it. For example, the HOBBIT (HBT) gene is required for the correct development of the hypophysis; in strong hbt mutants both the QC and cap columella are lacking (Willemsen et al., 1998). This effect was not confined to embryos; similar phenotypes relating to cap and QC were found in adventitious roots formed from callus. Additional hypophyseal genes, such as ORC and GREMLIN (Scheres et al., 1996), have been identified in Arabidopsis, but their effects remain to be examined in detail. Other evidence against a strict mosaic, determinative type of embryogenesis is that suspensor cells, which usually do not proliferate, can develop into additional proembryos when growth at the embryonic pole is caused to fail (Haccius, 1978). This can also occur during normal embryogenesis (Masand, 1963). A number of mutants of Arabidopsis also show this feature, notably the mutations at the TWIN locus (Vernon and Meinke, 1994), where it is associated with abnormalities in the primary embryo. Because the embryo develops in the nonrestrictive space of the former embryo sac, the cell patterns associated with the early stages of embryogenesis probably have significance for the internal mechanical stability of the embryo only (see Thomson and Hull, 1934). Any more sophisticated physical property of the wall pattern arising out of the polarity of the embryo and its cells is held in readiness for when the radicle begins to penetrate the soil. An interesting question is whether the division program in the embryo runs without any regulation by the external environment; such would

Barlow

accord with the suggestion of Lintilhac (1974) that the embryo sac is a compression-free space within the ovule and, hence, division patterns are regulated internally. However, in the fern Phlebodium aureum, Ward and Wetmore (1954) showed that relief of stresses imposed by prothallial tissues on the developing embryo disturbed the pattern of divisions during early embryogeny, suggesting that in this case the normal course of development did depend on external mechanical influences. The distinction between the precise, even stereotyped, embryonic cell division patterns of Petunia hybrida (solanad embryogeny) and the more irregular pattern of Vitis vinifera (asterad embryogeny), at least in the early embryogenic stages (Fig. 7A,B), was remarked upon by Vallade (1989). He suggested that

Figure 7 Contrasting cellular patterns in embryos of (A) Petunia hybrida (cf. Fig. 4) and (B) Vitis vinifera. Although the number of cells is different in the two cases, the disposition of cells appears less ordered in B. (From Vallade, 1989; with permission.) (C, D) A similar pair of embryos (C, Brassica napus, and D, Gossypium hirsutum) with contrastingly ordered cellular patterns. (From Tykarska, 1979, and Reeves and Beasley, 1935.)

Cellular Patterning in Root Meristems

the difference between the two embryogenic patterns had its basis in the relationship between embryonic volume growth and the rate of cell proliferation. In Petunia—and the same seemed to hold for Arabidopsis and Brassica (Fig. 7C)—the embryo grew little in size during the early proliferative stages, whereas in Vitis there was relatively more volumetric growth (Fig. 8). A similar relationship is evident in the early embryogeneses of Capsella bursa-pastoris and Gossypium hirsutum (Fig. 8). The Gossypium embryo was initially larger and had a less regular pattern of cell division (Fig. 7D) than did the smaller embryo of Capsella. From this point of view, it may be that the cellular patterns are the outcome of adherence to Errera’s rule for cell division—i.e., that new division walls should be of minimal area (Errera, 1886; see also Steward, 1958; Korn and Spalding, 1973). Given this rule, together with an additional rule defining where the new division wall will be attached (e.g., Hofmeister’s rule of equal volume partitioning of the

Figure 8 Relationship between growth of the embryo (log10 volume,
m3 ) and cell number (plotted as log2 values, but with the actual values also inserted on the inside of the ordinate for reference). Data points are mean values of each variable for five species: A, Arabidopsis thaliana (from Mansfield and Briarty, 1991); B, Brassica napus (from Tykarska, 1976); G, Gossypium hirsutum (cell numbers estimated from Pollock and Jensen, 1964); P, Petunia hybrida; and V, Vitis vinifera (data for these last two species from Vallade, 1989). Note that embryonic volume is greater per cell number, especially at the 4- to 16-cell stage, in species V and G than it is in species A, B, or P. This may enable alternative orientations of future divisions (cf. Fig. 7).

59

dividing cell), it is possible to arrive at predictable and invariant cellular patterns, such as are evident in Petunia and Brassica. In Vitis and Gossypium, on the other hand, the division patterns are more variable simply because, owing to their more rapid embryonic growth, there are alternative positions at which the new division walls can be inserted without the two mentioned division rules being violated. The relationship between the rates of growth and division of the various embryogenic cellular groups may be influenced by the physicochemical properties of the boundary wall of the embryo (Ward and Wetmore, 1954). This could explain some of the irregular embryonic division patterns found in certain mutants of A. thaliana. The effects of gnom (Mayer et al., 1993) and fass (Torres-Ruiz and Ju¨rgens, 1994) were evident at the earliest stages of embryogenesis; here, the usual asymmetry of the first zygotic division was upset. Another interesting mutant is monopteros (Berleth and Ju¨rgens, 1993), in which the divisions of cells descended from h2 and d2.1 and d 0 2:1 (and their homologs in the other half of the root) were switched from their usual longitudinal orientation to a transverse orientation (see Fig. 3). The result was that a column of cells penetrated the basal, or root, end of the embryo, and that the cap and QC failed to develop normally. Cell lineages have been dismissed as irrelevant to plant tissue differentiation (Dawe and Freeling, 1991; Irish, 1991, 1993). But this conclusion was derived in relation to plant structures formed late in development, even though these structures could have already been determined by a lineage-based differentiation system at an earlier stage. The last-mentioned system may operate in embryos, where the structural plan for the future adult tissues is being established. Many of the bifurcations in the embryogenic cell genealogies of Figs. 3 and 4 correspond to quantal mitoses (Holtzer et al., 1975) in which there is supposed to be a partitioning or segregation of differentiated cytoplasmic domains and areas of preexisting cell wall. Both of these features could have the potential to influence the course of subsequent differentiation in the dissimilar daughter cells. Another possibility is that, at certain times during embryogeny, epigenetic modifications occur in the nuclei of sister cells. This would also manifest as a quantal mitotic event. Both situations would lead to the establishment of cell lineages with unique and heritable characteristics. It is known, for instance, that nuclei can inherit specific proteins through a succession of mitoses (Raff et al., 1994) and, moreover, within a growing root, cells of

60

each tissue have characteristic patterns of nuclear chromatin (Barlow et al., 1982; Balus˘ ka, 1990). Thus, each of the four histogens of the root, as well as the QC, may, in the process of their establishment, have come to possess (and then maintain) different cytoplasmic complements and/or epigenetic modifications. The transposon excision in Arabidopsis embryos mentioned earlier (Scheres et al., 1994) is one such epigenetic event, except that it occurs at random (or so it was assumed). A more usual type of epigenetic change associated with tissue differentiation is the methylation of the cytosine bases of nuclear DNA. Methylation results in nuclei which vary in the amounts of 5-methylcytosine (Holliday, 1987; Cedar and Razin, 1990). During the development of somatic carrot embryoids from a progenitor cell type, for example, the degree of DNA methylation increases (LoSchiavo et al., 1989; Munksgaard et al., 1995). So far, this rather general finding correlates neither with the changing spectrum of cellular proteins known to occur concurrently in this system (Racusen and Schiavone, 1988) nor with the development of specific tissues. However, LoSchiavo et al. (1989) found that a small number of embryoids were able to develop in the presence of the hypomethylating drug 2-amino5ethoxy-carbonyl-pyrimidine (ECP), and that, although hypocotyl and shoot development took place, these embryoids lacked roots. It seems that ECP and the associated low level of DNA methylation affected the establishment of proper root–shoot polarity in the embryoid. Division of a cell with a predetermined asymmetry has been proposed as crucial for the initiation of cortex and endodermis in Arabidopsis roots (Gallagher and Smith, 1997). Evidence gathered from the mutants scarecrow and shortroot showed that the corresponding dominant alleles are regulatory elements in this quantal division process, and are of especial importance for the establishment of endodermis (Fig. 9). This tissue normally originates from the inner daughter cell of a periclinally-divided cortex–endodermis progenitor cell (CEP) and may be derived soon after the division which gives birth to the cells labelled CoR in Fig. 3. The CEP is itself the product of unequal transverse division of a mother cell (an autoreproductive stem cell). The two mentioned mutants permit the unequal transverse division in the stem cell, but not the unequal periclinal division in the CEP. The lack of the periclinal division is hard to understand, unless this particular division orientation is dependent upon a predivisional asymmetry of the CEP. If so, then both mutants fail to support this asymmetry. Interestingly,

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Figure 9 Scheme for the division of a mother cell that intiates files of cortex and endodermis in roots of wild-type and mutant (scarecrow and shortroot) seedlings of Arabidopsis thaliana. (A) In the wild-type, an autoreproducing stem cell (a) divides unequally at step (i) to produce a cortex-endodermis initial (CEP). At step (ii), the CEP divides periclinally to produce endodermis and cortex cells. The two divisions at steps i and ii continue to increase the number of endodermis and cortex cells; the a cell maintains itself at each division. (B) In the mutants, step ii is abolished. As a result, in scarecrow a chimerical cortical-endodermal daughter cell is produced, while in shortroot the daughter shows no characteristics of endodermis. These mutants indicate that some type of asymmetry of either wall or peripheral cytoplasm becomes established in the a cell and in the CEP. (Modified from Gallagher and Smith, 1997; with permission from Elsevier Science.)

the CEP and its descendents are revealed by immunofluorescence binding of the CCRC-M2 antibody (which recognises a cell wall carbohydrate epitope) to be chimeras for characteristics of both cortex and endodermis (Di Laurenzio et al., 1996). Another type of asymmetric division is that which initiates the differentiation of nonhair and hair cells in the root epidermis. The Arabidopsis mutant werewolf abolishes this asymmetry of cell fate and accordingly all cells become hair cells (Lee and Schiefelbein, 1999). Both the WEREWOLF and SCARECROW proteins are transcription factors. B.

Lateral Roots

The earliest stages of lateral root initiation on a parent root (or shoot) axis (see also Chapter 8 by Lloret and Casero in this volume) appear to trace the same crucial rhizogenetic steps that occurred earlier in the zygotic

Cellular Patterning in Root Meristems

embryo. This must also be true of the pattern of gene activity necessary for imparting identity to the various root tissues which differentiate within a growing lateral root primordium. Whether the patterns of divisions are similar in the two systems is unclear, but presumably each must construct a cellular template which enables rhizogenesis to become self-maintaining (Malamy and Benfey, 1997; Barlow et al., 2000). Rhizogenetic regions within cellular aggregates grown in vitro also share the same steps of cell patterning. Reorientation of cell growth and the establishment of four histogenetic tiers of cells seem to be key events in establishing the precursor of a primordial apex (Tylicki et al., 2000). Because lateral root primordia of higher plants have a multicellular origin, no single pair of cells in the pericycle is directly equivalent to the cell pair ca and cb of the embryonic lineage. It is possible that the new axis of a primordium, oriented in the radial plane with respect to the longitudinal axis of the parent root, could commence with cells that are equivalent to cells h1, q2.1, q2.2, q2.3, and q2.4 (in species with onagrad embryogeny) (see Fig. 3). The four quadrants arising from the q2 cells are identifiable in lateral roots of tomato and primary roots of Brassica napus (Kuras, 1980). Transverse divisions occur in pericycle cells before the periclinal divisions which accompany the

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outgrowth of the primordium (Lloret et al., 1989; Casero et al., 1993; Malamy and Benfey, 1997) (Fig. 10). They may be needed to provide these small primordial q2.1–q2.4 cells. Also, recognition of these early transverse divisions raises the possibility that synthesis of new proteins required for lateral root initiation may occur earlier than anticipated hitherto (e.g., Keller and Lamb, 1989; Neuteboom et al., 1999). When JIM antibodies were applied to transverse sections of roots of carrot, one of them, JIM4, an anti-arabinogalactan protein, was shown to recognize pericycle cells opposite xylem where lateral root primordia would be expected to form (Knox et al., 1989). In soybean, a hydroxylproline-rich glycoprotein is associated with the xylem arcs (Ye and Varner, 1991), and in Arabidopsis parenchyma cells neighboring the xylem arcs express transcripts of a cyclin A gene which is involved in cell cycle regulation (Burssens et al., 2000). However, in all cases, the relevance of these proteins to primordium development is unknown, but does suggest a degree of preformation of the sites for potential primordia. The conditions leading to the origin of lateral root primordia in angiosperms will no doubt become better known when the problem is examined more closely. In the fern Ceratopteris thalictroides, however, the cellular origin of each lateral root primordium is strikingly

Figure 10 Scheme of division of a pair of elongated pericycle cells in the root of Allium cepa during the initiation of a lateral root primordium. These cells would be located opposite a xylem pole. (A) Nuclei of two contiguous cells move toward each other. (B) The cells divide transversely (with respect to the cell and root axis) and unequally. (C, D) Unequal divisions continue until four cells are present within the walls of each original mother cell. (E) The first periclinal division occurs in a central cell of this group of eight cells. This division represents a change in the orientation of the growth axis which permits a primordium to form. This sequence of divisions, A–E, occurs over a distance of 15 mm, commencing 7 mm behind the tip. Although only two cells are shown, neighboring pericycle cells also undergo some unequal divisions. However, the pair of cells that underwent the first of such divisions has precedence and serves as the focus for primordium development opposite the xylem, but some of the other unequally dividing cells also contribute to this process. (From Casero et al., 1993; with permission of Springer-Verlag and of Prof. P. J. Casero.)

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clear. A cell in the endodermis enlarges and then immediately undergoes a sequence of four divisions in different planes that thereby create three root sectors and one cap sector (Lachmann, 1907). This sequence of divisions repeats, and the derivatives (merophytes) continue to divide in planes characteristic of the homologous merophytes in the parent root apex. The fivecelled primordium thus has already the cellular pattern of a new root (Fig. 11). The parent root develops in a similar way from superficial cells in stem and petiole (Lachmann, 1907; see also Gunning et al., 1978a, for details of the origin of shootborne roots of Azolla pin-

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nata). Thus, throughout the ontogeny of the plant, once a lateral root-forming apical cell is created, it immediately commences functioning in a root-specific fashion. According to Vladesco (1935), who worked mainly with embryos of Gymnogramme sulphurea, apical cells arise but do not necessarily persist. Then, a new generation of apical cells comes into existence. It is as though, in this species, a number of ‘‘trial runs’’ of cell patterning are required before a stable pattern of organogensis with an apical cell is instituted. The apical cells that structure the lateral roots of leptosporangiate ferns may be determined to develop

Figure 11 Development of a lateral root primordium in a shootborne root of the water fern, Ceratopteris thalictroides. (A, B) The precursor of an apical cell (a) enlarges in the endodermal layer. (C–F) The apical cell then divides in regular sequence and generates a tetrahedral form. The apical cell and the resulting primordium are here emphasized by thicker boundary walls, but in reality these walls are not so prominent. A–E, Transverse sections; F, longitudinal section. (From Lachmann, 1907.)

Cellular Patterning in Root Meristems

in their characteristic fashion in accordance with both PI and a lineage program (Barlow, 1984, 1989a). PI specifies which endodermal cell lying on the circumference of the stele will develop as an apical cell, whereas lineage specifies which group of cells within a merophyte will respond to PI. Whether such a means of primordium specification applies to angiosperm roots is not known, but observations on the first periclinal divisions in the pericycle of tomato roots shows a strict relationship between the order number of the cells in the primordial row and the vasculature of the parent root (Barlow et al., 2000). Most theories of lateral root formation implicate PI (Torrey, 1986; Vuylsteker et al., 1998) and favor a dependency upon critical levels of cytokinin and auxin (Zhang and Hasenstein, 1999); but lineages may also play a part, in the way suggested by Barlow and Adam (1988). In fact, it is probable that the centre of the primordium lies at the junction of four pericyclic cellular complexes (cf. Fig. 10), where the four oldest walls join to form a cross (Barlow et al., 2000). IV.

CELLULAR PATTERNS IN GROWING ROOT APICES

A.

Patterns of Meristematic Cell Division

Most embryos undergo a period of dormancy or quiescence which interrupts their growth shortly after their organogenetic phase has been completed. Imbibition cancels this interruption and revitalizes the growth process. When the radicle protrudes, the cells in the apical meristem may already be actively dividing. This pattern of activity applies to species with epigeal germination. In species with hypogeal germination, cell division commences after the rootlet has emerged (Schatt et al., 1985; Obroucheva, 1999). In both epigeal and hypogeal species, the onset of mitoses seems dependent upon the presence of a critical number of elongating cells (Obroucheva, 1999). Growth and mitotic division within the germinating radicle commence at different locations and at different times (Pukhal’skaya, 1949). The sequence of activation probably reflects the changing pattern of water availability within the radicle and this can be marked indirectly by soluble radioactive tracers of DNA and RNA synthesis (Stein and Quastler, 1963; Payne et al., 1978). Refined molecular probes now enable temporal and spatial patterns of the onset of various processes to be monitored: the pattern of nuclear DNA synthesis, for example, has been shown to coincide with the re-establishment of the cortical microtubular cytoskeleton (Jing et al.,

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1999), which is a major regulator of cell wall synthesis. New tubulin synthesis and tubulin gene activation occur concurrently with the onset of DNA replication, though some of the microtubules seen early in germination may be elaborated from a granular form of tubulin stored within the dry seed. The number of dividing cells along the length of the meristem of the germinating radicle is small at first and reflects the size of the meristem at the end of embryogenesis prior to dormancy. This number is increased during germination by new rounds of transverse, proliferative divisions until a meristem of stable size is achieved (Deltour et al., 1989). In Arabidopsis thaliana, the resumption of root meristem activity is controlled by two genes, RML1 and RML2, which map to different chromosomes (Cheng et al., 1995). Mutation at either of these loci results in determinate root growth in which the potentially meristematic cells at the apex become completely differentiated. Usually, all preformed cells of the embryonic meristem are replaced by postformed cells produced by divisions within the emerging rootlet, but in the RML mutants it seems as though the switch between preformed and postformed cell growth and division is blocked or subject to a checkpoint. A similar checkpoint seems to occur naturally in determinate root growth systems, such as those of cactus (Dubrovsky, 1997) and in the nodule roots of Myrica gale (Torrey and Callaham, 1978). In roots with indeterminate growth, where the preformed/postformed checkpoint is passed, the question arises as to how it comes about that there are characteristically different numbers of proliferative cells along the cell files of the various tissues (Luxova´, 1975). It is possible that a repressor of mitotic activity passes into the proliferative cells from the older, nondividing and maturing cells. Some of the latter cells would already have been present within the embryonic radicle. In dicot seedlings, preformed, mitorepressive cells might also be located within the hypocotyl. As the meristem becomes increasingly active at germination, these mature (or maturing) cells are replaced by a new population of mitorepressive cells. Thus, a stable feedback system is set up between the meristem and the maturing compartment (Fig. 12). Other feedback systems have been proposed to regulate division and differentiation in the root apex (Barlow, 1984; Van den Berg et al., 1995) while yet another, governed by the CLAVATA gene, operates upon central and rib meristem cells in the shoot apex (Schoof et al., 2000). Action of the CLAVATA gene should be borne in mind in investigations of the interrelationship between QC and the rest of the meristem. And with regard to

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Figure 12 Proposed negative feedback between the growing, but nondividing, cellular compartment and the meristematic compartment where successive rounds of cell division occur (indicated by tailed arrows). A putative mitorepressive factor is produced by the nondividing cells and inhibits (angled arrow and ‘‘x’’) division at the basal end of the meristem. Cells so affected enter (straight arrow) the nondividing compartment where they in turn acquire the putative mitorepressive property. As long as the rate of mitorepressive action keeps pace with the rate of cell production, the meristem remains a constant size.

the QC, upon germination a variable proportion of cells in this zone traverse the cell cycle from G1, through S phase, to mitosis (most QC cells in Pisum sativum make this traverse [Jones, 1977], fewer do so in Malva sylvestris [Byrne and Heimsch, 1970a]). The significance of this cell division event within the QC is not clear. However, in Picea glauca, germination brings about a slight but significant alteration to the cellular configuration within the root apex (Yeung et al., 1998). Prior to germination the pole of the Picea root is occupied by a layer of nondividing cells. Following germination, these cells divide once and the daughters then comprise a larger, multilayered group of cells. It is as though the mitotic event ensures the genetic and metabolic activation of these ‘‘preinitial’’ cells, and also finalizes their spatial arrangement as true functional initials. Transgenic Arabidopsis plants with additional cyclin gene DNA (cyc1At) controlled by the cdc2aAT promotor were shown to have enhanced root growth (Doerner et al., 1996). Whether this was due to an increased rate of cycling in all meristematic cells or to an increased number of cycling cells was not revealed. It might have even been a result of the relaxation (due to the additional cyclin genes) of the mitorepressive activity of the new maturing cells (see Fig. 12), just as the RML1 and RML2 phenotypes could be due to an enhancement of this repressive system. Whatever the mechanism, it is possible that the relative abundance of cyclin gene product enables flexible growth control (Doerner et al., 1996). This plastic response might naturally be initiated by the level of

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available internal auxin or external nutrients. Nitrate (NO 3 ) supply is one such exogenous root growth regulator which can stimulate the rate of root elongation (Zhang et al., 1999) and might achieve this through an effect on meristem size and/or degree of mitotic repression, apparently acting in the same manner as (or as a substitute for) auxin (Poddar et al., 1997). The longitudinal, formative divisions which initiate all the cell files of the root occur within the apex of the meristem (Fig. 1). As already discussed, the pattern of these cell divisions is established during embryogeny; the actively growing meristem then perpetuates this pattern as the root continues its growth. However, as the primary root lengthens, the numbers of cells which initiate cell files can either increase (Hayat, 1963), remain the same, or decrease (Litinskaya, 1993). There can also be some moderate alteration to the overall pattern, as indicated by the change from open to closed meristematic organizations in the roots of certain species of Asteraceae as they grow older (Armstrong and Heimsch, 1976). The same occurs in primary roots of Malva sylvestris as they lengthen from 0.3 cm to 33 cm (Byrne and Heimsch, 1970b). A QC also becomes evident (it is absent until the root is 3–6 cm long, perhaps because of the nuclear DNA synthesis that accompanies germination) and increases in size and cell number (up to 700 cells) as root diameter increases (Byrne and Heimsch, 1970a). There need not be a direct positive correlation between the number of initial cells and the diameter of the root: roots that become thinner as they grow do not necessarily show a corresponding decrease in the number of initial cells; in some cases the number can actually show an increase (e.g., Hayat, 1963; Chiang and Tsou, 1974). In primary roots of Phaseolus radiatus, the initial zone was reported as being four or five cells wide at germination (0 h), while 24–48 h later, three additional cells were present (Fig. 13), indicating the occurrence of approximately one new longitudinal division of each cell in this zone (Chiang and Tsou, 1974). The changing number of initials was also associated with an increasing complexity of the histogenetic program (Fig. 14). During the emergence of lateral roots the number of initial cells may increase (Popham, 1955a) and the roots become thicker. Unfortunately, the corresponding behavior of the QC is unknown in these roots. Conversely, the QC in lateral roots of Vicia faba was found to develop shortly after their emergence (MacLeod and McLachlan, 1974) but, in this case, no information was given concerning the number of initial cells. The relaxation of quiescence during germination might permit the increase in the number

Cellular Patterning in Root Meristems

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Figure 13 Changing cellular patterns at the apex of a primary root of Phaseolus radiatus during germination and early growth. In the dormant radicle (0 h), there are no mitoses; the preformed central initial cells are marked with open circles. By 12 h, some of these cells have begun to divide (daughter cells are stippled) and initiate a central calyptrogen. Later, other central initial cells divide and contribute to the plerome and thence to the stele. At this time, a set of peri-initials (stippled circles) has also become established around the central group. By 48 h and 120 h, further divisions have occurred and the peri-initials have increased in number. They constitute a periblem and a lateral portion of the calyptrogen, and accordingly contribute to cortex and epidermis and peripheral root cap, respectively. For further detail, see Fig. 14. P marks the pericycle cell layer; the outer wall of P is also drawn thicker. (Modified from Chiang and Tsou, 1974; with permission.)

of initials, but this increase may slow once the QC has become established. Any condition that alters the relative rates of growth and cell division in the three planes of the root (axial, radial, and tangential) will affect the proportions of the respective types of division (Barlow and Adam, 1989). This suggests that selection of division planes is a response to Hofmeister’s rule that cells should divide perpendicular to the plane of maximum growth. Probably the effect on cell shape arose owing to rearrangements of the cytoskeleton in response to altered levels of endogenous growth regulators (Balus˘ ka et al., 1999). Similar effects of growth regulators may also operate in the dividing zones of root meristems, being especially significant in regions where formative divisions are taking place. Both the proliferative and formative postformed cells of the growing apex continue to obey the same division rules as applied earlier during root embryogenesis. As mentioned, these rules relate to the rates and directions

of wall growth of a mother cell (Hofmeister’s rule) as well as to the relative dimensions of the cell (Errera’s rule). All this was realized long ago by Sachs (1887; 447). He believed that the laws of embryonic growth, once established, were perpetuated in the meristems of roots and other organs, and that it is the formative zone of apical meristems that retains these embryonic properties. A further description of the pattern of cell growth within a root apical meristem can be achieved by means of a growth tensor which makes use of the meristem’s natural pattern of anticlinal and periclinal walls in specifying a coordinate system. Two types of tensors have been devised to simulate two types of root apex (Hejnowicz, 1989; Hejnowicz and Hejnowicz, 1991). One of them leads to a root with a QC, the other to a root in which there is an active growth site in place of a QC. The resulting pattern of proliferative activity in the latter case corresponds to that found in fern roots in which there is a mitotically active apical cell. Part of

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Figure 14 Schematic interpretation of the changing population of initial cells of germinating roots of Phaseolus radiatus and their histogenic derivatives (cf. Fig. 12). (A) In the dormant root (0 h). (B) At the 12th hour of germination. Initials on only one side of the root are represented. (Modified from Chiang and Tsou, 1974; with permission.)

the problem of simulating stable patterns of division within an embryonic apex is to develop a suitable growth field. How this comes about in a real meristem is unknown, but presumably it is regulated by the fluxes of growth regulators which specify the relative amounts of cell wall extension in each plane. The importance of hormone fluxes in maintaining the closed apical division pattern of maize roots is reinforced by the findings of Kerk and Feldman (1994). They fed the auxin transport inhibitor, triiodobenzoic acid, to roots which, as a consequence, showed many breaks in the cap boundary and signs of cell divisions in the QC. Additional layers of cortical cells were also found at the pole of the root. However, these two reported anomalies occur occasionally in untreated roots of maize, the first-mentioned type occurring upon germination of certain cultivars, the last-mentioned during the ageing of primary roots (Barlow and Rathfelder, 1984). Variation in auxin transport may account for interspecific variation in the number of tiers of cortical initials—for example,

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in primary roots of species within the genus Linum (Byrne and Heimsch, 1968). Interestingly, experimental inhibition of the auxin-transporting system in pine roots resulted in extensive dichotomous and coralloid branching (Kaska et al., 1999). Probably these two types of branching resulted from the activation of growth and division at the lateral edge of the quiescent center. Activation of groups of cells on either side of an inactive zone at the tip of the rhizophore of Selaginella spp. resulted in the formation of two or three triangular apical initials which, in turn, led to di- or trichotomous branching and subterranean root formation (Lu and Jernstedt, 1996). In fact, our unpublished observations from Selaginella suggest an evolutionary rationale for the establishment of a QC in higher plants: in some ancestral plant(s) the onset of quiescence in cells at the apex during root growth promoted root branching from primordia which had, as a consequence of this quiescence, been initiated at the apex. The evolutionary fixation of a permanent quiescent zone in roots of modern-day plants then enabled the changeover from dichotomous to herringbone type of root branching. Despite modifications to the cellular pattern which occur in the roots of some species during the immediate postgermination period, the patterns at this stage are descended largely from the pregermination embryonic patterns. Voronin (1969) described a set of seven cellular patterns in roots from a range of taxa within the pteridophytes, gymnosperms, and angiosperms (Fig. 15). Voronin’s classification, however, was intended to reveal phylogenetic relationships by showing how one pattern could have been transformed into another. The idea of a phylogeny of cellular patterns has been developed more generally by taking into account the number of wall faces (3, 4, 5, etc.) displayed by the structural initial cells from which cell files and tissues ultimately arise (Barlow, 1994c). Differing cellular patterns issue from initial cells with increasing grades of geometrical complexity. Each pattern would constitute an archetype of cellular construction (Lu¨ck and Lu¨ck, 1993). Each archetype might be associated with a distinct energy costs (Barlow, 1994d). Then, if the organ is taken as the reference level, the paraboloidal form of angiosperm root apices (see Barlow and Rathfelder, 1984) would correspond to an archetype of organ construction. The distinction between open and closed meristems has been discussed elsewhere (Clowes, 1981; Barlow, 1995a). It relates to the dynamics of cells at the junction between root and cap tissue. In terms of the

Cellular Patterning in Root Meristems

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Figure 15 Schematic drawings of cellular patterns in root apices from a range of taxa with indications of the putative evolutionary/morphological transformations (arrows) that might convert one type of apex into another. (A) Marratiaceous fern. (B) Leptosporangiate fern. (C) Gymnosperm. (D) Dicotyledon (open type of meristem). (E) Monocotyledon (closed type of meristem). (F) Dicotyledon (closed type of meristem). Filled arrowheads indicate pericycle; open arrowheads indicate the boundary between cap and root tissue. Small arrows indicate the directions in which new cells are produced by the initial cells, each of which are marked with a dot. (Redrawn from Voronin, 1969.)

growth tensor and the growth field which it specifies, whether a meristem is open or closed depends on the stability of the growth field. When the growth field is stationary, a closed meristem develops, but if it shifts in an axial direction, the meristem does not assume closedness and hence remains open. A shifting growth field can be inferred from certain experimental results (Nakielski and Barlow, 1995). Roots of tomato, mutant at the GIB-1 locus, when cultured in vitro, exhibit cycles of growth and shrinkage of the QC which are also associated with cycles of root cap regeneration (Barlow, 1992). Wild-type tomato roots maintain a stable closed structure. Their growth field is thus inferred to be fixed. In roots of the gib-1 mutant, the QC is active on its acropetal face and a new cap meristem begins to replace the old one (Nakielski and Barlow, 1995); hence, the growth field is inferred to shift axially in a basal direction. The GIB-1 locus of tomato regulates gibberellin biosynthesis, and mutant plants have a lower gibberellin content than wild-type plants. How this relates to the stability of the cellular patterns within their roots is unknown. A shifting

growth field may also account for the change from closed to open root apical organization during germination. Another situation in which the growth field is inferred to shift axially is after excision of the root cap (Nakielski, 1992). The center of the field then moves a small distance from the apex, whereupon the growth field is reestablished in a new site. The original pattern of cell growth and division is eventually regained, but only after it has been redirected to regenerate the cellular pattern associated with the root cap. If slightly more tissue than just cap is removed, subsequent regeneration of the field takes place with the development of a new meristem (or sometimes two new meristems) within the remaining basal portion of the original meristem (Reihman and Rost, 1990). It might be conjectured that the initiation of each new lateral root primordium within the pericycle and endodermis also depends on the periodic redevelopment of a new growth field in these two tissues. The above lines of evidence suggest that the patterns of cell enlargement and division during embryogenesis,

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radicle development, and lateral root primordium formation are the consequence of establishing a particular growth field. These patterns conform to a growth tensor with either a natural or an orthogonal coordinate system (Hejnowicz, 1989). The tensor, though based on actual rates of cell displacement, is, in effect, a representation of a physicochemical system which coordinates growth within the root apex. This may be equivalent to PI specified by hormonal fluxes. The organizing center, with its wandering property, is a point within the apex holding a particular positional value. B.

Cellular Patterns and Tissue Differentiation

One of the main functional aspects of the cellular patterns generated within the root apex relates to the wellknown, but little understood, process of tissue differentiation. All roots conform to a common histological pattern—stele to the interior, epidermis to the exterior, cortex in between, with a cap at the summit. In certain situations, the way in which differentiation proceeds can be modified during the course of root growth. For example, aerial roots of Ficus benghalensis show anatomical changes upon entering the soil, enabling them to be redesignated as terrestrial absorbing roots (Kapil and Rustagi, 1966). Although the same tissues are present in each type of root, there are alterations to the relative numbers of cells in the tissues (e.g., pith cells are plentiful in aerial roots of F. benghalensis but are absent in the terrestrial roots) and to the degree of tissue development (e.g., periderm is well developed in aerial roots whereas endodermis is poorly developed; the converse applies in terrestrial roots). Similar modifications occur within prop roots of Piper auritum as they enter the soil (Greig and Mauseth, 1991). By contrast, rather few quantitative differences were found between the tissues of aerial and subterranean roots of Monstera deliciosa (Hinchee, 1981). Whatever the changes, they occur when one set of external, abiotic environmental conditions is replaced by another during root growth. They are examples of the plastic developmental response of root apices. At another level of organization, different internal conditions impinging on root primordia at the time of their development bring into being dimorphic root systems (Barlow, 1986). Three species of Cyclanthaceae provide examples of dimorphic systems (Wilder and Johansen, 1992). The anatomy of their absorbing and anchoring roots differs in at least 15 quantifiable anatomical characteristics, as well as in more gross mor-

phological features. A second example is the system of air and water roots of Ludwigia peploides (Ellmore, 1981). Here, hormonal conditions at the time of root initiation, which also correlate with the position of the root primordia on the stem, influence which of the two types of root subsequently develops. Presumably, the determining event selects one of a number of alternative combinations of gene activity that accordingly leads to one particular pathway of rhizogenesis (see Barlow, 1994a). The stage of development at which a primordium becomes responsive to determining conditions is not known but, in roots of Convolvulus arvensis, it must occur quite early, as was shown by the experimentally induced switch to shoot development of primordia that would otherwise have formed new lateral roots (Bonnett and Torrey, 1966). As a result of the determining event, young primordia showed different patterns of cell divisions, one pattern being associated with the shoot-forming pathway, the other with root formation. The first group of cells to show differences in the division pattern was in the endodermis of the parent root. In the usual root-forming pathway, these cells divided and contributed to the emerging lateral root, whereas when shoot formation was induced, their contribution to the primordium was much more limited. There were also differences in the way in which cells of the growing primordium of each type made contact with the vascular system of the parent root. Another aspect of cellularity relates to its mechanical properties. The division of cells within a root elaborates a system of turgid protoplasts and semirigid walls which minimizes the force experienced by the root as it grows in mechanically resistant soil. Equally, the cellular pattern might maximize the root’s physiological response to an environment of this type should the walls be capable of transmitting mechanosensory information. When roots meet impenetrable soil, the cells of the cortex swell radially and those of the epidermis stretch tangentially. Together, these features enable the root apex to force apart the resistant soil structure and thereby maintain root elongation. The differential sensitivity of root cells to ethylene (Balus˘ ka et al., 1993) leads to the promotion of radial growth of the cortical cells, similar to the effects occurring in soil. The consequent swelling of the root amplifies their natural wedging ability due to the paraboloidal form of the apex. Whether different arrangements of cells within the cortex affect a root’s response to impedance is not known. For example, would roots with concentric rings of cortical cells arranged in radial rows (see Williams, 1947) be more

Cellular Patterning in Root Meristems

or less efficient in overcoming impedance than roots with less regular radial cortical cell patterns? The ever-present impedance exerted upon roots by soil, with the greatest pressure exerted upon their tips, is probably the reason for root meristems being apical rather than intercalary (as often accompanies shoot growth in monocots), because an apical location minimizes the chance of a root with circular cross section buckling as it grows. A further, mechanical attribute of the cortex is that, under anaerobic conditions, it contributes a system of air channels (aerenchyma), which helps maintain the growth of the root tip. Aerenchymas often develop lysigenously as a result of the selective death of certain cortical cells (Kawai et al., 1998). In extreme types of lysigenous aerenchymas, the cortical cells which remain alive comprise narrow, radial strips of tissue that provide mechanical struts linking the superficial dermal tissue (epidermis plus exodermis) to the vascular cylinder. These struts also channel water and solutes from the dermal tissues to the endodermis. The means by which some but not all cells are selected to die, and how this is regulated in a patterned way, is not known. It is theoretically possible for the pattern to be imprinted in the cortical tissue at the time of the latter’s construction in the meristem (i.e., it is prepatterned). Another way is through more local controls whereby already differentiated cells are protected from dying. For example, within both maize and willow root aerenchymas, living cortical cells are located on radii that extend from the protoxylem poles (Konings and Verschuren, 1980). Some cell-death-limiting influence may therefore emanate from the protoxylem, implying that cortical cells opposite the phloem may be more likely to die. Here, it is interesting to note that the oil reservoirs found in the inner cortex of roots of Solidago canadensis are also located opposite the phloem (Curtis and Lersten, 1990) and that these idioblasts have a schizogenous origin (which is perhaps a mild form of cellular lysis that results in a weakening of contact between cell walls). A third possibility (not incompatible with those previously mentioned) is that, within a region of cortex subjected to cellular lysis, the lytic process commences at random, but once started, it becomes self-regulating, stopping short of completely destroying all the competent cortical cells. One might speculate, therefore, that during the evolution of aerenchyma-bearing plants a fine balance has been achieved in the regulation of cortical cell death to ensure that enough autolysis occurs to provide the necessary number and volume of air channels but not

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so much as to threaten complete cortical tissue collapse. Observations of Seago et al. (1999) on roots of the aquatic plant Hydrocharis morsus-ranae strongly suggest that the cell death necessary for lysigenous aerenchyma development in the root cortex is correlated with, and may even be regulated by, the cell division pattern (Fig. 16). Two divisions, one radial the other periclinal, produce a quartet of small cells which are destined to die and thereby form an aerenchymatous cavity in the cortex. Other interesting patterns of cell division were also observed by Seago et al. (2000) to be associated with the formation of a schizogenous cortical aerenchyma in roots of Nymphaea odorata (Fig. 17). Both in this last-mentioned species and in H. morsus-ranae, the geometric regularity with which large and small cells are distributed in the cortex is striking and begs for explanation not only in terms of the cell biological questions which they raise (relating to cell division control, positional information, cell separation), but also in relation to the advantages that such precise patterns of aerenchyma formation confer on these water-growing roots. This question of the geometry of cellular packing (square or hexagonal cell perimeters in root cross sections) in relation to waterlogging responses was examined in the root cortex of a number of species (Justin and Armstrong, 1987). A square packing arrangement (where the cells are arranged in radial rows) seems better for aeration of the cortical tissue on account of the relatively more voluminous intercellular space. Secondary tissues are often associated with specialized physical functions within the root. For example, the development of a protective periderm follows from the sloughing of their cortex. But notably, secondary vascular tissue develops from a cambium present within the stele of dicots species. The resulting secondary thickening, involving the differentiation of cambial derivatives as either secondary xylem or secondary phloem, occurs far behind the root apex in a zone where tissues have recently reached maturity (cf. Popham, 1955b). There are parallels (but also some striking differences) between the initiation of both the cambium and the lateral root primordia. One similarity is that, in both cases, the predominant direction of new growth is radial. Also, the first divisions are localized, rather than scattered, within the initiating tissue (Fig. 18). The cambium is derived initially from narrow bands of stelar parenchyma cells. Pericycle cells opposite the protoxylem poles are among the last to be recruited into the dividing cambial cell population. Interestingly, the first ray parenchyma cells to develop

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Figure 16 Cellular patterns associated with lysigenous aerenchyma formation in roots of Hydrocharis morsus-ranae. (A) Within a cross section of the root meristem (0.4 mm from tip of meristem), groups of one, two, or four cells are evident, the number of cells in the group depending on the number of previous periclinal and radial divisions. (B) Three millimeters further back along the meristem, the small cells comprising the groups of four have autolysed (stars). This has given rise to the air spaces. Groups of two larger cells remain (darts). These cells widen radially and form bridges across the aerenchymatous cortex. The first division (as seen in cross section) that led to the groups of two cells was periclinal; both these two cells survive. The first division on the pathway to the groups of four cells was radial and was then followed by a periclinal division; all four cells autolyse. (C) Radial bridges of cortical cells seen in longitudinal section (1.2 mm behind tip of the meristem). Mitoses are still present (darts). Scale bar ¼ 50
m; same magnification for all photos. (From Seago et al., 1999; with permission of the authors and Canadian Journal of Botany.)

are sited opposite the xylem poles (e.g., Loomis and Torrey, 1964; Torrey and Loomis, 1967). In woody species, rays are often initiated by the repeated anticlinal division of an elongated cambial cell. In this respect there is similarity with the early stages of lateral root primordium initiation (see Casero et al., 1993; Chapter 8 by Lloret and Casero in this volume). The new ray initials then divide periclinally. The ray parenchyma becomes conspicuous because the cells retain their cytoplasmic contents. Also, cambial ray cells do not divide frequently, and so they form files of cells greatly elongated in the radial plane (Fig. 19). A final aspect of longitudinal divisions in roots which deserves mention is the development of secondary, or supernumerary, cambia. This can lead to macroscopic features of root development which have both functional, structural, and sometimes eco-

nomic implications. In sugar beet (Beta vulgaris), for example, a number of secondary cambia develop simultaneously in the outer part of the stele (Artschwager, 1926). Their cellular descendants differentiate as xylem and phloem, but many cells remain parenchymatous. The parenchyma fills with sucrose, sometimes to a considerable degree (20% of sugar beet root fresh weight is sucrose), and the root as a whole increases in girth to give the familiar root storage organ. In this, and similar bulbous storage roots, the investment of resources in root growth is considerable and contributes to a strategy of ‘‘anchorage by root mass’’ for such soil-rooted plants (Ennos and Fitter, 1992). Often, a shallow groove runs from base to apex on the surface of roots of beet (Artschwager, 1926). This groove is due to a retardation of cambial growth and cell division opposite one of the two

Cellular Patterning in Root Meristems

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Figure 17 Aerenchyma developing in the meristematic zone of the root cortex of Nymphaea odorata. Triangular cortical cells (S) divide unequally (open arrows) to produce a small daughter cell. The pattern of division is such that where the short walls of the small cells meet, the intercellular spaces open up by schizogeny. Scale bar ¼ 20
m; same magnification for all photos. (From Seago et al., 2000; with permission of the authors and the Annals of Botany Company.)

Figure 18 The first cell divisions that mark the initiation of vascular cambium in the tap root of Aesculus hippocastanum. Radially oriented groups of newly divided cambial cells, C, can be recognized by their thin cell walls. P is a primary phloem element. Scale bar ¼ 25
m. (Original. By courtesy of Dr. N. J. Chaffey.)

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Barlow

Figure 19 Vascular cambium (C) and its derivatives (xylem, X, and phloem, P) in a tap root of Aesculus hippocastanum. Rays (R) are cells elongated in the radial plane. Scale bar ¼ 25
m. (Original. By courtesy of Dr. N. J. Chaffey.)

phloem poles. In economic terms, the groove is undesirable as it leads to significant amounts of unwanted soil being harvested along with the root. However, the groove does improve anchorage of the beet in the soil. Such a grooved root may be evolutionarily related to a type of root which, after a few seasons of growth, splits longitudinally, just as do (and using the same mechanism of differential cambial growth, but working over a longer period of time) the trunks of certain trees (e.g., Taxus baccata) and tap roots of Taraxacum koksaghyz (Bulgakov, 1944), and thus aids vegetative propagation.

V. ASYMMETRICAL CELL DIVISIONS Asymmetric formative divisions were discussed earlier (see Section III.A), especially in relation to embryonic root development. However, the production of unequally sized daughter cells by asymmetric division also occurs in the proliferative compartment of roots during the postgermination phase of their growth. In addition to the unequal partitioning of cytoplasm following mitosis, there can also be unequal repackaging of the nuclear proteins within the sister interphase

nuclei (Armstrong and Davidson, 1982). Just as may be the case in formative divisions, an asymmetric proliferative division may be evidence for some asymmetry of cytoplasm (polarization) in the mother cell. Unequal proliferative divisions are a feature of the root epidermal cells of some species (Clowes, 2000). An outcome of this is that one cell out of each pair of daughter cells subsequently gives rise to a root hair, whereas the other cell remains hairless. A similar inequality is also evident in the root cortex of Monstera deliciosa, where trichosclereids (internal hairs) develop from the smaller daughter cell of some divisions (Bloch, 1946; Barlow, 1984). In addition, asymmetric cortical cell division is a prelude to schizogenous aerenchyma formation in roots of Nymphaea odorata (Seago et al., 2000) (see Fig. 17). However, the chain of cause and effect that links inequality of cell size to different cell fates must remain circumstantial until more detail accrues on the consequences of unequal division for the partitioning of preexisting gene products within the two daughter cells and the subsequent patterns of gene expression. Nevertheless, the few experiments that have been performed are suggestive of such a link. They have been aimed, for example, either at suppressing hair development by means

Cellular Patterning in Root Meristems

of the promotion of equal, rather than unequal, division or, alternatively, at inducing all cells, irrespective of their size, to become hairs. One simple experiment involved the epidermis of maize roots. Equal divisions were induced by their growth in water: hair development, normally associated with unequal cell division, was suppressed (Fig. 20A) (Ivanov and Filippenko, 1979; Filippenko, 1980). Unequal divisions also naturally occur in the cortex of primary roots of Zea mays (cv. LG11), where, apart from their size, there is no other evident difference between the two sister cells (Barlow, 1987). However, this inequality was found to be restricted to the first few divisions immediately following germination. Later, as the roots lengthened, divisions became more equal. As in the cases mentioned above, the direction of unequal cell production in the maize cortex is not left to chance: the daughter cell in an apical position was usually longer than the basal daughter. One consequence of such a size differential is that the larger daughter cell divides before the smaller cell (Fig. 21). Similar findings have been reported for the cortex

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of wheat roots (Demchenko, 1975; Demchenko and Ivanov, 1978). Persistence of this directed pattern of unequal cell productions means that, after a few cell generations, a particular distribution of cell lengths is noticeable in each cortical cell file. Such patterned division sequences have been analyzed in the young primary root of maize (Lu¨ck et al., 1994a,b; Barlow, 1987) but, as noted above, the initial pattern of inequalities ceded to one in which daughter cells were not so unequal in size (Lu¨ck et al., 1997). A possible contributory factor in the change in division pattern is that the cells in which unequal divisions were displayed were preformed embryonic cells and, later, they were displaced by descendents of postformed cells that originated close to the root tip. Interestingly, paired files of cells created in the outer cortex by an early longitudinal division subsequently showed a pattern of cell productions that was the inverse (i.e., divisions follow a basipetal sequence within the file) of cells in neighboring files which lack the longitudinal division (their cells show acropetal division sequences). The reason for this is obscure, but these inverted sequences

Figure 20 Evidence of unequal cell division and subsequent cell growth in the primary root of Zea mays. (A) Percentage of newly divided pairs of cells with varying ratios of apical to basal (a/b) cell lengths in the epidermis of roots grown either in air (where hair cells develop from the epidermis) or in water (where the epidermis is hairless). The a/b ratio shifts to a value closer to 1 (indicative of equal division) in the water-grown roots. The cells were measured in a zone of the meristem in advance of hair initiation, so it is not certain, in the case of air-grown roots, if all pairs of cells would have formed hairs or not. (B) Similar data to A, except that the values denoted by line 1 refer to newly divided pairs of cells in the most proximal tier of the root cap columella. Values denoted by line 2 refer to the a/b ratio for the same pair of cells, but measured just prior to division of the most proximal (b) cells. The shift in the percentages (with respect to the values denoted by line 1) suggests that the more distal (a) daughter cell of a division grows much less than the proximal cell. (A modified from Ivanov and Filippenko, 1979; B modified from Ivanov and Larina, 1976.)

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Barlow

Figure 21 Unequal divisions in the cortex of a primary root of Zea mays. Usually, the more apical cell is the larger in a pair of daughter cells. As a consequence, it enters mitosis before the smaller basal daughter cell. This pattern of division persists for a number of divisions until these cells and their descendants are displaced from the meristem. The cells in this photograph are the products of the first two rounds of division following imbibition. The arrow points toward the root apex, emphasizing the polarity of the division sequence. Scale bar ¼ 10
m.

account for some of the low-frequency division pathways found in an analysis aimed to discover how the various cortical cell division patterns arose (Lu¨ck et al., 1994b). The presence of differently sized cells within a population of otherwise apparently homogeneous cells indicates that the division process cannot be taken for granted as being one from which equally sized cells will inevitably emerge. Little is known of how the size of each daughter could be regulated, so no ready answer is available to the important question of what factors regulate the siting of the new cell wall within the mother cell. Hofmeister’s division rule and a critical aspect ratio seem, at a first approximation, to be sufficient (see Barlow and Adam, 1989), but genetic control over divisional asymmetry has been discovered in many systems (Horvitz and Herskowitz, 1992; Way et al., 1994), and this type of control may override others which seem to be based on cell geometry. Genetic regulation of cell size may be related to subsequent cell differentiation programs which depend for their inception on the occurrence of quantal mitoses at precise times within a cell lineage (e.g., the scarecrow mutation of Arabidopsis). Unequal division presents a problem that is rather different from, but nevertheless

related to, that concerning whether a cell within a meristem is to divide longitudinally or transversely. The siting of the new division wall not only relates to local conditions within the dividing mother cell but, in turn, is also related to conditions within the tissue as whole, whereas the plane of division depends more on this broader, tissue-based set of conditions which includes growth polarities already established in the axial and radial planes (Lynch and Lintilhac, 1997). Both local and more global controls of division impinge on the siting of the preprophase band (PPB) of microtubules, a component of the cytoskeleton which seems invariably to predict where the developing cell plate will attach to the walls of a dividing mother cell (Wick, 1991). The siting of the PPB therefore has a direct influence on the overall pattern of mitosis within tissues (Gunning et al., 1978b; Lloyd, 1986). In the case of unequal transverse divisions in the epidermis, the PPB is, as expected, sited asymmetrically within the cell (Gunning et al., 1978b). This is presumably a response to asymmetry of cytoplasm and nuclear positioning during preprophase, if not beforehand. The unequal early transverse divisions in the maize cortex can be equalized by exposing the roots to methanol (Barlow, 1989b). This same chemical also equalizes

Cellular Patterning in Root Meristems

the early divisions of fern spores and, in so doing, changes the course of differentiation of one of the daughter cells (Miller and Greany, 1976; Vogelmann and Miller, 1981)—both daughters subsequently differentiate as rhizoids rather than as a rhizoid and protonema initial (which are the products when methanol is absent). It is not known how methanol affects either cell polarity or PPB positioning. In maize, the usual asymmetry of cytoplasmic RNA staining within stelar cells was also minimized by methanol (Barlow, 1989b), and this disturbance to cytoplasmic partitioning may have affected the location of the PPB. Transversely dividing cells in the cortex of maize roots lie within a growth field that has a more or less uniform rate of longitudinal extension. Hence, any pattern of differential cell sizes is a reflection of size at birth and is not a product of subsequent elongation. However, in the columella of the root cap (and the same may be true of cells on the basiscopic face of the QC), the meristematic cells lie upon a steep growth gradient. Those cells within the proximal, first tier of the short cap meristem have a faster elongation rate than cells in more distal tiers (Barlow, 1977). Thus, although divisions in the first tier may be equal, the two daughter cells quickly become unequal in length (Fig. 20B) (Ivanov and Larina, 1976; Ivanov, 1979). Such a gradient makes it difficult to judge, in microscope preparations of cap tissue, whether divisions are equal or not without examining cell sizes immediately after cytokinesis. Asymmetrical divisions in roots endow each of the two resulting daughter cells with different potentialities for their subsequent structure and function. The already mentioned root-hair-inducing division in epidermis is an example of this. At the time of their origin, the smaller hair cell initials (trichoblasts) are located in a region of root where elongation is ceasing. The hair initials, having acquired an RNA-rich cytoplasm following the unequal division, retain the ability for massive cytoplasmic and cell wall growth. The new cytoplasmic material bursts out from a small area of the external periclinal wall of the trichoblast, this being the only site where any further increase in wall area can take place (though some mutations can alter the location of this site, or even abolish it). Trichosclereids of Monstera roots likewise extend their side walls into the intercellular air spaces within the maturing zone of the cortex. That the division that creates hair cells is often the ultimate or penultimate one in a series of proliferative divisions means that the resulting hair cell initial will be endowed with numbers of plasmodesmata on each of its walls that are different from those asso-

75

ciated with nonhair cells. Hence, the hair and nonhair cells have different degrees of symplasmic connection with each of their neighboring cells, as occurs, for example, in the water plant Trianea bogotensis (Kurkova and Vakhmistrov, 1984). In part, the differential is due to the diminution of plasmodesmatal density brought about by the continuing expansion of the walls. A similar mechanism might explain the increasing symplasmic isolation of maturing epidermal cells on the Arabidopsis root and the hair cells to which they give rise (Duckett et al., 1994). Similar considerations also hold for roots that possess an exodermis (internal to the epidermis) consisting of short passage cells, a type of cell which seems to be developmentally less mature than the longer, nonpassage, sister cell. Although the anatomy of onion root exodermis suggests a function similar to that of endodermis, the short passage cells seem able to enhance the viability of overlying epidermal cells during stressful conditions (Barrowclough and Peterson, 1994). The patterning of the dimorphic exodermal cells can be variable with long and short cells arranged either irregularly or regularly. In the latter case, the long and short cells alternate within a given file (Peterson, 1989). Likewise, epidermal hair cells can show various patterns. In Azolla pinnata, the pattern of hairs results from a lineage-based differentiation program in which asymmetrical epidermal cell division is one of the final steps (Gunning et al., 1978a). In members of the Brassicaceae, the patterning of the differentiated epidermal cells is not only the outcome of asymmetrical transverse divisions but also results from a specific type of contact between their anticlinal walls and the underlying cortical cells (Bu¨nning, 1951; Barlow, 1984; Volkmann and Peters, 1995). Moreover, in Potomageton natans, there is a relationship between the asymmetrical division pattern in epidermis and exodermis. Here each tissue forms long and short cells late in the divisional history of the respective cell files. Exodermis cells underlying the short hair initials divide asymmetrically, but when exodermis cells overlie long, nonhair cells, they do not divide and, hence, remain long (Tschermak-Woess and Hasitschka, 1953). When considered in the light of the function that such morphologically specialized cells possess, the root epidermal hair system might justifiably be considered a paradigm for investigating the patterns of gene control and physiology within the context of a specific tissue structure–function relationship (Dolan, 1996). Not all the elements in this system of interacting cells are by any means certainly identi-

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fied. Moreover, the function of root hairs is manifold: they participate in solute uptake and they stabilize the root in soil, to name but two evident functions. But to what extent are they dispensable in either of these processes? Whatever the emerging view on this question, it is clear that patterned cell division has a fundamental role not only in the specific case of the epidermis but also in the more general process of establishing root form during embryogenesis, as well as for the continued maintenance of form and function of roots in their natural environment.

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5 Root Hairs: Hormones and Tip Molecules Robert W. Ridge and Masayuki Katsumi International Christian University, Tokyo, Japan

I.

INTRODUCTION

supply them to root hairs. In addition, under natural conditions, root hairs are exposed to microorganisms that can also produce plant hormones (Wang et al., 1982; Pegg, 1985). (2) Stimulation of the normal process of development by exogenous supply of the hormone and/or suppression of the process by application of inhibitors. (3) Suppression of the process in mutants related to the hormone such as those deficient in the hormone or with a defective hormone response. Recent findings of many mutants of Arabidopsis, associated with root hair formation, morphology, and growth, implicate plant hormones, especially auxin and ethylene, are involved in these processes (Schiefelbein and Somerville, 1990; Baskin et al., 1992; Su and Howell, 1992; Schiefelbein et al., 1993; Galway et al., 1994; Masucci and Schiefelbein, 1994; 1996; Cernac et al., 1997; Grierson et al., 1997; Schneider et al., 1998; see also Aeschbacher et al., 1994).

The past 5 years have seen a significant increase in interest in root hair biology, especially in their cell and molecular biology. Current major areas in root hair research can be loosely grouped into four parts: cell biology, physiology, genetics, and plant–microbe interactions, though the last group encompasses aspects of the previous three (Ridge and Emons, 2000). Attempting to summarize these areas in a short chapter will give none of them justice and only repeat much of what is available in that monograph. Thus, we have decided to discuss two topics not previously covered in root hair chapters in Plant Roots (cf. Ridge, 1996), the role of hormones and the phenomenon of root hair tip molecules.

II.

ROOT HAIRS AND PLANT HORMONES

A.

The pattern of root hair growth and development is under the influence of various factors including genetic, physiological, and environmental factors (see Ridge, 1995b; Peterson and Farquhar, 1996). There are at least three criteria to be considered in whether or not a plant hormone is required for normal progress of the developmental process of root hairs: (1) Production of the hormone by root hairs or supply of the hormone from other sources to root hairs. For this, though it is not easy to demonstrate that root hairs per se produce plant hormones, we know that other root tissues can

Hormones in Root Hair Formation

Auxin and ethylene affect root hair formation. Stimulation of root hair formation by ethylene was probably first described by Borgstro¨m (1939) in Pisum sativum, Vicia faba, and Lupinus luteus (cf. Abeles et al., 1992; see also Chapter 27 by Hussain and Roberts in this volume). Masucci and Schiefelbein (1994) have demonstrated that an Arabidopsis mutant, rhd6, shows three distinct defects in root hair formation—a reduction in the 83

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number, an overall basal shift in the site of formation, and a relatively high frequency of epidermal cells with multiple root hairs. These phenotypes can be rescued by application of exogenous indole-3-acetic acid (IAA) or 1-aminocyclopropane-1-carboxylic acid (ACC), an ethylene precursor. Ethylene-induced root hair formation in Arabidopsis has also been reported (Baskin and Williamson, 1992). Dark-grown wild-type Arabidopsis seedlings, which are largely root hairless and produce little ethylene, can develop root hairs by exogenous treatment with ethylene and ACC (Cao et al., 1999). Treatment of wild-type Arabidopsis with aminovinylglycine (AVG), an inhibitor of ACC synthase in the pathway of ethylene biosynthesis, can phenocopy the rhd6 phenotype. Abolishment of root hairs by treatments with AVG (Tanimoto et al., 1995; Masucci and Schiefelbein, 1996) or with silver nitrate, an ethylene action inhibitor (Baskin and Williamson, 1992) has also been reported for Arabidopsis. Additional supportive evidence for the involvement of ethylene/ACC in root hair production is ACCinduced ectopic root hair formation in wild-type seedlings (Tanimoto et al., 1995; Masucci and Schiefelbein, 1996). Ectopic root hairs are also present in ctr1, an ethylene-response mutant (constitutive triple responses) (Dolan et al., 1994; Tanimoto et al., 1995). Arabidopsis root hairs develop preferentially on trichoblasts overlying the cortical anticlinal cell walls, whereas nonhair cells develop on atrichoblasts overlying the cortical periclinal cell walls. Since in etiolated Arabidopsis, ethylene and ACC can induce root hairs on trichoblasts, and ethylene-overproducing mutants (eto1, eto2, eto3, abd, eto4) preferentially develop root hairs on trichoblasts, these cells should be more sensitive to ethylene than atrichoblasts (Cao et al., 1999). Exogenous auxins such as IAA and 2,4dichlorophenoxyacetic acid (2,4-D), can induce ethylene production but do not cause ectopic root hair formation. In addition, the fact that the number of root hairs in the ethylene response mutants etr1 and ein2 are normal, suggests that ethylene may not be necessary for absolute numbers of root hairs (Masucci and Schiefelbein, 1996). Lack of ectopic root hair formation due to ACC treatment has also been reported in wild-type and etr1 Arabidopsis seedlings (Pitts et al., 1998). Masucci and Schiefelbein (1996) have suggested that ethylene acts independently of TTG and GL2, positive regulators of trichome development (Hu¨lskamp et al., 1994), and possibly downstream of those genes. Cao et al. (1999) have also come to the same conclusion by a study with CTR1 gene, a negative regulator of the

Ridge and Katsumi

ethylene response (Kieber et al., 1993; Roman et al., 1995). An increase in root hair number by kinetin treatment has also been reported in Raphanus sativus (Bittner and Buschmann, 1983). B.

Hormones in Root Hair Elongation

1.

Auxins/Ethylene

Elongation of root hairs of Agrostis alba is stimulated by a low concentration of 0.1 pM IAA that inhibited at high concentrations (0.1–1 mM) (Jackson, 1960). Bates and Lynch (1996) reported that root hair elongation of the axr2 mutant of Arabidopsis is stimulated by low phosphorus availability, but an auxin antagonist, 2-(p-chlorophenoxy)-2-methylpropionic acid inhibits root hair elongation in low-phosphorus plants. Concomitantly, application of exogenous IAA stimulated root hair elongation in the wild-type high-phosphorus plants. If root hairs require auxin for normal growth, the level of endogenous auxin must be extremely low. This might be confusing since Pitts et al. (1998) obtained promotion of root hair growth of Arabidopsis with a relatively high concentration (10 nM to 10 mM) of 2,4-D. However, since they measured the effect 7 days after treatment, the root hairs might have adapted to the high concentration. If the effect would have been measured soon after the treatment, the effect could have been inhibitory. One of the major effects of auxin at subcellular levels, which leads to elongation of ordinary cells, is cell wall loosening due to the acidification of the cell wall matrix via proton secretion from the cytosol into the apoplast (Cleland, 1987; Cosgrove, 1986, 1993; see also Chapter 30 by Pilet in this volume). Though root hairs elongate by tip growth (see Ridge, 1993), it is possible that auxin controls root hair elongation through a similar mechanism. It has been demonstrated that tip-growing cells like pollen tubes and root hairs require a continuous influx of Ca2+ at the growing tip and an internal Ca2+ gradient with higher levels at the tip for their continued elongation (Reiss and Herth, 1979; Clarkson et al., 1988; Schiefelbein et al., 1992; de Ruijter et al., 1998; see also Chapter 31 by Poovaiah et al. in this volume). Auxin is known to affect cytoplasmic streaming in various types of plant cells including root hairs (see Ayling et al., 1994). Cytoplasmic streaming in Avena root hairs, is stimulated by low concentrations of auxin, though it is inhibited at high concentrations (Sweeney, 1944). A similar effect of IAA has also

Root Hairs

been shown by Ayling et al. (1994). IAA (10–100 nM) stimulates cytosolic streaming in tomato root hairs within 1–2 min after application, whereas concentrations of IAA > 1
M are inhibitory. Inhibition of streaming by a high concentration (10 mM) of the synthetic auxinNAA, has also been reported for root hairs of Hydrocharis dubia (Tominaga et al., 1998). Such inhibition was shown to be a result of acidification of the cytoplasm that disturbed the orientation of actin filaments and disrupted cortical microtubules. On the other hand, Tretyn et al. (1991) reported that auxin regulates cytoplasmic Ca2+ levels and pH in Sinapis alba root hairs through changes in membrane potential. They suggested that a high concentration (100 nM) of IAA inhibits root hair growth by depolarizing root hairs to diminish Ca2+ gradient. But, it is argued that auxin-induced changes in Ca2+ levels and in pH are too fast to correlate with auxin-induced stimulation of cytoplasmic streaming (Ayling et al., 1994). Cytoplasmic streaming is not indispensable for ordinary cell elongation, because cytochalasin B, which inhibited cytoplasmic streaming completely, did not entirely inhibit auxin-induced growth of Avena or maize coleoptiles (Cande et al., 1973). Recent studies on the molecular biology of Arabidopsis have revealed some auxin/ethylene related genes associated with root hair growth and development. Many auxin mutants of Arabidopsis have shorter root hairs than those of the wild-type. The aux1 (Okada and Shimura, 1995; Pitts et al., 1998), axr1 (Lincoln et al., 1990; Pitts et al., 1998), and axr2 and axr3 (Leyser et al., 1996) genes all affect root hair growth. The axr1 mutants of Arabidopsis have reduced numbers of root hairs (Cernac et al., 1997). On the other hand, a normal number of root hairs was found for the weak axr1-3 allele (Masucci and Schiefelbein, 1996). Pitts et al. (1998) examined this in detail and found that the discrepancy is due to the methods of measurement they employed; the lengths of the root hairs of axr1-12 and axr1-7 were clearly shorter than wild-type hairs, while axr1 mutations had little effect on root hair initiation. In the double mutants of sar1 and axr1-12 the length of root hairs was the same as that of the wild-type (Cernac et al., 1997; Pitts et al., 1998). SAR1 has been shown to act after AXR1 in the pathway of auxin response (Cernac et al., 1997). Pitts et al. (1998) have found that both 2,4-D, and ACC can stimulate root hair elongation of the wildtype, and of mutants deficient in auxin response (aux1) or in ethylene response (etr1) of Arabidopsis thaliana. Nevertheless, these mutations have no significant effect

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on root hair initiation (Masucci and Schiefelbein, 1996). Since the AUX1 gene product was suggested to be an auxin flux carrier in Arabidopsis roots (Bennett et al., 1996), shorter root hairs of axr1 mutant might be attributed to a defect in auxin supply to trichoblasts which is indispensable for root hair elongation. On the other hand, the AXR1 gene product is similar to the ubiquitin-activating enzyme, E1, which is necessary for the activation of Rub1 protein in Arabidopsis (del Pozo et al., 1998). Modification of nuclear proteins in the root hair by Rub is necessary for root hair growth, and auxin probably stimulates root hair elongation via this AXR1 pathway. Whatever the mechanism is, the presence of auxinbinding protein in root hairs of Zea mays (Radermacher and Kla¨mbt, 1993) might indicate that auxin has some controlling role in root hair growth and development. Indeed, Baskin and Williamson (1992) have reported that ethylene increases root hair growth in Arabidopsis seedlings. 2.

Gibberellin

Application of GA3 had practically no growth stimulating effect on clover root hairs, in a concentration range from 1 nM to 10
M. However, uniconazole (10
M), an inhibitor of gibberellin biosynthesis, markedly inhibited root hair growth, suggesting that endogenous gibberellin was involved in the normal growth of clover root hairs (Izumo et al., 1995). Exogenous GA3 (0:1
M) could completely overcome the uniconazole-induced growth inhibition (Izumo et al., 1995). These results suggest that normal root hairs are dose saturated with endogenous gibberellin, and that the effective gibberellin concentration is very low. The results are also consistent with the finding of Tanimoto (1987, 1991) obtained by similar experiments using another gibberellin biosynthesis inhibitor, ancymidol. The conclusion is that roots require a very low level of endogenous gibberellin for normal growth (see Chapter 24 by Tanimoto in this volume). Gibberellininduced stimulation of root hair elongation has also been reported for Pisum sativum (Pecket, 1960) and Agrostis alba (Devlin and Brown, 1969). It is very likely that gibberellin is required for normal growth of root hairs, but in very small quantities. Gibberellin controls cortical microtubule orientation, thus affecting the arrangement of cellulose microfibrils (Ishida and Katsumi, 1991, 1992; Shibaoka, 1994). This effect correlates with GA-induced cell elongation (Ishida and Katsumi, 1991). Since in root hairs

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microtubules are associated with tip growth (Emons and Wolters-Arts, 1983; Emons et al., 1990; Ridge, 1995a; Bibikova et al., 1999; Ridge and Emons, 2000), GA-involved control of root hair growth might be related to this cytoskeleton protein. 3. Cytokinin/Abscisic Acid/Brassinosteroid Roots of Arabidopsis seedlings are very sensitive to cytokinins. By treatment with 5 nM to 10
M benzyladenine (BA), the growth of primary roots was reduced, while that of root hairs was stimulated (Su and Howell, 1992). Su and Howell (1992) have isolated cytokinin response mutants (ckr1) of Arabidopsis which have longer primary roots and shorter root hairs than the wild-type in the absence of BA. Growth of root hairs of ckr1 was slightly stimulated by BA and reached to the same length as that of the control wild-type in the presence of 50–500 nM BA. In normal Arabidopsis seedlings, primary roots, and root hairs may counterresponse the endogenous cytokinins (Su and Howell, 1992). It is likely that cytokinin is involved in root hair growth. Since cytokinin can induce ethylene production (Abeles et al., 1992; see also Chapter 25 by Emery and Atkins in this volume), some of the cytokinin effects observed might be ascribed to ethylene. Endogenous ABA is known to mediate plant water stresses (Davies and Mansfield, 1983; Hartung and Davies, 1992; see also Chapter 26 by Hose et al. in this volume). The effect is expressed in a significant reduction in overall growth. Worrall and Roughley (1976) and Vartanian (1981) reported that drought stress caused existing root hairs to become fragile and thin walled, and produced short and swollen new root hairs in Trifolium subterraneum and Sinapis alba, respectively. This suggests that ABA is involved in the formation of deformed root hairs. Schnall and Quatrano (1992) have demonstrated that treatment of Arabidopsis with ABA resulted in the formation of similar deformed root hairs. But ABA had no significant effect on the total number of root hairs. However, the ABA-insensitive mutants abi1 and abi2 did not respond to exogenous ABA (no deformed root hairs). Nevertheless, an ABA-sensitive mutant, abi3, displayed a response similar to that of the wild-type (Schnall and Quatrano, 1992). It is unlikely that ABA is required for normal growth and development of root hairs. 4. Concluding Remarks About Hormones Cumulative data in the literature provide strong evidence that plant hormones are involved in the process

Ridge and Katsumi

of root hair development and elongation. Findings of various auxin/ethylene-associated mutants in Arabidopsis and their molecular biological studies have demonstrated that auxin and ethylene, in particular, are important endogenous factors which control not only the elongation but also the development of root hairs. Cytokinin, gibberellin, and brassinolide may also endogenously influence these processes, though evidence is still circumstantial. The mechanism of the hormonal control of root hair elongation is not clearly understood. Whatever it is, it must be different from the mechanism of ordinary cell elongation which depends on cell wall loosening, because root hair elongation proceeds by tip growth.

III.

MOLECULES AT THE ROOT HAIR TIP SURFACE

The first reports of distinct molecules at the tips of root hairs were in the middle to late 1980s (Werner and Kuhlmann, 1985; Diaz et al., 1986; Ridge and Rolfe, 1986; Diaz, 1989) (Fig. 1). Since then, there have been only a few publications directly addressing the role of known tip molecules or announcing the discovery of new ones. Indeed, the few molecules known to be specific for the tips of root hairs are lectins, sugars, glycoproteins, and a spectrinlike molecule. Extensin is probably present, though not visualized at the tip (Arsenijevic-Maksimovic et al., 1997). A.

Lectins at the Tips of Root Hairs

The role of lectins in legume nodulation was recently reviewed (Hirsch, 1999). Lectin on the surface of pea root hairs was detected, using an antibody against pea seed lectin (Diaz et al., 1986). Fluorescent-labeled lectins of root hair tips were found to be distributed in dense small patches rather than uniformly over the tip. Diaz et al. (1986) also showed that lectins are not present on all hairs, and gave evidence that lectin labeling of tips was detected predominantly opposite protoxylem poles. The significance of this finding is that early development of symbiotic nodules is usually opposite the protoxylem poles of the young root, and that infectivity of root hairs (as shown by the presence of lectin) is enhanced by unknown (hormonal?) factors transported from within the root. In the zone of mature hairs, where lectins are rarely found in hairs, inoculation with rhizobia produced few nodules compared to the immature root hair zone. The latter had high levels of lectin labeling and produced many nodules. This

Root Hairs

87

Figure 1 Lectin binding to root hair tips demonstrating the presence of different sugar moieties at the tip surface. (A) Macroptilium atropurpureum (Leguminaceae) labeled with MPA-FITC (from Maclura pomifera, haptens: N-acetyl galactosamine, -D-galactose). (B) Rafanus sp. (Brassicaceae) labeled with RCA-1-FITC (from Ricinus communis, hapten: -D-galactose). (From Ridge et al., 1998.)

proves that root lectins are likely to play a role in the early stages of nodulation. Further work in this area showed that lectins were involved in host specificity of nodulation (Diaz et al., 1989a,b, 1995). The pea lectin gene was transformed into white clover plants, which were then able to be nodulated by Rhizobium leguminosarum bv. viciae. These investigators also altered the carbohydrate-binding domain of the pea lectin by sitedirected mutagenesis and when this mutant gene was introduced into white clover plants, the Vicia rhizobia failed to nodulate them. More recently, Van Rhijn et al. (1998) showed similar results by transforming Lotus with soybean lectin gene. Soybean lectin was detected at the tips of young growing root hairs of transgenic Lotus plants. Inoculation with Bradyrhizobium japonicum resulted in nodulation of Lotus, which is normally not nodulated by B. japonicum. An interesting additional result of their work showed that exopolysaccharide of the bacteria is likely to be involved in early interactions with the root hair (see also Hirsch, 1999). This suggests that lectins in the root hair tip may directly bind the

bacteria to the root hair tip at the onset of the earliest interactions between plant and rhizobia. Certainly it is known that binding is an essential prerequisite for the colonization and infection of many kinds of tissue (Beuth et al., 1996), and also that lectins mediate many biological recognition processes (Sharon and Lis, 1995). A lectin detected on root hair surfaces carrying enzyme activity similar to apyrase (ATP diphosphohydrolase) and that binds Nod factors has also been reported, although it was not detected solely at root hair tips (Etzler et al., 1999). The lectin was called LNP by the authors (lectin nucleotide phosphohydrolase). Etzler et al. (1999) reported that this lectin binds Nod factor and that an antibody to LNP inhibits the ability of rhizobia to deform root hairs and to form nodules. However, their results are limited by single determination points in their Nod factor-binding experiments, difficult-to-interpret micrographs, and lack of explanation of some of their techniques. This is a potentially exciting result, but we must await confirmation from other laboratories.

88

B.

Ridge and Katsumi

Sugar Molecules at the Tips of Root Hairs

Lectins are widely used as tools for detecting specific sugar molecules, particularly on cell surfaces. Ridge and Rolfe (1986) found that RCA-I lectin (from Ricinus communis, specific for -D-galactose) bound to the root hair tips of siratro (Macroptilium atropurpureum), a cowpea-type legume known to be nodulated by several groups of rhizobia. Preincubation of roots, using a spot inoculation technique in the most susceptible zone (smallest emerging root hair zone) of the root for infection, showed that lectin bound to root hair tips prevented infection and nodulation by rhizobia. The authors concluded that carbohydrate molecules at the surface of the root hair tips could be directly involved in the initiation of the symbiosis. More recent work (Ridge et al., 1998) has shown that the broad host range of siratro has a multiplicity of sugars at its root hair tips, whereas narrow host range plants such as clover and alfalfa have usually one (RCA-I binding), sometimes two kinds of sugars at their tips. The authors also showed that some nonlegumes have sugars at their tips, though their study was limited to the Brassicaceae. Such a defined area of sugar molecules at the tips of root hairs suggests that the molecules are probably the sugar moieties of membrane-bound glycoproteins, Apparently they are recycled at the tip, where there is a large presence of clathrin at the base of the apical dome. Otherwise they would be detectable below the apical dome. Indeed, in the case of one lectin, VFA, which is specific for -Dmannose, this is true. In addition the results show that there may be a conserved sugar expression (or default expression) of one sugar, -D-galactose, present on narrow host range legumes and on one member of the Brassicaceae. More recently, Samaj et al. (1999) have shown specific localization of arabinogalactan-protein epitopes at the surface of maize root hairs, where strongest expression was at the bulge of root hair initiation (cf. Ridge and Rolfe, 1986) and at the tips of growing hairs. Samaj et al. (1999) also agreed with previous suggestions that such glycoproteins may be involved in plant-microbe interactions (by signaling events). What is the significance of sugar molecules at the tips of root hairs? In the case of root hairs that apparently have several kinds of sugars, it is asked whether they are the same glycoprotein molecule, or is each on different molecules, or variations thereof? Only recently it was begun to be understood how plants

use the structural diversity of oligosaccharides to regulate important cellular processes such as growth, development, and defense (John et al., 1997). These oligosaccharides are known to be biologically active at extremely low concentrations. In the case of sugar molecules at the tips of root hairs, especially those in legumes, it is imperative to understand if they are involved in early interactions with microorganisms in the rhizosphere. Indeed, the earlier work of Ridge and Rolfe (1986) showed that by blocking the sugar sites of siratro root hair tips, nodulation was prevented. Etzler et al. (1999) have recently reported a similar result whereby an antiserum to a lectin at the Dolichos root hair surface prevented nodulation by rhizobia. Despite the limited amount of information available, this is an important area of research for root hairs, because distinct molecules at the tips suggest many possibilities: control of polarity, communication to the rhizosphere through receptor activity, passive and active plant pathogen interactions. Indeed, the very nature of the growing tip of this cell, where the cell wall is expanding in direct exposure to the rhizosphere, makes it a likely site for many kinds of interactions. In this connection, it has been shown that in a nodulation-minus soybean, the stimulation of cell division in the cortex of the root by the synthetic auxin 2,4-D and the consequent cell wall expansion, allowed rhizobia-induced infection thread development directly in these cells rather than in root hairs (Akao et al., 1991, 1999) with a subsequent successful symbiosis. This means that for the wildtype soybean, invasion of root hairs by rhizobia requires an expanding cell wall rather than tip growth per se, and that symbiotic-specific communication molecules or receptors, if they exist, can also be expressed in other cells.

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Ridge and Katsumi eds. Arabidopsis. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, pp 665–684. Pecket RC. 1960. Effects of gibberellic acid on excised pea roots. Nature 185:114–115. Pegg GF. 1985. Pathogenic and nonpathogenic microorganisms and insects. In: Pharis RP, Reid DM, eds. Hormonal Regulation of Development III. Berlin; Springer-Verlag, pp 599–624. Peterson L, Farquhar ML. 1996. Root hairs: Specialized tubular cells extending root surfaces. Bot Rev 62:1–40. Pitts RJ, Cernac A, Estelle M. 1998. Auxin and ethylene promote root hair elongation in Arabidopsis. Plant J 16:553–560. Radermacher E, Kla¨mbt D. 1993. Auxin-dependent growth and auxin-binding proteins in primary roots and root hairs of corn (Zea mays L.). Plant Physiol 141:698– 703. Reiss H, Herth W. 1979. Calcium gradients in tip growing plant cells visualized by chlorotetracycline fluorescence. Planta 146:615–621. Ridge RW. 1993. A model of legume root hair growth and Rhizobium infection. Symbiosis 14:359–373. Ridge RW. 1995a. Microvesicles, pyriform vesicles and macro-vesicles associated with the plasma membrane in the root hair of Vicia hirsuta after freeze-substitution. J Plant Res 108:363–368. Ridge RW. 1995b. Recent developments in the cell and molecular biology of root hairs. J Plant Res 108:399–405. Ridge RW. 1996. Root hairs: cell biology and development. In: Waisel Y, Eshel A, Kafkafi U, eds. Plant Roots: The Hidden Half. 2nd ed. New York; Marcel Dekker, pp 127–147. Ridge RW, Emons AMC. 2000. Root Hairs: Cell and Molecular Biology. Tokyo; Springer-Verlag. Ridge RW, Rolfe BG. 1986. Lectin binding to the root and root hair tips of the tropical legume Macroptilium atropurpureum Urb. Appl Environ Microbiol 51:328–332. Ridge RW, Kim R, Yoshida F. 1998. The diversity of lectindetectable sugar residues on root hair tips of selected legumes correlates with the diversity of their host ranges for rhizobia. Protoplasma 202:84–90. Roman G, Lubarsky B, Kieber JJ, Rothenberg M, Ecker JR. 1995. Genetic analysis of ethylene signal transduction in Arabidopsis thaliana: five novel mutant loci integrated into a stress response pathway. Genetics 139:1393–1409. Samaj J, Braun M, Balus˘ ka F, Ensikat H-J, Tsumuraya Y, Volkmann D. 1999. Specific localisation of arabinogalactan-protein epitopes at the surface of maize root hairs. Plant Cell Physiol 40:874–883. Schiefelbein J, Galway M, Masucci J, Ford S. 1993. Pollen tube and root-hair tip growth is disrupted in a mutant of Arabidopsis thaliana. Plant Physiol 103:979–985. Schiefelbein JW, Somerveille C. 1990. Genetic control of root hair development in Arabidopsis thaliana. Plant Cell 2:235–243.

Root Hairs Schiefelbein JW, Shipley A, Rowse P. 1992. Calcium influx at the tip of growing root-hair cells of Arabidopsis thaliana. Planta 187:455–459. Schnall JA, Quatrano RS. 1992. Abscisic acid elicits the water-stress response in root hairs of Arabidopsis thaliana. Plant Physiol 100:216–218. Schneider K, Mathur J, Boudonck K, Wells B, Dolan L, Roberts K. 1998. The ROOT HAIRLESS 1 gene encodes a nuclear protein required for root hair initiation in Arabidopsis. Genes Dev 12:2013–2021. Sharon N, Lis H. 1995. Lectins—proteins with a sweet tooth: function in cell recognition. Essays Biochem 30:59. Shibaoka H. 1994. Plant hormone-induced changes in the orientation of cortical microtubules: alterations in the cross-linking between microtubules and the plasma membrane. Annu Rev Plant Physiol Plant Mol Biol 45:527–544. Su W, Howell S. 1992. A single genetic locus, Ckr1, defines Arabidopsis mutants in which root growth is resistant to low concentrations of cytokinin. Plant Physiol 99:1569–1574. Sweeney BM. 1944. The effect of auxin on protoplasmic streaming in root hairs of Avena. Am J Bot 31:78–80. Tanimoto E. 1987. Gibberellin-dependent root elongation in Lactuca sativa: recovery from growth retardant-suppressed elongation with thickening by low concentration of GA3. Plant Cell Physiol 28:963–973. Tanimoto E. 1991. Gibberellin requirement for the normal growth of roots. In: Takahashi N, Phinney BO, MacMillan J, eds. Gibberellins. New York; SpringerVerlag, pp 229–240.

91 Tanimoto M, Roberts K, Dolan L. 1995. Ethylene is a positive regulator of root hair development in Arabidopsis thaliana. Plant J 8:943–948. Tominaga M, Sonobe S, Shimmen T. 1998. Mechanism of inhibition of cytoplasmic streaming by auxin in root hair cells of Hydrocharis. Plant Cell Physiol 39:1342– 1349. Tretyn A, Gottfried W, Felle HH. 1991. Signal transduction in Sinapis alba root hairs: auxins as external messengers. J Plant Physiol 139:187–193. Van Rhijn P, Goldberg RB, Hirsch AM. 1998. Lotus corniculatus nodulation specificity is changes by the presence of a soybean lectin gene. Plant Cell 10:1233– 1249. Vartanian N. 1981. Some aspects of structural and functional modifications induced by drought in root systems. In: Brouwer R, Gasparikova O, Kolek J, Loughman BC, eds. Structure and Function of Plant Roots. The Hague; Maritinus/Nijhoff/Dr. W Junk, pp 309–318. Wang TL, Wood EA, Brewin NJ. 1982. Growth regulators, Rhizobium and nodulation in pea. Indole-3-acetic acid from the cultured medium of nodulating and nonnodulating strains of R. leguminosarum. Planta 155:345–349. Werner D, Kuhlmann KP. 1985. Legume root hair proteins. In: Magnien E, de Nettancourt D, eds. Genetic Engineering of Plants and Microorganisms Important for Agriculture. Dordrecht, Netherlands: Nijhoff and Junk Publ, pp 66–67. Worrall VS, Roughley RJ. 1976. The effect of moisture stress on infection of Trifolium subterraneum L. by Rhizobium trifolii Dong. J Exp Bot 27:1233–1241.

6 Secondary Growth of Roots: A Cell Biological Perspective Nigel Chaffey IACR-Long Ashton Research Station, University of Bristol, Long Ashton, Bristol, England

I.

INTRODUCTION

body, which in the past 10–20 years has benefited greatly from the development of new techniques in cell and molecular biology. The application of those techniques, largely concentrated upon the ‘‘model plant’’ Arabidopsis thaliana (e.g., Somerville, 2000), has generated a tremendous amount of information concerning plant development. Because of problems—real or perceived—inherent in studying the cambium of trees (see Chaffey, 2001c), there has been a marked reluctance to pursue such investigations, and it is only recently that the techniques of modern cell biology have begun to be applied to this secondary meristem (e.g., Lachaud et al., 1999).

The primary apical meristems of the root and shoot produce the cells and tissue systems of the primary plant body (Cutter, 1978), such as the epidermis, cortex, and primary vascular tissues, and give rise to elongation growth. Secondary growth (synonymous with secondary thickening) is the province of the two laterally situated secondary meristems, the vascular cambium (herein simply referred to as the cambium) and the cork cambium, and results in increase in girth of the organ. Products of these two cambia together constitute the secondary plant body—secondary xylem and secondary phloem, and periderm, respectively. The terms primary and secondary indicate the order in which these two forms of growth occur; they should not be seen as an indication of their relative importance. Indeed, both are necessary for the successful growth and establishment of the majority of vascular plants, and secondary growth is essential to the development of the tree habit. Furthermore, although separated in space, both types of growth can occur at the same time. At the heart of secondary growth is the cambium. Notwithstanding the numerous reviews of this meristem (almost solely of the shoot) over the past 40 years (cf. Larson, 1994; Catesson, 1994; Lachaud et al., 1999), there has been little new cambial cell biology to review since the heyday of cambial ultrastructural studies in the 1960s and early 1970s. That contrasts markedly with the situation for the primary plant

II.

SCOPE OF THIS REVIEW

Aspects of primary growth and the biology of the primary body of roots are considered in other chapters in this volume. Background information on the anatomy and secondary growth of roots can be found in most standard plant anatomy texts. General aspects of the secondary growth of roots have previously been reviewed, most notably by Fayle (1968), Wilson (1975), and Woods (1991). As is proper, those reviews reflected the prevailing knowledge and interests of the time, and generally stood outside of the root ‘‘looking in.’’ However, since the last of those reviews was published, new aspects of the cell biology of secondary growth in roots have been studied, and it is therefore appropriate now to consider the inner workings of the 93

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system. Since most is known about the ultrastructure of, and the dynamics of the cytoskeleton during, the cambial seasonal cycle and secondary vascular differentiation of the angiosperm trees, Aesculus hippocastanum L. (horse-chestnut), and Populus tremula L:  P. tremuloides Michx. (hybrid aspen), this review will concentrate upon those topics in those taxa. Accordingly, this chapter summarizes the published work of Chaffey et al. (1996, 1997a–d, 1998a, 1999, 2000a,b) and Chaffey and Barlow (2000, 2001), and the work in progress of Chaffey, Barlow, and Sundberg, and Chaffey and Barlow.

III.

identify a region of similar-looking cells—the ‘‘cambial zone’’—wherein the true cambium lies. However, it is confidently predicted that suitable markers will soon be forthcoming as a result of the current interest in the molecular biology of cambial growth and activity (e.g., Hertzberg and Olsson, 1998; Newman and Campbell, 2000). The established cambium consists of two morphologically distinct cell types: fusiform initials (which are greatly elongated in the axis of the root) and ray initials (which are cuboid) (Fig. 3). Both are thin-walled, highly vacuolate cells (Fig. 4) with the usual complement of organelles one would expect for a cell in the root (see Table 1).

SECONDARY GROWTH: A RECAP

Even the most cursory glance at a transverse section of a tree root (Fig. 1; see also Fig. 18 in Chapter 4 by Barlow in this volume) shows the essential features of its secondary vascular system (SVS), concentric tissuezones—the phloem toward the outside, the cambial zone, and the xylem—and radial files of cells that traverse those zones. Both the radial and concentric features are products of periclinal (synonyms in the literature: tangential, additive) cell division of the cambial initials (Fig. 2), and of their daughter cells. Subsequently these cambial derivatives grow and differentiate as phloem cells or xylem cells in a positiondependent manner. Several subdivisions of the tissue zones are recognized in arboreal plants. For example, Bailey (1952) identified six zones related to wood formation: the cambial zone; the zones of cell enlargement, cell maturation, mature sapwood, and sapwood-heartwood transition; and the inner core of heartwood. That those same zones are still recognized today is both a testament to the insight of those earlier workers, and an acknowledgement of how little our understanding of the SVS has advanced. As our methods of analysis and inquiry become more refined, we will probably have cause, and need, to reevaluate those zones, possibly defining them more specifically and precisely by use of ‘‘molecular markers.’’ The powerhouse that drives all this growth activity within the SVS is the cambium. Unfortunately, although the cambium undeniably exists, there are currently no known markers that allow us unambiguously to identify true cambial initial cells (e.g., Catesson, 1994). Indeed, it is still a matter of debate as to whether the cambium proper is comprized of one or more tiers of cells (cf. Catesson, 1974; Larson, 1994; Iqbal, 1995). For that reason, the best we can presently do is to

Figure 1 Light micrograph of a transverse section of the root of hybrid aspen showing the arrangement of tissues of the secondary vascular system. Key: CZ, cambial zone; f, fusiform cambial cell; F, fiber; r, ray cambial cell; SE, sieve elements; SPh, secondary phloem; SXy, secondary xylem; VE, vessel element. Scale bar ¼ 20
m.

Secondary Growth

Figure 2 Transmission electron micrograph of a transverse section of the cambial zone of the taproot of horse chestnut showing a recently periclinally divided ray cambial cell. Key: p, plastid; rw, radial wall; tw, tangential wall; v, vacuole; star, nascent division wall. Scale bar ¼ 2:5
m.

Fusiform initials give rise to the cells of the vertical, or axial, component of the SVS, which, in horse chestnut and hybrid aspen roots, are: axial parenchyma, fibers, sieve elements, companion cells, and vessel elements (Table 2; Chaffey et al., 1999). Note, however, that transverse cell divisions of axial cambial derivatives within the cambial zone are necessary to produce axial parenchyma cells in both the xylem and phloem. The ray initials produce the ray cells (Fig. 1), which, in the xylem, can be further distinguished as two subtypes (Braun, 1984). Contact ray cells, where they lie next to vessel elements, have large-diameter pits, which are mirrored by the adjacent vessel element. Isolation ray cells, which may also lie next to vessel elements, have only small, elliptical simple pits (as for fibers and xylem axial parenchyma). All phloem ray cells appear to be of the same type. Although the rays are predominantly uniseriate, biseriate rays are occasionally found in the xylem and phloem of hybrid aspen (particularly in the root). Additionally, phloem rays are subject to dilatation growth, which effectively makes the rays multiseriate in this tissue.

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Figure 3 Light micrograph of a tangential longitudinal section through the cambial zone of the taproot of horse chestnut showing the ray (r) and fusiform (f) cambial cells; the encircled region indicates a relatively recent pseudotransverse division wall. Scale bar ¼ 20
m.

At maturity, and relative to the precursor cambial cell, all secondary vascular cells have different shapes, their cell walls are thicker, variously elaborated, and, in the case of all xylem cells and phloem fibers, also lignified (Table 2; Chaffey et al., 1999). Many ultrastructural changes also accompany secondary vascular differentiation (Iqbal, 1995).

IV.

ROOT AND SHOOT CAMBIA ARE ALIKE

Superficially, secondary growth in roots appears the same as it does in shoots, and for many years it has been almost an article of faith that root and shoot cambia were the same. However, in the absence of definitive information regarding the cell biology of root cambia, and in light of the differences in wood anatomy between roots and shoots of angiosperm trees (Patel, 1965), this view was difficult to sustain.

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Figure 4 Transmission electron micrograph of a transverse section of the taproot of horse chestnut showing a file of phloem ray cells. Key: n, nucleus; r, ray cambial cell; R 0 and R 00 , successive stages in differentiation of phloem ray cells; arrow indicates direction of differentiation. Scale bar ¼ 5
m.

To my knowledge, the only work on the cell biology of tree root cambia is the series of articles by Chaffey et al. (1996, 1997a–d, 1998a, 1999, 2000a,b), which examined the ultrastructure of, and cytoskeletal dynamics within taproots of Aesculus hippocastanum. Very little difference between the cambia of roots and shoots of horse chestnut was found in the ultrastructure of ray and cambial cells, in their seasonal cycle (Table 1), and in the behavior of their microtubule and microfilament cytoskeleton, both during the cambial seasonal cycle and during differentiation of secondary xylem and phloem. The major benefit of this revelation is that shoot and root cambia do indeed appear to be similar, and information derived from one can be integrated with that derived from the other. However, lest we be tempted to ignore root cambia completely, it should be stressed that so few taxa have been studied that to conclude that all shoot and root cambia are the same may be premature. Certainly, much more work is needed to cover the range of tree and wood types, which embraces angiosperms—gymnosperms, temperate—tropical, ring-porous—diffuseporous, storied—nonstoried cambia.

Table 1 Seasonal Changes in Ultrastructure of Fusiform and Ray Cambial Cells Within Taproots of Aesculus hippocastanum L. Feature Cell walls Nucleus1 Mitochondria Plastids Microtubules2 Microfilaments2 Vacuome Endoplasmic reticulum Dictyosomes Secretory vesicles Coated vesicles PCR/TGN6 Plasmalemma Oleosomes Microbodies Ribosomes7

Active cambium Thin, amorphous Single þþ þþ (little starch) Random3 Axial4 Single, large vacuole þþ (rough only) þþ (active) þþ þþ þ Undulating þ þ þþ

Dormant cambium Thick, lamellate Single þþ þþ (more starch) Oblique and helical3 Axial4 Dispersed, small vacuoles þþ (rough mainly, some smooth5 ) þþ (less active) þþ þðþÞ þ Undulating þþ þðþÞ þþ

Key: 1 Occasionally containing microtubule-like structures; 2 cortical elements; 3 endoplasmic microtubules particularly prominent in ray cells; 4 random, but rare in ray cells; 5 smooth endoplasmic reticulum only seen in fusiform cells; 6 partially coated reticulum/trans Golgi networklike structure (Chaffey et al., 1997d); 7 frequently in polysomal configurations; þ, þðþÞ, þþ, increasing abundance.

Secondary Growth

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Table 2 Comparison of Some of the Characteristics of Cells of the Secondary Vascular System in the Taproot of Aesculus hippocastanum Tissue/cell type Phloem Companion cell Sieve element Fiber Axial parenchyma Ray cell Cambium Ray cell Fusiform cell Xylem Fiber Axial parenchyma Vessel element Contact ray cell Isolation ray cell

Walla ;  lignified; type of elaboration

Widtha

Lengtha

Wider Wider Wider Wider Equald

Shorter Equal Longer Shorter Longere

Thicker, Thicker, Thicker, Thicker, Thicker,

N/A N/A

N/A N/A

Thin, nonlignified; pit fields only Thin, nonlignified; pit fields only

Wider Wider Wider

Longer Shorter Equal

Equald Equald

Longere Longere

Thicker, lignified; simple pits, obliquely angledc Thicker, lignified; simple pits, transversely angledc Thicker, lignified; bordered and contact pits, tertiary thickenings, simple perforations Thicker, lignified; contact pits, interray cell pits Thicker, lignified; simple pits, interray cell pits

nonlignified; pitted nonlignified; sieve areasb lignified; simple pits, obliquely angledc nonlignified; pitted nonlignified; pitted

Key: a Relative to precursor cambial cell; b at sieve plate and within lateral cell walls; c orientation of long axis of elliptical pit relative to cell’s long axis; d measured axially; e measured radially.

V.

CAMBIAL CELL DIVISION AND GROWTH

A.

The Arithmetic Cambium: Division and Multiplication

As a result of the combination of meristematic activity of the cambium and the growth of the daughter cells, the cambial cylinder moves outward. If radial growth were to continue unchecked, it is likely that the pressures so generated within the cambium and the phloem would result in these tissues being torn apart. Clearly, this does not happen; the radial expansion is somehow accommodated. The integrity of the SVS is maintained by at least three sites of cell division/growth in the radial plane, which together lead to an increase in girth. The main ‘‘pressure relief valve’’ is anticlinal (synonyms in the literature: radial, multiplicative) division of cambial initials to produce new cambial initials circumferentially, which subsequently grow and develop their own radial files of secondary vascular cells. In nonstoried cambia, such as those of horse chestnut and hybrid aspen, where the ends of adjacent cambial cells are at different levels, these anticlinal divisions vary from nearly transverse to nearly longitudinal, and are termed ‘‘pseudotransverse.’’ Because the division wall is offset from the vertical, both daugh-

ter cells are shorter than the mother cell. Thus, to increase the circumference of the cambial cylinder, not only do the daughters have to increase in diameter, but they also have to elongate to regain the original parental length. This contrasts with periclinal cell division, which is essentially vertical, longitudinal division that gives rise to two daughter cells of approximately equal length. In storied cambia, such as that of Robinia, where all fusiform cambial cells are the same length, anticlinal cell division is truly longitudinal and two equal-length daughters are produced, which only have to increase in diameter to achieve increase in cambial circumference. A second line of defense against unchecked radial expansion, but which is more pronounced in the stem, which increases in girth much more than the root, is so-called dilatation growth (e.g., Larson, 1994) of phloem rays. In hybrid aspen the typically uniseriate rays undergo anticlinal radial divisions and cell growth to broaden into multiseriate rays. Thirdly, the combination of anticlinal cell division in the cork cambium (Y. Waisel, personal communication, 2000) and cell growth also serve to relieve any buildup of pressure. The conditions that promote and sustain these anticlinal cell divisions are unknown, but it seems plausible that some sort of pressure/stretch-detection mechanism exists at the cellular level, which subsequently sets off an appropriate number of rounds of cell divi-

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sion/enlargement sufficient to dissipate the immediate danger. However, a further consideration is that in dilatation growth, parenchymatous phloem ray cells, which are differentiated cambial derivatives, can subsequently be encouraged to undergo cell division. How similar is this cell division to that which takes place within the cambium proper? Do the differentiated ray cells have to dedifferentiate to a meristematic state before they can undergo division? Is this cell division also influenced by auxin flow, as considered to be the case for the cambium (e.g., Aloni et al., 2000)? If so, how is the appropriate level/flow of auxin generated in this specialized region of the phloem? The presence of a radial gradient of auxin across the tissues of the SVS in stems of angiosperm and gymnosperm trees has recently been established (Sundberg et al., 2000). Extension of this study to a consideration of root cambia, dilatation growth, and the much understudied cork cambium, should prove quite rewarding. However, mere production of more initial cells by anticlinal cell division in the cambium is not enough. The right sort of cambial initial has to be produced. A certain ratio of fusiform:ray cells needs to be maintained for proper functioning of the SVS (Larson, 1994). To maintain that ratio it will be necessary to produce more ray cells. Ray cells can generally be produced in one, or all, of three ways. Transformative cell divisions can occur in which either a part, or the whole, of a fusiform cambial cell becomes subdivided, by anticlinal, transverse cell divisions, to produce ray initial(s) (e.g., Phillips, 1976). Thirdly, a most unequal lateral cell division of a fusiform cambial cell can occur whereby a ray initial is cut off from the side of the fusiform cell (e.g., Phillips, 1976). Understanding the conditions that promote the production of ray initials represents a considerable challenge. B.

Breaking the Rules

Of the two types of cambial initials, understanding cell division of the fusiform cells is the most problematic. They are long cells (165–329
m in horse chestnut root—Chaffey et al., 1999) but can be up to several millimeters long in conifers (Larson, 1994), whose periclinal or pseudotransverse divisions ‘‘do not obey the usual laws of cell division’’ (Cutter, 1975). For example, they violate Errera’s Law (Cutter, 1978), which states that cells divide by a wall of minimal surface area. As if that were not intriguing enough, they also appear to lack a preprophase band (PPB). The PPB is a circumferential aggregation of cortical microtubules (MTs) that appears to define both the plane of subse-

quent cell division and the site of attachment on the parental cell wall of the new division wall (e.g., Wick, 1991). However, ultrastructural study of cell division in stem cambia of Robinia (Farrar and Evert, 1997) has failed to identify a PPB in fusiform initials, as has my extensive search by immunofluorescent techniques for MTs in roots of horse chestnut and hybrid aspen. If the PPB performs the role ascribed to it, how do fusiform initials manage without it? What guides the advancing cell plate to the correct place on the parental cell wall? Indeed, is the cell plate ‘‘guided’’ in the usual sense? Ultrastructural study of cell division in fusiform cambial cells of Fraxinus (Goosen-de Roo et al., 1984) identified a previously unsuspected class of MTs, within the phragmosome, which advances before the phragmoplast. They suggested that these longitudinally oriented (i.e., parallel to the developing cell wall) MTs guide the growing cell plate to the ‘‘parental cell wall site previously marked by the preprophase band of microtubules’’ (Goosen-de Roo et al., 1984). However, my own immunofluorescent work, admittedly on different species and using a different visualization technique, has not demonstrated any MTs in this location in dividing fusiform cells. Perhaps the best we can say at present is that it is uncertain whether a PPB is present in such cells, or whether such phragmosomal MTs can perform the role suggested by Goosen-de Roo et al. Of course, it might be that a PPB is unnecessary and a wide variety of daughter cell lengths can be tolerated—ranging from those that result from near-transverse to those from near-longitudinal variants of pseudotransverse cell division. The system then would be reliant upon elongation growth of both daughter cells to reconstitute the original cell length, rather than the accurate placement of the division wall at a specific site on the parental wall. On this basis, it is proposed that a PPB is only essential to cells in situations where it is important to preserve a particular cell shape or to maintain specific cell-cell geometry. An example of this in the SVS could be the retention of the regular, radial symplasmic transport route, which is provided by the xylem and phloem rays. Consistent with this suggestion, we have identified a PPB in ray cambial cells by immunolocalization techniques. Nevertheless, it is necessary to establish in a more rigorous way whether a PPB really is absent from fusiform cells. What do we currently know about the cell biology of cambial cell division in the root? To date, cytoskeletal dynamics have only been examined during periclinal cell division and the transverse cell divisions that

Secondary Growth

generate axial phloem parenchyma cells in roots. Those studies, which have so far considered immunofluorescent localization of tubulin (for MTs), actin (for microfilaments—MFs), unconventional myosin VIII (‘‘myosin’’) (characterized by Reichelt et al., 1999) and callose, have revealed that karyokinesis is accompanied by development of a mitotic spindle, which contains tubulin, but in which neither myosin nor actin have yet been detected. During subsequent cytokinesis, both tubulin and actin are immunolocalized either side of the new division wall (Fig. 5a,b, see color insert), within the phragmoplast, whereas myosin and callose are found within the wall (Fig. 5c,d). Interestingly, cell walls at this very early stage of development are apparently unstained by Calcofluor (Chaffey, 2001d), indicating that they are almost devoid of cellulose, and in agreement with earlier reports of a callosic phase in cell wall development (Waterkeyn, 1967). Whether callose is made in situ, as is assumed for subsequent cellulose synthesis, or is transported there in secretory vesicles, is not yet known. As for cell division in other cell systems, in longitudinal section it is evident that the tubulin and actin are concentrated within the phragmosomes at the two ends of the centrifugally advancing cell plate. The MTs here, which lie at right angles to the plane of the developing cell wall (Fig. 6), can be viewed as providing guides for transport of secretory vesicles to the developing cell wall, as can the associated MFs. Once vesicles arrive at the phragmoplast, the dense MT-MF array could help to trap such vesicles there, ensuring that their contents are released at the correct place. Immunolocalization of actin, and possibly tubulin, is also evident along the nascent cell wall between these two phragmoplasts, as is myosin and callose within the wall. The actin and myosin can be envisaged as acting in concert here as a motile cytoskeletal system (e.g., Reichelt and Kendrick-Jones, 2000), which could translocate those dictyosome-derived secretory vesicles that arrive at the developed cell plate to the phragmoplasts, where they can contribute to the development of the nascent division wall. Soon after this stage, the callose-staining throughout the wall (Chaffey, 2001e) becomes lost and the cell walls stain strongly with Calcofluor, indicating the presence of cellulose. Whether callose stainability disappears because the callose is replaced, or is altered in some way so that its antigenicity is masked or lost, is not known. In line with current interest in the chemistry of cambial cell walls, and the enzymes that modify it (e.g., Follet-Gueye et al., 2000; Micheli et al., 2000),

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Figure 6 Transmission electron micrograph of a transverse section of the cambial zone of the taproot of horse chestnut showing a recently periclinally divided fusiform cambial cell undergoing cell wall formation. Key: rw, radial wall; tw, tangential wall; star, nascent division wall; arrows, microtubules. Scale bar ¼ 2:5
m.

further study of this phenomenon is clearly warranted. However, neither callose nor myosin is completely absent from the mature cell wall; both are retained at the pit fields, presumably in connection with the plasmodesmata at those sites (e.g., Radford et al., 1998; Radford and White, 1998). The association of callose with early stages of development of otherwise cellulosic structures, such as cambial division walls and the peripheries of bordered and contact pits, suggests that the deposition of this polysaccharide may be a necessary precursor to cellulose deposition. Perhaps the colocalization of callose and myosin at these sites is necessary to attract and/or retain and/or anchor the MTs or MFs that are related to further wall elaboration at those sites.

VI.

CYTOSKELETAL CONSIDERATIONS OF THE SECONDARY VASCULAR SYSTEM

Although many changes take place as cambial derivatives differentiate as secondary vascular cells (Table 2) (Iqbal, 1995; Chaffey, 2001b), alterations of their size,

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shape, and walls are among the most obvious. In view of the roles ascribed to cellulose microfibrils in influencing the direction of cell expansion (cf. Green, 1969), and to the cytoplasmic MTs in influencing the arrangement of those microfibrils within the cell wall (reviewed by Giddings and Staehelin, 1991), to date the cell walls and the cytoskeleton are the most studied aspects of root cambial cell biology. Using a combination of conventional transmission electron microscopy and immunofluorescence localization of the cytoskeletal proteins tubulin (for MTs) and actin (for MFs), the changes taking place to these cell components during secondary vascular differentiation have been well characterized in taproots of A. hippocastanum (Chaffey et al., 1996, 1997a–d, 1998a, 1999, 2000a,b; summarized for wood formation in this tree in Table 3). Our immunofluorescent work in progress has now extended that study to roots of hybrid aspen. A recent review (Barlow and Balus˘ ka, 2000) which concentrates on the primary plant body also deals in detail with the MT cytoskeleton in roots.

A.

Cambium

In active cambia, the cortical MTs of both ray and fusiform cambial cells are randomly organized, and frequently overlap. Although cambial cell wall structure has not specifically been examined in horse chestnut or in hybrid aspen, the literature shows that the primary walls of secondary xylem cells—which are derived from the cambial cell wall—contain disorganized cellulose microfibrils (e.g., Awano et al., 2000). Thus, the similarly randomly arranged MTs can be seen as circumstantial evidence for the view that MTs influence the orientation of microfibrils. What is not obvious, however, is the significance of the overlapping MTs. Do they accord with corresponding production of similarly overlapping cellulose microfibrils? Or do such images record the changeover from one MT/ microfibril orientation to another? Perhaps of more immediate significance is that the random arrangement of MTs and microfibrils in both cambial cell types potentially permits growth in any direction, i.e., isotropically. However, cambial derivatives grow anisotropically, but this takes place principally along one or both of two axes—longitudinal and radial—and the combination of the two varies quite

Table 3 Behavior of the Cortical Microtubule and Microfilament Components of the Cytoskeleton During the Cambial Seasonal Cycle and Xylogenesis in the Taproot of Aesculus hippocastanum Cell type Ray cambial (active) Ray cambial (dormant) Fusiform cambial (active) Fusiform cambial (dormant) Fiber (early stages—phase I) Fiber (middle stages—phase II) Fiber (maturity) Axial parenchyma (early stages) Axial parenchyma (maturity) Vessel element (early stages) Vessel element (middle stages) Vessel element (maturity) Contact ray (early stages) Contact ray (maturity) Isolation ray (early stages) Isolation ray (maturity)

Microtubules

Microfilaments

Random Parallel-helical Random Parallel-helical Parallel-helical, wavy Parallel-helical, dense Absent Parallel-helical Axial-helical Random Various3 Absent Random, rings5 Axial-helical Random Axial-helical

Random Random Axial Axial Axial1 Axial2 Absent Axial1 Axial2 Axial Axial2 , various3;4 Absent Axial, rings5 Axial2 Axial1 Axial2

Key: 1 Ellipse associated with developing simple pits; 2 branched, but overall net-axial orientation maintained; 3 rings at peripheries of bordered and contact pits, and perforation plate, and at aperture of developing bordered pit, parallel-aligned arrays associated with developing tertiary thickenings; 4 meshwork over perforation plate; 5 rings at periphery of developing contact pit.

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dramatically depending upon the cell-type. Thus, fibers grow considerably along both axes, sieve and vessel elements grow more radially than longitudinally, and ray cells grow almost solely radially. Further complications arise from the facts that ray cambial cells can ‘‘transform’’ into fusiform cambial cells (e.g., Larson, 1994), fusiform cambial cells can give rise to ray cells, daughters of pseudotransverse division of a fusiform initial must grow in both axes (although considerably more longitudinally) to regain the parental cell volume, and fusiform cambial cells can undergo transverse divisions to produce axial parenchyma cells. In other words, the randomness of the MTs and microfibrils within the cambial cells can be interpreted as an indication that these cells are as yet uncommitted to a particular cell fate. Potentially, each cambial derivative could follow one of several pathways of differentiation. Nevertheless, it is unusual to find random MTs in a cell whose shape is other than that of a sphere (cf. cultured tobacco protoplasts—Vissenberg et al., 2000b). However, as for the protoplast, which will subsequently develop new MT arrangements as it differentiates (Vissenberg et al., 2000b), the most likely explanation for the random MTs is the same in each case. By contrast, the prominent MFs within fusiform cambial cells are found in axially oriented bundles throughout the cambial seasonal cycle. In ray cambial cells, however, the MF bundles are much less common and apparently randomly oriented, although they too persist in these cells throughout cambial activity and dormancy. Nowhere in cambial cells have MFs been observed co-oriented with MTs. In both cambial cell types the MF bundles are implicated in cytoplasmic streaming during the active part of the cambial seasonal cycle (Chaffey et al., 2000a).

B.

Early Stages of Secondary Vascular Differentiation

As cambial derivatives differentiate, their MTs generally adopt new arrangements, and it is tempting to suggest that this may indicate commitment of a cambial derivative to a particular cell fate (e.g., Chaffey et al., 1997b). However, since the earliest stages of differentiation are more easily identified by the increase in cell diameter, and relocation of the nucleus to a tangential wall (e.g., Fig. 4; Chaffey et al., 1999), it is not yet possible to decide whether changes to the MT cytoskeleton are more cause or effect of the differentiation process.

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1.

Fibers

a.

Biphasic Fiber Growth

Although both normal wood and gelatinous fibers are found within roots and shoots of hybrid aspen, only the cytoskeletal dynamics of normal wood fibers have so far been studied in such roots. Accordingly, only normal wood fibers are considered further. Two phases of development of wood fibers are apparent. Phase I commences with the establishment of a parallel alignment of MTs, but although the individual MTs themselves appear wavy and may overlap, crosslinks between MTs and the plasmalemma have not been observed. It is suggested that this phase is concerned with cell growth, hence with production of primary cell wall, and takes place whilst the cells are within the enlargement zone of the secondary xylem. The succeeding phase II shows an increase in the number of MTs relative to phase I, but they are still present as a layer that is almost exclusively one MT deep. The MTs are all at the same angle within a cell (Fig. 7a, see color insert), and in places observed to be crosslinked to the plasmalemma, but not to each other. It is suggested that this phase occurs in the maturation zone when fibers have ceased enlargement, and are undergoing the main stage of cell wall thickening. Although crosslinks between MTs and the plasmalemma have so far been seen only in ultrastructural study of hybrid aspen stem material, a similar association between MTs and plasmalemma in horse chestnut root material is concluded from study of plasmolysed fibers. In such material, the parallel alignment of MTs is still seen, despite the fact that the plasmalemma has retracted from the cell wall. Presumably, these crosslinks act to hold the MTs in a specific orientation to each other, which thereby promotes a more ordered orientation to the cellulose microfibrils (or to other components of the cell wall). It is widely acknowledged that fibers, and other wood cells, have highly orderd microfibrils in the various S(econdary) wall layers. Presence of plasmalemmal crosslinked MTs in fibers during phase II of growth, but not during phase I or in fusiform cambial cells, can be related to this apparent need for a much more ordered secondary cell wall, the better to perform its strengthening role. By contrast, for a primary cell wall, which is laid down in a cell that is still growing, the emphasis is more on flexibility and the ability of the wall to accommodate further increase in cell volume. In such a case, a less ordered microfibril arrangement will perhaps be of more use, and may indeed be favoured by the presence of non-plasmalemmal-crosslinked MTs.

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If we accept that MT orientation has a role in determining the arrangement of cell wall microfibrils, then our observed variation in MT orientation from fiber to fiber, which is inferred to relate to successive stages of fiber differentiation, could be related to the deposition of different layers of the secondary cell wall (S1, S2, or S3), or to successive development of different lamellae within a single wall layer (Barnett et al., 1998). Clearly, further work is required to decide between these possibilities. It is noteworthy that development of phloem fibers appears to be similar to that of their xylem counterparts. Certainly, they undergo a growth phase that is the same as the phase II of xylem fibers (Fig. 7b, see color insert). However, what we don’t yet know is the full sequence of phloem fiber differentiation. This largely relates to the fact that more effort has been concentrated upon understanding xylogenesis in the SVS, but partly to the apparent inability to identify phloem fibers at earlier stages of their differentiation (e.g., Chaffey et al., 2000b). Parallels are also evident between development of xylem fibers and cotton ‘‘fibers’’ (which, strictly speaking, are seed coat trichomes). Detailed ultrastructural work by Seagull (1992) with in vitro grown cotton fibers has established that MTs are generally randomly oriented during fiber initiation and early elongation, reorienting to shallow pitched (near-transverse) helices as elongation and primary wall deposition continue. Finally they give way to steeply pitched (near longitudinal) MT helices during secondary wall deposition. Accompanying these changes were increases in MT length, number, proximity to the plasmalemma, and a decrease in variability of MT orientation. With the exception of a shallow helical phase, and in the absence of data regarding MT length, the similarity of cotton fiber and wood fiber MT dynamics and the associated phases of growth is remarkable. Although the economic importance of cotton means that research on this crop is more highly funded than that on wood fibers, in view of the similarity of the cell biology of growth between the two cell types, a bonus of the research on cotton is likely to be its relevance to a greater understanding of wood fiber biology. b. Bipolar Fiber Growth Over many years it has been established that wood fibers grow primarily, if not solely, at their tips (Larson, 1994). Although the mechanism of this tip growth is not yet known, it has been suggested that it may be facilitated by secretion of wall-degrading enzymes at the tips (Wenham and Cusick, 1975). The

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MFs retain their net axial orientation (Figs 7c,d) throughout all phases of fiber development. This is consistent with a role in supplying precursors or secretory vesicles, either via cyclosis or by directed transport (Boevink et al., 1998) of enzymes, to the growing tips of these cells. Support for this view has been provided by the polar distribution of expansin mRNAs found in putative xylem fiber cells in stems of Zinnia elegans (Im et al., 2000). Along with xyloglucan endotransglycosylases (Vissenberg et al., 2000a), expansins are a class of enzymes that are implicated in ‘‘wall-loosening’’ events (Cosgrove, 1998), such as are necessary to facilitate tip growth of xylem fibers. Citing work of Roberts and Uhnak (1998) using the in vitro Zinnia system, in which leaf mesophyll cells are induced to differentiate as tracheary elements, Im et al. (2000) proposed that longitudinally oriented MTs might play a role in the translocation of the mRNAs to the fiber tips. In planta, in cells of the SVS of angiosperm trees, longitudinally oriented MTs have only so far been seen in gelatinous fibers of hybrid aspen (see also ultrastructural studies in Populus (Mia, 1968; Fujita et al., 1974) and Salix (Robards and Kidwai, 1972), and the immunofluorescent studies of Fraxinus (Prodhan et al., 1995). It therefore seeems more likely that the axially (i.e., longitudinally) oriented MFs are a better candidate for any polar distribution of mRNAs that may be found in angiosperm trees. This interpretation also emphasizes the need for caution in making inferences about the natural system from an in vitro one. Is the growth of fibers really ‘‘tip growth?’’ How does it compare with that in the more usual tip-growing systems of fungal hyphae, pollen tubes, root hairs, and fern protonemata (Geitman and Emons, 2000; see also Chapter 5 by Ridge and Katsumi in this volume)? The straightforward answer is that wood fiber growth has not yet been studied in sufficient detail to permit a proper comparison with other tip-growing systems. However, some observations are pertinent. Wood fibers are single cells, both ends of which grow (i.e., they undergo bipolar growth—Chaffey et al., 1999), and whose MTs (Fig. 7a) and MFs appear to extend to the tips of the cell. Other such systems are unipolar and their apical regions usually have different MT and MF distributions from the rest of the cell. The growing tip of a wood fiber is long tapered and more varied in shape than the ‘‘hemisphere’’ of, e.g., a pollen tube, and may bifurcate (Chaffey et al., 1999). Wood fibers bear wall elaborations—pits—which are absent from fungal hyphae or root hairs. During early development, wood fibers are in contact with other wood

Secondary Growth

cells via plasmodesmata; all the other tip-growing systems are essentially isolated from other cells. The wall of fibers is probably much more structurally elaborate than that of most of the other systems, and is lignified. Taken together, wood fibers have a number of features that serve to distinguish them from the unipolar tip-growing systems. However, fibers are such an important economic cell type, e.g., for wood pulp, that further study of their mode of growth is warranted, and may even contribute to a greater understanding and appreciation of more conventional tip growth. c.

Pits in Fibers

The only deviation from the axial MF array in wood and phloem fibers is seen in the association of MFs with sites of simple pit formation in these cells (Fig. 7d), where ellipses of antiactin reactive material are found. This is similar to the situation in axial and isolation ray parenchyma in both Aesculus and hybrid aspen. It may be significant that these ellipses are not seen in isolation, but are usually attached to MF bundles. Such an association might indicate that the MF bundles act as guides for the transport of enzymes of cell wall modification, or precursors for cell wall synthesis, to the site of pit development. That no specific array of MTs has been seen at these sites implies that this cytoskeletal component is not directly involved with either the siting or the development of the pits. Furthermore, the absence of MTs from the pit membrane of simple pits—and all other pit types in the secondary xylem—which remain as regions of unthickened primary wall middle lamella, is consistent with the view that presence of MTs is required for secondary wall thickening to take place (Chaffey et al., 1999). d.

Waste Not, Want Not

Notwithstanding 50 years of ultrastructural study of the SVS, new discoveries continue to be made. A good example of this is the role of plasmatubules in wood cells. Plasmatubules (PTs) (Harris et al., 1982) are small-diameter, tubular evaginations of the plasmalemma found at sites where there is an inferred, but relatively short-term, high flux between the apoplasm and the symplasm (Harris and Chaffey, 1985, 1986). During ultrastructural studies of the SVS of Aesculus root, PTs were only seen in differentiating xylem cells (Chaffey et al., 1999; Chaffey, 2001e). The plasmalemma-bound symplasmic compartment of an individual differentiating xylem cell is surrounded by a large apoplasmic space, which consists of its own cell wall, the walls of other cells, and the lumina of fully

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differentiated fibers and vessel elements. The observed distribution of PTs can thus be related to their inferred role in symplasm–apoplasm exchange, facilitating the resorption of the products of xylem cell lysis, which might otherwise be lost, from the extracellular medium. In this way PTs act as an adjunct to the symplasmic intercellular fluxes within and between the longlived ray and axial parenchyma cells. This notion of PT-facilitated recycling receives experimental support from a study of the uptake of radioactively labeled lysine by dwarf mistletoe from its host (Coetzee and Fineran, 1989), in which PTs were implicated. Furthermore, absence of PTs from differentiating phloem cells is consistent with this suggestion for xylem, since here symplasmic routes between adjacent cells are maintained via plasmodesmata. However, even in situations within the phloem where such symplasmic continuity is lost, e.g., during the differentiation of bast fibers, PTs have been recorded (Sal’nikov et al., 1993). Elsewhere, PTs have been illustrated and discussed in the study of tracheid differentiation in Cryptomeria sp. (Takabe and Harada, 1986), and differentiating tracheary elements of Salix dasyclados (Sennerby-Forsse and Von Fircks, 1987). PTs are also evident in xylem fibers of Acer (Cronshaw, 1965) and Aesculus (Barnett, 1981). 2.

Vessel Elements

Vessel elements possess several distinct arrays of MTs and MFs in association with the numerous wall elaborations found in this cell type (Table 3); however, I shall only consider the development of the perforation. The perforation plate is a highly specialized region of the vessel element wall. It is an MT-free zone (Chaffey et al., 1999) that is devoid of callose (unpublished observations), and almost devoid of cellulose (Benayoun et al., 1981), and which neither secondarily thickens nor becomes lignified (Chaffey et al., 1999). Since removal of the perforation plate facilitates longdistance water transport throughout the tree, it has been much studied (Meylan and Butterfield, 1981; Butterfield, 1995), although the details of its formation and subsequent loss are not fully known. From a cytoskeletal point of view, we know that in roots (and shoots) of Aesculus, the periphery of the perforation plate is marked by a ring of both MTs and MFs, although in hybrid aspen, the MF ring appears to be absent, and a dense meshwork of MFs lies over the perforation plate itself. In the context of formation of the perforation, most significance is attached to the MF meshwork. In accordance with

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the view that enzymatic dissolution may play a role in the removal of the perforation plate (Benayoun, 1983), it is suggested that transport of the secretory vesicles containing the necessary enzymes to the perforation plates is facilitated by the net axial MF bundles within these cells. However, once there, the MF meshwork effectively traps them so that their lytic cargoes are released only at the perforation plate. Related to the development of the perforation, and to the more general consideration of how the various types of pits in vessel elements and other wood cells are sited, is the question of establishment of the ‘‘perforation domain.’’ Chemically, the perforation domain is characterized by the absence of cellulose, which elsewhere is a common component of the cell wall and which was apparently present within the precursor cambial cell wall. How is the cellulose removed from this specific location? How does the subsequent organization of MFs and/or MTs at the periphery of the domain come about? Is it due to the presence of particular MT- and/or MF-associated proteins at this site? If so, how do they get there? If one considers the tip of an axial cambial derivative, it can be likened to the two sides that form the apex of a steeply angled triangle. The two sides look the same, yet only one will go on to develop a perforation. What is different between these two sides of the cell tip? One way of approaching this question is to examine these cells with a wide range of antibodies against cell surface epitopes such as the John Innes monoclonal antibodies (JIMs) (Willats et al., 2000), looking for differences between and within the walls. Both cell wall and plasmalemmal epitopes should be considered, since it is not clear if the domain is a product of one or other or both of these cell compartments. 3. Multifaceted Microfilaments A net axial orientation of MFs is retained in the fusiform cambial cells and the axial components of the secondary xylem. However, there is a pronounced increase in the degree of branching of the MF network in axial xylem cells as cells differentiate from fusiform cambial cells into fibers or into vessel elements. Thus, there appears to be a correlation between the degree of radial growth of the cell and the extent of branching of the MF network. In keeping with the notion that axial MFs are related to tip-directed cell wall growth/elaboration/modification, it is suggested that the branched MF elements are involved with delivery of enzymes (such as expansins or xyloglucan endotransglycosylases) and/or wall precursors to specific sites on

Chaffey

the side walls. There it may be necessary to break intermolecular bonds, such as xyloglucan crosslinks (Fujino et al., 2000), so facilitating wall growth, or to permit wall elaboration such as development of simple pits in fiber cells. The degree of branching can be viewed as being at a maximum for the axial cell components in the case of transversely oriented MFs that are seen in vessel elements. Although MFs appear not to have a role in the change of orientation of MTs (Chaffey et al., 1997b), their putative involvement in formation of all pit types and perforations, and in directional cell expansion suggests that they have important roles in cell differentiation generally, and secondary vascular differentiation specifically. Generally, work on MFs lags considerably behind that on MTs, particularly in plant cells. It is hoped that studies such as the one reviewed here, and a recent book devoted to MFs in plants (Staiger et al., 2000), will improve awareness of this remarkable cytoskeletal component. In particular, given the responsiveness of MTs to plant hormones (Shibaoka, 1994; Balus˘ ka et al., 1999) and the role of both MTs (Chaffey, 2000; Funada, 2000) and plant hormones (Aloni et al., 2000) in wood formation, further studies of the effects of plant hormones on MFs is desirable. C.

Seasonal Cycle of Cambial Activity and Dormancy: A Cytoskeletal Angle

It is well known that cambial cells of the stems of temperate tree species undergo a seasonal cycle of activity and dormancy, which is paralleled by changes in cell wall thickness (Catesson, 1994). A similar seasonal cycle was found within root cambia of horse chestnut (Chaffey et al., 1998a). However, associated with the changes in cambial wall thickness was a previously unexpected cycle of MT reorientation. During cambial dormancy, when the cell walls are thickened, the MTs are arranged in a parallel-aligned helical orientation. When growth resumes and the walls become thin again, the random array of MTs reappears. These observations raise a number of questions. First, how do the MTs, which appear to persist throughout the life of the cambial cells, survive dormancy? Although it is widely reported in the literature that MTs are depolymerized by low temperatures, their presence in cambia of roots from frozen soil (Chaffey et al., 1998a) contradicts the view that MTs are absent from dormant cambia (Savidge, 1993). Are the MTs in both active and dormant cambial cells naturally resistant to cold depolymerization? Or are the MTs of dormant cambial cells comprized of a cold-resistant type

Secondary Growth

of tubulin, possibly an isoform of - or -tubulin, heterodimers of which constitute the MT, or a posttranslationally modified form of tubulin? Although our preliminary immunofluorescent examination shows presence of acetylated and tyrosinated tubulins in stem SVS material of horse chestnut and hybrid aspen, no similar studies have been undertaken with roots. What is the significance of the helical orientation of the MTs during dormancy? Certainly, there appears to be an association of parallel, helical MTs with wall thickening throughout the SVS. It is seen during differentiation of fibers, vessel elements, and phloem cells. Therefore, by comparison with secondary wall formation in those other cells of the SVS, and in keeping with the definition of a secondary wall as that which is laid down over an existing primary wall in a nongrowing cell, this ‘‘dormancy wall’’ of the cambium is considered a secondary wall (Chaffey et al., 1998a). Although it seems likely that the change of orientation of MTs from random to helical is related to the wall thickening during dormancy, whether this orientation of cytoplasmic MTs is mirrored by the orientation of cellulose microfibrils within the cell wall remains to be determined. It is tempting to suggest (Chaffey et al., 1998a) that the dormancy-associated wall differs somehow from the primary wall which surrounds active cambial cells, and that it is this difference that allows this dormancy wall to be lysed during spring cambial reactivation (Funada and Catesson, 1991), but prevents the ‘‘active cambial primary wall’’ from suffering a similar fate. However, once wall thickening has taken place, the MTs persist. Why? Do they serve other functions unrelated to wall-elaboration? For example, do they act as a protein store that can be subsequently degraded and reutilized? This suggestion is superficially attractive for horse chestnut, which, unlike hybrid aspen, appears not to store protein in the SVS. However, we do not know if the MTs are used in that way. What seems less questionable is that the partial wall lysis in springtime will have several effects: it will reduce a major physical constraint to cell expansion, and it will also produce oligosaccharides. These sugars could serve as substrates for energy production and synthesis of cell components, and/or act as solutes to raise cell turgor facilitating cell growth (Boldingh et al., 2000). It is also possible that oligosaccharins may be produced which can act as signaling molecules, which can have a number of roles in plant growth and development (Dumville and Fry, 2000). Indeed, the simple sugars produced generally as part of wall digestion

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could have profound roles in aspects of growth and differentiation far beyond their use as substrates (Sheen et al., 1999; Smeekens, 2000), and, potentially, far beyond the immediate vicinity of the cambium. In this regard it is worth recalling the role attributed to a ‘‘secreted factor,’’ possibly an oligosaccharide, in coordinating cell expansion and differentiation in the Zinnia mesophyll system (Roberts et al., 1997). Superficially, the enzymatic digestion that appears to accompany cambial reactivation has similarities with some aspects of the breaking of seed dormancy and fruit ripening. A more detailed comparison of all three phenomena is likely to provide further insights into cambial dormancy and spring activation. Another important question arises in the context of cambial dormancy. Should dormant cambial cells be considered cambial cells? By definition, the cambium is a meristematic tissue. Clearly, dormant cambium is not meristematic, nor does it look like active cambium. This consideration is more than a semantic nicety, since it raises the further question of whether a dormant cambial cells undergoes a more profound ‘‘transdifferentiation’’ to become meristematic again, rather than simply wall thinning and vacuolation. D.

Keeping It All Going

To maintain the meristematic activity of the cambium, and the growth and differentiation of its derivatives, input of materials is needed, both as respirable substrates to support the high energy demand of cell division and growth, and as structural raw materials. Although hydrolysis of reserve materials and cambial wall-lysis in situ may contribute to the cambium’s demand for substrates, it is assumed that the majority of the required material is ultimately derived from photosynthesis within the leaves. For stem cambia it is also likely that local bark—or even pith—photosynthesis can supplement this leaf supply of photosynthate. Of course, that is a luxury not available to roots. However, although it is well established that the axial pathway of photosynthate transport is within the sieve tubes of the phloem, and that the rays are implicated in phloem–xylem exchange (Van Bel, 1990), the pathway from sieve tube to cambium is less clear (Sauter, 2000; Van Bel and Ehlers, 2000). Recent examination of the cytoskeleton within the long-lived cells of the SVS, particularly the ray cells, has helped to shed some light on the problem of photosynthate transfer to the cambial sink (Chaffey and Barlow, 2001). At the final stages of xylem ray cell differentiation, when wall thickening and elaboration

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are complete, MFs and MTs are both bundled and netaxially oriented. At maturity, the MFs and MTs of axial xylem parenchyma cells are similarly oriented, parallel to the long cell axis. However, since the long axes of axial parenchyma and ray cells are at right angles to each other, so are the axes of orientation of the MT and MF cytoskeletons. Similarly, in mature phloem ray and axial parenchyma cells, although the MTs are more helically oriented (Fig. 8a,b, see color insert), a net-axial orientation of both MTs and MFs (Fig. 8c) is attained. Since cell differentiation has finished, and given that such cells live for many months (up to several years in the xylem), what is the role of MTs and MFs here? In view of the established importance of the long-lived ray cells as the symplasmic transport pathway between xylem and phloem (Van Bel, 1990) and their role in storage of reserve products (e.g., Sauter and Van Cleve, 1990; Ho¨ll, 2000), we have proposed involvement of both cytoskeletal components in intracellular transport (Chaffey and Barlow, 2001). Additionally, unconventional myosin VIII and callose (a marker for plasmodesmata) have been immunolocalized to the pit fields between adjacent cells within the rays of the phloem and xylem, and between ray cells and adjacent axial parenchyma. This apparent plasmodesmatal coupling of living parenchyma cells thus provides an extensive 3-dimensional symplasmic network, which extends radially from the central pith to outer bark, circumferentially within the axial parenchyma of the xylem and phloem, and axially via the sieve tubes of the phloem, which ramifies throughout the tree. The apparent colocalization of actin and myosin to the plasmodesmata of the membranes of the pits has led us to speculate further that intercellular transport, between ray and axial parenchyma cells, may be mediated by plasmodesmata whose diameter can be increased/reduced via relaxation/contraction of an actomyosin complex. It is thus envisaged that this network is responsible for not only transport of photosynthate and other materials around the tree, but also, by appropriate degrees of opening/closing of plasmodesmata, the establishment of symplasmically isolated domains (Lucas et al., 1993), which may have importance for coordination of developmental events throughout the tree. This view of the radial-axial symplasmic pathway also provides an additional role for the dilatation growth that can occur within phloem rays. Seen in a transverse section of the root (or shoot), dilatation growth appears like a triangle, with its base toward

Chaffey

the outside and its apex pointing toward the center of the organ. In that way it can be considered to act like a funnel, receiving photosynthate and other materials at its base and sides, from the sieve elements and associated cells, and channeling it toward the bottom of the funnel to enter the uniseriate ray that then extends from the phloem into the cambial zone. The cytoskeleton-facilitated solute transport is envisaged to break down as the helical/axial arrays of MTs and MFs are lost within the cells of the cambial zone, i.e., in the cells where the transported material is required to fuel respiration and cell growth. One of the attractions of this idea is that transport can be bidirectional, from either the phloem side or the xylem side. Potentially, cambial reactivation in spring can be ‘‘kick-started’’ by release of reserves from the axial and ray parenchyma cells of the xylem; thereafter, leaf photosynthate can be channeled to the cambium via the phloem route.

VII.

QUO VADIMUS?

Where do we go from here? Since ‘‘here’’ is a state of comparative ignorance about the cell—and molecular—biology of tree root cambia, further study in almost any direction will generate useful information. In nature, roots grow in the soil. That makes them messy and more difficult to access than aerial portions of the plant. Trees are rather large, and working with their roots is even more problematical than with smaller plants. It often takes a long time to dig up a tree, or even a sapling, clean the roots, and subsequently fix them, etc. It is quite likely that cell changes will have taken place during that time, compromising the interpretation of results. It is thus no wonder that aerial portions of plants are a more attractive alternative. Can we make root work a little easier? Probably, yes (see also Chapter 19 by Waisel in this volume). However, as with so many other aspects of modern plant biology, it is likely that we will have to turn to Arabidopsis to help us. Amongst the many virtues of this crucifer are that its roots can be grown in agar or another, similar nonsoil medium, permitting more ready harvest, etc. Nevertheless, how can a small, ephemeral weed help us to understand secondary growth? Although most work with Arabidopsis is currently directed at its primary plant body, under appropriate conditions it can undergo quite substantial secondary thickening and wood formation, particularly in its hypocotyl and root (e.g., Dolan and Roberts, 1995; Regan et al., 1999; Zhao et al., 2000).

Secondary Growth

Ultrastructurally, the cambial cells of Arabidopsis appear like those of angiosperm tree roots and shoots, and the fibers and vessel elements of its wood are morphologically similar to those of hybrid aspen and horse chestnut. Undeniably, there are limits to the relevance of wood formation in Arabidopsis to the process in trees. However, there is a lot of fundamental work that can be done, and Arabidopsis will have an important role to play in future development of hypotheses, which can subsequently be tested in the tree (Chaffey, 1999a). Ultimately, however, one would like techniques of investigation that will enable us to study the living processes in situ, in vivo and in planta. Unfortunately, although recent advances have been made in applying the techniques of modern cell and molecular biology to the cambium (Savidge et al., 2000; Chaffey, 2001a), until such work can be carried out with living cambia it will be necessary to use alternative models. In that regard, using intact roots of Arabidopsis to understand secondary growth of trees has obvious attractions over the other main model system for studying tracheary element formation, isolated Zinnia mesophyll cells (McCann et al., 2000). In this regard, we should recall that xylogenesis has long been viewed as a model system for the understanding of plant cell differentiation, and that one of the earliest model systems proposed was roots (Torrey et al., 1971). What tree species should be used as the ‘‘model species’’? Most of the fundamental groundwork on the ultrastructure and cytoskeletal dynamism of the root SVS has been carried out in Aesculus hippocastanum (Chaffey, 2000). However, although it is a majestic tree when in flower, it is more sensible to concentrate upon poplar/aspen and other members of the genus Populus, which by general consensus are the model angiosperm trees (Chaffey, 1999a, 2001c). As reviewed here, hybrid aspen and horse chestnut roots appear similar, certainly in respect of their cytoskeletal dynamics during wood formation. It is to be hoped that concentration on a single tree in this way will generate the critical mass of interest that has been so successful with Arabidopsis.

VIII.

THE NEXT 10 YEARS

These are exciting times for biology. The near-daily advent of technical advances means that previously intractable systems, such as the SVS, are beginning to give up their secrets, and we can now dare to ask

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questions we couldn’t even think of 20 or 10 years ago. What advances might we expect in cambial cell biology in the next 10 years? Just as any developmental phenomenon can be studied at many different levels, consistent with the questions being asked and the availability of the necessary techniques, for the SVS we can identify anatomical, biochemical, and molecular-genetic levels (Chaffey, 2001b). We have barely begun to make inroads into the basics of the anatomical-biochemical (i.e., ‘‘cellular’’) changes that accompany secondary vascular differentiation, and there is much still to do in this area. Further documentation of that will help to establish hypotheses that can be tested, no doubt in tandem with Arabidopsis (e.g., Chaffey et al., 1998b; Chaffey, 1999b). However, the most dramatic developments are expected at the molecular-genetic level of inquiry, particularly with regard to differentiation of individual cell types and to the overall control of the SVS. Several groups are interested in identifying cambialspecific genes or genes that are involved in differentiation of secondary vascular cells. For example, Sterky et al. (1998) have recently generated a library of nearly 5700 expressed sequence tags (ESTs) from the woodforming tissues of hybrid aspen. Using a subtractive technique, Bossinger and Leitch (2000) have produced a library that is enriched in gene fragments that are differentially expressed in woody tissues, from in vitro–grown stem explants of Eucalyptus globulus. Over the next few years, we shall see many more such DNA libraries created, with a corresponding increase in the exploitation of the opportunities that they will provide for investigating the SVS. Understanding the ‘‘master genes’’ that control pathways of differentiation, such as xylogenesis in angiosperm trees, has already begun (Hertzberg and Olsson, 1998). All developmental processes depend for their success on the regulated and coordinated expression of many genes. In turn, those genes are ‘‘controlled’’ at the gene transcription level, which is mediated via transcription factors. Newman and Campbell (2000) recently presented evidence from loblolly pine that MYB proteins could act as transcription factors regulating some aspects of xylogenesis. Rather satisfyingly, they have identified orthologs of their pine genes in Arabidopsis, paving the way for use of both systems in tandem to understand this aspect of cambial activity. Homeobox genes (genes coding for transcription factors, which are involved in ‘‘switching on’’ other genes that control particular pathways of development), particularly in regard to vascular differentia-

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tion, are being investigated in Arabidopsis (Baima et al., 2000). One such gene, ATHB-2, is of considerable interest because elevated levels interfere with auxin response pathways and appear to affect secondary thickening by acting as a negative regulator of gene expression (Steindler et al., 1999). This work highlights another area where our understanding is likely to increase dramatically in the near future, the molecular basis for the involvement of plant hormones in influencing the activity of the SVS. IX.

CONCLUDING COMMENTS

Although it is probably more satisfying to have a complete story, the action-packed thriller, which ends with a ‘‘to be continued . . .’’ is arguably the more exciting. I believe that The Story of Cambium falls into that latter category. We currently know just enough to make the subject interesting, but the questions that it raises, and their relevance to the really big issues of modern biology—cell differentiation and determination—should make us all keen to tackle the next instalment. ACKNOWLEDGMENTS I thank John Barnett and Peter Barlow for their patience in trying to teach me ‘‘trees and cambium’’ and ‘‘roots and cytoskeleton,’’ respectively, and both for lively discussions of those subjects over the past few years. I also thank Peter and the editors for their helpful comments on an earlier version of this chapter. The IACR receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the U.K. REFERENCES Aloni R, Feigenbaum P, Kalev N, Rozovsky S. 2000. Hormonal control of vascular differentiation in plants: the physiological basis of cambium ontogeny and xylem evolution. In: Savidge R, Barnett JR, Napier R, eds. Cell and Molecular Biology of Wood Formation. Oxford, UK: Bios Scientific Publishers, pp 223–236. Awano T, Takabe K, Fujita M, Daniel G. 2000. Deposition of glucuronoxylans on the secondary cell wall of Japanese beech as observed by immuno-scanning electron microscopy. Protoplasma 212:72–79. Bailey IW. 1952. Biological processes in the formation of wood. Science 115:255–259. Baima S, Tomassi M, Matteucci A, Altamura MM, Ruberti I, Morelli G, 2000. Role of the ATHB-8 gene in xylem

formation. In: Savidge R. Barnett JR, Napier R, eds. Cell and Molecular Biology of Wood Formation. Oxford, UK: Bios Scientific Publishers, pp 445–455. Balus˘ ka F, Volkmann D, Barlow PW. 1999. Hormone-cytoskeleton interactions in plant cells. In: Hooykaas PJJ, Hall MA, Libenga KR, eds. Biochemistry and Molecular Biology of Plant Hormones. Amsterdam; Elsevier, pp 363–390. Barlow PW, Balus˘ ka F. 2000. Cytoskeletal perspectives on root growth and morphogenesis. Annu Rev Plant Physiol Plant Mol Biol 51:289–322. Barnett JR. 1981. Secondary xylem cell development. In: Barnett JR, ed. Xylem Cell Development. Tunbridge Wells, UK: Castle House Publications, pp 47–95. Barnett JR., Chaffey NJ, Barlow PW. 1998. Cortical microtubules and microfibril angle. In: Butterfield BG, ed. Microfibril Angle in Wood. Christchurch, New Zealand: IAWA/IUFRO, pp 253–271. Benayoun J. 1983. A cytochemical study of cell wall hydrolysis in the secondary xylem of poplar (Populus italica Moench). Ann Bot 52:189–200. Benayoun J, Catesson AM, Czaninski Y. 1981. A cytochemical study of differentiation and breakdown of vessel end walls. Ann Bot 47:687–698. Boldingh H, Smith GS, Klages K. 2000. Seasonal concentration so non-structural carbohydrates of five Actinidia species in fruit, leaf and fine root tissue. Ann Bot 85:469–476. Boevink P, Oparka K, Cruz SS, Martin B, Betteridge A, Hawes C. 1998. Stacks on tracks: the plant Golgi apparatus traffics on an actin/ER network. Plant J 15:441–447. Bossinger G, Leitch MA. 2000. Isolation of cambium-specific genes from Eucalyptus globulus Labill. In: Savidge R, Barnett JR, Napier R, eds. Cell and Molecular Biology of Wood Formation. Oxford, UK: Bios Scientific Publishers, pp 203–207. Braun HJ. 1984. The significance of the accessory tissues of the hydrosystem for osmotic water shifting as the second principle of water ascent, with some thoughts concerning the evolution of trees. IAWA Bull ns, 5:274– 294. Butterfield BG. 1995. Vessel element differentiation. In: Iqbal M, ed. The Cambial Derivatives. Berlin; Gebru¨der Borntraeger, pp 93–106. Catesson A-M. 1974. Cambial cells. In: Robards AW, ed. Dynamic Aspects of Plant Ultrastructure. London; McGraw-Hill, pp 358–390. Catesson A-M. 1994. Cambial ultrastructure and biochemistry: changes in relation to vascular tissue differentiation and the seasonal cycle. Int J Plant Sci 155:251– 261. Chaffey NJ. 1999a. Cambium: old challenges—new opportunities. Trees 13:138–151. Chaffey NJ. 1999b. Wood formation in forest trees: from Arabidopsis to Zinnia. Trends Plant Sci 4:203–204.

Secondary Growth Chaffey NJ. 2000. Cytoskeleton, cell walls and cambium: new insights into secondary xylem differentiation. In: Savidge R, Barnett JR, Napier R, eds. Cell and Molecular Biology of Wood Formation. Oxford, UK: Bios Scientific Publishers, pp 31–42. Chaffey NJ, ed. 2001a. Wood Formation in Trees: Cell and Molecular Biology Techniques. Amsterdam; Harwood Academic Publishers (in press). Chaffey NJ. 2001b. Introduction. In: Chaffey NJ, ed. Wood Formation in Trees: Cell and Molecular Biology Techniques. Amsterdam; Harwood Academic Publishers (in press). Chaffey NJ. 2001c. An introduction to the problems of working with trees. In: Chaffey NJ, ed. Wood Formation in Trees: Cell and Molecular Biology Techniques. Amsterdam; Harwood Academic Publishers (in press). Chaffey NJ. 2001d. Wood microscopical techniques. In: Chaffey NJ, ed. Wood Formation in Trees: Cell and Molecular Biology Techniques. Amsterdam; Harwood Academic Publishers (in press). Chaffey NJ. 2001e. Conventional (chemical-fixation) transmission electron microscopy and cytochemistry of angiosperm trees. In: Chaffey NJ, ed. Wood Formation in Trees: Cell and Molecular Biology Techniques. Amsterdam; Harwood Academic Publishers (in press). Chaffey NJ, Barlow PW. 2000. Actin in the secondary vascular system of woody plants. In: Staiger C, Balus˘ ka F, Volkmann D, Barlow PW, eds. Actin: A Dynamic Framework for Multiple Plant Cell Functions. Dordrecht, Netherlands: Kluwer, pp 587–600. Chaffey N, Barlow PW. 2001. The cytoskeleton facilitates a three-dimensional symplasmic continuum in the longlived ray and axial parenchyma cells of angiosperm trees. Planta (in press). Chaffey NJ, Barlow PW, Barnett JR. 1996. Microtubular cytoskeleton of vascular cambium and its derivatives in roots of Aesculus hippocastanum L. (Hippocastanaceae). In: Donaldson LA, Butterfield BG, Singh PA, Whitehouse LJ, eds. Recent Advances in Wood Anatomy. Rotorua, NZ: New Zealand Forest Research Institute, pp 171–183. Chaffey NJ, Barlow PW, Barnett JR. 1997a. Formation of bordered pits in secondary xylem vessel elements of Aesculus hippocastanum L.: an electron and immunofluorescent microscope study. Protoplasma 197:64–75. Chaffey NJ, Barlow PW, Barnett JR. 1997b. Microtubules rearrange during differentiation of vascular cambial derivatives, microfilaments do not. Trees 11:333-341. Chaffey NJ, Barnett JR, Barlow PW. 1997c. Visualization of the cytoskeleton within the secondary vascular system of hardwood species. J Microsc 187:77–84. Chaffey NJ, Barnett JR, Barlow PW. 1997d. Endomembranes, cell walls and cytoskeleton: aspects of the biology of the vascular cambium of Aesculus hippocastanum L. Int J Plant Sci 158:97–109.

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7 The Kinematics of Primary Growth Wendy Kuhn Silk University of California, Davis, California

I.

KINEMATIC ANALYSES ILLUMINATE ROOT FUNCTION

shape change in fluids and continuua, are particularly relevant. (A distinction is made between kinematics and ‘‘dynamics,’’ the study of the forces and energies that produce the observed motions and shape changes.) Applications of kinematic analyses include consideration of the ‘‘material’’ aspects of root development to elucidate spatial–temporal relationships in growing tissue. Growth itself can be characterized in terms of relative elemental rates, analogous to strain rates used in continuum mechanics. In structural studies, kinematic expressions can be used to find basic physical relationships among growth rates, cell division rates, anatomy, and morphology. In studies of mineral nutrition, kinematic expressions allow us to find local nutrient deposition rates in expanding tissue from spatial concentration patterns. In ecological studies, the growth strain rate field provides a quantitative characterization of effects of environmental variation on growth. And some basic ideas from growth kinematics, including the use of a moving reference frame attached to the growing root tip, allow us to understand the relationship between the growth zone and its rhizosphere.

Symbols used to represent kinematic variables and parameters: x z r t X xðX,tÞ Z zðZ,tÞ v vx and vr u L g @V=V@t vf ; k; n

Longitudinal distance from root tip Longitudinal distance from soil surface Radial distance from root center Time Particle found x mm from root tip at initial time Location, in co-moving reference frame, at time t of particle X Particle found z mm from soil surface at initial time Location, in stationary reference frame, at time t of particle Z Rate of displacement from root tip Components of growth velocity in axial and radial directions Rate of displacement from soil surface Length of root Relative elemental growth rate (REG rate or growth strain rate) Local relative rate of increase in volume Parameters to fit velocity field v(x) to logistic function

II.

The concepts and numerical methods from fluid dynamics and continuum mechanics are powerful tools for solving problems in root physiology. The methods of ‘‘kinematics,’’ i.e., the study of motion or

ROOT DEVELOPMENT HAS SPATIAL AND MATERIAL ASPECTS

As one looks back from the root cap and root apex one sees a zone of cell division (with slow growth), then a zone of rapid expansion that overlaps a zone of tissue 113

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differentiation. Differentiation may be further characterized by the locations of specialized cells. For example, under constant environmental conditions in a particular species the formation of phloem, root hairs, and xylem may occur at identifiable distances from the root apex. In botany textbooks the pattern along the root axis is shown to represent a developmental gradient, and the root tip is described in terms of anatomically and functionally distinct zones. However, to understand the root we must also recognize that the developmental zones are formed from a procession of tissue elements each of which first grows slowly while producing new cell walls, then elongates rapidly, and then further differentiates specialized cells. When the root is viewed over time, a marked cohort of cells appears to flow from the root tip through the standing developmental pattern. The existence of a form that is unchanging (or slowly changing) in time, and that is composed of elements that are experiencing rapid change, is reminiscent of fluid structures. And indeed, allusions to rivers, fountains, and the wakes of boats appear often in classical botanical literature. Root growth zones, like boat wakes, are composed of changing ‘‘material elements’’—cells, analogous to water droplets in the boat wake. To understand root structure it is important to appreciate both spatial patterns, as observable in photographs, and the ‘‘material’’ aspects or real elements, i.e., the properties of the cells that comprise the spatial pattern as they move through it. This distinction between spatial (or site-specific) and material (or cell particle-specific) descriptions of developmental variables is central to the understanding of plant form and to clarifying the relationships among spatial and temporal patterns in developing roots (Green, 1976; Silk and Erickson, 1979; Gandar, 1980, 1983; Silk, 1989, 1992).

III.

ROOT GROWTH CAN BE VISUALIZED IN STATIONARY AND MOVING REFERENCE FRAMES

In the discussion of material specifications the emphasis was on the continuing flow of cells away from the root tip. From another point of view, it is the pattern that moves through a constantly extending sequence of cells. If we consider spatial coordinates attached to the soil surface, each cell is displaced only a small distance from where it is formed. But the various zones move from one place to another down the growing root, keeping a constant distance behind the moving tip. This view of root organization arises when we use a

stationary reference frame and is useful, for instance, for understanding the relationships between the developing root surface and the stationary soil particles that surround it. In contrast, the use of a ‘‘comoving’’ reference frame attached to the root tip allows us to find the many steady (time-invariant) or quasi-steady patterns that characterize root development. Historically, botanists have observed that primary extension growth is confined to apical regions of roots and shoots. Marks placed far from the apex do not separate from each other, although they are found progressively farther from the growing apex. One way of specifying growth is to plot the positions of cellular particles, or applied marks, versus time (Fig. 1). The resulting ‘‘pathline’’ (Gandar, 1983) or ‘‘growth trajectory’’ (Silk and Wagner, 1980; Hejnowicz, 1984) is a material specification of growth, because material particles are followed. If distance is measured from the apex, the slope of the growth trajectory increases monotonically from small values near the apex to a constant value at the base of the growth zone. To characterize the growth pattern, a family of curves can be obtained to show growth trajectories of particles at different initial positions (Fig. 1). The relationships between the growth trajectories in the stationary and comoving reference frames can be understood with the use of some formalisms from continuum mechanics (Silk, 1989). To connect the stationary and comoving reference frames, we can use two different variables for distance: Let x denote the distance from the root tip, and z denote the distance from the soil surface (or any other stationary reference origin). To distinguish properties associated with moving cellular particles from properties associated with spatial locations (instantaneously occupied by a particular particle), we can use capital letters for the material properties and lowercase letters for spatial properties. In particular, let X denote the material or real cellular particle located initially at location x0 ¼ X. Then xðX,tÞ represents at time t the spatial location occupied by particle found initially at the location X. This is the growth trajectory or particle pathline followed over time by the cellular particle, specified by its initial distance from the root tip. The expression zðX,tÞ represents the position of the same particle in the stationary reference frame. We can see that xðX,tÞ þ zðX,tÞ equals the length of the root at time t. Formally, for the material particle X it is easily seen that zðX,tÞ þ xðX,tÞ ¼ LðtÞ where LðtÞ represents root length at time t.

ð1Þ

Kinematics of Primary Growth

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Figure 1 Growth trajectories of cellular particles in the maize root, shown in two reference frames. The root has a growth zone 12 mm long and extends initially (at time 0) 12 mm below the soil surface. The stationary reference frame is indicated with dashed lines showing zðZ,t) where z is distance from the soil surface of cellular particles initially located, respectively, at 10, 10.5, 11, and 11.5 mm from the soil surface. These particles are also initially located, respectively, at 2.0, 1.5, 1.0, and 0.5 mm from the root tip. On the graph the material particle located initially z ¼ 10 and x ¼ 2 is displaced from the soil surface as shown by the lowest dashed line in the figure and also displaced from the root tip as shown by the highest solid line on the figure. With time each particle decelerates away from the soil surface to a constant depth in the soil and also accelerates to a constant rate of displacement from the root tip.

A similar relationship can be found to connect the growth velocities in the two reference frames. Let v represent the velocity of displacement from the tip, and u represent the velocity of displacement from the soil surface. Then vðX,tÞ þ uðX,tÞ ¼ @L=@t

ð2Þ

where the capital letters are used to denote velocities associated with the material particle. Equation (2) says the rate at which a particle moves away from the soil

surface plus the rate at which it moves away from the apex equals the elongation rate of the root, i.e., the time rate of change of root length. If instantaneous point-particle interchangeability is invoked, equation (2) also implies that at time t the approximately steady field vðxÞ can be used to compute uðx,tÞ as the difference between the overall elongation rate and velocity in the moving reference frame. These uðx,tÞ can then be assigned to the appropriate z values via equation (1). The characteristics of the velocity field (Erickson and Sax, 1956) are apparent in Fig. 2. The velocity

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Figure 2 The growth velocity field, shown in two reference frames. When plotted as a function of x, distance from the root tip, displacement velocities are time invariant (upper figure). That is, rate of displacement, from either the tip or the base of the root, is constant at a particular location. When plotted in a stationary reference frame, the velocity field is shifted to the right over time (lower figure).

of displacement from the root tip increases monotonically with position, because progressively more expanding tissue is found between the origin and progressively more basal locations (Fig. 2, solid line). At the base of the growth zone, where expansion is no longer occurring, the growth velocity acquires a uniform value equal to the elongation rate of the organ. In

our experiments, in the maize primary root growing at 28 C, individual particles accelerate to a final growth velocity (rate of displacement from the apex) of 3:2 mm h1 . Velocity of displacement from the soil surface is minimum at the base of the root (Fig. 2, dashed lines). Thus the uðxÞ and uðzÞ fields give us a physically intuitive picture of the growth velocity, since we know

Kinematics of Primary Growth

the root tip moves most rapidly away from the soil surface while the basal regions of the root are not being displaced. With time the curves of vðzÞ and uðzÞ are shifted to the right (Fig. 2, bottom), while uðxÞ and vðxÞ remain unchanged (Fig. 2, top). It is the time invariance of vðxÞ that makes it so useful. Graphs of growth trajectories can be constructed in the moving and stationary reference frames by integrating particle velocity over time, as shown in Fig. 1. In the moving reference frame we see that a cellular particle accelerates through the growth zone to achieve a constant velocity of displacement from the tip. As long as the root continues to grow, the particle will increase its distance from the tip with time. In the stationary reference frame, cellular particles decelerate to zero velocity of displacement. The final location of the particle depends on its initial location; particles initially close to the root tip move deeper into the soil than particles initially farther from the tip. The growth trajectories of the comoving reference frame show quantitatively how the ‘‘stationary pattern’’ on the root axis must be occupied by a changing population of cells that flows away from the tip. And the growth trajectories in the stationary reference frame show how the ‘‘moving pattern’’ on the root axis is maintained a constant distance from the root tip. IV.

A.

ROOT GROWTH IS QUANTITATIVELY DESCRIBED IN TERMS OF PARTICLE TRAJECTORIES, VELOCITIES, AND RELATIVE ELEMENTAL GROWTH RATES (STRAIN RATES) Specification of Axial Growth in One Dimension

Contemporary growth analysis emphasizes the relationships among growth trajectories, growth velocities, and relative elemental growth rates. We have seen that the growth trajectory provides a material specification of growth; a family of curves represents the displacements of the many cellular particles during the tissue expansion. The growth trajectory can be used as a space–time map. Tabulated values of position and time can be used to infer the time course for any developmental variable with a known spatial distribution along the root axis (Silk and Erickson, 1979). The velocity field is a spatial, or Eulerian, specification of growth because it involves the movements of many particles found instantaneously at different locations. The velocity field can be calculated from shortterm marking experiments. It is used in many impor-

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tant physiological applications, including calculations of biosynthesis rates, cell production rates, and material derivatives in growing tissue (Silk and Erickson, 1979; Gandar, 1980). For the physiologist the most useful growth descriptor is probably the relative elemental growth (REG) rate (Erickson and Sax, 1956), called the growth strain rate by engineers. In one dimension the REG rate, g, is the velocity gradient; mathematically, g ¼ @v=@x ¼ @v=@z. The REG rate is also approximated by the local relative growth rate of a segment of initial length L, g
L=L
t. The local relative growth rate approaches the REG rate as
L and
t become small. The REG rate field gives a quantitative description of the location and magnitude of growth within the root. In our experiments in a maize root elongating at 3:2 mm h1 , for example, growth occurs within the region extending 12 mm from the tip (Fig. 3, top curves). The REG rate maximum, 0:45 h1 , is found 4–6 mm from the apex. The REG rate profile gives a quantitative characterization of growth and is thus ideal to show the effect of environmental variation on growth. The REG rate also appears in theoretical studies, including the basis for axial curvature (Silk and Erickson, 1978) and growth sustaining water potential (Silk and Wagner, 1980). It provides the spatial pattern for determining the ultrastructural control of growth (Sugimoto et al., 2000). B.

Radial Growth Rates

A comprehensive analysis of growth including bending and twisting may involve the use of natural coordinate systems and be quite complex (Hejnowicz, 1984; Silk, 1989; Jirasek et al., 2000). Simpler analyses can be made for the many cases in which the root is radially symmetrical, has steady morphology and cell size distribution, and grows without bending or twisting. In such cases a two-dimensional analysis in cylindrical coordinates can provide a comprehensive description of the growth. We can use the coordinate system (x; r), where x is longitudinal distance from the root tip, and r is radial distance from the root center. The components of growth velocity can be given as vx and vr . As described in the section on axial growth, the pattern of vx can be determined with an axial marking experiment. If root morphology is steady, the maximum radial growth velocity (at the root surface) can be expressed in terms of the spatial pattern of root radius and longitudinal growth velocity. That is, at the root surface vr ¼ vx @r=@x. However, to find the spatial pattern of radial expansion, we need to know the spatial

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increase in cell widths at two longitudinal positions and dividing by the time required for displacement between the two positions. The longitudinal component of the growth strain rate @vx =@x can be obtained as usual with an axial marking experiment, and a circumferential relative growth rate is given by vr =r. Note that even in the absence of twist the local, relative growth rate in volume, V, includes the circumferential as well as the radial strain rates: @V=V@t ¼ @vx =@x þ @vr =@r þ vr =r

Figure 3 REG rate fields fit to an asymmetric logistic function for maize roots grown at different temperatures (upper figure) and soil water potentials (lower figure). (From Pahlavanian and Silk, 1988; Sharp et al., 1988; and Morris and Silk, 1992.)

distribution of vr along the root radius. This can be difficult to obtain noninvasively. For the case of steady cell size distributions we can infer the radial growth velocity distribution by comparing cell widths in the same file at different longitudinal positions. For this simple case we see that a longitudinal marking experiment can be combined with a determination of the profile of cell widths at different distances from the tip to obtain the growth strain rates in three dimensions. Now vr ¼ vx @r=@x, where r is the radial location of the cell file at position x. A radial growth rate @vr =@r can be approximated by measuring the relative

ð3Þ

Relatively few two-dimensional analyses of root growth can be found in the literature. Hejnowicz and colleagues (1984, 1993) pioneered the use of curvilinear coordinates to show relationships among growth trajectories and isovelocity curves within root apices. Barlow et al. (1991) characterized the effects of a mutation on components of the growth rate in tomato roots. Water stress has been shown to affect radial growth rates differently from longitudinal growth rates in the root meristem (Sharp et al., 1988; Van der Weele et al., 2000). The osmotic adjustment under water stress involves a decrease in radial growth rates, related to an overall decrease in osmoticum deposition rates (Sharp et al., 1990; but see Ober and Sharp, 1994). These studies directed attention to the control of growth anisotropy. Baskin and Bivens (1995) found that different toxins affect longitudinal and radial growth rates in Arabidopsis roots in different ways. And studying the ultrastructural basis for the different effects of water stress on the radial and longitudinal components of the growth rates, Baskin and Sharp and colleagues (Liang et al., 1997; Baskin et al., 1999) showed that the degree of growth anisotropy in maize roots was not correlated with the degree of alignment of either microtubules or microfibrils. The growth analyses combined with spatial patterns of ultrastructural detail have suggested the need for a new paradigm to replace the multinet theory of cell expansion.

V.

FLAWS IN EXPERIMENTAL DESIGN AND INTERPRETATION CAN OCCUR IN THE STUDY OF ROOT GROWTH ZONES

Physiological research on growing roots is tricky. Physiologists are trained in chemistry where the most commonly held assumptions are what Paul Green called ‘‘the assumptions of the well-stirred beaker.’’ In the famous Michaelis-Menton model for enzyme kinetics, for example, spatial homogeneity and tem-

Kinematics of Primary Growth

poral variation are assumed. In the root growth zone, by contrast, the simplest possible set of assumptions includes spatial variation (the presence of the developmental gradient) and temporal constancy (if the apex is chosen as origin of the coordinate system). Experiments on growing tissue must be designed to take account of cellular displacements during growth. A.

Assessing Environmental Effects

A naı¨ ve, but not uncommon, experimental design is to subject the root tip to an environmental perturbation, such as immersion in a toxic chemical. After a day or two the treated and control roots are compared. To make physiologically meaningful comparisons, the fates of treated cells should be mapped so that the properties of the tissue generated during the treatment can be compared to the tissue generated in the absence of the treatment. Thus, good experimental design requires that growth trajectories as well as spatial patterns in developmental variables should be determined in studies of growing parts of roots. Assessment of the effect of environmental variation on root properties must include identification of the tissue produced during the environmental perturbation. B.

Designing a Meaningful Growth Analysis

Marking experiments to obtain growth velocities and REG rates also need some attention to find meaningful spatial and temporal scales. A single marking experiment, with proper spatial and temporal resolution, can be used to infer the growth trajectory (by following identifiable particles on the root surface) and the REG rate profile (by differentiation of particle velocity with respect to position). To determine the REG rate profile, the marking experiment must be designed with spatial resolution sufficient to evaluate the REG rate at perhaps 10 locations within the growth zone. Adequate temporal resolution requires that marks not move too far from their initial positions during the observation period (Erickson and Silk, 1980; Silk, 1984). As longer time intervals are used, then the calculated REG rates become progressively overestimated, and the length of the growth zone becomes badly underestimated. If the calculated local relative growth rate is assigned to the final midpoint of a segment rather than the initial apical location, then the observation period can be somewhat longer, but the magnitude of the REG rate becomes progressively underestimated (Peters and Bernstein, 1997). A good rule of thumb is that

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< 20% of the length of the growth zone should be displaced past the boundary of the growth zone during the observation period. Then, whether assigned to initial, midpoint, or final locations during the growth analysis, the local relative growth rates will approximate the instantaneous values of the REG rate. To determine whether growth is steady, growth trajectories or velocity profiles should be evaluated at different times. If growth is steady, then many developmental regularities occur: successively formed growth trajectories can be superimposed, and velocity and REG rate profiles coincide for observations made at different times. If growth is known to be steady, then a single determination of the growth velocity field can be integrated (in a special way, following a particle as it moves through the growth zone) to obtain the growth trajectory. However, a growth trajectory obtained by numerical integration of the growth velocity will be subject to some uncertainty. Experimental error in measured growth velocities near the root tip becomes amplified by the integration process through the growth zone. This is a reflection of a physical variability inherent in the growth process. In fact, small differences in growth velocity near the root tip do result in large differences—for instance, in final segment length as a root segment is displaced through the growth zone. This has led to some controversies in interpretation of effects of genetic and hormonal variation on growth patterns (Peters et al., 1999). C.

Measurement of Local Biosynthesis and Uptake Rates in Growing Tissue

A related artifact involves the design of radioactive labeling experiments to measure biosynthesis or incorporation rates. In designing and interpreting radioactive labeling experiments, one confronts the difficulty that the experiment is not instantaneous. Some time is required for penetration of label into the tissue, and in this time period the cells initially at one location will be displaced. The problem can be illustrated by considering the deposition rate profile calculated using continuity equations for uronide incorporation into cell walls (Fig. 4 from Silk et al., 1984). In the roots of this study, a cell located initially at 4 mm from the tip will be located 1 h later at 5.2 mm, where the incorporation rate is quite different. In a labeling experiment, as the incubation period increases, the amount of label found at a location is less likely to be an accurate representation of the local deposition rate; the cells at a given location, having arrived from

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growing roots. Thus, in labeling experiments, as in growth analysis, it is important that the cells at a particular location remain near the attributed sites during the time interval for the observation of change.

VI.

Figure 4 The apparent incorporation rate of uronide into the growth zone of the maize root for different times of incubation in radioactive label. Top curve (asterisks) shows rates calculated using a continuity equation with data on uronide content and growth velocity. If this curve is assumed to be the true, instantaneous rate of uronide incorporation, then incubation in label for 15 min, 2 h, and 5 h would produce the set of progressively flatter curves. To use labeling experiments to localize uptake patterns within the growth zone, the investigator must choose incubation times short enough to minimize the growth displacements. (From Silk et al., 1984.)

progressively more apical sites, will present an integration of the incorporation rates associated with the different positions traversed en route. A computer simulation of the deposition process reveals that for these fast-growing roots, only incubation periods < 30 min will reveal an accurate profile of the local deposition rates. Before 2 h of incubation, the important characteristics of the deposition rate profile are lost: the peak at 3 mm is not readily observable, and the low values at the basal end of the growth zone are shown erroneously large. After 5 h of incubation in radioactive label, the deposition rate profile would appear almost flat. In light of published observations that 3–6 h is required for good penetration of polysaccharide precursor into the corn root, it can be concluded that it is not possible to obtain incorporation rate profiles with radioactive label supplied to rapidly

EFFECTS OF ENVIRONMENTAL AND GENETIC VARIATION ON GROWTH HAVE BEEN QUANTIFIED WITH REG RATES

It has long been recognized that the REG rate field can be used to describe quantitatively the spatial pattern of root growth within the root (Goodwin and Stepka, 1945; Erickson and Sax, 1956). Thus the REG rate field provides the basic phenomenology for the physiological understanding of growth. By now, many kinematic studies have established the effects on the growth rate distribution of environmental factors, including temperature (Pahlavanian and Silk, 1988), water stress (Sharp et al., 1988; Van der Weele et al., 2000), irradiance (Muller et al., 1998), salinity (Zhong and La¨uchli, 1993), and ambient pH (Peters and Felle, 1999; Walter et al., 2000). REG rate profiles have also been used to quantify growth patterns at different developmental stages (Beemster and Baskin, 1998). Most recently, with the explosion of work on mutant plants, the REG rate field has been used to infer the mechanisms for genetic control of growth (Sugimoto et al., 2000; Beemster and Baskin, 2000). A number of different spatial patterns and corresponding physiological mechanisms have been found to underlie environmentally induced variation in root elongation rates. An interesting contrast was observed between the effects of water availability and temperature. Pahlavanian and Silk (1988) concluded that within a certain range, temperature affected the rates of developmental processes, including REG rates, without much affecting the coordination of other processes. In maize roots growing at particular temperatures between 19 C and 29 C, there were similar patterns of cell length. Furthermore, the length of the growth zone appeared invariant although root elongation rate varied threefold over this range of incubation temperatures. In contrast, Sharp et al. (1988) found that as water stress increased, the length of the growth zone became shorter, while the magnitude of the REG rates was conserved in the apical parts of the growth zone. These effects were summarized by Morris and Silk (1992) who fitted a flexible, asymmetric logistic function to the primary data sets. The velocity fields were well fit in the spatial domain using the function

Kinematics of Primary Growth

vðxÞ ¼ 

vf 1 þ ekðxx0 Þ

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leagues (e.g., Ober and Sharp, 1994;) revealed that the maintenance of the REG rate during water stress depended on increased levels of abscisic acid, promoting increased deposition rates of proline as an osmoticum. The abscisic acid acts by decreasing ethylene production under water stress (Spollen et al., 2000).

ð4Þ

1=n

where vðxÞ represents the velocity at position x, vf is the velocity at the base of the growth zone, and x0 is chosen as that spatial position satisfying ½vðx0 Þn ¼

vnf 2

ð5Þ

VII.

The effects of temperature and water stress were displayed by plotting the longitudinal REG rate field, @v=@x against x (Fig. 3). Then the environmental effects were summarized by tabulating values of the root elongation rate, vf ; the parameter k, a measure of the spread of the velocity curve along the x-axis; the parameter n, related to the position of the inflection point of the REG rate curve; and the maximum REG rate for the different environmental conditions (Table 1). For the chosen range of temperatures and soil water potential, environmental variation produced similar changes in overall root elongation rate for the two factors. However, water stress and temperature affected the spatial growth patterns in different ways. Confirming the conclusions of the empirical studies, the fitting parameters indicate that temperature does not much affect the spread of the velocity or longitudinal REG rate curves, while increasing water stress causes a progressive shortening of the growth zone and a basal displacement of the point of maximum REG rate. The maximum longitudinal REG rate changed in proportion to incubation temperature but was not much affected by water stress. Thus, the physiology of the responses to the two environmental factors must be different. Subsequent work by Sharp and col-

KINEMATIC ANALYSES REVEAL INTERACTIONS BETWEEN GROWTH ZONES AND THEIR RHIZOSPHERES*

Symbols used to represent variables and parameters in model for pH of rhizosphere: F a pH1 bHS DHS D r t 0; t r 0; r z0 z V L R

Radial flux of H þ (from the root surface) Root radius Initial soil pH Soil buffering capacity Soil acidity diffusion coefficient Diffusivity of H þ Gradient operator Time Radial distance from root center Longitudinal distance from soil surface Longitudinal distance from root tip Growth velocity Distance parameter Rhizosphere or Pe´clet number

The ability of the root to change the pH of the soil in its immediate vicinity affects the uptake of both beneficial nutrients and phytotoxic metals, as described in Chapter 33 by Gerendas and Ratcliffe in this volume. Thus, an understanding of root-induced pH patterns in the rhizosphere is central to the study of plant nutri-

Table 1 Effects of Incubation Temperature and Soil Water Potential on the Growth Velocity of the Primary Maize Root Temperature ( C) 29 24 19 16 29 29 29

Water potential (MPa)

Elongation rate (mm h1 ) vf

k

n

Maximum REG rate ðh1 Þ

0:03 0:03 0:03 0:03 0:20 0:81 1:60

3.14 2.12 1.62 1.18 2.04 1.58 1.13

0.55 0.53 0.58 0.55 1.06 1.25 1.52

0.83 0.80 0.75 0.68 1.66 1.66 2.19

0.46 0.30 0.25 0.18 0.45 0.41 0.32

The parameters gave the best fit of the velocity field to an asymmetric, logistic function. Source: Morris and Silk (1988). *Section VII is adapted from Kim et al., 1999, and Nichol and Silk, in press, with permission from Blackwell Science Ltd.

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tion and to the physiology of root growth. This is recognized in many studies of genetic variation in capacity to modify the pH of the rhizosphere (e.g., Gollany and Schumacher, 1993; Yan et al., 1996; Degenhardt et al., 1998). Most of the models of root–rhizosphere interactions consider the mature part of the root, since the largest proportion of root tissue is no longer growing. However, recent work recognizes that many of the physiologically important uptake processes occur in the soil surrounding the meristem and growth zone (e.g., Henriksen et al., 1992; Jaillard et al., 1996; Felle, 1998). Many of the ideas from growth kinematics, including the importance of a moving reference frame attached to the root tip and the recognition of spatial variation within the growth zone, can be used to extend our understanding of the rhizosphere of the growth zone. Recently, we have used growth kinematics in a mathematical model to predict pH in the rhizosphere of a growth zone (Kim et al., 1999).

A.

Classical Theory and Modifications Needed to Understand Growth Zones

Classically, a diffusion theory has been used to predict the plant-induced pH fields in the rhizosphere (Nye, 1981, and references therein). Pointing out that a charge balance is axiomatic, Nye hypothesized that the root would release protons if more cations than anions are absorbed from the soil. Assuming the hydrogen ions would diffuse according to a modified Fick’s Law, Nye used an analytic solution for the diffusion equation with flux over the surface of a cylinder to find rhizosphere pH as a function of distance, r (from the root surface), and time, t:     aF 2:25DHS t pH ¼ pH1  ln ð6Þ 2bHS DHS r2 where F is the flux of Hþ (from the root surface); a is root radius; pH1 is the initial soil pH; bHS is soil buffering capacity; and DHS is the soil acidity diffusion coefficient. The existing theory is powerful in explaining how hydrogen ion fluxes associated with root metabolism affect soil properties. However, it is one-dimensional and assumes constant, spatially uniform Hþ flux. Thus, while it works well for the rhizosphere around mature root tissue, it needs modification to be used for growth zones. This is because growing tissue is characterized by spatial patterns that correspond to changes in developmental stage. Primary growth is

localized in small (cm-scale) zones at the tips of roots. The pH at the surface of growth zones has often been observed to be as much as 2 pH units higher or lower than neighboring mature root tissue (e.g., Weisenseel et al., 1979; Ha¨ussling et al., 1985; Gollany and Schumacher, 1993; Taylor and Bloom, 1998). Thus axial gradients in ion deposition rate and proton flux should be resolved on the scale of millimeters within the growth zone. In Kim et al. (1999), several conceptual modifications are made to adapt the Nye theory to growing tissue. The Hþ flux is plotted as a function of distance from the root tip. In this reference frame the pattern of Hþ flux can be taken to be steady (independent of time) if environmental conditions are held constant. (The soil next to the root above the growth zone is strongly affected by transpiration. However, since there is no functional xylem in the growth zone, we hypothesize transpiration has only indirect effects on the rhizosphere of the growth zone.) Thus, in the plantbased reference frame the Hþ flux is steady (like the classical theory) but nonuniform (in contrast to the classical theory). The root tip is propelled at a constant velocity of, say, 2 mm h1 through bulk soil. From the point of view of a soil particle next to the root, the Hþ fluxes corresponding to the different root locations will be encountered in a predictable sequence. The fixed soil particle will absorb the flux associated with a neighboring root element. Since the root tip is moving 2 mm h–1 downward, every half-hour the flux into the soil particle will be from a root element that is located 1 mm farther from the root tip. Thus, we can simplify the problem by imagining a stationary root, surrounded by a moving soil medium that is flowing 2 mm h1 upward. Solving for the pH field around the growth zone involves following a slice of soil as it ‘‘moves’’ away from the tip upward, keeping track of the history of the radial pH profile and updating the Hþ flux over time as the soil encounters the older tissue elements. B.

The Convection Diffusion Model

To derive our model, we began with the diffusion model (as described by Nye, 1981).      @ Hþ ð7Þ ¼ r: Dr Hþ 0 @t In this equation t 0 is time, D is the diffusion coefficient, and r is the gradient operator. (In this general formulation, in terms of proton flux, the diffusion coefficient,

Kinematics of Primary Growth

123

D, is determined empirically from the measured diffusion of protons in soil or other growth substrates of interest and includes the soil buffering capacity.) Assuming that the solution is radially symmetric, equation (7) can be reduced to the following axisymmetric diffusion equation, written in cylindrical coordinates;    þ     þ  @ Hþ @H 1 @ @ 0@ H þ 0 D ð8Þ ¼ 0 0 Dr r @r @z @t 0 @r 0 @z 0 where r 0 is the radial distance from the center line of the root, and z 0 is the vertical distance from the soil surface. If the diffusion coefficient, D, is constant then equation (8) can be rewritten as "        # þ @ Hþ @2 Hþ @2 Hþ 1@ H ¼D 0 þ þ ð9Þ r @r 0 @t 0 @z 02 @r 0 2 To take into account the root growth rate, we pick our reference frame to be stationary relative to the tip of the root. In other words, our reference frame moves with the root growth zone at the growth rate of the tip of the root. In this reference frame, the soil would appear to move around the root just as a person on a moving glass bottom boat would observe, in looking at the water through the glass, that the water is moving around the boat. As described in Section III, this transformation is familiar to those who do root growth analysis. Mathematically, in this reference frame we have the following coordinate transformation: t ¼ t 0;

r ¼ r 0;

z ¼ z 0  Vt

ð10Þ

where V is the growth rate (and direction) of the tip of the root. Here we assume that the root only grows in the axial direction, z 0 . So our model, taking into account the motion of the growth of the root, is given by; @½Hþ  @½Hþ  ¼V @t @z " # 1 @½Hþ  @2 ½Hþ  @2 ½Hþ  þ þ þD r @r @r2 @z2

ð11Þ

The solution to equation (11) can be characterized by a dimensionless number, R ¼ VL D , called the rhizosphere number, analogous to the Pe´clet number of transport theory (Tennekes and Lumley, 1994). The variable L represents a characteristic length scale; for most roots L can be taken to be 1 mm. Physically, large R implies convection (i.e., root elongation) is more important than diffusion in dispersing the protons; small R implies that diffusion is relatively more important. Dimensional analysis reveals that the rhizosphere

number can be used to predict the extent of the rhizosphere of the growth zone and the time required for it to become steady. C.

Radial pH Patterns in the Rhizosphere of the Growth Zone

Our convection diffusion model predicts that after several hours of growth in soil a steady pH pattern will surround the moving root tip, and that acidification of soil pH will be detectable only within 1 mm from the root surface (Kim et al., 1999). A second prediction is that after just 50 min of incubation an agar medium, the root will acidify the surrounding substrate to a distance of > 5 mm. The wider zone of root influence in agar is due to the 2000-fold increase in proton diffusivity in agar relative to soil. The time to achieve a steady-state pH pattern around the tip of a root growing in agar or aqueous solution is on the order of 1 day. These predictions were obtained by solving equation (11) using our empirical data for diffusivities in sandy soil (Nichol and Silk, in press) and Felle’s data (Felle, 1998) for proton flux across roots growing in solution culture. Recently we made some empirical tests of the published predictions of the convection diffusion model (Nichol and Silk, in press). We could not make a precise comparison between our measured pH fields and the predictions of our model, because our boundary conditions (initial substrate pH and proton flux across the root surface) were different from those in Felle’s experiments. However, we were able to test the robust conclusions for rhizosphere dimensions. We measured pH at different radial distances from the root surface to test these predictions of the theory (Table 2). The sandy soil was found to be acidified at r ¼ 0:2 mm but not at r ¼ 1, 3, or 5 mm from the root surface at the locations z = 4 and 10 mm behind the root tip. In contrast, the agar was acidified at all measured radial locations, r = 0.2, 1, 3, and 5 mm from the root surface. Thus, at least qualitatively, the empirically determined dimensions of the rhizosphere in the different substrates support the theoretical predictions of the convection diffusion model. In summary, to study pH in the rhizosphere of growth zones we needed to modify the classical model to include the growth rate and a spatially varying flux of Hþ ions from the root surface. Use of a moving reference frame attached to the root tip simplified the problem. We retained the idea that diffusion is the main process for the distribution of Hþ in the rhizosphere. Our major conclusion from preliminary

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Silk

Table 2 Empirical Verification of Rhizosphere Dimensions in Different Substrates (initial bulk substrate pH
6:2) Soil pH measurements after 18 h Radial distance from surface (mm) Axial distance from tip (mm)

4 10

0:2a 5.43b 5.74b

(SD) (0.23) (0.14)

0–2 6.17 6.20

(SD) (0.12) (0.10)

2–4 6.31 6.11

(SD) (0.19) (0.31)

4–6 6.10 6.13

(SD) (0.14) (0.16)

0 10

5.44b 5.64b

(0.15) (0.09)

5.62c 5.75

(0.14) (0.17)

5.79c 5.84c

(0.11) (0.08)

5.82c 5.92c

(0.11) (0.09)

Agar pH measurements after 50 min

a

pH measurements in substrate directly behind the growing root. Sign test shows rhizosphere is more acidic than the bulk soil (P < :01). c Friedmann test shows an influence of the root on the pH of the substrate (P < :05). Source: Nichol and Silk, in press. b

microprobe measurements, as well as from the numerical simulations, is that the inclusion of the root growth rate leads to a steady (time invariant) pH pattern on the root surface and in the rhizosphere of the root growth zone. In contrast, the stationary soil particles change in pH with time, as the root tissue next to a particular soil element is located progressively farther from the moving root tip. D.

Implications of the Convection Diffusion Model for Plant Growth and Nutrition

For the soil, the effect of a buffered growth zone is transient. This is because the growth zone moves rapidly—on the time scale of hours to a day—through a soil layer. However, for the growth zone the pH can be constant during the time required for many populations of cells to be displaced through the structure. This conservation of surface pH in the growth zone is probably important for the physiology of the root extension. It may also have an important effect on nutrition of the growing tissue, as suggested in Chapter 36 by Neumann and Ro¨mheld on rhizosphere chemistry. A branched rooting system extends, manufacturing a collection of locally acidified (or alkalinized) microenvironments that surround the tips as they penetrate to new locations. The conceptual framework for our recently published convection diffusion model draws attention to the inadequacy of the classical ideas for predicting properties of the rhizosphere around the growth zone. The soil adjacent to the mature part of the root experiences continuing proton flux, so that, according to the classical model, pH of the rhizosphere will change logarithmically with time (Nye, 1981). However, unlike the nongrowing part of the root, the growth zone, located at the moving root tip, continu-

ally changes its position with respect to the stationary soil particles. Furthermore, the expansion zone is extremely active as a sink for nutrient deposition, so that fluxes of cations and anions are an order of magnitude higher in the growth zone than in the mature part of the plant (compare Mengel and Barber, 1974, to Silk et al., 1986). Thus even on a time scale of hours, the growth zone can cause changes in the chemistry of the adjacent soil. The convection diffusion model is of general interest. For any substance that is secreted or taken up by growing tissue and that moves down a concentration gradient in the soil, the model can be used to characterize the spatial and temporal distribution patterns within the rhizosphere. Thus, from an ecological perspective, our model gives insight into many interactions between moving growth zones and the soil. Solution of the model equations revealed the possibility for steady patterns to exist in the rhizosphere of the growth zone, if the soil is homogeneous (Kim et al., 1999). This leads to the insight that as it moves through successive soil layers the root tip can surround itself with a constant pH field. However, this paradigm needs to be tempered by the realization that proton and bicarbonate fluxes across the root surface may be strongly affected by environmental conditions, especially soil physical and chemical properties. Recent work has already shown that the axial pH pattern on the root surface depends on external pH if the root is grown in solution culture (Peters and Felle, 1999). Preliminary work in our laboratory suggests that proton fluxes across the surface of roots grown in solution culture may differ in magnitude from those for roots grown in soil. Dimensional analysis indicates that the results of the model are robust in predicting a larger rhizosphere and longer time dependence in agar (or solution culture) relative to soil (Kim et al., 1999),

Kinematics of Primary Growth

and these predictions have been empirically verified (Nichol and Silk, in press). However, it may not be possible to solve the convection diffusion model for one growth medium, and scale the results according to the proton diffusivity of other media to obtain an accurate description of the pH field in particular soils. Quantitative analysis of the important problem of rhizosphere chemistry around the root tip will require both an understanding of the model relating flux to pH field and detailed empirical studies to characterize the proton flux patterns in different soils. The technology for measurement of pH gradients on the millimeter scale is now available, but work remains to show the interactions between rhizosphere pH and the nutrition of the growth zone and to find the effects of important environmental variables such as soil moisture content, mechanical impedance, and nutrient availability.

VIII.

TECHNOLOGY IS AVAILABLE TO MAKE KINEMATIC ANALYSES FASTER AND SIMPLER

Kinematic analysis of plant growth, involving analysis of particle trajectories, velocities and relative elemental growth rates, is facilitated by the technology of automated image analysis. This decade has seen the commercial availability of digital cameras that can be interfaced with high-speed computers with gigabytes of memory. Flexible software packages can now be developed to replace the laborious digitizing techniques of the last century (Schmundt et al., 1998). This should streamline kinematic studies and lead to an extensive database quantifying genetic, developmental, and environmental effects on growth. A comprehensive database of the growth patterns will provide a sound basis for the understanding of the physiology of root growth.

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125 tubules and cellulose microfibrils. Plant Physiol 119:681–692. Beemster GTS, Baskin TI. 1998. Analysis of cell division and elongation underlying the developmental acceleration of root growth in Arabidopsis thaliana. Plant Physiol 116:1515–1526. Beemster GTS, Baskin TI. 2000. STUNTED PLANT1 mediates effects of cytokinin, but not of auxin, on cell division and expansion in the root of Arabidopsis. Plant Physiol 124:1718–1727. Degenhardt J, Larsen PB, Howell SH, Kochian LV. 1998. Aluminum resistance in the Arabidopsis mutant alr-104 is caused by an aluminum-induced increase in rhizosphere pH. Plant Physiol 117:19–27. Erickson RO, Sax KB. 1956. Elemental growth rate of the primary root of Zea mays. Proc Am Phil Soc 100:487– 498. Erickson RO, Silk WK. 1980. The kinematics of plant growth. Sci Am 242:134–151. Felle HH. 1998. The apoplastic pH of the Zea mays root cortex as measured with pH-sensitive microelectrodes: aspects of regulation. J Exp Bot 49:987–995. Gandar P. 1980. The analysis of growth and cell production in root apices. Bot Gaz 141:131–138. Gandar P. 1983. Growth in root apices. I . The kinematic description of growth. Bot Gaz 144:1–10. Gijsman AJ. 1990. Rhizosphere pH along different root zones of Douglas-fir (Pseudotsuga menziesii), as affected by source of nitrogen. Plant Soil 124:161–167. Gollany HT, Schumacher TE. 1993. Combined use of colorimetric and microelectrode methods for evaluating rhizosphere pH. Plant Soil 154:151–159. Goodwin RH, Stepka W. 1945. Growth and differentiation in the root tip of Phleum pratense. Am J Bot 43:36–46. Green P. 1976. Growth and cell pattern formation on an axis. Critique of concepts, terminology and modes of study. Bot Gaz 137:187–202. Ha¨ussling M, Leisen E, Marschner H, Ro¨mheld V. 1985. An improved method for nondestructive measurements of the pH at the root–soil interface (rhizosphere). J Plant Physiol 117:371–375. Hejnowicz Z. 1984. Trajectories of principal directions of growth, natural coordinate system in growing plant organ. Acta Soc Bot Polon 53:29–42. Hejnowicz Z, Karczewski J. 1993. Modeling of meristematic growth of root apices in a natural coordinate system. Am J Bot 80:309–315. Henriksen GH, Raman DR, Walker LP, Spanswick RM. 1992. Measurement of net fluxes of ammonium and nitrate at the surface of barley roots using ion-selective microelectrodes. 2. Patterns of uptake along the root axis and evaluation of the microelectrode flux estimation technique. Plant Physiol 99:734–747. Jaillard B, Ruiz L, Arvieu J-C. 1996. pH mapping in transparent gel using color indicator videodensitometry. Plant Soil 183:85.

126 Jirasek C, Prusinkiewicz P, Moulia B. 2000. Integrating biomechanics into developmental plant models expressed using L-systems. In: Spatz H-C, Speck T, eds. Plant Biomechanics. Stuttgart, Germany: Georg Thieme Verlag, pp 615–624. Kim TK, Silk WK, Cheer AY. 1999. A mathematical model for pH patterns in the rhizospheres of growth zones. Plant Cell Environ 22:1527–1538. Liang BM, Sharp RE, Baskin TI. 1997. Regulation of growth anisotropy in well-watered and water-stressed maize roots. I. Spatial distribution of longitudinal, radial, and tangential expansion rates. Plant Physiol 115:101–111. Morris AK, Silk WK. 1992. Use of a flexible logistic function to describe axial growth of plants. Bull Math Biol 54:1069–1081. Mengel DB, Barber, SA. 1974. Rate of nutrient uptake per unit of corn under field conditions. Agron J 70:695– 698. Muller B, Stosser M, Tardieu F. 1998. Spatial distributions of tissue expansion and cell division rates are related to irradiance and to sugar content in the growing zone of maize roots. Plant Cell Environ 21:149–158. Nichol SA, Silk WK. Empirical evidence for a convectiondiffusion model for pH patterns in the rhizospheres of root tips. Plant Cell Environ (in press). Nye P. 1981. Changes of pH across the rhizosphere induced by roots. Plant Soil 61:7–26. Pahlavanian A, Silk WK. 1988. Effect of temperature on spatial and temporal aspects of growth in the primary maize root. Plant Physiol 87:529–532. Ober ES, Sharp RE. 1994. Proline accumulation in maize (Zea mays L.) primary roots at low water potentials. I. Requirement for increased levels of abscisic acid. Plant Physiol 105:981–987. Peters WS, Bernstein N. 1997. The determination of relative elemental growth rate profiles from segmental growth rates: a methodological evaluation. Plant Physiol 113:1395–1404. Peters WS, Felle HH. 1999. The correlation of profiles of surface pH and elongation growth in maize roots. Plant Physiol 121:905–912. Peters WS, Fricke W, Chandler PM. 1999. XET-related genes and growth kinematics in barley leaves. Plant Cell Environ 22:331–332. Sacks MM, Silk WK, Burman P. 1997. Effect of water stress on cortical cell division rates within the apical meristem of primary roots of maize. Plant Physiol 114:519– 527. Schmundt D, Stitt M, Jahne B, Schurr U. 1998. Quantitative analysis of the local rates of growth of dicot leaves at a high temporal and spatial resolution, using image sequence analysis. Plant Journal 16:505–514. Sharp RE, Silk WK, Hsiao TC. 1988. Growth of the maize primary root at low water potentials. Plant Physiol 87:50–57.

Silk Sharp RE, Hsiao TC, Silk WK. 1990. Growth of the maize primary root at low water potentials. II. The role of growth and deposition of hexose and potassium in osmotic adjustment. Plant Physiol 93:1337–1346. Silk WK. 1984. Quantitative descriptions of development. Annu Rev Plant Physiol 35:479–518. Silk WK. 1989. Growth rate patterns which maintain a helical tissue tube. J Theor Biol 138:311–327. Silk WK. 1992. Steady form from changing cells. Int J Plant Sci 153:S49–S58. Silk WK, Erickson RO. 1978. Kinematics of hypocotyl curvature. Am J Bot 65:310–319. Silk WK, Erickson RO. 1979. Kinematics of plant growth. J Theor Biol 76:481–501. Silk WK, Wagner KK. 1980. Growth sustaining water potential distributions in the primary corn root. Plant Physiol 66:859–863. Silk WK, Walker RC, Labavitch J. 1984. Uronide deposition rates in the primary root of Zea mays. Plant Physiol 74:721–726. Silk WK, Hsiao TC, Diedenhofen U, Matson C. 1986. Spatial distributions of potassium, solutes, and their deposition rates in the growth zone of the primary corn root. Plant Physiol 82:853–858. Spollen WG, LeNoble ME, Samuels TD, Bernstein N, Sharp RE. 2000. Abscisic acid accumulation maintains maize primary root elongation at low water potentials by restricting ethylene production. Plant Physiol 122:967–976. Sugimoto K, Williamson RE, Wasteneys GO. 2000. New techniques enable comparative analysis of microtubule orientation, wall texture, and growth rate in intact roots of Arabidopsis. Plant Physiol 124:1493–1506. Taylor AR, Bloom AJ. 1998. Ammonium, nitrate, and proton fluxes along the maize root. Plant Cell Environ 21:1255–1263. Tennekes H, Lumley JL. 1994. A First Course in Turbulence. Cambridge, MA: MIT Press. Van der Weele CM, Spollen WG, Sharp RE, Baskin TI. 2000. Growth of Arabidopsis thaliana seedlings under water deficit studied by control of water potential in nutrient-agar media. J Exp Bot 51:1555–1562. Walter A, Silk WK, Schurr U. 2000. Effect of soil pH on growth and cation deposition in the root tip of Zea mays L. J Plant Growth Regul 19:65–76. Weisenseel MH, Dorn A, Jaffe LF. 1979. Natural H+ currents traverse growing roots and root hairs of barley (Hordeum vulgare L.). Plant Physiol 64:512–518. Yan X, Lynch JP, Beebe SE. 1996. Utilization of phosphorus substrates by contrasting common bean genotypes. Crop Sci 36:936–941. Zhong H, La¨uchli A. 1993. Spatial and temporal aspects of growth in the primary root of cotton seedlings: effects of NaCl and CaCl2. J Exp Bot 44:763–771.

8 Lateral Root Initiation Pedro G. Lloret and Pedro J. Casero Universidad de Extremadura, Badajoz, Spain

I.

INTRODUCTION

1. The first step is a division of the founder cells. This has initiated an interesting controversy. Do founder cells cease to divide as they leave the apical meristem but later recover the capacity to proliferate far from the apical meristem? Alternatively, does the proliferation of the founder cells not really stop? 2. Founder cells undergo transversal, periclinal, anticlinal, and oblique divisions giving rise to a group of cell derivatives in which it is difficult to recognize the future tissues of the LRs. Such cell divisions produce the cells in the LR primordium. This event includes induction of important changes in the cell walls, cytoskeleton, cytoplasm, nuclear displacement, genetic expression, etc. 3. Derivative cells differentiate acquiring the morphological features of the LR tissues that show the same features of the parent roots. 4. LRs originate endogenously from tissues lying inside the parent root. Therefore, they must grow through the parents root tissues in order to emerge. 5. LRs may originate either from a single parent tissue or from several tissues. 6. The parent tissues adjacent to the lateral roots can undergo major structural and functional changes to facilitate the growth of the lateral root inside the parent root and its emergence. 7. Regulation by hormones such as auxins and cytokinins. 8. The founder cells are similar to the neighbor cells but the lateral root primordia are delimited structures separated from the parent tissues. This constitu-

Lateral roots (LRs) as defined here are those that originate from some other root. This means that the lateral roots can be derived either from a seminal root, an adventitious root, or another lateral root. The lateral roots, therefore, constitute almost the whole root system. They influence many aspects that are so important for the plant’s development as its adaptability to the environment and its absorption capacity. Lateral roots affect plant size, plant production, vitality, etc. All these aspects justify the scientific interest that the study of lateral roots has always earned. Moreover, in many plants the lateral roots originate far from the apex. Therefore, cell proliferation during lateral root development does not overlap the cell proliferation of the apical meristem. The two processes are temporally and spatially separated. In these plants, the analysis of lateral roots is more relevant because these roots originate from a very few parent cells, called founder cells, which are very long and highly vacuolated mature cells. These cells proliferate and give rise to very short derivative cells, which are nearly isodiametric, with thinner cell walls, a marked increase in cytoplasmic basophilia and volume, a pronounced nucleolar enlargement, and numerous small vacuoles. These short cells show the typical morphological features of meristematic cells, suggesting that the founder cells undergo a dedifferentiation process. Lateral root initiation and development involve the following: 127

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Lloret and Casero

tes a very attractive developmental model because the founder cells must be targets for the hormones, diffusing molecules, etc. 9. The LRs are located in a specific position in relation to the vascular structures, forming longitudinal ranks along the parent roots. 10. The distribution of the lateral roots along the parent roots is not at random and is related to the environmental conditions, allowing optimal utilization of soil resources. Excellent reviews have been published on LR development (Van Tieghem and Douliot, 1888; Torrey, 1961, 1986; Von Guttenberg, 1968; McCully; 1975; Peterson and Peterson, 1986; Charlton, 1991, 1996). After Zobel’s (1975, 1991) general reviews on the genetics of root development, a more specific analysis of the genetics of LR development is lacking. Probably the reason has been that this research field has been in continuous growth during recent years. Now, we are witnessing the maturing of the great array of molecular, genetic and cellular techniques that constitute a very reliable and consistent research system which will ensure significant results in the near future leading to a better understanding of the control mechanisms of lateral root initiation and development. II.

EVENTS DURING LATERAL ROOT FORMATION

A.

Sequence of Formation

Roots elongate continuously due to the activity of their apical meristem. Since LRs are formed from relatively young parent root tissues and usually form in an acropetal sequence (Abadı´ a-Fenoll et al., 1986), they are initiated between the previously formed LRs and the apical meristem (Fig. 1). Nevertheless, the formation of LRs sometimes proceeds in an order that is not strictly acropetal (MacLeod, 1990; Pellerin and Tabourel, 1995). The acropetal origin of LRs should thus be taken as a trend rather than a rule. One possible cause for altering the basic scheme of acropetal formation might be that the activity of the different cell ranks is not synchronized. Charlton (1975) described in Pontederia cordata the existence of retarded ranks producing their distalmost new LR primordia at a greater distance from the apex than more advanced ranks. This results in small LR primordia intercalated between others at later stages of development. In some extreme cases, the competence of the parental root pericycle or endodermis to initiate LR primordia lasts for so long that in broad regions of

the parent root new and old LR primordia can coexist. This has been described in root systems of Zea mays. Initiation of LR primordia of Zea mays is not confined to the subapical region but new anlages grow up to as far as 5–8 cm from the apex (MacLeod, 1990). A similar situation occurs in Triticum aestivum: from the root apex to the base the frequency of LR primordia increases to a maximum located 40–50 mm from the apex (Bingham et al., 1997). New primordia of wheat also appear in tissues that already contain visible primordia. The appearance of small primordia between emerged growing LRs (Fig. 2) does not imply a nonacropetal sequence of development. It may happen that during the development of LR growth of some primordia is delayed. A slower growth of late-formed primordia would also yield images such as that of Fig. 2. Nevertheless, it appears that commonly all primordia eventually emerge even if initiated out of sequence (Lloret and Pulgarı´ n, 1992; MacLeod and Thompson, 1979).

Figure 1 Segment of primary root of Zea mays showing the development of LRs in an apparent acropetal order, with longer LRs at the basal most region and progressively shorter LRs toward more apical regions. Bar: 20 mm.

Lateral Root Initiation

129

B.

Figure 2 Intact maize root with young LRs showing short LRs between longer ones. This suggests that the sequence of formation is not strictly acropetal. Note that there is a clear LR dimorphism with narrow LRs (arrow) which are longer than broad LRs (arrowhead). Bar: 1 mm.

The existence of dormant or late-formed LR primordia presents a problem for studies of the LR distribution pattern. Indeed, if these minute anlages are not detected under the microscope, the pattern of externally apparent LR distribution in a given species may be obscured (Mallory et al., 1970). The possibility that LRs emerge out of sequence is a normal situation for roots developing under experimental conditions (Charlton, 1996). Nevertheless, the formation of LRs in an inverse order to the natural acropetal sequence is unusual. To the best of our knowledge, there has only been one report of such finding (Dyanat-Nejad and Neville, 1972). This basipetal sequence of LR development occured on the preformed root region in the germinative radicle of Theobroma cacao. This region has a poorly defined transition to the hypocotyl, and consequently these roots can really be adventitious roots originated from the hypocotyl rather than true LRs.

General Aspects of Lateral Root Development

In some plants, the LRs originate so close to the apical meristem that either none of the vascular tissues of the main root is differentiated or only the protophloem appears to be mature (Mallory et al., 1970; McCully, 1975). However, it is more common to find plants in which LRs are initiated from region including well differentiated tissues, away from the apical meristem (Bell and McCully, 1970; McCully, 1975; Blakely et al., 1982; Charlton, 1983b; Lloret et al., 1988, 1989; Pulgarı´ n et al., 1988; Casero et al., 1989, 1993, 1995, 1996; Seago and Marsh, 1990). It is generally accepted that the distance from the apex of the main root to the point of appearance of the LRs is fairly constant and is characteristic for each species (Torrey, 1961; Charlton, 1991). Plants in which the LRs originate far from the apex of the mother root are interesting for studies of LR initiation, because the cell proliferation involved in LR initiation does not overlap the cell proliferation in the apical meristem. The two phenomena are separated in time and space. However, new findings regarding LR initiation raises the question whether cell proliferation really ceases in the apical meristem and is then reactivated to give rise to the LRs. Root anatomy is relatively uncomplicated. Roots of primary growth are cylindrical with a diameter that is practically constant except at the apex. At the center of such roots is the vascular cylinder, which presents a characteristic alternating arrangement of the conducting tissues, xylem and phloem. In either case, the outermost cells of the two tissues, xylem and phloem, are in contact with the pericycle, that usually forms a continuum and delimits the outside of the vascular cylinder. LRs originate endogenously from the endodermis in ferns (Clowes, 1961; McCully, 1975; Lin and Raghavan, 1991; Charlton, 1996) and from the pericycle in gymnosperms and angiosperms (McCully, 1975; Peterson and Peterson, 1986; Fahn, 1990; Charlton, 1996). In some angiosperms, both the pericycle and endodermis contribute to the tissues of the LR, although in many cases the derivatives of the endodermis are short-lived (reviewed by McCully, 1975). Therefore, the early LR development occurs inside the parent roots (Fig. 3). The LRs are located in defined relation to the vascular pattern (Fig. 4). In some species, they appear close to the xylem poles whereas in other species they appear next to phloem poles (Van Thieghem and Douliot, 1888; Esau, 1977;

130

Figure 3 A lateral root primordia ( ) can be observed attached to an xylem rank inside an intact adventitious root. Bar: 200
m.

Nishimura and Maeda, 1982; Lloret et al., 1989; Fahn, 1990; Casero et al., 1995). Thus, it can be assumed that in the pericycle, cells located opposite either xylem or phloem poles (depending on the species) and capable of initiating LR primordia alternate with cells which are apparently unable to initiate LR primordia. What

Lloret and Casero

is the factor responsible of this difference is still far from being answered. In carrot, LRs originate from pericycle cells located next to the phloem poles where the pericycle is unilayered while pericycle in front of the xylem is bistratified (Esau, 1977; Lloret et al., 1989; Knox et al., 1989; Casero et al., 1995, 1998). However, in Pisum sativum, LRs initiate from a multilayered pericycle in front of the xylem while the pericycle is single-layered in front of the phloem (Lloret et al., 1989; Casero et al., 1998). In many plants, including Allium cepa, Raphanus sativus, Helianthus annuus, and Arabidopsis thaliana, in which the pericycle consists of only one layer of apparently uniform cells, initiation of LR primordia only occurs in the pericycle in front of the xylem (Blakely et al., 1982; Lloret et al., 1989; Casero et al., 1995; Laskowski et al., 1995). Therefore, the number of layers in the pericycle has its effects but does not determine its capacity to form LRs. In onion, carrot, and pea, species which show very different pericycle patterns, Lloret et al. (1989) and Casero et al. (1989b) have describe two types of pericycle cells distinguishable by their different length and position with respect to the vascular pattern. In onion and pea, the shorter cells were found opposite xylem poles, where LR initiation occurs, whereas the longer cells were opposite the phloem poles. This is true also

Figure 4 Transverse section of an onion adventitious root. After successive periclinal divisions, the pericycle (P) becomes multiseriate between three xylem poles (X). Bar: 30
m.

Lateral Root Initiation

for A. thaliana (Laskowski et al., 1995). In carrot, the shorter cells were found adjacent to the phloem poles, where LR initiation occurs. Thus, apparently the shorter pericycle cells seem to be better suited for LR initiation. Because roots normally exhibit symplastic growth, differences in mean length between pericycle cells located opposite xylem and phloem poles at any given distance from the tip reflect differences in rates of transverse division between these two cell types in the meristem (Luxova´, 1975; Webster and MacLeod, 1980; Rost et al., 1988; Casero et al., 1989b). Therefore, cell length, transverse proliferation in the meristem, and LR initiation could be related. While this is a very attractive hypothesis, it is necessary to remember that LR development is a spatially located phenomenon in which only a very limited number of grouped cells of the mother root are involved. This number varies considerably depending on the species (MacLeod and Thompson, 1979). Thus, in radish, a group of 30 pericycle cells originate a LR (Blakely et al., 1982), while in A. thaliana only 11 adjacent pericycle cells are involved in the formation of an LR (Laskowski et al., 1995). Like the rest of the root tissues, pericycle cells form vertical columns of cells that can be traced to initial cells in the root apical meristem. Many columns of such cells are involved in the process of LR initiation in the root. Therefore, it is difficult to

131

explain why at any transverse level only one LR is formed in front of one of the xylem poles, and why LRs are not initiated in front of the remaining xylem poles at the same distance from the apex although the corresponding opposite pericycle cells are just as short (Casero, unpublished data). Moreover, the longitudinal extension of the LR primordia suggests that very few pericycle cells in the same column can be involved in the initiation. Adjacent pericycle cells located both above and below those involved in the initiation are similar in length to the pericycle cells from which the LR is initiated, but they are unable to initiate a LR under normal conditions. All this suggests that several interacting factors regulate the initiation of the LRs by discriminating between adjacent pericycle cells. In angiosperms, periclinal division of a few pericycle cells is one of the most common morphological criteria used to define LR initiation (Esau, 1940, 1977; Clowes, 1961; Foard et al., 1965; Bonnett and Torrey, 1966; Bell and McCully, 1970; Charlton, 1975; McCully, 1975; Blakely et al., 1982; Lloret et al., 1989; Fahn, 1990; Seago and Marsh, 1990). Periclinal divisions are very noticeable in cross section in plants with an uniseriate pericycle because the two radially arranged derivatives can be easily recognized in contrast with the rest of the pericycle (Blakely et al., 1982; Lloret et al., 1989; Casero et al., 1993) (Fig. 5). Just before the first periclinal divisions, pericycle cells undergo a noticeable

Figure 5 Transverse section of an onion adventitious root showing one of the pericycle cells in front of the xylem undergoing a periclinal division (arrowhead). C, cortex; E, endodermis; P, pericycle; X, xylem. Bar: 30
m.

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radial enlargement (Popham, 1955; Clowes, 1961; Bonnett and Torrey, 1966; Charlton, 1975; Casero et al., 1993), a marked increase in cytoplasmic basophilia and volume (Bell and McCully, 1970; Karas and McCully, 1973), and a pronounced nucleolus enlargement (Seago, 1973). It is particularly interesting that pericycle cells undergoing periclinal divisions are very short (Van Tieghen and Douliot, 1888; Bell and McCully, 1970; McCully, 1975; Blakely et al., 1982; Seago and Marsh, 1990; Laskowski et al., 1995; Malamy and

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Benfey, 1997). In onion, for instance, they are only 30–50
m long (Fig. 6) (Lloret et al., 1989; Casero et al., 1993). Since LR initiation occurs after cell elongation has terminated, elongated and highly vacuolated pericycle cells must divide transversely to become short enough before dividing periclinally (Van Tieghem and Douliot, 1888; Bell and McCully, 1970; Blakely et al., 1982; Lloret et al., 1989; Malamy and Benfey, 1997). In the apical meristem, cells divide and elongate, but beyond the meristem zone cells elongate only. At a certain distance from the tip elongation ceases. In onion, no mitotic cells can be found beyond 1600–1800
m from the apex. At this level, the mean length of the pericycle cells is < 60
m (Casero et al., 1989a). Then, pericycle cells increase in length. Between 6 and 7 mm from the apex, all pericycle cells are highly vacuolated, containing a central nucleus and measuring over > 200
m in length. Transverse divisions are a necessary stage prior to the periclinal divisions. This led Casero et al. (1993, 1995, 1996) to focus their attention to the precise sequence of cellular events from which such short pericycle cells are derived in different plants. The resulting proliferation pattern is original and very attractive and allows clear identification of the site of LR initiation closer to the apex than previously documented.

C.

Figure 6 Longitudinal section of an onion adventitious roots showing a pericycle column (P) in front of one of the xylem poles (X). One short pericycle cell within a group of short cells has undergone periclinal division, forming two cells located at the same transversal level (arrowhead). Bar: 30
m.

Transverse Division in the Pericycle Subsequent to Elongation

The occurrence of transversal divisions in pericycle cells proximal to the elongation zone has been noted in onion and in other species in which LRs are initiated far from the apex (Van Tieghem and Douliot, 1888; Bell and McCully, 1970; Lloret et al., 1989). In roots of Allium cepa and Pisum sativum, Lloret et al. (1989) describe pericycle cells undergoing symmetric (Fig. 7) and asymmetric transversal divisions (Fig. 8) closer to the apex than where the periclinal divisions occur. Symmetric transverse divisions can occur in pericycle cells situated either opposite the xylem or opposite the phloem, but do not give rise to cells as short as those which can divide periclinally (Casero et al., 1993). Asymmetric transverse divisions occur only in pericycle cells situated in front of xylem, giving rise to short cells which can divide periclinally (Casero et al., 1993). The results strongly suggest that these asymmetric divisions are related to LR initiation.

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Figure 8 Longitudinal section of an onion adventitious root showing a pericycle cell undergoing an asymmetrical transverse division in front of one of the xylem poles. C, cortex; E, endodermis; P, pericycle; X, xylem. Bars: 20
m.

Figure 7 Longitudinal section of an onion adventitious root showing a pericycle cell undergoing a symmetrical transverse division in front of one of the xylem poles. C, cortex; E, endodermis; P, pericycle; X, xylem. Bars: 20
m.

D.

Asymmetric Transverse Divisions During the Earliest LR Development in Vascular Plants

The pericycle cells that are involved in the initiation and development of the LRs can be named ‘‘founder pericycle cells’’ (FPC) following the definition given by Laskowski et al. (1995), because they are activated and divide giving rise to derivative pericycle cells which differentiate into the apical meristem and the tissues of the lateral roots. Although Casero et al. (1993, 1995, 1996) had previously used the term ‘‘mother pericycle cells’’ to define them, FPC is now more generally used.

The first morphological event related to the LR initiation can be observed in just one pericycle column. Approximately when elongation ceases, two elongated and highly vacuolated adjacent pericycle cells (FPC) located in the same column undergo nearly synchronous asymmetric transverse divisions. This occurs in front of one of the xylem poles in onion, radish, and sunflower and near one of the phloem poles in carrot and corn (Casero et al., 1993, 1995, 1996). Two short pericycle derivatives are then produced, lying end to end in the same column flanked above and below by the two longer pericycle derivatives (Fig. 9). The short cells tend to have a central nucleus, while the longer cells have nuclei, which tend to be displaced toward the short cells. After these transverse divisions, it is easy to distinguish the original extension of the FPCs because the new transverse cell walls are noticeably thinner than the older ones. Moreover, a small intercellular space can be seen between the two FPCs and the cells of the adjacent tissues. No intercellular space can be distinguished at the level of the new cell walls (Casero et al., 1993, 1995, 1996). Observations by

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Figure 9 Longitudinal section of an onion adventitious root showing two short pericycle cells derivatives, lying end to end, which are flanked by two longer cells in front of one of the xylem poles. Arrowheads mark the original length of the founder pericycle cells. C, cortex; E, endodermis; P, pericycle; X, xylem. Bars: 30
m.

Demchenko and Demchenko (1996) in Triticum aestivum also strongly suggest that LR initiation occurs when two FPCs in front of the phloem undergo asymmetric transverse divisions like those described above. More recently, similar phenomena were observed in two adjacent FPC pericycle cells in front of the xylem poles in Lactuca sativa (Zhang and Hasenstein, 1999) and A. thaliana (Casimiro et al., unpublished results). These results allow us to generalize and assume that in angiosperms the polarized asymmetric transverse division of two adjacent FPCs is a general feature and can be considered the first recognizable stage of LR development. This objective morphological criterion allows clear identification of the site of LR initiation. In onion,

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radish, sunflower, corn, and carrot the pair of short pericycle cells appears
8–10 mm, 10–13 mm, 12–15 mm, 12–15 mm, and 12–15 mm, respectively, behind the tip, whereas the first periclinal division takes place at
21–23 mm, 15–18 mm, 19–21 mm, 21–24 mm, and 21–24 mm behind the tip, respectively (Casero et al., 1995). The distance behind the root apex where LRs are initiated is important when the proliferation of the pericycle cells from the apical meristem is considered. It is well known that in species in which LR initiation occurs opposite the xylem, the pericycle cells in front of the xylem divide further from the tip than the pericycle cells located in front of the phloem (Luxova´, 1975; Rost et al., 1988; Casero et al., 1989a). Therefore, the pericycle cells near the xylem are shorter than those next to the phloem. By contrast, in those species in which LRs appear next to the phloem, the pericycle cells located near the phloem are shorter than those next to the xylem (Lloret et al., 1989). This implies that, in these species, the pericycle cells close to the phloem divide further from the tip than those in front of the xylem. The question is whether or not the FPC cease to divide before LR initiation. The results presented by Jensen and Kavaljian (1958), Balodis (1968), Luxova´ (1975), Rost et al. (1988), and Casero et al. (1989a) regarding the occurrence of mitosis in the pericycle near the root apex, suggest that proliferation of the pericycle cells ceases, because no mitoses could be seen beyond a certain distance behind the tip. John et al. (1993) also suggested that proliferation ceases in the pericycle cells but they point out that the development of some pericycle cells is arrested in the G2 phase. These cells can, therefore, be induced to divide very quickly with auxin. Blakely et al. (1982) also speculate that the cycle of the pericycle cells stops at G2, except for that of the FPC. Our results indicating that LR initiation occurs nearer the tip support the hypothesis that pericycle cells do not leave the division cycle when they enter the elongation zone. Thus, in onion roots, the first periclinal divisions of the pericycle cells occur
21– 23 mm behind the tip, but the first asymmetric transverse divisions occur at
7 mm behind the tip (Casero et al., 1993). Assuming that an onion root increases its length by
15 mm/day (unpublished data) and that the cycle time lasts 13.5 h at 25 C (Gime´nez-Martı´ n et al., 1977), it is possible that a cell that undergoes its last division in the apex could divide again further behind the tip, for example,
7–8 mm behind the tip, where transversal divisions have indeed been seen. This observation strongly suggests that the pericycle cells involved

Lateral Root Initiation

in LR initiation continue dividing once they leave the meristem (Casero et al., 1993). Furthermore, the PFC show a very important characteristic, which distinguishes them from the rest of the pericycle cells: a regular pattern of cell divisions which is initiated with the coordinated asymmetric division of two neighboring cells (Casero et al., 1993, 1995). Asymmetric transverse division during the LR initiation is not exclusive to angiosperms. During LR initiation in ferns the nucleus of endodermal cells in front of xylem poles moves toward a position close to the wall and then divides asymmetrically giving rise to two cells of different sizes (Clowes, 1961; Lin and Raghavan, 1991). However, two main differences must be emphasized. In ferns, unlike higher plants, just one cell in front of the xylem divides asymmetrically and, furthermore, this is an endodermal cell. Studying polarized asymmetric transverse divisions during lateral root intiation in angiosperms opens up very attractive possibilities in plant development and cell biology. Recently, it has also been suggested, in comparing mutant and wild-type embryogenesis in A. thaliana, that GNOM gene activity is required for the asymmetric division of the zygote, and in its absence a symmetric division occurs which leads to other embryonic defects (Aeschbacher et al., 1994). The SCR (scarecrow) gene is required for the two distinct asymmetric cell divisons necessary for establishing the two ground tissue layers in the embryo and postembryonic root meristem (Dolan, 1997). Therefore, in root meristem of scr seedlings, the cortical/endodermal initial cells fail to undergo their asymmetric division which would produce the cortical and endodermal cell layers. These mutant roots have a single layer which shows attributes of both cortex and endodermis (Scheres et al., 1995; Di Laurenzio et al., 1996). The short-root (SHR) gene is required for the asymmetric cell division responsible for endodermis and cortex formation (Helariutta et al., 2000). These findings suggest that such asymmetric divisions depend on the expression of these genes. During LR initiation two pericycle cells, that appear identical to their neighbors, well differentiated and highly vacuolated, undergo asymmetric divisions almost simultaneously. A number of interesting questions arise from these observations: Are these asymmetrical divisions also conditioned by a gene expression? Why does it occur in just two cells out of so many? Does some signal induce the gene expression? Does the signal pass through both pericycle cells to synchronize them? Why do the two nuclei move in opposite directions?

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The hypothesis of intercellular communication during LR initiation seems more significant after observing that a wave of proliferation extends from the first FPCs and as neighbor pericycle cells become successively FPCs they also undergo asymmetric transverse divisions. This has been studied in detail in onion roots between 10 and 15 mm behind the tip (Casero et al., 1996). The adjacent FPCs were observed to undergo polarized asymmetrical division in relation to the first pair of short cells. This occurs in FPCs located in the same column above and below the first two FPCs. Thus, alternating sequences of short and long pericycle derivatives can be found in the same column. At that time, proliferation extends laterally to the adjacent columns. Therefore, pairs of short pericycle cell derivatives located at the same transverse level can be seen in longitudinal tangential sections (Fig. 10). Asymmetric proliferation also extends upward and downward in these other columns. This means that new adjacent FPCs are successively involved in the LR initiation reproducing the division pattern of the first two FPCs. Moreover, a clear polarization is shown in all the new FPCs in relation to the first pair of short cell derivatives. The longer pericycle cell derivatives now undergo asymmetric transversal divisions, giving rise to new short cells close to those formed previously. These follow the same sequence as the first asymmetric transverse divisions. This explains the formation of the groups of short cells observed in onion between 15 and 20 mm behind the tip. These groups of short cells show a gradual increase in their radial diameter towards the center. The radial diameter of the longer cells gradually increases toward the end nearer the short cells (Casero et al., 1993, 1996). In onion roots, the first periclinal divisions of LR initiation were seen 20–25 mm behind the tip. The centermost cells of a group of short cells, which show a noticeable radial expansion, are generally the first cells to undergo periclinal divisions, giving rise to two radially arranged cells. This means that the first periclinal divisions occur in the older short pericycle derivatives. Periclinal proliferation then extends to the neighboring short cells following approximately their formation sequence (Casero et al., 1993, 1996). Subsequently, successive waves of cell divisions emulating that in the two pairs of FPCs initially engaged in LR formation (i.e., asymmetric transverse cell division, formation of groups of short cells, and periclinal divisions) extend centrifugally as a wave from the center of the LR primordia.

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serve as a signal that moves between cells. One could speculate that pericycle cells, which are involved in LR initiation, would form a symplastically isolated group of cells. In such a case, the signal could not move farther above or below, and would be restricted to the limits of the LR anlage. Experiments carried out by Duckett et al. (1994) strongly suggest that differentiating epidermal cells can become symplastically isolated from other epidermal cells and from the underlying cortex cells, forming a ‘‘symplastic unit.’’ Indeed, the symplastic isolation of cells during the development of a tissue or an organ has been widely observed (Carr, 1976; Kwiatkowska, 1988; Duckett et al., 1994). If the group of founder cells indeed form a symplastic unit which becomes isolated from its neighbors during pericycle differentiation, LR initiation could be an excellent model with which to study cellcell interactions in plants. However, the pericycle cells of onion located near the xylem poles are activated before the intervening pericycle cells in front of the phloem poles (Casero et al., 1996). It is difficult to explain this observation on the basis of the assumption that the signal moves symplastically, via plasmodesmata, to neighboring cells. But it cannot be ruled out that the cell cycle of the cells near the phloem is much longer than that of the cells near the xylem. Another possibility is that each group of neighboring pericycle cells is independently activated.

Figure 10 Tangential longitudinal section of onion adventitious root showing pairs of short pericycle cell derivatives located approximately at the same transversal level in adjacent pericycle columns. C, cortex; E, endodermis; P, pericycle; X, xylem. Bars: 30
m.

The above results suggest that the FPC could be symplastically coupled, allowing small molecules to pass from cell to cell, as has been observed in root epidermal cells of Arabidopsis by using Lucifer Yellow and carboxyfluorescein (Duckett et al., 1994). After injection into an epidermal cell, the dye was seen in surrounding epidermal cells, having moved preferentially to cells within the same column. In onion roots, pericycle cells in the same column also tend to divide before cells in adjacent columns. Jackson et al. (1994) suggested that changes in gene expression patterns precede, or at least are concomitant with, the determination of the position of primordia in the shoot meristem. The proteins, which are products of the expression of these genes, e.g., KN1 protein, could

E.

Development of Lateral Roots After Initiation

Malamy and Benfey (1997) have described in detail the lateral root development after the occurrence of the periclinal divisions in the relatively simple roots of A. thaliana. The structure generated has a radial organization similar to that of the mature root tip. Fig. 11 is an attempt to summarize the main development stages following the results of Malamy and Benfey (1997). Stage I: The pericycle file contains many short cell derivatives (Fig. 11, St I). It is now known that they are produced after asymmetric transverse divisions of two adjacent founder cells as in other plants (Casimiro et al., unpublished results). Stage II: Lateral root primordia contain two layers of short-cell pericycle derivatives due to the periclinal proliferation of the previously formed short derivatives (Fig. 11, St II). The inner layer was termed IL, and the outer one, OL. Arabidopsis thaliana, Allium cepa and Raphanus

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137

Figure 11 Schematic representation to summarize the main development stages following the results by Malamy and Benfey (1997). St I: The pericycle file contains many short cell derivatives. St II: Lateral root primordia contain two layers of short-cell pericycle derivatives due to the periclinal proliferation of the previously formed short derivatives. IL, inner layer; OL, outer layer. St III: The lateral primordia contain three layers because the OL layer divides periclinally again giving rise to OL1 and OL2 layers. St IV: The lateral primordia contain four layers because the IL layer undergo periclinal divisions giving rise to IL1 and IL2 layers. St V: The centermost cell derivatives of OL1 and OL2 divide transversely. St VI: The centermost cells of OL1 undergo periclinal divisions. These cells will give rise to the future cap. The rest of the OL1 cells do not undergo periclinal divisions and will produce the future epidermis. The centermost cells of OL2 will produce the future quiescent center. The rest of the cells of OL2 undergo a periclinal division, forming the layers OL2a and OL2b that will form the future cortex and endodermis, respectively. IL1 will form the future pericycle. The centermost cells of IL2 undergo a noticeable radial expansion and periclinal divisions. They will give rise to the rest of the vascular cylinder. C, future cortex; E, future endodermis; Ep, future epidermis; St, stage; VC, future vascular cylinder.  Future cap cells.

sativus are examples of plants in which the periclinal divisions are symmetric (Blakely et al., 1982; Casero et al., 1993, 1995, 1996; Malamy and Benfey, 1997). However, in other plants, such as Zea mays, periclinal divisions are asymmetric. The daughters closer to the endodermis are narrower than the others (Bell and McCully, 1970; Ashford and McCully, 1973; McCully, 1975; Casero et al., 1995). In Malva silvestris, Byrne (1973) showed that cells in the two layers had very different behavior although they were formed after a symmetrical periclinal division. More recently, Malamy and Benfey (1997) have elegantly demonstrated in A. thaliana seminal roots that, although IL and OL are morphologically similar, they also present distinct identities as is shown by the differential gene expression after a promoterless GUS gene is inserted approximately 1 kb upstream of the start site of the SCR gene. Cells may also have different identities within a single layer (Malamy and Benfey, 1997). Results by Karas and McCully (1973) show major changes in the cell walls preceding the periclinal division indicating that in corn enzymatic hydrolysis must be involved in these changes.

Stage III: The OL layer divides periclinally again. The two new layers, OL1 and OL2, replace the OL layer, with OL1 being the outer one (Fig. 11, St III). Stage IV: The lateral primordia contain four layers because the IL undergo periclinal divisions giving rise to two layers (IL1 and IL2), with IL2 being the closer to the seminal vascular cylinder (Fig. 11, St IV). The pericycle probably arises from IL1 while the other stele cells arise from proliferation of IL2 (Malamy and Benfey, 1997). In Ipomoea purpurea, Seago (1973) also described the formation of four layers of pericycle derivatives with the inner layer dividing before the outer one. The author suggested that the initial cells were formed from these layers. The two inner layers gave rise to the vascular initial cells, the next one to the cortex, and the outermost to the initial cells of the capepidermis. Stage V: The centermost cell derivatives of OL1 and OL2 divide transversely. Proliferation then extends to the adjacent cell derivatives in these layers. The most centered IL2 cells undergo expansion and division distorting the shape of IL1 and OL2. (Fig. 11, St V).

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Stage VI: This development stage is very interesting because the main cellular layers of the future mature root are established (Fig. 11, St VI). Malamy and Benfey (1997) showed that these layers can be recognized by means of cell line markers. There are no marker lines that specifically stain the initial cells. However, in the stage VI primordium there are cells that can be tentatively identified as initial cells, based on their position at the ends of differentiated cell files and comparison with the positions of initial cells in the mature root tip. The centermost cells of OL1 undergo periclinal divisions. These cells will give rise to the future cap as is demonstrated by means of the cell marker LRC244, which is also expressed in the cap cell of the seminal root. The rest of the OL1 cells do not undergo periclinal divisions and will produce the future epidermis. The cell marker EpiGL2 expressed in these OL1 cells and in the epidermis of the seminal root the epidermal cells that will not produce hairs. The centermost cells of OL2 will produce the future quiescent center. The rest of the cells of OL2 undergo a periclinal division forming the layers OL2a and OL2b that will form the future cortex and endodermis, respectively. End195 marks the OL2b in the LR primordia and endodermis in the seminal roots. End195 also marks the cortex/endodermal initial cell which undergoes a periclinal division to generate cortex and endodermal cell files. The marker corAx92 identifies the OL2a cells in the LR primordia and the cortical cells in the seminal roots. IL1 will form the future pericycle. The centermost cells of IL2 undergo a noticeable radial expansion and periclinal divisions. They will give rise to the rest of the vascular cylinder. The end of development stage VI is reminiscent of the primary root tip. Other studies have also tried to identify the future mature root tissues in the lateral root primordia before emergence based on the cell location. In LR primordia of I. purpurea, Seago (1973) observed that the apical cells of the third layer of pericycle derivatives, counted outward, do not divide but the remaining third layer cells undergo periclinal divisions giving rise to the ground meristem. These results are interesting to compare with those of Malamy and Benfey (1997) because this third layer in I. purpurea would be equivalent to the OL2 layer in A. thaliana. Therefore, the endodermis and cortical cells of the lateral roots are formed from an

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equivalent layer in the two plants. Periclinal divisions were also observed in the flanks of the outermost pericyclic-derived layer of Typha glauca to produce the future ground meristem and protodermis (Seago and Marsh, 1990). The remaining pericycle derivative cells of this layer do not divide periclinally. In this species, pericycle derivatives are covered by a layer of endodermis derivatives. The endodermis derivative cells at the tip quickly undergo periclinal divisions like the outermost pericycle derivative layer in Arabidopsis. However, in T. glauca, cells formed from endodermis derivatives do not produce lateral root tissues. Cap cells will be formed from the ground meristem protodermis just after emergence (Seago and Marsh, 1990). Thus, the mature lateral root tissues of A. thaliana are exclusively formed from the pericycle cell derivatives. This was previously reported by Van Tieghem and Douliot (1888) for Convolvulus siculus and C. tricolor, by Dittmer and Spensley (1947) for Descurainia, by Davidson (1965) for Vicia faba, by Riopel (1966) for Musa acuminata, by Byrne (1973) for Malva sylvestris, by Seago (1973) for I. purpurea, by Charlton (1975) for Pontederia cordata, by McCully (1975) for Daucus, and by Seago and Marsh (1990) for Typha glauca. However, in many other plants, Zea mays for example (Bell and McCully, 1970; Karas and McCully, 1973), pericycle, endodermis, cortex, and stelar parenchyma divide during the lateral root initiation. All these cells undergo changes in the structure of cytoplasm and cell walls which are reviewed by McCully (1975). The endodermis outside the lateral root primordia of A. cepa, also undergo asymmetric transverse divisions like the pericycle. A similar situation occurs in Helianthus annuus (Fig. 1a of Charlton, 1996). Transverse proliferation would explain how endodermis cells incorporate 3H-thymidine prior to radial expansion and anticlinal divisions in Malva sylvestris (Byrne, 1973). Many authors have found that the endodermis shows similar morphological features to the pericycle during lateral root initiation: increasing cytoplasmic content (Bonnett and Torrey, 1966; Seago, 1973), increasing cytoplasmic basophilia (Bell and McCully, 1970), and enlargement of the nucleoli (Seago, 1973). The cell walls show major changes (Karas and McCully, 1973). Endodermis cells outside the lateral primordia undergo tangential expansions and anticlinal divisions to accommodate the growth of the pericycle derivatives (McCully, 1975; Clowes,

Lateral Root Initiation

1978). The new cells do not form Casparian strip (Peterson and Peterson, 1986). The original Casparian strip regions do not change and remnants of them can occasionally be seen on some of the anticlinal walls of the cells derived from the endodermis which cover the young primordium (McCully, 1975). The endodermis also undergoes periclinal divisions giving rise to several radially arranged layers (Popham, 1955; McCully, 1975; Charlton, 1996). Early authors (Tschermak-Woess and Dolezal, 1953; Gramberg, 1971) described proliferation of the cortical cells outside the lateral primordia. More recently, Casero et al. (1996) observed that the cortex outside the lateral root primordium of A. cepa undergoes asymmetric transverse divisions like the pericycle. In Glycine max, the cortical cells divide periclinally (Byrne et al., 1977). Several layers of cortical cells dedifferentiate becoming incorporated into the primordium in Cucurbita pepo (Charlton, 1996). Endodermis and cortex cells enter successively into proliferation after those of the pericycle (Casero et al., 1996). This allows us to extend the hypothesis of symplastic communication to the endodermis and the cortex. McCully (1975) suggested that a substance diffusing from the young primordia can influence cortical cells. Cells other than the pericycle also proliferate during LR initiation and can also contribute to the formation of the lateral root. The contribution of the parent root tissues to lateral root formation depends on the plant species. In maize, for example, the endodermis of the parent root gives rise to the epidermis of lateral roots. This is based on the fact that the epidermis of the parent root and endodermis derivatives of the lateral primordia show a similar morphology and secrete a thick coat of mucilage to the outer surface of their outer tangential wall. Moreover, a few endodermal derivatives at its tip divide periclinally to produce the root cap initial cells (cf. McCully, 1975). The contribution of the parent endodermis of pea roots is more extensive than in maize, producing the cortex, the epidermis and the cap of the lateral root (Popham, 1955). In Cucurbita maxima, the endodermis forms the cortex, the epidermis, the cap and the vascular tissue of the lateral root (Mallory et al., 1970). In maize (Bell and McCully, 1970; Ashford and McCully, 1973) and in Ipomoea purpurea (Seago, 1973), the parenchyma cells of the parent root contribute to the basal tissue of the lateral primordia, playing an important role in the vascular connection. Cell proliferation does not necessarily mean that the cell derivatives will form part of the future LR. In many examples, endodermis and cortical derivatives

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form a covering over the young primordium that is finally stretched, broken, and shed (McCully, 1975). The German term Tasche—pocket—was used by Von Guttenberg (1968) to refer to the structures derived from the endodermis. In some cases the tasche persists and forms a substitute root cap; in others the epidermis of the lateral root is derived from the inner layer of the endodermal derivatives and persists, while the remainder of the tasche is shed (Schade and Von Guttenberg, 1951). The review by Charlton (1996) also refers to the poche digestive of Van Tieghem and Douliot (1888) because it is also a structure formed of endodermis derivatives although it can also contain overlying cortical derivatives. The tasche and the poche digestive could have similar roles secreting enzymes to facilitate the passage of lateral primordia through the parent root tissues. Coated vesicles that fused to the plasmalemma of the outermost cells of the tasche of Convolvulus arvensis were observed, suggesting that they contained hydrolytic enzymes (Bonnett, 1969). In Z. mays, the outermost derivatives of the parent root endodermis show high levels of acid phosphatase and -glycosidase activity (Ashford and McCully, 1973). Peretto et al. (1992) and Bonfante and Peretto (1993) found polygalacturonases in the outer part of the primordium and tasche and in the pectic material between the LR and parent cortical tissues of Allium porrum. Enzymatic hydrolysis facilitates LR emergence. Pectinolytic enzymes are secreted by the lateral primordium to separate the parent cells and collapse them. However, Bell and McCully (1970) reported that the walls of hypodermis and epidermis of Z. mays are lignified and suberized, and are not easily penetrated. Some authors claim that a simple mechanical process is enough to explain the passage of the lateral primordium through the parent root tissues. Sutcliffe and Sexton (1968) suggested that the high levels of glycerophosphatase activity detected in the cortical cells adjacent to lateral root primordia during emergence are due to breakage of these parent root cortical cells induced by mechanical pressure. Peterson and Peterson (1986), Charlton (1991), Lin and Raghavan (1991), and Charlton (1996) observed that the cortical cells adjacent to primordia collapse as the primordium emerges. Charlton (1996) showed that in Cicer arietinum cortical cells outside the primordia separate. Therefore, it is reasonable to assume that both physical and enzymatic processes are involved in lateral root emergence (Bonnett, 1969) and that they can occur either simultaneously or separate in time.

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During early developmental stages the LR primordia are made up of similar proliferative cells, and it is sometimes difficult to distinguish the derivative cells from the parent pericycle, endodermis, and cortex. However, populations of cells can be distinguished in small primordia by their different mean cycle time (McCully, 1975). Early growth of primordia was mostly due to proliferation of cells occupying a central core (Friedberg and Davidson, 1971), while later, growth occurred preferentialy through proliferation of peripheral cells. Cells of Vicia faba undergo major changes 24 h before emergence, which depend on their location. The most proliferative central cells become almost completely quiescent while the peripheral cells increase their rate. Mitotic activity then resumes in the central cells and falls off in the peripheral cells (McCully, 1975). Laskowski et al. (1995) suggested that the formation of a meristem in A. thaliana is a two-step process involving the establishment of a population of rapidly dividing cells followed by the process of meristem organization, during which initial cells are established. Although in A. thaliana LR primordia are sufficiently organized before emergence to proceed with development of LRs even if isolated from the parent root (Laskowski et al., 1995; Malamy and Benfey, 1997), an active apical meristem must be established later (Cheng et al., 1995). These authors show that rlm mutants are unable to activate a root meristem. Therefore, growth of the LR of these mutants is normal until emergence, but is then arrested. Emergence appears to be largely due to enlargement of the basal cells rather than to cell division. Before emergence the basal cells elongate, being less proliferative than the more apical and superficial cells (Malamy and Benfey, 1997). The central basal cells contain small vacuoles in contrast with the highly vacuolated cells which surround them. The quiescent center is normally detectable in the apical meristem of emerged lateral roots (Clowes, 1958). The vascular connection between LR and the parent root is then formed. Phloem connections are formed from the pericycle and vascular parenchyma derivatives while the xylem connection is established through the parenchyma derivatives neighboring the parent xylem (McCully, 1975; Peterson and Peterson, 1986; Luxova´, 1990; Vidal-Bernabe´ et al., 1998). It is generally accepted that maturation of the vascular cell connectors into vessels proceeds acropetally into the lateral roots, and occurs either at the time of lateral root emergence or later. The vascular tissues produced by the apical meristem of the lateral roots mature basipetally towards the parent roots (Byrne et al., 1982).

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Oparka et al. (1995) have demonstrated the necessary symplastic connection between the primary root and the lateral ones in roots of A. thaliana. This was done by means of carboxyfluorescein and confocal laser scanning microscopy. Oparka et al. (1995) observed that the establishment of intercellular transport between the parent root phloem and the lateral root primordia was enabled by structural differentiation of adjacent phloem connector elements. This occurred very early, when the lateral root primordia were visible as small bulges protruding from the cortex. Phloem connectors were detectable so early because they showed intense point fluorescence on their walls. The sieve elements were structurally recognizable immediately after the emergence of the LR.

III.

TYPES OF LATERAL ROOTS

LRs are structures that originated by cell division in the parent root pericycle. When they emerge they have an apical meristem similar to that of the root axis that originated it (Torrey, 1986). This definition is perhaps too broad as the LR population in a specific axis is not always as homogeneous as expected. The first source of variation comes from the sequence of development that is not always perfectly acropetal. Frequently, poorly developed abortive or dormant LRs are interspersed between more-developed LRs, apparently breaking the acropetal sequence. They may be acropetal LRs whose development has stopped. Nevertheless, true new LR primordia can develop between older formed LRs (Charlton, 1991). Hence, in a first approach, the LRs can be classified into acropetal laterals and adventive late-forming laterals. The basal roots, often considered to be LRs, are a special type of roots. Genetically and physiologically it appears that they may really be different from LRs (Zobel, 1975). Basal roots were initially demonstrated in tomato and have later also been observed in other species (Stoffella et al., 1992; Bonser et al., 1996). In tomato, they are perfectly discriminated in homozygotic double tomato mutants dgt ro that in theory should not form LRs (dgt mutation) or adventitious roots (ro mutation), but do form roots probably from the hypocotyl (Zobel, 1975). Hence in strict terms, they cannot be regarded as LRs because of their shoot origin. Cluster rootlets (Peterson, 1992; Skene et al., 1996), root primordial masses and fasciated roots (Hinchee and Rost, 1992a), and hairy roots (Biondi et al., 1997) have a structure and origin similar to those of normal LRs. However, they still differ by being much

Lateral Root Initiation

closer to each other than normal LRs. Probably, all these types of LR result from response of the parent root to altered physiological conditions and overcoming possible inhibitions between adjacent LR primordia. The type of alteration hairy roots undergo is believed to be the result of auxin overproduction promoted by the insertion into their DNA of bacterial aux and/or rolB gene sequences plus possible auxin hypersensitivity (Biondi et al., 1997). Ectomycorrhizal roots are present in many gymnosperms and angiosperms (see Chapters 28 by Bacon et al. and 50 by Kottke in this volume). A detailed description of this association between LRs and symbiotic fungi in Pinus resinosa was given (Wilcox, 1968b). This gymnosperm shows a clear LR dimorphism with long and short laterals (Wilcox, 1968a). Long LRs have a greater diameter and faster growth than short LRs. It is these short roots that establish the symbiosis (Peterson and Peterson, 1986). As the name indicates, in an ectomycorrhizal association a mantle of hyphae encloses the root without penetrating into its cells. Nevertheless, the presence of such a mantle significantly modifies the structure and function of the affected LR (Peterson, 1992). The main structural differences between them and regular LRs are rounding of the root tip, reduced growth, meristem vacuolisation, inhibition of root hair formation, and in several cases also dichotomous branching (Peterson, 1992; Peterson and Peterson, 1986). Other particular LR types that were described in the literature are the narrow and wide LRs of maize, which differ not only in diameter (Fig. 2) but also in growth (MacLeod, 1990). Are all these patterns of development really very different? Probably such root types develop in response to particular physiological and/or environmental conditions, but at the cellular level they do not represent substantial deviation from the basic aspects of LR development. So many LR types rather support the concept of plasticity of development, which allows the LR, for example, to stay dormant when environmental conditions are adverse and resume growth when they become to favorable (Dubrovsky et al., 1998). LRs could also transform into clustered rootlets when grown under low P concentration (Johnson et al., 1996). The most impressive example of plasticity is the transformation of endogenous root primordia into endogenous buds. Such a phenomenon occurs in a small group of species (Bonnett and Torrey, 1966). The initial endogenous primordia originate from the pericycle at a specific location relative to the parent root vascular system which would usually give rise to

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LRs. Excised Convulvulus roots formed either lateral roots or endogenous buds. The initiation of an endogenous bud had a higher probability on the proximal part of the root. At early stages of development, root primordia were identical to endogenous buds. Later, the pathways of development differed between these two structures, mainly in their contribution of parent root tissues to the body of the primordia, in the growth rate, and in the orientation of cell divisions (Bonnett and Torrey, 1966). Despite the apparent initial similarity between root and shoot primordia, conclusive evidence of an undetermined primordium, or of an undetermined primordium site, was not obtained in this study. Hence, this situation deserves further analysis now that we have more powerful research tools. IV.

LATERAL ROOT INITIATION AND REGULATION OF THEIR DEVELOPMENT

A.

Lateral Root Development Appears to Be a Multiphasic Process

It has been estimated that a red oak tree has
500 million living root tips, most of which are LRs (Lyford, 1975). This impressive number of LRs shows how important it is for the plant to explore the soil and to acquire enough water and nutrients to stay alive. Evidently, given the importance of having a root system extending over large soil volume, the development of LRs must be under fine regulatory controls. The formation of LRs has a high adaptive value and is therefore under a strong selective pressure. The formation of LRs is a multiphase process consisting of two or three main stages under different development controls. Some authors have considered two stages: initiation and meristem formation (MacIsaac et al., 1989; Laskowski et al., 1995). The moment that marks the transition from initiation to the following stages of growth has been placed early, when the LR primordium is considered autonomous— i.e., grows in vitro without supplementary growth regulators (Laskowski et al., 1995). It could also happen later, when the primordia has emerged as a LR (MacIsaac et al., 1989). Others have proposed three stages: initiation, development of primordia into roots, and emergence coupled with subsequent growth (Van Staden and Ntingane, 1996; Baum et al., 1998; Zhang and Hasenstein, 1999). As the process of LR development can stop at initiation (Celenza et al., 1995) shortly before emergence (Mallory et al., 1970) or after it (Baum et al., 1998), perhaps this view reflects

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most accurately the fine physiological controls that determine the overall processes involved in the formation of LRs. The initiation phase is represented by the active proliferation of a few FPCs. During this stage reactivated pericycle cells continue to their proliferative activities and organize an anlage formed by a few tiers of cells. Frequently the pericycle-derived cells are partially surrounded by an endodermal covering originating from the parent root endodermis (Esau, 1940). During LR initiation pericycle cells proliferate actively and duplication times as short as 3–4 h have been described (MacLeod and Thompson, 1979). The second stage of development occurs later, when the LR primordium acquires sufficient physiological autonomy to grow in a culture medium (Laskowski et al., 1995). There are different populations of cells according to the length of the cell cycle (Friedberg and Davidson, 1971). As a culmination of the processes the LR primordium emerges in this phase. This occurs rather by the rapid elongation of cells at the base of the LR primordium than by cell divisions in the apical meristem (Bell and McCully, 1970). The growth of the emerged LR is considered the third stage of development. At this moment the LR apical meristem is very similar to the apical meristem of the parent root (Dolan et al., 1993). In most species the LR now acquires a quiescent center (MacLeod and McLachlan, 1974; McCully, 1975) and reaches its vascular connection with the parent root (MacLeod and Francis, 1976; Byrne et al., 1977). For sustained growth, the young LR requires auxin supply (Celenza et al., 1995). It has a root cap derived from the pericycle, which replaces the ephemeral endodermal covering (Clowes, 1978; Seago, 1973), and as a consequence it gradually develops a gravitropic response (Moore and Pasieniuk, 1984; see also Chapter 30 by Pilet and Chapter 3 by Sievers et al., in this volume). B.

Endogenous and Exogenous Factors Regulate Lateral Root Formation

As a result of their evolution, the higher plants show the capacity to regulate the development of LRs to fulfill efficiently all the functions assigned to root systems. The LRs have a decisive influence on root system capacity to anchor the plant to the soil and to acquire water and nutrients. Consequently, practically all vascular plants can develop LRs. There are a few exceptions of species with primary roots that cannot develop laterals. Noteworthy cases are the aquatic fern Azolla pinnata (Barlow, 1984) and the angiosperm Lemna

(Clowes, 1985). Several mutants with a more or less impeded capacity of LR formation, such as axr1, aux1, axr4, Dwf, alf4, tir3, and dgt, were described in Arabidopsis thaliana, Zea mays, and Lycopersicon esculentum, species that normally develop LRs (Mirza et al., 1984; Schiefelbein and Benfey, 1991; Hobbie, 1998). Some aerial roots do not develop LRs until they reach the soil (Zimmerman and Hitchcock, 1935). Finally, there are lettuce cultivars that show delayed LR formation (Zhang and Hasenstein, 1999). All these examples suggest that the capacity of LR formation can be modulated by exogenous and endogenous factors (see also Chapter 14 Feix et al., in this volume). Changes in the physical or chemical environment that modify the physiology of roots can strongly influence on LR development. Such changes probably interact with metabolic processes in the aerial parts of the plant or in the root itself (see also Chapter 9 by Waisel and Eshel in this volume), which in turn are necessary for root system development. By means of these mechanisms, physical factors such as temperature (Sattelmacher et al., 1990; Gladish and Rost, 1993; McMichael and Burke, 1998), mechanical impedance (Goss, 1977; Feldman, 1984; Singh and Sainju, 1998), or light (Furuya and Torrey, 1964; Reinhardt and Rost, 1995) can modulate LR formation. Heavy metals, NaCl, and other chemicals in the root environment may be toxic for LR development (Malone et al., 1978; Waisel and Breckle, 1987; Reinhardt and Rost, 1995; Baligar et al., 1998). A deficit of certain nutrients also has a strong effect on LR formation (Baligar et al., 1998). Most of these factors probably regulate LR development through systemic influences on the metabolism at the whole-plant level. It is well known that nitrates favor branching of root systems (Hackett, 1972; Drew, 1975; Granato and Raper, 1989; Robinson, 1994). Similar effects have been reported for other inorganic nutrients such as ammonia and phosphates (Drew, 1975). As phosphates promote first-order LR elongation rather than branching, they can bring about the same results as nitrate, having different developmental effects (Robinson, 1994). Frequently, LRs that find favorable soil patches grow and branch profusely, whereas other roots, initiated in less favorable sites, suffer reduced growth. Such a local response to inorganic nutrients operates by different mechanisms to stimulate branching by enhancing growth at the whole-plant level. It has been proposed that tissues that receive adequate nitrogen supply are preferred sinks for photosynthates compared to tissues receiving limited nitrogen

Lateral Root Initiation

(Sattelmacher and Thoms, 1991). Another possibility is that nutrients, particularly nitrates, regulate hormone metabolism or transport in localized regions or even act themselves as developmental signals (Cao et al., 1993). Because recent research has offered evidence for this type of control mechanism, the effects of nitrate on LR initiation are of special interest (Zhang et al., 1999). Those authors have demonstrated that in A. thaliana roots nitrate ions control the formation of LRs by means of a dual mechanism; primarily they constitute a signal which promotes LR development from pericycle cells. This effect is localized in the root regions directly affected by nitrate, and seems to affect the development of primordia into LRs rather than their initiation. At the same time, the assimilated nitrates affect LR development by controls traceable to the whole-plant level. Curiously, this systemic effect is inhibitory rather than stimulatory (Zhang et al., 1999; Zhang and Forde, 2000). Experiments with auxinresistant mutants of A. thaliana have provided evidence for an overlap between the localized stimulatory effect of nitrate on LR elongation and the mechanism of response to auxin (Zhang et al., 1999; Zhang and Forde, 2000). Increase of branching frequency in response to nutrient availability may occur not only on a localized scale of a single-root axis, but also on a general scale of whole-root system (Granato and Raper, 1989; Gersani and Sachs, 1992; Bingham et al., 1997). A split-root hydroponics system was designed to test the effects of a heterogeneous supply of nitrate to only one of two axes of maize (Granato and Raper, 1989). Total allocation of dry matter between shoot and root was not altered in this experiment, but partitioning between the two axes was strongly affected. The þN axis receiving increased dry matter and producing more LRs comparatively to N axis. Pea plants grown in hydroponic culture with one half of their root system bathed in nutrient-rich solution and the other half in a less rich solution, also showed that LR development was favored in the former and inhibited in the latter (Gersani and Sachs, 1992). Hence, a compensatory growth seems to control branching in deprived versus well-fed roots. Endogenous factors clearly condition LR initiation. Excised roots growing in culture medium require a wide range of substances for LR formation. For example, pea roots required, in addition to auxin and sucrose, thiamin, nicotinic acid, adenine, and one or more micronutrients (Torrey, 1956). For plants growing in more natural conditions, LR formation is an energy-consuming process that can only occur at the

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expense of available metabolic substrates, particularly of assimilates originating in the shoot (Philipson, 1988). Recent investigations have confirmed the importance of sugars in LR formation. Feeding halves of wheat root systems with glucose resulted in an increase of branching in the fed half (Bingham et al., 1997). Such results have suggested that one of the main limiting endogenous factors regulating LR formation is sugars. As they sustain root growth, sugars are a necessary component of growth media when excised roots are grown in vitro. Sugars also accumulate in cavities just outside the LR primordia and contribute to their growth (Friedberg and Davidson, 1971; MacLeod and Francis, 1976). As with inorganic nutrients, it is difficult to know whether sugars act by increasing metabolism, as true signals (cf. Bingham et al., 1998), or by both mechanisms. Assimilate partitioning is mainly controlled by the sink strength of the different organs. The main root apex and the apex of developing LRs clearly compete for assimilates. Hence, when the root apex is removed the assimilates are redirected to LRs. The mode of dry matter partitioning between the LR in Pinus pinea depends on the time of decapitation. Removing the root tip of young seedlings without emerged laterals results in a homogeneous redistribution of assimilates along the main root axis. When an older taproot is decapitated after it has begun to form LRs, then the assimilates accumulate in basal regions of the root system where the laterals are developing. These results strongly suggest that there is competition for assimilates between the taproot apex and developing laterals. They are also consistent with the hypothesis that assimilates are more necessary for the elongation of LRs than for their initiation (Atzmon et al., 1994). The plant hormone auxin plays various key regulatory roles during LR initiation, organization of the apical meristem of the LR primordia, and finally the emergence and subsequent growth of the LR. In most plant species, exogenous auxins promote the initiation of LRs (Torrey, 1962; Blakely et al., 1972, 1982; Webster and Radin, 1972; Wightman et al., 1980; Zeadan and MacLeod, 1984; Hinchee and Rost, 1986; Hurren et al., 1988; MacIsaac et al., 1989; Lloret and Pulgarı´ n, 1992; Vuylsteker et al., 1997, 1998; Baum et al., 1998; Zhang and Hasenstein, 1999; see also Chapter 23 Gaspar et al., in this volume). The typical response to exogenous auxin treatment is the formation of new LR primordia out of the acropetal sequence. These out-of-sequence primordia have been called adventive LRs to distinguish them from adventitious or shootborne roots (Barlow,

144

1986). Concerning the acropetal series of LRs, some species appear to be insensitive to exogenous auxins (Charlton, 1983a, 1991). Furthermore, it has not been possible to correlate the LR frequency and endogenous auxin contents of maize excised root fragments (Golaz and Pilet, 1987). Hence, the evidence that links auxins to the formation of LRs has been considered to be circumstantial (Charlton, 1996). Nevertheless, using transgenic plants and developmental mutants we now have more clues that link auxins and LR initiation (Celenza et al., 1995; Larkin et al., 1996). Studies of LR development based on classical experiments involving exogenous auxin treatments certainly have limited value. Indeed, most studies performed with exogenous auxin treatments have suffered from three main handicaps: (1) they have frequently not distinguished between acropetal and adventive LRs; (2) they have not demonstrated the final auxin concentration that affect the pericycle cells; and (3) no distinction between the effects of exogenously applied versus endogenous auxin was made. Nevertheless, the evidence obtained suggests that auxins are involved in the regulation of LR formation. Several explanations were given to those particular cases where auxins apparently did not promote LR. The apparent insensitivity to exogenous auxin in some species could result from the permeation barriers impeding the exogenous auxin from reaching the target tissue—i.e., the pericycle (Blakely et al., 1986). An alternative explanation for the negative results in exogenous auxin treatments takes into account the limited time in which the root pericycle can initiate LRs (Abadı´ a-Fenoll et al., 1986; Lloret and Pulgarı´ n, 1992). Hence, the current view is that endogenous auxins are the main regulators of LR formation (Hobbie, 1998). Moreover, not only the auxin concentration but also the sensitivity of the pericycle and the efficiency of the auxin transport is relevant for the development of LRs. Perhaps the most direct evidence that links auxin to LR development comes from the demonstration that plants that overproduce auxins have also increased numbers of LRs. The sur1 mutants of A. thaliana have high auxin contents in root tissues and are considered to be auxin overproducers (Boerjan et al., 1995). The sur1 mutants showed increased LR formation (Boerjan et al., 1995). Transgenic plants that overexpress bacterial iia genes and consequently have high auxin levels, also tend to form many LRs (Klee et al., 1987). On the other hand, the dgt mutant of Lycopersicon esculentum, which shows reduced auxin sensitivity, has also impeded capacity of LR develop-

Lloret and Casero

ment (Zobel, 1973; Kelly and Bradford, 1986; Muday et al., 1995). Similarly, in A. thaliana, the combination of two auxin resistance mutations (axr4 and axr1) results also in few LRs compared to the parental lines or single mutants (Hobbie and Estelle, 1995). These mutants have multiple phenotype alterations compared to wild plants other than their modified capacity for LR formation. Nevertheless, there are three mutants which show specific alterations in LR formation in A. thaliana (Celenza et al., 1995). These have been named alf mutants (alf1, alf4 and alf3) and show defects at different stages of LR development. The first, alf1, is probably defective in auxin catabolism in the root, leading to high auxin concentrations in root tissues, which in turn promotes increased LR formation. This mutant is thought to be the same as sur1, hls3, and rty (Scheres et al., 1996). The second, alf4, seems to have a defect in primary perception or response to auxin as its pericycle cells are unable to form LRs. The third, alf3, initiates LR primordia, but they abort later. The mutant alf3 is rescued by exogenous auxin treatments, but alf4 is not. All three mutants provide strong support for the hypothesis that endogenous auxin is the major regulator of LR initiation and subsequent growth of the primordia (Celenza et al., 1995). Auxin transport in higher plants is a directional and regulated process. It has been known for a long time that auxin applied to the shoot eventually reaches the root (Morris et al., 1969; McDavid et al., 1972). Once in the root, auxin is transported acropetally in the central cylinder (Mitchel and Davies, 1975; Tsurumi and Ohwaki, 1978; Kerk and Feldman, 1995). It can accumulate in developing LR primordia (Rowntree and Morris, 1979) or at the root apex (Kerk and Feldman, 1994), where it induces a zone with organized pattern and polarity properties (Sabatini et al., 1999). When the auxin reaches the apex the movement is reversed, and auxin moves basipetally between the root tip and the elongation zone probably through the root epidermis and/or the outer cortex (Tsurumi and Ohwaki, 1978; Yang et al., 1990; Balus˘ ka et al., 1994; Estelle, 1998). The acropetal transport of auxins in the root is dependent on metabolic energy (Torrey, 1976). Given its pathway inside the root, the acropetal flux of auxin might influence the formation of LR primordia in the pericycle. There are three lines of evidence which relate auxin transport to LR initiation. In Pisum sativum seedlings, exogenous auxin applied to the plant to substitute the excised cotyledons mimics the role of these organs in the regulation of LR initiation and emergence

Lateral Root Initiation

(Hinchee and Rost, 1986). The second line of evidence for this relationship comes from the study of the aux1 and tir3 mutants of A. thaliana that show reduced numbers of LRs owing to a probable defect in the auxin transport mechanism (Ruegger et al., 1997; Bennett et al., 1996, 1998). Nevertheless, these mutants did not show dramatically altered LR formation. This fact probably occurs because the genetic redundancy in the multigene family encoding auxin transport proteins attenuate the effect of a single mutant locus. Treatments with auxin transport inhibitors result in much stronger effects. These compounds, e.g., naphtylphtalamic acid or semicarbazone derivative 1, are effective in inhibiting the formation of LRs in tomato (Muday and Haworth, 1994) and A. thaliana roots (Reed et al., 1998). We do not know whether primordia are initiated under such treatments in these species but either they subsequently do not emerge or their initiation has also been inhibited. In Pisum sativum roots, it appears that under auxin inhibitor treatment, structures similar to laterals called ‘‘root primordial masses’’ are initiated but fail to grow out because they are not capable of organizing an apical meristem. Such structures are distributed in regions along the parent root axis which normally produce laterals. When treatment is suppressed or exogenous auxin is supplied, they can grow as clusters of LRs (Hinchee and Rost, 1992a). Some of the alterations promoted by auxin transport inhibitors seem to be similar to the case of alf3 mutants (Celenza et al., 1995). The results in P. sativum roots are also consistent with the hypothesis that auxin transport inhibitors cause auxin redistribution and apparent morphogenetic effects in the root (Sabatini et al., 1999). Nitrogen-fixing nodules of leguminous plants are structures a related to LR development. Characterization of the genetic and physiological control of nodule formation has demonstrated that mutants with altered nodulation also show altered LR formation (Kneen et al., 1994). All the available evidence points to an increased auxin sensitivity as a cause for the relationship between nodule and LR formation. Furthermore, in the model species for determinate nodule formation, Lotus japonica, it was shown that the induction of nodule organogenesis and the initiation of LRs share common regulatory elements (de Bruijn et al., 1998). Auxin is not the only growth regulator that modulates LR formation. Exogenous kinetin at low concentrations stimulates auxin-induced LR production in excised pea roots, but at higher concentrations (higher than 4:6  105 ) it is a powerful inhibitor (Torrey,

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1962). The cytotokinin concentrations that inhibited LR formation have been considered as physiological, or at least nontoxical (Torrey, 1962). On the other hand, the spontaneous formation of LRs also seems to be inhibited by exogenous cytokinin in lettuce roots (MacIsaac et al., 1989). Furthermore, the studies of Wightman et al. (1980) and Van Staden and Ntingane (1996) have demonstrated that exogenous cytokinins inhibited not only initiation but also the emergence of LRs. As cytokinins are if not produced at least accumulated at the primary root apex and the apices of developing LRs (Feldman, 1975; Van Staden and Davey, 1979; Forsyth and Van Staden, 1981; but see Chapter 25 by Emery and Atkins in this volume), and the decapitation of primary roots transitorily stimulates LR formation (Wightman and Thimann, 1980; Forsyth and Van Staden, 1981; Lloret et al., 1988), it has been propossed that these hormones are the inhibitors that explain why LRs are not initiated near the root apex (Van Staden and Ntigane, 1996). The concept of apical dominance applied to roots has recently been revisited by Zhang and Hasenstein (1999) in lettuce roots (Lactuca sativa, cv. Baijianye). The seedlings of this cultivar do not normally produce LRs, but removal of the primary root tip triggers a rapid response and LR primordia are initiated in
9 h. As LR primordia are newly initiated in response to root decapitation and growth regulators that usually promoted the growth of shoot buds inhibited LR primordia development, it appears that root apical dominance is not a quite similar phenomenon to its counterpart in shoot development, although the same growth regulators, auxin and cytokinin, are involved (Cline, 1997). In fact, auxin and cytokinin concentrations along the parent root are thought to directly control LR initiation and emergence (Hinche and Rost, 1986). Webster and Radin (1972) reported interaction between auxin and cytokinins in excised pea roots growing in culture medium. In such roots the pericycle can initiate LRs or contribute to the formation of a cambium. The behavior of the pericycle depends on the ratio of exogenous auxin to cytokinins. Auxins alone promote LR initiation. Cytokinins alone bring about formation of a multiseriate pericycle. The combination of both growth regulators organizes a functional cambium. The control of LR formation would be better understood if it were analyzed in terms of interactions between hormones rather than by considering the effects of an isolated growth regulator. The exact nature of such interactions is for the moment unknown, and this is an aspect that deserves further research.

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Several Arabidopsis mutants with altered response to cytokinins have been isolated. These pas mutants show abnormal root system development. The mutants pas1 and pas3 develop short primary roots with no or with very few LRs, whereas pas2 shows long primary roots and increased numbers of LRs. Physiological and biochemical analyses show that cytokinins are involved in pas phenotypes. The pas mutants are not cytokinin overproducers but are probably altered in their sensitivity to cytokinin (Faure et al., 1998). If cytokinins are supposed to inhibit LR initiation, one important question remains unresolved: What happens in plant species that initiate their LR primordia close to the root apex. This occurs, for example, in Ceratopteris thalictroides and Cucurbita maxima (Mallory et al. 1970) or Pontederia cordata, Pistia stratiotes, and Musa acuminata (Charlton, 1987). We still have no clear explanation for this. One possibility is that LR formation is a constitutive capacity of the root apex and proceeds in these species even under high cytokinin concentrations at the site of initiation. Another is that the sensitivity to or the actual concentration of cytokinins differs from one species to another. Other phytohormones have a less clear influence and often give rise to inconsistent or even contradictory reports about their effects on LR development. Perhaps the reason is that they regulate this process no directly but through interaction with auxins and cytokinins. One phytohormone that is produced at the root apex and that inhibits LR initiation and emergence when applied exogenously is abcisic acid (ABA) (Hooker and Thorpe, 1998). It is interesting to note that the treatment of excised tomato roots cultured in vitro with fluoridone, an inhibitor of ABA biosynthesis, stimulates the initiation of LRs but not their emergence and subsequent growth (Hooker and Thorpe, 1998). The overall evidence suggests that gibberellic acid plays at most a minor role in regulating LR initiation (Torrey, 1976). The same is true for the ethylene, although this hormone might affect LR initiation through its well-known effect on auxin lateral transport (Lee et al., 1990). It has recently been reported that silver ions, known inhibitors of ethylene action, promote the elongation of LRs but have no effect on LR initiation in Lactuca sativa roots (Zhang and Hasenstein, 1999). From a cellular point of view, the initiation of a LR primordium at a specific site implies that pericycle and/ or endodermis cells can perceive signals that are responsible for organized LR development. Furthermore, they are able to integrate such signals

Lloret and Casero

with promotive or inhibitory physiological inputs. All these interactions between cells are far from being understood. Nevertheless, the concept is arising that root cells react to stimulus according to their location on a positional framework, which is used as a reference for patterning. One factor that contributes to this framework is polar auxin transport (Sabatini et al., 1999; Doerner, 2000). Hence, auxin is beginning to be viewed not as a classical morphogen as defined for animal organisms, but as somehow important in letting the cells know where they are located. V.

LATERAL ROOT PATTERNING

A.

Relationship of Lateral Root Initiation Sites with the Vascular Pattern

The most evident aspect of LR arrangement is that the LR primordia are initiated at specific positions related to the vascular pattern of the parent root. Hence they form ranks along the parent root. In some instances these ranks run along the protoxylem poles in others along the protophloem poles (McCully, 1975). B.

Arrangement of Laterals Along the Parent Root

When roots grow under controlled environmental conditions, they tend to form LR primordia at a regular rate. Consequently, the total number of LRs is positively correlated with the root length (Lloret et al., 1988), and similar numbers of roots per centimeter are observed along wide LR-bearing regions (Lloret and Pulgarı´ n, 1992). In some instances, the number of LRs formed decreases toward distal regions of intact roots (Hummon, 1962; Granato and Raper, 1989). Near the apex and in some instances also near the root base, there are two zones that lack LRs (Mallory et al., 1970; Lloret and Pulgarı´ n, 1992). The reason for the absence of LRs near the apex has been explained in general to the age of pericycle cells located in this region (Abadı´ a-Fenoll et al., 1986) or to the accumulation of cytokinins (Atzmon and Van Staden, 1994). What causes the basal LR-free region is unknown. Under experimental conditions we can promote or inhibit LRs in specific regions of the parent root. Exogenous auxin treatments result in accumulation of LRs at the distal end of the root (Lloret et al., 1992; Vuylsteker et al., 1998). Tritiated thymidine treatments suppress the initiation of LRs at regions of active formation, whereas they do not affect older

Lateral Root Initiation

root regions where LR formation has finished (Hummon, 1962). This means that not all cells along the root are equally reactive to the treatment, being more sensitive the younger they are. There is a gradient of reaction to experimental treatments, which suggests that the capacity for LR formation is limited in time and space (Abadı´ a-Fenoll et al., 1986).

C.

Pattern of Distribution Within Ranks

Some studies have analyzed the LR ranks individually (Mallory et al., 1970; Charlton, 1975, 1977, 1982, 1987; Barlow and Adam, 1988; Lloret et al. 1988, 1998; Pulgarı´ n et al., 1988; Hinchee and Rost, 1992b; Newson et al., 1993). In general, it is accepted that along each rank the LRs are rather regularly arranged (Charlton, 1991, 1996). Nevertheless, the distance between LRs is highly variable in most species. Perhaps the data that fit best a regular mathematical model are those obtained in Ceratopteris thalictroides. In this fern the root is diarch. As growth retracts from the root apex, the root is twisted and the LR primordia that develop opposite the xylem poles form two helical series. Within each series the LR primordia are regularly spaced (Mallory et al., 1970). Excised tomato roots show an interesting feature. The histogram of interlateral spacing of ranks belonging to individual roots of the same rank seems to be multimodal (Barlow and Adam, 1988). The peaks of frequency occur at multiples of a fundamental distance, or ‘‘quantum.’’ This opens up the possibility that segments or metamers could be at the basis of root development (architecture). When the available data were tested statistically they did not always show a significant trend (Newson et al., 1993). Nevertheless, this view of roots with metamers marked by LR positions should be further tested (see also Chapter 4 by Barlow in this volume). When LR primordia are initiated near the apex the spacing within ranks seems to follow a logarithmic scale. This is a consequence of the nearly exponential elongation of roots behind the apex. Hence, in Pontederia cordata and Pistia stratiotes, Charlton (1975, 1983b) analyzed root spacing in the form of log (p2/p1), where p2 and p1 are the positions of two successive LRs behind the root tip and p2 is the smaller value. This parameter was defined as the rhizotron ratio and has been used for the study of LR patterning in Musa acuminata (Charlton, 1982; Draye et al., 1999). All these species also showed a regular spacing of successive LRs along each rank.

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Between different ranks, an apparent interaction could arise from a persistent synchrony of LR initiation in different ranks (Fig. 12). The simplest example of such a synchrony is the root of C. thalictroides (Mallory et al., 1970). In this fern, two protoxylembased ranks of LRs initiate LR primordia sequentially, thus forming pairs of LRs. These LR primordia are slightly displaced with respect to each other in the longitudinal plane but away from other pairs. Hence groups of two LRs can be discerned in this species. The regularity in LR spacing seems to be a consequence of the regular segmentation of the root apical cell and consequently could be due to a mechanism of synchrony between ranks similar to that shown in Fig. 12. The LR positioning in another fern with diarch roots has also been related to the pattern of segmentation of the apical cell (Charlton, 1983a). Higher-plant roots, of Musa acuminata, Pistia stratiotes, and Pontederia cordata, have shown apparent interactions between ranks, which finally have been demonstrated to be spurious (Charlton, 1987). It appears that in tomato roots the two main ranks of laterals also form primordia independently (Barlow and Adam, 1988). On the other hand, there were some references to clumping of LRs (Yorke and Sagar, 1970; Lloret et al., 1988), which suggest possible interaction between ranks in P. sativum and A. cepa. The tendency to clump may lead to the formation of LR clumps at the same transversal level of the parent root. The number of groups and the number of members of each group of onion roots widely exceed what one would expect based on random coincidence (Pulgarı´ n et al., 1988; Lloret et al., 1988, 1998). The correlation between ranks is also statistically significant for roots of Potentilla palustris (Charlton, 1987). The possibility of interaction between ranks, is intriguing but difficult to prove. The fact that in some species LR primordia inhibit the formation of new primordia in the longitudinal plane, whereas they tend to be formed grouped at the same transversal level, supports the hypothesis of the control by a growth regulator that is transported in a polar pathway. The pattern of LR distribution may be set by the root apex as part of the general process of cell production and differentiation. Alternatively, the pattern of LRs can be determined later, by stimulating mother cells by hormones or by other mobile morphogens. In reality, the two models are not mutually exclusive. Regular spacing within ranks may arise from differences in the longitudinal distribution of nutrients or

148

Lloret and Casero

Figure 12 The possible theoretical relationships between two ranks forming LR primordia (arrowheads) in a physiological background of stimulating or inhibiting factors. When LR primordia coincide at the same transverse level (left center), it can occur because of a simple coincidence without interaction between LR primordia belonging to both ranks (upper left drawing), because of a local stimulus originated in surrounding tissues (left middle) or because of a stimulating effect originated within the proper primordia which tend to be formed grouped at the same transverse level (left bottom). When the LR primordia do not coincide (right center), then it also can occur randomly (upper right) or by an inhibiting influence originated in initiating LR primordia which would move through the vascular cylinder impeding the formation of LR primordia at the same transverse level (bottom right).

growth regulators within the pericycle and/or endodermis cells (Riopel, 1966, 1969; Pulgarı´ n et al., 1988). Initially, such chemical differences do not have to be large as they can undergo self-amplification by a sort of autocatalytic process (Meinhardt, 1984). This would break the uniformity of pericycle and/or endodermis cells in the longitudinal direction. Once generated, the more or less regularly spaced periodic chemical patterns of primordial initiation would proceed at predictable distances from each other and with apparent mutual repulsion. This is similar to the classical view of the order of leaf arrangement on the shoot (Chapman and Perry, 1987). Another possibility, not necessarily incompatible with the former, is that the pattern of LR distribution would be set by the root apex by the formation of a series of mother cells at regular intervals (Barlow and Adam, 1988). These intervals may be considered on the basis of distances between successive LR primordia or better as intervening cells between them. The FPCs initially commited to develop LR primordia could eventually do it or not, depending on the physiological conditions at the moment of LR initiation (Charlton, 1983a; Barlow and Adam, 1988). Hence, more sites for LRs may be determined than actually develop. In fact, an investigation of the relationships between pericycle cell length and the number of LR primordia in decapitated onion

roots has shown that decapitation stimulated LR formation in distal regions to a greater extent than it inhibited pericycle cell elongation (Lloret et al., 1985). D.

Asymmetry of Lateral Root Development

Not all ranks of a given root necessarily form LRs at the same rate. The LRs of adventitious roots of onion are initiated near two neighboring ranks (Lloret et al., 1988; Pulgarı´ n et al., 1988). This pattern is altered if the roots are treated with auxins (Lloret et al., 1998). Asymmetric distribution of laterals between ranks has also been described for the development of early laterals (probably basal roots) on the pregerminative root of Theobroma cacao L. (Dyanat-Nejad and Neville, 1972). The ranks with the greatest number of LRs of such roots are connected with the central xylem poles of the cotyledons. A similar relation was found for the LR primordia of P. sativum preformed on the embrionary radicle (Hinchee and Rost, 1992b). In this case, the primordia were most abundant on the vascular strand connected with one of the cotyledons Apparently, the capacity for LR formation of each individual rank is related to the liberation of some growth regulator by the underlying vascular strands. Sometimes the asymmetry occurs in more distant locations along the root. For example, in curved root

Lateral Root Initiation

segments, more LR primordia arise on the convex side and none on the concave side (McCully, 1975; Fortin et al., 1989). For the moment we have no clear explanation for the reason of all these asymmetries in LR development.

VI.

CONCLUDING REMARKS

Noticeable results have recently been obtained by using new techniques. However, a lot of very important questions about LR formation yet remain unresolved and deserve particular attention in future research. Now, it is known that in angiosperms the first morphological event associated to LR initiation is the polarized asymmetrical transverse division of a pair of pericycle cells. Nevertheless, other molecular and cellular phenomena should precede the activation of FPCs to initiate LR development. These events are under study and it is certain that new cues about them will emerge in the next few years. The transition from the initial phase of development to the organization of the apical meristem of the LR has been shown to involve changes in the expresion of several cell markers and in the physiology of the cells of LR primordia. The interactions between relevant cells for LR development are partially understood. Concerning the analysis of the factors controlling LR initiation, the situation is no better. Although we are aware that this event is related to the effect of various growth regulators, the mode of action of phytohormones on LR development is to be understood at the cellular level. Moreover, it is not known if LR spacing within ranks is due to the distribution of nutrients and growth regulators within pericycle cells, or if it is traceable to the divisional history at the apex producing FPCs at regular intervals, or if it is due to a combination of these two possibilities. It is also unknown why in some species LRs arise opposite xylem parent root poles, whereas in others they do it opposite phloem poles. Partial answers to some of these problems are available. However, a full understanding is still far away. To optimize resources it is particularly important to concentrate research effort on a few well-known models—i.e., Allium, Arabidopsis, Lycopersicon, Pisum, and Zea. As more genes regulating LR development become available, specific steps of LR development will be targets for genetic modification to dissect accurately the whole process.

149

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Lateral Root Initiation Laskowski MJ, Williams ME, Nusbaum HC, Sussex IM. 1995. Formation of lateral root meristems is a twostage process. Development 121:3303–3310. Lee JS, Chang WK, Evans ML. 1990. Effects of ethylene on the kinetics of curvature and auxin redistribution in gravistimulated roots of Zea mays. Plant Physiol 94:1770–1775. Lin B-L, Raghavan V. 1991. Lateral root initiation in Marsilea quadrifolia. I. Origin and histogenesis of lateral roots. Can J Bot 69:123–135. Lloret PG, Pulgarı´ n A. 1992. Effect of naphthaleneacetic acid on the formation of lateral roots in the adventitious root of Allium cepa: number and arrangement of laterals along the parent root. Can J Bot 70:1891– 1896. Lloret PG, Vidal MR, Casero PJ, Navascue´s J. 1985. The relationship between lateral-root distribution and endodermis and pericycle cell length in Allium cepa L. adventitious roots. Ann Bot Lond 56:189– 195. Lloret PG, Casero PJ, Navascue´s J, Pulgarı´ n A. 1988. The effects of removal of the root tip on lateral root distribution in adventitious roots of onion. New Phytol 110:143–149. Lloret PG, Casero PJ, Pulgarı´ n A, Navascue´s J. 1989. The behaviour of two cell populations in the pericycle of Allium cepa, Pisum sativum, and Daucus carota during early lateral root development. Ann Bot Lond 63:465– 475. Lloret PG, Pulgarı´ n A, Casimiro I, Molina M, Casero PJ. 1998. Effect of a treatment with naphthalene-acetic acid on the arrangement of lateral roots in onion (Allium cepa L.): general pattern and coordination between ranks. Bot Acta 111:55–61. Luxova´ M. 1975. Some aspects of the differentiation of primary root tissues. In: Torrey JG, Clarkson DT, eds. The Development and Function of Roots. London; Academic Press, pp 73–90. Luxova´ M. 1990. Effect of lateral root formation on the vascular pattern of barley roots. Bot Acta 103:305– 310. Lyford WH. 1975. Rhizography of non-woody roots of trees in the forest floor. In: Torrey JG, Clarkson DT, eds. The Development and Function of Roots. London; Academic Press, pp 179–196. MacIsaac SA, Sawhney VK, Pohorecky Y. 1989. Regulation of lateral root formation in lettuce (Lactuca sativa) seedling roots: Interacting effects of -naphthaleneacetic acid and kinetin. Physiol Plant 77:287–293. MacLeod RD. 1990. Lateral root primordium inception in Zea mays L. Environ Exp Bot 30:225–234. MacLeod RD, Francis D. 1976. Cortical cell breakdown and lateral root primordium development in Vicia faba. J Exp Bot 27:922–932.

153 MacLeod RD, McLachlan SM. 1974. The development of a quiescent centre in lateral roots of Vicia faba L. Ann Bot Lon 38:535. MacLeod RD, Thompson A. 1979. Development of lateral root primordia in Vicia faba, Pisum sativum, Zea mays and Phaseolus vulgaris. Rates of primordium formation and cell doubling times. Ann Bot Lond 44:435– 449. Malamy JE, Benfey PN. 1997. Organization and cell differentiation in lateral roots of Arabidopsis thaliana. Development 124:33–44. Mallory TE, Chiang SH, Cutter EG, Gifford EM Jr. 1970. Sequence and pattern of lateral root formation in five selected species. Am J Bot 57:800–809. Malone CP, Miller RJ, Koeppe DE. 1978. Root growth in corn and soybeans: effects of cadmium and lead on lateral root initiation. Can J Bot 56:277–281. McCully ME. 1975. The development of lateral roots. In: Torrey JG, Clarkson DT, eds. The Development and Function of Roots. London; Academic Press, pp 105– 124. McDavid CR, Sagar GR, Marshall C. 1972. The effect of auxin from the shoot on root development in Pisum sativum L. New Phytol 71:1027–1032. McMichael BL, Burke JJ. 1998. Soil temperature and root growth. Hortscience 33:947–951. Meinhardt H. 1984. Models of pattern formation and their application to plant development. In: Barlow PW, Carr DJ, eds. Positional Controls in Plant Development. Cambridge, UK: Cambridge University Press, pp 1–32. Mitchell EK, Davies PJ. 1975. Evidence for three different systems of movement of indoleacetic acid in intact roots of Phaseolus coccineus. Physiol Plant 33:290–294. Mirza JI, Olsen GM, Iversen TH, Maher EP. 1984. The growth and gravitropic responses of wild-type and auxin-resistant mutants of Arabidopsis thaliana. Physiol Plant 60:516–522. Moore R, Pasieniuk J. 1984. Graviresponsiveness and the development of columella tissue in primary and lateral roots of Ricinus comunis. Plant Physiol 74:529–533. Morris DA, Briant RE, Thompson PG. 1969. The transport and metabolism of 14C-labelled indoleacetic acid in intact pea sedlings. Planta 89:178–197. Muday GK, Haworth P. 1994. Tomato root growth, gravitropism, and lateral development: correlation with auxin transport. Plant Physiol Biochem 32:193–203. Muday GK, Lomax TL, Rayle DL. 1995. Characterization of the growth and auxin physiology of roots of the tomato mutant, diageotropica. Planta 195:548–553. Newson RB, Parker JS, Barlow PW. 1993. Are lateral roots of tomato spaced by multiples of a fundamental distance? Ann Bot Lond 71:549–557. Nishimura S, Maeda E. 1982. Cytological studies on differentiation and dedifferentiation in pericycle cells of excised rice roots. Jpn J Crop Sci 51:553–560.

154 Oparka KJ, Prior DAM, Wright KM. 1995. Symplastic communication between primary and developing lateral roots of Arabidopsis thaliana. J Exp Bot 46:187–197. Pellerin S, Tabourel F. 1995. Length of the apical unbranched zone of maize axile roots. Its relationship to root elongation rate. Environ Exp Bot 35:193–200. Peretto R, Favaron F, Bettini V, De Lorenzo G, Marini S, Alghisi P, Cervone F, Bonfante P. 1992. Expression and localization of polygalacturonase during the outgrowth of lateral roots in Allium porrum L. Planta 188:164–172. Peterson RL. 1992. Adaptations of root structure in relation to biotic and abiotic factors. Can J Bot 70:661–675. Peterson RL, Peterson CA. 1986. Ontogeny and anatomy of lateral roots. In: Jackson MB, ed. New Root Formation in Plants and Cuttings. Dordrecht, Netherlands: Martinus Nijhoff, pp 1–30. Philipson JJ. 1988. Root growth in Sitka spruce and Douglas-fir transplants: dependence on the shoot and stored carbohydrates. Tree Physiol 4:101–108. Popham RA. 1955. Zonation of primary and lateral root apices of Pisum sativum. Am J Bot 42:267–273. Pulgarı´ n A, Navascue´s J, Casero PJ, Lloret PG. 1988. Branching pattern in onion adventitious roots. Am J Bot 75:425–432. Reed RC, Brady SR, Muday GK. 1998. Inhibition of auxin movement from the shoot into the root inhibits lateral root development in Arabidopsis. Plant Physiol 118:1369–1378. Reinhardt DH, Rost TL. 1995. Primary and lateral root development of dark- and light-grown cotton seedlings under salinity stress. Bot Acta 108:457–465. Riopel JL. 1966. The distribution of lateral roots in Musa acuminata ‘‘Gros Michel.’’ Am J Bot 53:403–407. Riopel JL. 1969. Regulation of lateral root positions. Bot Gaz 130:80–83. Robinson D. 1994. The responses of plants to non-uniform supplies of nutrients. New Phytol 127:635–674. Rost TL, Jones TJ, Falk RH. 1988. Distribution and relationship of cell division and maturation events in Pisum sativum (Fabaceae) seedling roots. Am J Bot 75:1571–1583. Rowntree RA, Morris DA. 1979. Acumulation of 14C from exogenous labelled auxin in lateral root primordia of intact pea seedlings (Pisum sativum L.). Planta 144:463–466. Ruegger M, Dewey E, Hobbie L, Brown D, Bernasconi P, Turner J, Muday G, Estelle M. 1997. Reduced naphthylphthalamic acid binding in the tir3 mutant of Arabidopsis is associated with a reduction in polar auxin transport and diverse morphological defects. Plant Cell 9:745–757. Sabatini S, Beis D, Wolkenfelt H, Murfett J, Guilfoyle T, Malamy J, Benfey P, Leyser O, Bechtold N, Weisbeek P, Scheres B. 1999. An auxin-dependent dis-

Lloret and Casero tal organizer of pattern and polarity in the Arabidopsis root. Cell 99:463–472. Sattelmacher B, Thoms K. 1991. Morphology of maize root systems influenced by local supply of nitrate or ammonia. In: McMichael BL, Persson H, eds. Plant Roots and Their Environment. Amsterdam; Elsevier, pp 149– 156. Sattelmacher B, Marschner H, Ku¨hne R. 1990. Effects of the temperature of the rooting zone on the growth and development of roots of potato (Solanum tuberosum). Ann Bot Lond 65:27–36. Schade C, Von Guttenberg H. 1951. U¨ber die Entwicklung des Wurzelvegetations Punktes der Monokotyledonen. Planta 31:170–198. Scheres B, Di Laurenzio L, Willemsen V, Hauser MT, Janmaat K, Weisbeek P, Benfey PN. 1995. Mutations affecting the radial organisation of the Arabidopsis root display specific defects throughout the embryonic axis. Development 121:53–62. Scheres B, McKhann HI, Van den Berg C. 1996. Roots redefined: anatomical and genetic analysis of root development. Plant Physiol 111:959–964. Schiefelbein JW, Benfey PN. 1991. The development of plant roots. New approaches to underground problems. Plant Cell 3:1147–1154. Seago JL. 1973. Developmental anatomy in roots of Ipomoea purpurea. II. Initiation and development of secondary roots. Am J Bot 60:607–618. Seago JL, Marsh LC. 1990. Origin and development of lateral roots in Tipha glauca. Am J Bot 77:713–721. Singh BP, Sainju UM. 1998. Soil physical and morphological properties and root growth. Hortscience 33:966–971. Skene KR, Kierans M, Sprent JI, Raven JA. 1996. Structural aspects of cluster root development and their possible significance for nutrient acquisition in Grevillea robusta (Proteaceae). Ann Bot Lond 77:443–451. Stoffella PJ, Lipucci Di Paola M, Pardossi A, Tognoni F. 1992. Seedling root morphology and shoot growth after seed priming or pregermination of bell pepper. Hortscience 27:214–215. Sutcliffe JF, Sexton R. 1968. -Glycerophosphatase and lateral root development. Nature 217:1285. Torrey JG. 1956. Chemical factors limiting lateral root formation in isolated pea roots. Physiol Plant 9:370–388. Torrey JG. 1961. The initiation of lateral roots. In: Recent Advances in Botany. Toronto; University of Toronto Press, pp 808–812. Torrey JG. 1962. Auxin and purine interactions in lateral root initiation in isolated pea root segments. Physiol Plant 15:177–185. Torrey JG. 1976. Root hormones and plant growth. Annu Rev Plant Physiol 27:435–459. Torrey JG. 1986. Endogenous and exogenous influences on the regulation of lateral root formation. In: Jackson MB, ed. New Root Formation in Plants and

Lateral Root Initiation Cuttings. Dordrecht, Netherlands: Martinus Nijhoff, pp 31–66. Tschermak-Woess E, Dolezal R. 1953. Durch Seitenwurzelbildung induzierte und spontane Mitosen in den Dauergeweben der Wurzel. Ost Bot Zeit 100:358–402. Tsurumi S, Ohwaki Y. 1978. Transport of 14C-labeled indoleacetic acid in Vicia faba root segments. Plant Cell Physiol 19:1195–1206. Van Staden J, Davey JE. 1979. The synthesis, transport and metabolism of endogenous cytokinins. Plant Cell Environ 2:93–106. Van Staden J, Ntingane BM. 1996. The effect of a combination of decapitation treatments, zeatin and benzyladenine on the initiation and emergence of lateral roots in Pisum sativum. S Afr J Bot 62:11–16. Van Tieghem P, Douliot H. 1888. Recherches comparatives sur l’origine des membres endoge`nes dans les plantes vasculaires. Ann Sci Nat (Bot), 7ie`me se´rie 8:1–660. Vidal-Bernabe´ MR, Garcı´ a de la Puerta-Lo´pez P, Lo´pezGarrido E. 1998. Origin, formation and maturation of vascular tracheary elements between the main root and the lateral root of Allium cepa. Phyton Int J Exp Bot 63: 211–220. Vuylsteker C, Leleu O, Rambour S. 1997. Influence of BAP and NAA on the expression of nitrate reductase in excised chicory roots. J Exp Bot 48:1079–1085. Vuylsteker C, Dewaele E, Rambour S. 1998. Auxin induced lateral root formation in chicory. Ann Bot Lond 81:449–454. Waisel Y, Breckle SW. 1987. Differences in responses of radish roots to salinity. Plant Soil 104:191–194. Webster PL, MacLeod RD. 1980. Characteristics of root apical meristem cell population kinetics: a review of analyses and concepts. Environ Exp Bot 20:335–358. Webster BD, Radin JW. 1972. Growth and development of cultured radish roots. Am J Bot 59:744–751. Wightman F, Thimann KV. 1980. Hormonal factors controlling the initiation and development of lateral roots. I. Sources of primordia-inducing substances in the primary root of pea seedlings. Physiol Plant 49:13–20. Wightman F, Schneider EA, Thimann KV. 1980. Hormonal factors controlling the initiation and development of

155 lateral roots. II. Effects of exogenous growth factors on lateral root formation in pea roots. Physiol Plant 49:304–314. Wilcox HE. 1968a. Morphological studies of the roots of red pine, Pinus resinosa. I. Growth characteristics and patterns of branching. Am J Bot 55:247–254. Wilcox HE. 1968b. Morphological studies on the root of red pine, Pinus resinosa. II. Fungal colonization of roots and the development of mycorrhizae. Am J Bot 55:686–700. Yang RL, Evans ML, Moore R. 1990. Microsurgical removal of epidermal and cortical cells evidence that the gravitropic signal moves through the outer cell layers in primary roots of maize. Planta 180:530–536. Yorke JS, Sagar GR. 1970. Distribution of secondary root growth potential in the root system of Pisum sativum. Can J Bot 48:699–704. Zeadan SM, MacLeod RD. 1984. Some effects of indol-3-ylacetic acid on lateral root development in attached and excised roots of Pisum sativum L. Ann Bot Lond 54:759–766. Zhang HM, Forde BG. 2000. Regulation of Arabidopsis root development by nitrate availability. J Exp Bot 51:51– 59. Zhang NG, Hasenstein KH. 1999. Initiation and elongation of lateral roots in Lactuca sativa. Int J Plant Sci 160:511–519. Zhang HM, Jennings A, Barlow PW, Forde BG. 1999. Dual pathways for regulation of root branching by nitrate. Proc Natl Acad Sci USA 96:6529–6534. Zimmerman PW, Hitchcock PW. 1935. The response of roots to ‘‘root forming’’ substances. Contrib Boyce Thompson Inst 7:439–445. Zobel RW. 1973. Some physiological characteristics of the ethylene-requiring tomato mutant diageotropica. Plant Physiol 52:385–389. Zobel RW. 1975. The genetics of root development. In: Torrey JG, Clarkson DT, eds. The Development and Function of Roots. London; Academic Press, pp 261– 275. Zobel RW. 1991. Genetic control of root systems. In: Waisel Y, Eshel A, Kafkafi U, eds. Plant Roots: The Hidden Half. New York; Marcel Dekker, pp 27–38.

9 Functional Diversity of Various Constituents of a Single Root System Yoav Waisel and Amram Eshel Tel Aviv University, Tel Aviv, Israel

environments (Caldwell, 1994; Fitter, 1996; see also Chapter 53 by Nobel in this volume.) Very few of the numerous roots that constitute one root system are exposed to the same conditions. Usually, different roots within each root system are exposed to different physical or chemical conditions that prevail in various microsites within the rooting volume. Moreover, the roots themselves increase the environmental variability by depleting certain zones of the soil of minerals and water (Grime, 1994; Stark, 1994), by secreting organic compounds that are utilized by microorganisms and by changing the ionic composition of their immediate rhizosphere (see Chapter 36 by Neumann and Roemheld in this volume). One source of variation is the gradual maturation and aging of root tissues. As the root system develops, younger parts are added while others mature and eventually senesce and die. In most cases the meristem of the root apex can continue to generate young undifferentiated cells for a long period of time. Therefore, at any instance in time, root segments of different ages and degrees of development can be found along the same root axis (see Chapter 7 by Silk in this volume). These stages of maturation correlate with differences in associated microbial and mycorrhizal activities (Stark, 1994). Such differences in microbial associations between tap and lateral roots can be found even in roots of the

Because the Faculty or Power of a Body, lieth not in any of its Principles apart; but is a Resultance from them all; or from their being, in such peculiar sort and manner, United and Combined together. So the several parts of a Clock, it is their Form, by which they are, what they are; yet it is the setting together of such Parts, and in such a way only, that makes them a Clock. Nehemia Grew (1672)

I.

INTRODUCTION

Root systems are congregates of several individual components that together constitute the functional ‘‘hidden half’’ of plants (cf. Bohm, 1979; Feldman, 1984; Eshel and Waisel, 1996). This attribute is of special importance for roots of terrestrial plants that occupy heterogeneous environments that vary spatially and temporally (Caldwell and Pearcy, 1994). The ability of a plant to produce different types of roots is an inherent aspect of its plasticity which has an important adaptive characteristic (Barlow, 1993; Bell and Lechowicz, 1994). Variation in traits among the various components of plant root systems affects the capability of these plants to cope with their complex 157

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same plant that are grown in aeroponics, i.e., in an absolutely uniform environment (Waisel, 1996). As the individual parts of a root system develop at different microsites, under different internal and environmental conditions, and are of a variety of ages, variations in growth and physiological characteristics among them should be expected. However, though the structure and function of ‘‘typical’’ roots have been amply investigated, little attention has been given to the fine distinction in physiological traits that exist among them (Waisel and Breckle, 1987). In certain plants, several types of roots originate from different tissues and organs. Root systems of grasses are composed of a few basic types of roots: embryonic roots (including the primary root and the other seminal roots that develop adventitiously from the embryonic nodes) and roots that develop later from the lower stem nodes (adventitious, nodal, or crown roots). Roots of both systems remain active for long periods, and some of them may support the plant during the entire course of its life (Klepper et al., 1984). Nevertheless, during different stages of development, each of the two groups of roots supports different allotments of the shoots: the seminal roots support mainly the primary shoot, although some support is also given to the tillers. On the other hand, adventitious roots are basically connected only with one, or with very few of them. The two groups also differ in their physiological performance, and the contribution of seminal roots to the whole plant exceeds what would have been implied from their fractal mass. However, because of their ramified connections with various parts of the shoot, seminal roots are more important for the survival of whole plants than the adventitious roots. Many prostrate plants grow adventitious nodal roots from aboveground stems, from stolon, and from rhizomes. This additional variation of functional roots allows such plants to expand the soil area which they exploit. This may be an important trait for adaptation to habitats where the soil is shallow or waterlogged. The development of different root classes determines eventually the shape of the root system of mature plants. Root systems of various forage grasses (e.g., Lolium multiflorum, Phleum pratense, Festuca arundinacea, or Dactylis glomerata) develop bilayered root systems. Such plants have one group of surface roots and a second group of roots that penetrate deep into the soil. The roots of the first group are relatively uniform in length and diameter and exhibit high tensile strength (Kobayashi, 1977; Stokes and Mattheck,

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1996; Nicoll, 2000). The deep roots are more heterogeneous but generally have a lower tensile strength. A similar phenomenon is found in trees (e.g., Ficus sp., Tamarix aphylla) and other woody spieces (e.g., Retama roetam). There are also differences among roots of dicotyledonous plants, composed of main roots and of several types of laterals (see Chapter 2 by Fitter in this volume). Annual crop plants like beans (Phaseolus vulgaris) (Lynch and van Beem, 1994), sunflowers (Helianthus annuus) (Tamir, Eshel, and Waisel, unpublished data), tomato (Lycopersicon esculentum), and others have in addition to their taproot and the laterals that emerge from it, a group of basal roots which originate in the hypocotyl immediately beneath the soil surface (Zobel , 1986). Such basal roots are less sensitive to gravity and extend the root system horizontally. They branch as much as the taproot or even more, and increase the specific root density at the upper soil layers. This root mass exploits the most fertile portions in agricultural soils and can perform its physiological roles efficiently. Surface roots of wheat (Triticum aestivum) and rape (Brassica campestris) are outstanding in their capability to absorb nutrients. Most of the nutrient requirements of the mature plants of these species were satisfied by the upper roots. Only a fraction of the nutrients were absorbed from deeper soil layers (Bole, 1977). Such behavior was also observed for water uptake by wheat plants. The differences in uptake between the surface roots and the deeper ones are typical of the species; they were found for plants grown in root boxes, despite the uniform water content, soil composition, bulk density, and temperature of the medium (Bole, 1977). The adaptation of Lupinus albus plants to heterogeneous soils depends, in part, on their capability to produce short root clusters in response to acidic soil conditions. The development of cluster roots in acid soil horizons enables the plants to exploit the whole depth of the soil profile (Karley, 2000). Explanation of root functions at the level of the physiological mechanism requires knowledge of the composition and characteristic of the population of roots (Aguirrezabal et al., 1993). From an agricultural point of view, knowledge of the differences in behavior that exist among various root types, and of the magnitude of their variations, is most important. It determines the functional efficiency of the whole root system. However, not enough is known about these characteristics to serve as a basis for breeding or for genetic engineering of new cultivars for more efficient root systems. Among a list of some 1400 officially

Functional Diversity

released mutant varieties only two were for root architecture (Maluszynski et al., 2000). In this chapter we will address the following questions: What are the inherited differences among roots? What characteristics are involved? How do different types of roots respond to their environment?

II.

STRUCTURAL DIFFERENCES

A.

Architecture

Every root system comprises several components. In dicots the taproot is the first leader, which bears laterals. In most cases, some of the first-order laterals and the basals become leaders that elongate fast, persist for a long time, and thicken with time. These leaders contain most of the biomass of the root system and form the long-distance transport pathways that conduct water and nutrients. The higher-order laterals are usually fine roots that make up most of the surface area of the root system. These fine roots have a limited elongation period and they are usually short-lived (see Chapter 13 by Eissenstadt and Yanai in this volume for discussion of root lifespan). Differences in the thickness of the cortex and the number of xylem vessels exist between the main axis and the laterals of tomato plants (Eshel et al., 2000). The most obvious differences among the various groups of roots are the structural differences. It was noted that even in the simple and conservative root system of Arabidopsis thaliana the anatomical structure of lateral roots is different from that of the taproot (Aeschbacher et al., 1994). Differences in structure among individual roots can be traced to the site of initiation, to the size of the primordia, and to the age of the roots. The dimensions of the proximal meristem of primary roots of maize vary within a relatively wide range. The size of the meristems has an important impact on rates of their elongation and on their performance. The size of primordia of adventitious roots was shown to have a greater impact on subsequent development than the time of their initiation (cf. Luxova and Lux, 1981). The size of the primordia affects not only the subsequent growth rates of roots but also their longevity. Certain plant genera have an additional type of roots: they have dense bottle-brush-like clusters of short lateral roots covered with a dense mat of root hairs. The archetype of this so-called Proteid roots is found in the Proteaceae (see Chapter 55 by Pate and Watt in this volume). Lupinus albus plants growing on

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P deficient soil formed such proteoid roots as well (Skene and James, 2000). Short-root clusters are also common in pines (Wilcox 1967). Short roots of Pinus resinosa plants seem to be rapidly aborted, presumably because of the small size of their meristems. Primordia of adventitious roots of various water plants are also dimorphic: The ventral primordia of Nymphaea candida and of Nuphar lutea are capable of developing into full-size roots, whereas the upper ones either remain undeveloped (Kadej and Kadej, 1983), or develop into short roots or into aerenchymatous ones (Ellmore, 1981; Waisel and Agami, 1996). B.

Anatomy

Various roots differ in their capability for lateral transport of nutrients. To a certain degree, this has a structural basis and may be caused by variations in the conductance of the plasmodesmata. Indeed, the pore size of the plasmodesmata was found to differ among different cell types of one organ as well as among different organs (Erwee and Goodwin, 1985). Roots also differ in the structure of various mature cells. Casparian strips (i.e., the endodermal structures that control ion transport into the stele) are formed several millimeters beyond the tips of fast-growing leader roots but appear much closer to the tips in fine laterals. Thus, transport of ions into fast-growing roots would be less selective because solutes would be drawn into the xylem via the apoplast of the gap between the mature endodermis and the root tip. Ion selectivity of fine roots is much better, because of the smaller gap between the mature endodermis and the tips. Plant strategy may follow one of two alternatives for root architecture: production of long, strong, fastgrowing roots, thus sacrificing some of the selective capability; or production of fine, slowly growing roots, with the gain of better control of ion movement into the tops. In salt-treated plants, the second option seems to be preferred (cf. Hajibagheri et al., 1985). Thus, ion content of the plant seems to be determined not only by the physiological traits of the roots but also by the ratio of long roots to short ones. A series of studies McCully and colleagues (McCully and Canny, 1988; Wenzel et al., 1989; Varney and McCully, 1991; Varney et al., 1991; McCully and Mallett, 1993) described in detail the structure of maize root systems and of their distinct components. The younger parts of the nodal roots, and of their laterals, were shown to have closed immature metaxylem vessels. These root portions were

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covered by soil sheaths. As the roots mature, their metaxylem elements open and lose their sheaths. McCully and Canny (1988) attributed the shedding of this sheath to the drying of the soil surrounding the root surface. Most of the lateral roots of maize are short and lose their active meristems as they mature. Such lateral roots have been classified into four different types on the basis of their thickness and anatomy (Varney et al., 1991). The frequency distribution of diameters of roots of maize was bimodal with a minimum diameter of 0.6 mm; 97% of axile roots were larger than this value and 98% of laterals were smaller (Chan et al., 1989). There was a correlation between root diameter and elongation rate, with laterals having a steeper slope. The data have also indicated that laterals elongated during 2.5 days only, while axile roots continued to grow throughout the plant’s life. Jordan et al. (1993) recognized 14 different models of xylem organization in roots of maize plants grown in soil or in aeroponic culture. The number of differentiated metaxylem vessels increased in nodal roots and in long laterals with internode rank, with the nodal roots having higher proportion of xylem and higher calculated hydraulic conductivity. The short laterals stopped their development at the time of late metaxylem differentiation. Thus, 92% of the short roots of maize had only one differentiated vessel. Short laterals also had smaller-diameter vessels than long ones. Significant variation in diameter was also found among long laterals of nodal roots that were initiated from various nodes. Variations among individual roots in the numbers of xylem and phloem strands of the vascular bundles were also reported for certain species of trees. Variations between three and six strands per root were reported for seedlings of Abies alba, between four and six strands for individual roots of Quercus rubra, and between six and eight strands for seedlings of Quercus robur (Rypacek et al., 1976). No doubt that roots with the larger number of vascular strands would have a better capability for long-distance transport of water and of minerals than would roots with a lower number of strands. Moreover, the larger number of phloem strands seems also to affect the subsequent development of such roots. This would have a selfenhancement effect, first increasing the translocation of assimilates to the phloem-rich roots, and consequently increasing their size and accelerate their growth and requirement for additional assimilates. An increase in the mechanical resistance of the soil reduced very much the elongation rate of main root

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axes of pea (Pisum sativum) but did not affect elongation of the laterals (Tsegaye and Mullins, 1994). It also brought about a larger reduction in the diameter of main axes and first-order laterals than in the diameter of the second-order laterals. Distribution of lateral roots along the axes was not uniform with some segments of the root axes that were devoid of laterals and did not have soil particles clinging to them. Those were interpreted as root portions which grew across air gaps within the soil. It can be concluded that the structural differences among the various root types are tightly connected with their functional differences. The ability of a root to absorb and transport nutrients and water is governed by the morphological and anatomical characteristics of this organ, by its position within the root system, by the vascular connections with the other parts of the plant, and by its other physiological traits. The effects of structural differences among roots should be given more emphasis in future research, and attempts to quantitize such effects to explain the operation of entire root systems should be made. III.

FUNCTIONAL DIFFERENCES

A.

Growth

Elongation of main roots, initiation of lateral roots, and extension growth of laterals of various orders were frequently summed up under the general terms of root mass or root growth. This was done despite of the fact that each of the processes mentioned above reacts differently to various environmental and physiological determinants and consequently affects differently the characteristics of the entire plant (Eshel et al., 2001). One of the topics that attracted attention was the possible effect of the global increase in CO2 level on plant growth. Soybean plants grown under elevated CO2 level had larger root mass than those grown under current atmospheric conditions (Del Castillo et al., 1989). However, their treatment did not affect the rate of elongation of individual root axes. Instead, there was a significant increase in the number of actively growing roots. Thus, the root length density increased but the volume of the soil explored by the roots did not. Different growth rates for several components of the root systems of hydroponically grown sunflowers were reported (Aguirrezabal et al., 1993). Their results have indicated that the control of carbon partitioning among various components of a single root system is

Functional Diversity

determined by a combination of the distance of each sink from the source, and by its level of branching. Different portions of the root system had different rates of energy consumption, with the highest values exhibited by the growing tissue fractions (Bingham and Stevenson, 1993). The time course of initiation of lateral roots may follow different patterns: it can be linear in some roots but exponential or logistic in others. Averaging both processes for an entire root system would conceal the actual nature of development of some types of roots and mask that interesting source of variability (Buwalda et al., 1984; Waisel and Breckle, 1987; Eshel and Waisel, 1996). The subsequent development of lateral roots may also differ considerably in different species. For example, the lateral roots of spruce seedlings (Picea glauca) differ so much in their growth pattern that they actually form three different classes (Johnson-Flanagan and Owens, 1985). The growth patterns of roots may vary with their parent material. Roots of Ipomoea batatas that have developed from stored cut sprouts were longer, their cambium became active earlier, and the rate of their xylem lignification was faster than roots that have developed from fresh sprouts (Nakatani et al., 1987). In the developing system of young pea seedlings, the main root is supported by current photosynthates of the growing shoots, whereas the laterals are supplied more by the cotyledons. Changes in the structure of the root caps of tea plants cause changes in root gravitropism of main and of lateral roots and by that affect the architecture of the whole root system (Yamashita et al., 1997). Mineral status of the soil is one of the important factors affecting root growth. Heterogeneous distribution of nitrogen within the root zone of black birch (Betula lenta) brought about changes in whole root system architecture, increased link length, and induced simpler branching patterns (Crabtree and Berntson, 1994). It was concluded that in this case plant integration of the environmental responses may override local control of root growth. The proteid roots of Lupinus albus secreted acid which increased P availability in the immediate vicinity of these root clusters (Dinkelaker et al., 1989). Exposure of Lolium perenne to a low-P medium has reduced the number of adventitious roots but increased the length of each individual root (Troughton, 1977). Nitrogen nutrition and the mode of its application had specific effects on the number of lateral roots (Marriott and Dale, 1977). Abundant production of laterals is highly important for root growth in heterogeneous media. It affects the nutrient supply

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of the plants, the allocation of assimilates, and the production and supply of growth substances (Waisel and Shapira, 1971; Marriott and Dale, 1977). Lynch and coworkers have shown that low P affects several characteristics of bean and Arabidopsis roots. The basal roots were less gravitropic under low P leading to more efficient utilization of the upper soil horizon (Bonser et al., 1996; Liao et al., 2001; Lynch and Brown, 2001). Density of root hairs was higher and their elongation was faster under low P, allowing a lager soil volume exploited by each root axis (Bates and Lynch, 1996, 2000a,b; Zhong, 2001). Growth of roots of a divided root system depends on the supply of assimilates from the tops as well as on the spatial nutrient supply from the soil. In this respect, various roots of seedlings of Picea sitchensis have developed competitive relationships; growth stimulation of some of them caused an equivalent growth inhibition of the others (Philipson and Coutts, 1977). Subjection of single roots of Suaeda monoica (Waisel and Wilcox, unpublished data) and of Rhodes grass (Chloris gayana) (Waisel, 1985) to high concentrations of NaCl or of PEG has accelerated their extension growth. Such an acceleration of growth involved a larger allocation of assimilates to the treated roots and increased supply of construction material as well as of osmotically active organic substances. Indeed, tracing the movement of 14C-labeled assimilates into single roots of Pinus contorta, or to the water-stressed half of a Picea root system (Wolswinkel, 1985; Lang and Thorpe, 1986), has proven that more assimilates have moved into such stressed roots (Reid and Mexal, 1977). This seems to present an adaptation of the plant which helps it to cope with local environmental water stresses. However, in some cases, such adaptive changes persist for a short time only. When roots were given sufficient time for adjustment the direction of flow was reversed, and more assimilates were preferentially transported to the unstressed fraction (Farrar and Williams, 1988). B.

Water Transport

The soil–root interface is highly variable with respect to water and ion availability. However, because water conductivity of the moist bulk soil can be several orders of magnitude higher than that of roots, the main bottleneck for water uptake by plants lies in the water flux across the root tissues up to the xylem. The resistance to such a flux is affected by the age of the roots, by the degree of their development, by the magnitude of their suberization, and by the resistance of

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their endodermis (cf. Moreshet et al., 1996; Chapter 38 by Sperry et al. and Chapter 39 by Nardini et al. in this volume). Seminal roots of maize were shown to be much more efficient water suppliers to the shoots than are nodal roots (Navara, 1987). Thus, the contribution of individual roots to the water flux into a plant depends very much on the type and structure of roots and on the quantitative contribution of each of the developmental classes to the total performance of the root system. A most important characteristic that varies as root tissue mature is the degree of differentiation of the xylem vessels. In the youngest parts of the root, the xylem vessels are full of cytoplasm and other cell components. At that stage they do not conduct water as efficiently as when they are fully differentiated. The hydraulic conductivity of each root segment depends on the degree of differentiation of various xylem vessels and their respective diameters (Wenzel et al., 1989; Nobel, 1991; Wang et al., 1994; Oertli, 1996; Eshel et al., 2000). The ability of maize roots to take up water, as measured by movement of a dye into the tissue, also varied in accordance with root maturation and size (Varney and Canny, 1993; Canny and Huang, 1994). The vascular connections between the branch roots and the nodal roots is thought to have a significant role in connecting phloem and xylem conduits enabling the recycling of nutrients and organic compounds in the plant. These connections also contain tracheid barriers that are thought to reduce the chance of air bubbles from blocking the main vessels thus maintaining high hydraulic conductivity even under low water potentials (McCully and Mallett, 1993). Wan et al. (1994) compared shallow and deep lateral roots of Gutierrezia sarothrae. The deep roots were nonsuberized and had 40% more large-diameter xylem vessels than the shallow suberized laterals. As a result of these anatomical differences, the hydraulic conductivity of the deep roots was severalfold higher. Magnetic resonance imaging (MRI) was used to measure water uptake by individual roots of loblolly pine (Pinus taeda) seedlings. On the basis of weight and surface area, the tertiary fine roots were more efficient than the lateral or taproots in water uptake (MacFall et al., 1991). The so called Hydraulic lift phenomenon takes place in desert plants whenever water is taken up by the deep-reaching part of the root system, and supplied to the shallow roots which are surrounded by dry soil. The water is exuded to the upper soil layers through the night and is taken up again during the day (Richards and Caldwell, 1987). It was recently shown

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that it may also play a role in dryland agriculture (Wan et al., 2000). Different roots differ also in the direction of water flux. Reverse flow of water was reported for vertically descending roots, while water uptake by lateral roots continued. The continuation of water uptake by lateral roots enabled a downward siphoning of water induced when the surface soil is wetted (Smith et al., 1999). Efficiency of water uptake and transport is highly dependent on individual root structure. Water uptake is directly proportional to root surface area. Water transport, on the other hand, is proportional to diameter, and degree of differentiation of the xylem vessels. When compared on the basis of carbon cost, each root is capable of performing only one of the two functions at the most efficient way. These considerations are of prime importance, when plant adaptation to arid environment is considered (see Chapter 53 by Nobel in this volume). Individual roots of grapevines supply water and nutrients to different twigs along the same branch. There was no compensation by other roots, for the loss of supply capability of any root subjected to local adverse edaphic conditions (Shani et al., 1993). This might be characteristic also of other vine-like plants as well. C.

Ion Metabolism

One aspect of the heterogeneity of root environment is the spatial and temporal variation of mineral supply. The reaction of plants to localized higher concentration of nutrients have been the subject of many studies (cf. Robinson, 1994). Those which have employed the split-root system technique either in solution or soil media, imposed a constant uneven mineral distribution conditions. Others, which have used banded fertilization or decomposing organic matter sources, had a temporary concentrated source which might have either been used up or dispersed by water movement and diffusion. The initial response of the plants in both cases is thought to be the same, but the overall ecological implication is of course different. The general trends that have been observed are localized enhancements of root proliferation at the site of higher mineral concentration, and concomitant reduction in root growth elsewhere in the rooting medium. This is, then, not a mere localized response but a change in the pattern of development of the whole root system resulting in a clustered root distribution. Such a change might have a large effect on the functional capability of the root system. The reaction of the

Functional Diversity

roots to a localized increase in mineral availability is very rapid and was shown to result from changes in lateral initiation (Gersani and Sachs, 1992), change in size of first-order laterals, change in the size of the second-order laterals, or overall change in root relative growth rate (Robinson, 1994). Different species exhibited different types of responses, and also varied in their response to the different nutrients. It was demonstrated that several grass species responded by proliferation of roots in microsites fertilized with a full nutrient solution (Larigauderie and Richards, 1994). In general, ammonium, nitrate, and phosphate increased root growth at the higher supply zone, but potassium failed to induce such a reaction (Brouder and Cassman, 1994). It was therefore concluded that banded fertilization of potassium will not be of benefit for cotton, unless it is accompanied by banded application of one of the other nutrients. From measurements of uptake and accumulation of nitrate by various parts of the maize root system, Lazof et al. (1992) concluded that in addition to nitrate uptake by the root tip it also receives nitrogen from more mature parts of the root (cf. Brady et al., 1993). Over the whole root system, the lateral roots accounted for 60% of nitrate influx. A rapidly exchanging translocation pool was located within the laterals, from which nitrate was transported to the shoot. A comparison of the uptake kinetics of nitrate by seminal and crown roots of barley, revealed that Vmax of the seminal roots decreased while that of the crown roots did not. Thus, the contribution by the seminal roots to total nitrate uptake decreased from > 50% in vegetative plants to
20% just after main shoot anthesis, and to < 5% during grain filling stage (Mattsson et al., 1993). Pate and Jeschke (1993) compared the ion content of xylem sap from various components of the root system of Banksia prionotes. In this proteaceous plant they distinguished among the taproot, which grows straight down to the underground watertable, lateral roots, and clusters of proteoid—short roots characteristic to this family. Cluster roots were found to be principal xylem donors of malate, phosphate, chloride, sodium, potassium, and amino acids. Other parts of the root system served as major sources of other cations, nitrate, and sulfate (see also Chapter 55 by Pate and Watt in this volume). Despite the ostensible coordination among the various components of root systems, the independent behavior of individual roots is obvious. Chaillou et al. (1994) studied uptake of ammonium and nitrate

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by different root parts of hydroponically grown soybeans. They concluded that proportionality of uptake by the different parts was not governed by the shoot, which only limited the total uptake of nitrogen by supply of carbohydrates. Translocation of phosphorus from various roots to stolons of white clover (Trifolium repens) depended on the relative position of each root with respect to the stolon and on its age (Chapman and Hay, 1993). A stolon that originated from the same node as the root was the most significant sink, and its importance as a sink increased the older and larger it became (Kemball and Marshall, 1994). Nutrition of the shoots can also be affected by the storage capabilities of the roots. For example, cortex cells of maize roots delay the nutrient transport to the shoots by intercepting the transported P and by storing it. As roots age, the accumulated P is released out of their cells and eventually reaches the shoots. However, this does not occur simultaneously for all roots. Senescence of the cortex of lateral roots of wheat was shown to be faster than that of seminal roots (Fusseder, 1987). Therefore, such roots release P out of their cortex before the main axes and constitute a better P source for the shoot under conditions of marginal P supply. Behavior of shoots may also depend on the function of various root types. Salt-treated grasses contain higher levels of NaCl in the primary shoots than in their tillers. The increase in salt accumulation by the primary shoots is presumably a result of the decrease in selectivity of their older roots. On the other hand, roots of the younger tillers discriminate more efficiently against undesirable ions and therefore keep a lower salt content in their tillers. Salt content of the tillers remains low and the shoots remain unharmed until their own roots also age. The capability for ion uptake by seminal roots of maize differs from that of the adventitious ones (Fig. 1). Rates of uptake of N out of a mixture of NO3 and NH4 by the nodal roots of wheat were two to six times higher than by seminal roots (Kuhlmann and Barraclough, 1987). The uptake rates of K by taproots and by laterals of faba beans (Lev, 2000) also differed but to a smaller extent (Fig. 2). In both cases the question of whether uptake is affected by the type of the roots or by their age and developmental stage calls for additional research. Roots induce changes in the pH of their rhizosphere. Differences in pH were shown to occur along the root axis (see Chapter 33 by Gerandas and Ratcliffe in this volume) and among roots within the same root system. Such changes are determined by the

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Figure 2 Uptake of RbK by taproots and by lateral roots of Vicia faba plants in the presence and absence of NaCl.

Figure 1 (a) Inflow and (b) specific absorption rates of N and K by seminal and by nodal roots of maize. (From Kuhlmann and Barraclough, 1987.)

species characteristics as well as by the metabolic responses of the individual roots to environmental influences, for example, to the ionic form of supplied N (cf. Chapter 36 by Neumann and Ro¨mheld in this volume). However, beyond the taxonomic and the environmental effects, various roots of one plant grown in a uniform soil differ in a typical way. Three examples have been presented (cf. Marschner et al., 1986): 1. Main roots of maize have alkalized their rhizosphere, raising the pH from 6.0 to 7.5. However, lateral roots of the same axis have acidified the same soil, lowering the pH from 6.0 to pH 4.5. 2. Roots of Picea abies vary with regard to their acidification capability: some of them have lowered the pH of their rhizosphere, whereas others have remained unaffected. 3. Normal roots of Lupinus albus have raised the pH of the medium from 6.3 to 7.5. However, proteoid roots of the same root system, which had been formed

under P deficiency, have acidified the medium to below pH 4.5. Such changes in pH are, of course, crucial for the acquisition of nutrients from the soil and affect the N, P, Fe, and Mn economy of the entire plant. Thus, the use of average values of rhizosphere pH, for studies of nutrient dynamics in a plant–soil system, could be inaccurate and misleading. A similar case was demonstrated for kinetic analyses of ion uptake. Such analyses have been calculated for years using average values of heterogeneous batches of roots, despite the fact that such averages may have led to erroneous and misleading conclusions. Robinson et al. (1991) estimated that only a small fraction of the roots take part in nutrient uptake. Consequently, plant nutrient uptake rates based on total root length grossly overestimate the true values. Yanai (1994) presented a modification of the BarberCushman nutrient uptake model (Barber and Cushman, 1981; see also Chapter 37 by Silberbush in this volume) by introducing a separate term to account for nutrient uptake by newly grown roots. Further developments in that direction are hindered by the lack of quantitative data regarding the variation of ion uptake kinetic parameters among roots of different ages and different positions in the root system. Proofs that the rates of ion uptake, the modes of ion interactions, the electrical properties of root cells (Ishikawa et al., 1984), and the nature of the uptake mechanisms differ along roots of maize, barley, and beans have been available for a long time (Waisel and Eshel, 1992). Variability among the individual roots did not end with differences in uptake patterns. Such roots have also shown different magnitudes of

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165

uptake and a different balance between the metabolic uptake mechanism and the nonmetabolic ones. Variations along roots are not unique for ion transport and a similar phenomenon was also shown for the uptake of tritiated water. Such data provide additional arguments against the use of unspecified root batches without a detailed knowledge of their individual characteristics. Developments in microelectrode techniques allow accurate measurements of ion fluxes to be made at different locations among and along the roots with high resolution. Henriksen et al. (1992) used such a technique to measure ammonium and nitrate fluxes at various locations along seminal roots of barley. Their measurements revealed that uptake rates varied among the roots, spatially along the root, and temporally for every root. Therefore, different flux patterns were recorded during successive scans along the same portion of the root. Highest fluxes were found at the tip or within the apical two centimeters. They suggested that metabolic state of the cells at various portions of the apical region of the root may regulate these fluxes. Kochian et al. (1992) described the application of vibrating microelectrode technique to studies of ion fluxes at various sites along the root. Such a vibrating probe together with its associated computerized control and software allows for measurement of ion fluxes across the boundary layer of the root surface. Its high sensitivity enables measurements of ion fluxes at the surface of a single cell to be performed accurately (see Chapter 20 by Porterfield in this volume).

D.

Responses to Stress

Differences among individual roots, or among various root types, are usually smallest under optimal condi-

tions; that is, the conditions under which many root investigations are conducted. However, differences among roots become much more pronounced under the suboptimal conditions of salinity (Table 1), when roots are stressed (Waisel and Breckle, 1987). This is also true for O2 stresses. Oxygen-stressed roots of sunflower plants are short but have a larger density of laterals than do nonstressed roots (Wample and Reid, 1975). Eucalyptus camaldulensis plants develop fine lateral roots (<0:5 mm) in well-drained soil (Lev-Ha’ari, 1999). At the early stage of development, the primary xylem of such laterals occupies the center of the stele and is surrounded by a layer of three or four thinwalled parenchyma cells (Fig. 3A). As such roots mature, a periderm develops in the cortex and the phelem is sloughed off (Fig. 3B). When the soil is flooded, the same plants develop another type of laterals that are 2–4 mm thick with four to six primary xylem archs and a central parenchymateous pith (Fig. 3C). The thick cortex of these roots persists for a long time and the development of the periderm is delayed even though the vascular tissues are well differentiated (Fig. 3D) (Eshel, Lev-Ha’ari, and Efrat, unpublished data). The behavior of seminal roots of wheat also differs upon exposure to low pO2 stress. Roots that have developed from the basal internodes of the embryo under low pO2 grew slower than roots from the upper internodes (Erdmann et al., 1988). When studying the effect of hypoxia on wheat roots, respiration rates expressed on root length or weight basis were highest at the apex (Weidenroth, 1993). Similar responses to stress were reported by Waisel (1985) for single roots of Rhodes grass (Chloris gayana). Growth was severely inhibited when all roots were subjected to a 200 mol m3 NaCl treatment. However, when only a few of the roots were subjected

Table 1 Rates of Appearance of Lateral Roots and Rates of Extension Growth of Taproots and Lateral Roots of Bean Seedlings (Phaseolus vulgaris), in the Light and in the Dark, Under Various NaCl Treatments

Treatment solution 0.25 Hoagland’s solution 50 mol m3 NaCl þ Hoagland’s solution 100 mol m3 NaCl þ Hoagland’s solution

Light Dark Light Dark Light Dark

Addition of laterals (roots h1 )

Extension growth of laterals (mm h1 root1 )

Extension growth of taproots (mm h1 )

0:63  0:54 0:60  0:42 0:39  0:42 0:25  0:02 0:23  0:20 0:20  0:16

0:77  0:16 0:41  0:53 0:38  0:40 0:08  0:18 0:11  0:16 0:08  0:11

0:17  0:12 0:15  0:11 0:12  0:02 0:06  0:04 0:09  0:02 0:10  0:02

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Figure 3 Cross sections of two types of fine lateral roots of Eucalyptus camaldulensis. (A) Young and (B) mature roots in a well-drained soil (bar ¼ 100
m). (C) Young and (D) mature roots that develop under waterlogging (bar ¼ 500
m). (From Eshel, Lev-Ha’ari, and Efrat, unpublished.)

to such a solution, while the rest of them were kept in a nonsaline soil, growth of the salt-treated individuals was stimulated rather than inhibited. Apparently, one of the aspects of such stimulation involves an increase in water flow toward those roots, probably accompanied with an improvement of their carbon allotment. Similar results were obtained for roots of the halophyte Suaeda monoica (Waisel, 1972). Prevailing temperatures may also differentiate between the extension growth of various roots. Extension growth of primary roots of soybeans decreased with a rise in temperature, at a certain range, whereas that of the laterals remained steady. This was postulated to be a result of a preferred allocation of carbohydrates to the lateral roots as com-

Waisel and Eshel

pared to that of the primary roots (Stone and Taylor, 1983). Differences in allocation can also be implied from the responses of bean roots to day and night conditions. Under nonsaline conditions, extension growth of lateral roots was significantly reduced during the night, but that of the main roots was practically unaffected. Differences were intensified in salttreated plants (Eshel and Waisel, 1996). This seems to indicate that structural carbohydrates are allotted to each of the two types of roots from a different source. Gersani et al. (1993) studied the effect of subjecting part of a root system of Opuntia ficus-inidica to salinity stress. They found that the influence of salt on the growth of the main root was different from the effect on growth of the first and second order laterals. Dissimilar responses of various root types to NaCl were also found for seedlings of radish. In this case the taproot was less inhibited by salinity than the laterals (Waisel and Breckle, 1987). Various roots respond differently to the effects of heavy metals. The taproots of Fagus sylvatica and of peas are severly inhibited by relatively low concentrations of Pb, Zn, or Cd. Nevertheless, under identical conditions, the extension growth of the first-order laterals, of the same axes, was practically unaffected (see also Chapter 43 by Hagemeyer and Breckle in this volume). Other types of stress were also shown to affect root architecture. Tester et al. (1986) found that the main effect of decreased irradiance of the shoot on growth of the root system was a reduction in the numbers of initiated first- and second-order lateral roots. Morita and Okuda (1994) studied the effect of soil moisture content on root systems of wheat. Under high moisture content the plants had more nodal roots, but those were quite short. Low soil water content first inhibited elongation of the main axis of the seminal roots, then promoted the production of first-order laterals, and finally increased the length of these laterals. High moisture content of the soil is intertwined with hypoxic conditions. Under high pO2 in the root medium, uptake of K and of nitrate was higher by seminal roots. However, under hypoxia, uptake by the seminal roots was reduced to the level exhibited by the nodal roots (Kuiper and Walton, 1994). The effect of limited water supply on the topological characteristics of three range grasses was studied by Johnson and Aguirre (1991). Generally, branching of the roots decreased with decreasing water supply. Bromus tectorum was less sensitive than the other species studied. The ability of the seedlings of this species to grow with little water was thought to be related to

Functional Diversity

the greater order of branching of the seminal roots, the branching density of the main axis, and the length of lateral roots and external links. Drought-induced roots that developed on the main root of Brassica napus were longer than those that developed on the laterals (Balestrini and Vartanian, 1983). Zhang and Davies (1989) reported that ABA in the xylem sap from that part of the root system that was in drying soil was higher than that in xylem sap of the other parts. In spite of an overall high water potential of the shoot, this increased level of ABA from only a portion of the roots brought about stomatal closure in all leaves. Such differential responses by parts of the root system may serve as an adaptation mechanism that will reduce water uptake by the whole shoot, as soil drying begins at the top layers of the profile. Preliminary investigation of ABA content of the taproot and the laterals of faba bean plants (Fig. 4) yielded striking differences, in spite of the fact that the plants were grown in a uniform hydroponic culture (Hartung and Waisel, unpublished data). Hormone content of the root tips of two orchids (Aranda and Vanda) also varied with the root position along the stem (Zhang et al., 1995) E.

Mechanical Strength

Maintenance of an erect position (standability) of plants is a function of the mechanical properties of the individual roots that compose the root system. Large differences in tensile strength and in the vertical pulling resistance were observed among different types of maize roots. Highest resistance was found either for

Figure 4 ABA content of taproots and of lateral roots of Vicia faba plants. (From Hartung and Waisel, unpublished data.)

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crown roots or for roots with well-developed laterals (Beck et al., 1988). Adventitious roots of grasses also vary in the direction of their growth, a fact that influences the mechanical stability of young seedlings. The three first seminal roots of Poa pratensis grow at an angle of 120 between them, whereas roots that have developed later grow in other angles. Variations in the direction of growth were also reported for Phalaris arundinacea and Paspalum notatum Poir (Hirota and Kushida, 1985). The multidirectional growth pattern of roots, which is achieved by the orientation of certain individuals, contributes to the pulling resistance of the rhizomes and to the stability of the parent plants, especially under grazing stresses. Clear differences between leading roots and lateral ones in response to mechanical impedance was reported by Thaler and Pages (1999). Their findings show that the growth potential of different roots is limited by the mechanical environment. Growth of thick roots with large meristems was impeded more than growth of thin roots. The result is that high soil impedance affects more the growth of thick leader roots than that of thin laterals, giving an advantage to highly branched root systems. F.

Correlative Effects and Responses to Hormones

Although the behavior of roots of one root system is individually controlled, in the long run, their growth must be somehow intercorrelated. This is mediated by various growth hormones that are dispensed from a common pool (cf. Lamond et al., 1983). Changes in hormone allocation, at the individual root level, affects the structural and physiological characteristics of the entire system. For example, blocking the growth of taproots of Quercus robur seedlings, thus inducing a change in their hormonal balance, had caused thickening of the apices of the lateral primordia. Such laterals develop later into thicker roots whose extension growth was faster than that of similarly positioned normal laterals (Lamond et al., 1983). Differences in root cell contents were also noted. Lateral roots of beans were shown to contain less calmodulin and different actin-related proteins than leader roots (Westberg et al., 1994). This suggests that differences in polypeptide composition may contribute to the specific graviresponses which each type of those roots displays and eventually to the root architecture. Such effects cannot remain unbalanced. Gersani and Sachs (1992) used the term ‘‘correlative inhibition’’ to describe the fact that enhancement of lateral devel-

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opment at one part of the root system was accompanied by reduction in the other parts. They assumed that it is mediated by the hormonal balance of the various regions of the root system. G.

Aging and Senescence

The life span of individual roots differs greatly: main roots usually survive throughout the lifetime of plants, whereas laterals, especially those of higher orders, were reported to be short-living. Some roots survive for short periods only, whereas others have remained alive for many years. Various segments of roots, and various constituents of each root system, senesce at their own pace (cf. Bloomfield et al., 1996). This can also be observed at the tissue level. The cortex of primary roots of maize plants remains alive during most of the plant’s life. Senescence of the cortex of lateral roots is much faster than that of main roots (Fusseder, 1987). Thus, although the two types of roots might look alike, their physiology certainly differs. Sloughing off of some roots occurs naturally in several plant species, removing old roots or such roots as have stranded into unfavorable sites (cf. Caldwell, 1979). Removal of some senescent roots delays the senescence of neighboring laterals and prolongs their life span. Early shedding of such roots saves the plant structural carbohydrates, respiration substrates, and excretion materials. This is another indication of the benefits that can be gained by plants that have the proper physiological mechanism to allow them to distinguish between individual roots and to invest only in roots that operate efficiently. H.

Hormonal and Enzyme Differences

Plant hormones play a significant role in plant adaptation to their specific environment. This applies in general but also as a specific response of various parts of a single root system. Do different root types differ in their hormone production and signal transmission? The information that is conveyed by scouting roots (root signal) regarding the specific conditions at the site that they have reached, is transferred to all other parts of the plant and determines the root/shoot ratio, the site of lateral initiation, the number of initiated laterals, and the characteristics of the developing roots (Hartung and Heilmeier, 1992; Tradieu et al., 1992). ABA is a key hormone in plant root signaling and in plant stress responses. This hormone is not equally distributed among different root types of Vicia faba.

ABA content of the taproots being over four times higher than that of the lateral roots. Such differences were maintained in freshwater-grown plants as well as in salt-stressed ones (Fig. 4; Waisel and Hartung, unpublished data). This is further supported by the finding that slow-growing roots of maize are strongly inhibited by zeatin and have a higher ABA content than fast growing ones (Bourquin and Pilet, 1990). Different roots also have different relative water content and vary in their ABA content (Simonneau et al., 1998). As different roots are exposed to different environmental conditions, at least some of the response systems have to be differently expressed. Indeed, of 35 enzymes that were investigated in fava bean roots, some 13 differed in activity between the taproot and the laterals. Those are constituents of two basic metabolic pathways: glycolysis fermentation and nitrogen metabolism. The responses were not constant and under low aeration of the root media (
4 ppm) the differences between the two types of roots were amplified (Lev, 2000). A different number of isoenzymes was found for some enzymes in tap and in lateral roots of Vicia faba. The most conspicuous enzyme was alcohol dehydrogenase (Fig. 5): under normal conditions two isoenzymes were expressed in the taproot, but only one was expressed in lateral roots. However, under impaired aeration three isoenzymes were traced in the taproots whereas only one (maybe traces of second) was found in the lateral roots. GOT is another enzyme that showed a difference in the number of expressed isoenzymes; three isoenzymes were observed in the taproots, but only one was observed in the lateral roots. Proteoid roots of Lupinus albus, developed under low P supply, exhibited a higher specific activities of citarte synthase, malate dehydrogenase, and

Figure 5 Gel electrophoresis of ADH extracted from taproots and from laterals of Vicia faba plants grown in aerated and in nonaerated solutions. (From Lev, 2000.)

Functional Diversity

PEPcarboxylase than normal roots (Gilbert et al., 1999; Watt and Evans 1999). Thus, such results support our basic hypothesis that differences exist in several potential activities of the various types of roots even of one plant. Such differences may constitute an answer to the environmental conditions that such root types may confront. In most cases of our study the activities were higher in the taproot. Only acid phosphatase was expressed at higher levels in the lateral roots.

169

seedlings. This difference in oxygen consumption was probably a result of different rates of exudation of degradable carbon compounds by different roots (Hojberg and Sorensen, 1993) and by differences in bacterial colonization. This can be demonstrated even when different roots develop in a uniform environment. Differences in bacterial colonization of faba bean roots were demonstrated, using the GP and GN ‘‘Biolog’’ plates (Waisel, unpublished data).

V. IV.

INTERRELATIONSHIPS WITH BIOTIC FACTORS

Symbiotic relationships differ among various roots, and it is common to observe more than one mycorrhytic association for various roots of one root system. These may develop as one fungus—one root association, but more often will form clusters of several fungi on one root. Such multiple associations change along roots with time and place (cf. Wilcox, 1996). Some of the fungi have appeared individually, each on one root, whereas in other cases several fungi had developed simultaneously on a single root (see also Chapter 50 by Koettke and Chapter 49 by Sieber in this volume). Fungi and bacteria identify parts of roots but also different root types. For example, ectomycorrhizae of Eucalyptus dumosa were more successful in colonizing lateral roots than leading roots (Chilvers et al., 1987). A similar phenomenon was also shown for various pathogens that operate on taproots of Trifolium subterraneum but not on the lateral roots (Barbetti et al., 1987). The influence of mycorrhizae may also bring about a nonuniform root development and variation in the function of various roots depending on the development of their associated fungi (Berta et al., 1993). Excretion of organic substances (root exudation) varies greatly among roots of different types. Individual roots of seedlings of Pinus contorta subjected to media of various water potentials excreted much more organic materials under low water potentials (400 kPa) than under high ones (Reid and Mexal, 1977). In a heterogeneous environment, this means that induced excretion of organic substances, which varies among roots, is followed by changes in the biological activity of the rhizosphere and eventually causes differences in nutrition, hormone balance, and growth of plants. Microbial respiration around roots and oxygen uptake by them differed for different roots of barley

CONCLUSIONS

Analysis of a functional biological system is founded on three basic principles: knowledge of the structure and composition of the system examined, knowledge of the behavior of each constituent, and knowledge of the relative contribution of each fraction to the overall performance of the examined group. Some investigators have ignored such demands and treated plant organs as if they were absolutely uniform. Such an approach does not allow an investigator to identify unique types of roots or to pick up the ‘‘odd occurrences’’ (cf. Popper, 1972). In many investigations, the basic nature of the type of roots examined, their intrapopulation variability, or the specific contribution of each fraction of roots to the total behavior of the system was lost. Moreover, in many other cases, where different classes of roots can definitely be distinguished, their distinct nature was ignored and the unique behavior of each class was treated as part of the general variability of the examined root system. Average values of biological parameters are useful tools for the description of populations with continuous variations. However, in cases where traits do not yield a continuum, averages may distort the meaning of important data. Quantification of the mechanisms of growth, water uptake, or nutrient supply of various root types, by calculating average rates of uptake from the bulk soil, will make sense only if various classes of roots are well defined and if the relative contribution of each type of root will be quantitatively and qualitatively evaluated. Any attempt to describe or simulate the growth and function of a root system should take into account the differences among its various components. The message that is conveyed in the present chapter can be summarized by the following points: (1) root systems comprise several types of constituents, although not all of them can be identified morphologically; (2) each of these constituents has its own functional and/or structural characteristics; and (3) it is

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essential to know the relative contribution of each constituent in terms of the total function of a coordinated root system. Future research of root biology should employ such principles more decisively. Classification of roots down to the smallest distinguishable units, and treating them as distinct entities, would hopefully reveal their unique features that have been lost in the past among ‘‘averages.’’ This kind of studies will certainly employ new measurement techniques, with higher resolution and sensitivity. The capability for identifying the genes that are responsible for the production of functional proteins in specific roots exists. Nevertheless, so far little progress in that area of differential component analysis has been reported. Such studies should enable us to understand the molecular mechanisms responsible for the variations among the constituents of each root system and, on the other hand, the function of those elements which in concert make Nehemia Grew’s clock click.

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10 Biomechanics of Tree Root Anchorage Alexia Stokes Laboratoire de Rhe´ologie du Bois de Bordeaux, Cestas Gazinet, France

I.

INTRODUCTION

where the soil needs to be stabilized—e.g., on steep slopes subject to avalanches, on hillsides where erosion occurs after forest fires, or even on motorway embankments prone to mudslides (Barker, 1995). The major advantages of using vegetative material over inert engineering components are not only those related to environmental and ecological aspects, but are also the reduced cost and facility of installation. This chapter will provide a short synopsis of the recent work carried out in the domain of root biomechanics, with an emphasis on tree stability and ecoengineering.

Tree root systems serve three major functions; absorbing water and nutrients, storing carbohydrates, and anchoring the tree in the ground. Although the physiological aspects of water and nutrient uptake have been extensively investigated over many decades (see Waisel et al., 1996), the study of tree root biomechanics has only begun to be developed in the past 15 years (Coutts, 1983; Mattheck and Breloer, 1994; cf. Stokes, 2000). The increased interest in this subject has been fueled by the intensity and frequency of violent storms in Europe over the past decade (Quine et al., 1995), including the 1999 hurricane which hit Northern Europe and resulted in over 15 million hectares of wind-blown trees in France alone (source: Inventaire Forestier National). Studies of root system architecture and root mechanical properties of trees that were felled in this most recent storm should yield important information as to the characteristics that are important for tree stability. Not only is the understanding of tree root biomechanics vital for the improvement and selection of windthrow-resistant forest and urban trees, this knowledge is also important for the correct planting of species in zones where soil fixation is a priority. A new and rapidly developing discipline, ecoengineering, uses data on the mechanical behavior of individual roots, as well as of entire root systems, to help foresters, ecologists, and landscape engineers decide where and what to plant. This technique is specifically required in areas

II.

ANCHORAGE MECHANICS

The notion of determining tree stability characters was first suggested by Coutts (1983, 1986). Studying the root systems of Sitka spruce (Picea sitchensis), Coutts (1983) decided to establish which components of anchorage were the most important for tree stability. Sitka spruce in Scotland is frequently subjected to waterlogging, thus hindering the growth of deep vertical roots and rendering the tree highly unstable (Figs. 1, 2B). When the stem of a tree is displaced laterally, the windward side of the tree is lifted up and held in tension, whereas the lateral roots on the leeward side are pushed downward into the soil (Fig. 3). Vertical roots may be pulled upward or displaced sideways, and are discussed later in this chapter. Roots perpendicular to the direction of displacement are held in torsion and 175

176

Stokes

Figure 1 (a) Shallow-rooted Sitka spruce (Picea sitchensis). The growth of vertical roots has been impeded owing to an increase in the water table level, caused by seasonal waterlogging of peaty gley in Scotland. Photo by B.C. Nicoll, Forestry Commission, Scotland. (b) 50-year-old tap-rooted Maritime pine (Pinus pinaster), with vertical root growth also impeded due to both seasonal waterlogging and a layer of impenetrable hard pan, 1 m below the soil surface. Measurements and analysis of root architecture were carried out by C. Espagnet, B. Issenhuth, and F. Danjon, using the technique described in Danjon et al. (1999).

offer little resistance to uprooting. By repeatedly pulling trees during a sequence of cutting or breaking the roots and soil, the total resistive turning moment afforded by the anchorage was resolved into four components (Coutts 1986): (1) The weight of the root–soil plate (i.e., the roots and adhering soil) provide the initial resistance to overturning (Fig. 3a). (2) During uprooting, the soil underneath and around the edge of the plate is broken and provides further resistance (Fig. 3b). (3) The tensile strength of the roots on the windward side of the plate produces a third component (Fig. 3c). (4) The bending strength of the leeward roots and soil offers a fourth component (Fig. 3d). For 35-year-old shallow-rooted Sitka spruce, Coutts (1983, 1986) determined that the most important component of anchorage resisting overturning was that of the roots at the windward perimeter. This component accounted for
60% of the total anchorage. Secondly,

Figure 2 Different types of root system architecture. (A) Heart system with many horizontal and vertical roots. (B) Plate or sinker system with large lateral roots and some smaller vertical roots. Taproot system with one major central root and smaller horizontal and vertical roots. (From Stokes and Mattheck, 1996.)

the weight of the root–soil plate represented 13–45% of the total. This figure is highly variable, and is therefore an important factor to consider when trying to manipulate and improve tree root anchorage. The bending resistance on the leeward side of the tree accounted for only
20% of the total, and the breaking of roots underneath the soil–root plate even less. If a root is considered to be a circular cantilever beam, then its stiffness is related to the second moment of area (a function of radius to the fourth power). If the root branches into two forks of an even size with a total cross-sectional area equivalent to that of the par-

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Figure 3 The different components of root anchorage. (a) The weight of the root–soil plate (i.e., the roots and adhering soil) provide the initial resistance to overturning. (b) During uprooting, the soil underneath and around the edge of the plate is broken and contributes a further resistance. (c) The tensile strength of the roots on the windward side of the plate produces a third component. (d) The bending strength of the leeward roots and soil offers a fourth component. (From Coutts, 1983, 1986.)

ent, and the material properties stay the same, the total stiffness of the beam will be halved. As the leeward roots of a tree act under compression, the point at which the tree is levered out of the ground would occur closer to the stem, thus reducing stability, especially in trees with wide and shallow plate root systems. A similar study to that of Coutts (1983, 1986) was carried out on 16-year-old larch trees (Larix europea  L. japonica), by Crook and Ennos (1996).

These authors tried to examine the anchorage mechanics of a species with a deeply rooted ‘‘heart’’type system (Fig. 2A; Ko¨stler et al., 1968), unlike that of Sitka spruce, which has a shallow ‘‘plate’’ or ‘‘sinker’’ type root system (Figs. 1a, 2B). Crook and Ennos (1996) measured the bending strength of lateral roots to estimate the importance of root anchorage. Despite the differences in root system architecture, similar results were found between the two species, with the

178

contribution of the windward roots, combined with that of the taproot, providing 75% of the total anchorage, and the leeward laterals
25%. The contribution of the taproot alone could not be determined owing to the difficulties in carrying out such an experiment. However, later studies revealed the way in which taproots are displaced during overturning. Crook and Ennos (1997) studied the anchorage mechanics of the tropical taprooted species Mallotus wrayi (Fig. 2C) and found that during overturning, the tree rotates and bends on the windward side of the taproot. The tree can be said to act like a stake, with the taproot the point of that stake. The taproot itself pushes into the soil on the leeward side, the top half rotating, and the bottom half remaining reasonably well anchored (Fig. 4a). A crevice is then formed on the windward side, becoming larger as the tree is pulled over. Hintikka (1972) also found that the lower half of the taproot of Pinus sylvestris may make a semicircular movement and push into the soil on the windward side (Fig. 4b). It was therefore concluded that the taproot was firmly attached to the soil at its distal end, and that the lateral roots held the stem so rigidly that the taproot had to move in the opposite direction. Trees with well-developed taproots usually do not fail with the taproot slipping out of the ground, as in certain herbaceous species (Ennos 1989). However, the mode of failure does appear to depend on tree age. The anchorage of the taprooted species Maritime pine (Pinus pinaster Ait.) was investigated by pulling tests of trees of different ages (Stokes, 1999). Five-yearold pine trees did not fail during overturning, and after pulling, returned to the vertical position. Older trees, however, broke in the stem or at the base of the taproot, with sounds of lateral roots breaking also being heard. Therefore it was confirmed that only young trees can be completely anchored by a taproot, whereas older trees need well-developed lateral roots in order to resist overturning (Crook and Ennos, 1997). A third type of root system exists in addition to the shallow and taproot type systems described above. ‘‘Heart’’ root systems have both lateral and sinker roots (Fig. 2A; Ko¨stler et al., 1968) and are considered to be the most efficient types of root system when considering tree stability (Stokes et al., 2000). In overturning tests carried out on several adult forest trees, Stokes et al. (2000) determined that heart and taprooted species, such as oak (Quercus robur) and Douglas fir (Pseudotsuga menziesii), tended to fail in the trunk or stem base. Shallowrooted Norway spruce (Picea abies) uprooted at much lower loads, often with the root plate being

Stokes

Figure 4 (a) During overturning in taprooted trees, the taproot may be pushed into the soil on the leeward side of the tree, leaving a crevice in the soil on this side of the tree. (From Crook and Ennos, 1997.) (b) In certain cases, the taproot may push into the soil on the windward side of the tree, but still be firmly anchored in the soil by its distal end. (From Hintikka, 1972.)

completely lifted out of the ground (Fig. 5a). When trees with heart root systems did fail in the root system, the root–soil ball slid into the soil and was not lifted out of the soil (Fig. 5b). In static pulling tests, such as those carried out above, if trees are well anchored by the root system, and the moment required to resist overturning is greater than that needed to cause stem failure, the trunk will break (Putz et al., 1983). Therefore, species with heart and taproot systems, which fail in the trunk under loading, must all be better anchored than shallow platetype systems.

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179

tical roots exist, the lower surface of the root–soil plate is often very smooth (Fig. 1), with little cohesion or bonding between the root–soil ball and the surrounding soil. As the strength of the large lateral roots remains relatively constant along the length of the lateral roots (Stokes and Mattheck, 1996), the whole root system is lifted out of the ground (Fig. 5a), in contrast to heart root systems, where the roots can break when uprooted.

IV.

Figure 5 (a) Trees with shallow-plate systems uproot with the root plate being completely lifted out of the ground. (b) In trees with heart root systems which fail in the root system, the root–soil ball slides into the soil and is not lifted out of the soil.

III.

SOIL–ROOT INTERACTION

The mechanical behavior of roots with regard to anchorage depends very much on the nature of the soil in which the roots are embedded. The tensile strength of soil is 3–5 orders of magnitude weaker than that of roots under tension (Coutts, 1983). Soil shear strength decreases with increased moisture content and increases with stress normal to the shear plane. Windthrow most often occurs in waterlogged soils when soil is so wet that critical shear stress  is reached and soil particles simply slide over each other. When wind forces act on the tree crown, the windward roots are lifted up, decreasing frictional resistance on this side of the tree, and increasing it on the leeward side, as roots are pushed downward onto the hard bearing surface of the soil. In trees with heart root systems, where compression and bending strength decrease along the lateral roots (Stokes and Mattheck, 1996), horizontal roots bend their flexible distal ends as well as the root bases which are held more rigidly within the root ball, and root breakage is often incurred (Mattheck and Breloer, 1994). The root plate slides against the surrounding soil when ground friction is overcome (Fig. 5b). The ground transfers shear stresses only if sliding surfaces are pressed against each other. Therefore, in plate root systems, where few or no ver-

ROOT ANCHORAGE ON SLOPES

Not only do roots serve as anchors for trees, but they also play an important role in the reinforcement of soil, which is vital in fixing soil, especially on steep slopes (Gray and Leiser, 1982; O’Loughlin and Watson, 1979; Watson et al., 1999). Roots provide this reinforcing effect to the relatively plastic soil, through their tensile resistance and frictional or adhesive properties. Shear stresses in the soil mobilize tensile resistance in the roots, which in turn imparts greater strength to the soil. Lateral roots in the soil mass act like guy ropes to transfer stress from place to place, and solidify the soil matrix by preventing soil movement (Greenaway, 1987). The efficiency of this traction effect depends on the strength of the soil and roots and the strength of the root–soil bond (the maximum bonding force per unit area on the soil–root interface). As roots are less stiff than the surrounding soil matrix, tension applied to the top of the root will cause the root to stretch at its upper part and shear past the soil as it is pulled upward. Tension will thus be transferred to the soil. The greater the applied force, the greater the area of soil around the root that will fail, and the greater the length of root that will be stretched. However, the tensile strength of roots must be fully mobilized during failure; i.e., the roots must be long enough and/or frictional enough that the frictional bond between the roots and soil matrix exceeds the tensile strength of the roots. Too short a root will slip, or pull out before mobilizing the maximum tensile resistance and breaking in tension (Ennos, 1994). If the root system is subjected to sliding or pulling forces greater than the maximum resistance by the roots, roots will either be broken (failure in tension) or pulled out of the soil (bonding failure). The forces promoting shallow slope failure depend on the weight of soil and trees, slope angle, and wind forces (Fig. 6). During anchorage failure in a storm, cracks appear initially in the soil surface. Roots cross these cracks and will be pulled downward, thus preventing further downslope movement due to their tensile strength and through root–soil cohesion (Fig. 6a). However, during slope failure, roots on the

180

Stokes

Figure 6 The forces promoting shallow slope failure depend on the weight of soil and trees, slope angle, and wind forces. (a) during anchorage failure in a storm, cracks appear initially in the soil surface. Roots cross these cracks and are pulled downward, thus preventing further downslope movement due to their tensile strength and through root–soil cohesion. (b) During slope failure, roots on the upslope side of the tree anchor the sliding mass to the stable side and prevent further movement. (From Zhou et al., 1998; Watson et al., 1999; Fourcaud and Lac, 1996; and Danjon et al., 1999.)

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181

upslope side of the tree will be held in tension, and anchor the sliding mass to the stable side, to prevent further movement (Fig. 6b; Zhou et al., 1998). The efficiency of tree roots in reinforcing soil thus depends on both root tensile strength and root system morphology. The greater the tensile strength, the better the soil will be reinforced. Although the cross-sectional area of roots in a given unit area of soil is a factor which determines soil shear strength, the morphology of each root plays a role just as important. In a series of pulling tests, straight roots of Pinus yunnanensis were easier to extract from soil than root segments of the same length, but which were twisted or irregular (Zhou et al., 1998). Not only is the morphology of individual roots an important factor that influences their contribution to soil shear strength, but also the architecture of the entire root system. A root system composed of a large taproot is more likely to develop the full tensile strength of the taproot. However, in plate- or heart-type root systems examined by Wu et

Table 1

al. (1999), many of the roots would not fail in tension at shear displacements up to 40 cm. Therefore, the full tensile strength of these roots is not utilized when shear strength is estimated. In conclusion, it can be stated that the deeper the root system, the larger the precipitation that a slope can withstand without failure.

V.

ROOT STRENGTH

Root strength varies enormously, not only inter- and intraspecies, but also within the same root system, and may depend on the mechanical role of the root. Tensile strength has been considered to be the most important factor governing soil stabilization and fixation, and has therefore been studied in great detail (Table 1). Tensile strength decreases with increasing root size (Fig. 7), and this phenomenon has been attributed to differences in root structure, with smaller roots possessing more cellulose than older roots, cellulose being more

Root System Type of Different Forest Tree Species Type of root system

Plate Fraxinus excelsior Picea abies Picea sitchensis Pinus strobus (Populus sp.)a Populus tremula (Robinia pseudoacacia) Sorbus aucuparia

a

Heart Acer campestre Acer platanoides Acer pseudoplatanus Alnus glutinosa Alnus incana Betula verrucosa Carpinus betulus Castanea sativa (Fagus sylvatica) Larix decidua Larix leptolepsis (Populus sp.) Prunus avius Pseudotsuga menziesii Pseudotsuga taxifolia Quercus petraea Quercus robur Quercus rubra Tilia cordata Tilia platyphyllos Ulmus effusa Ulmus glabra Ulmus montana

Tap Abies alba Pinus contorta Pinus nigra Pinus pinaster Pinus sylvestris (Quercus sp.) (Robinia pseudoacacia)

Names in brackets can commonly be found with that type of root system, depending on local conditions. Source: Bu¨sgen et al. (1929), Ko¨stler et al. (1968), Eis (1978), Danjon et al. (1999).

182

Stokes

Figure 7 Relationship between tensile strength (MPa) and diameter in roots of Pinus radiata. Tensile strength decreases with increasing root diameter (y ¼ 268:0 0:02 þ2:41, R2 ¼ 0:66). (From O’Loughlin and Watson, 1979.)

resistant than lignin in tension (Turmanina, 1965; Commandeur and Pyles, 1991). Other factors that may govern root strength include the mode of planting: naturally regenerated Scots pine had roots more resistant in tension than those of planted pines (Lindstro¨m and Rune, 1999). The soil environment may also determine root strength: roots of Zea mays growing in weak soil were stiffer than those growing in strong soil (Goodman and Ennos, 1999). The time of year may also affect tensile strength. In temperate regions, roots are stronger in winter than in summer, due to the decrease in water content (Turmanina, 1965). In arid regions the opposite should occur. The data presented in Table 1 should therefore be considered as indicative of tensile strength only for a given species, and should be used with caution. Contrary to the increase in tensile strength with decreasing root size, compression and bending strength decrease with decreasing root size, this being more pronounced in species with heart and taproot systems compared to lateral roots from trees with plate root systems (Stokes and Mattheck, 1996; Stokes and Guitard, 1997). Depending on the mechanical role of a root in a system, wood strength will change to resist better the forces acting on that root; e.g., leeward roots are more resistant than windward roots. This increase in strength probably being due to a greater lignin content (Stokes et al., 1998). Root strength may even increase at certain points along a root, in order to resist rupture as that root repeatedly bends during wind sway (Stokes and Mattheck, 1996; Stokes, 1999). In trees growing on

slopes, tensile strength is greater in uphill roots than in downhill and horizontal lateral roots (Table 1; Schiechtl, 1980). Such changes in wood strength have been attributed to changes in wood anatomy, although an extensive study has yet to be carried out.

VI.

CONCLUSION

There is little literature regarding the existence of different types of root systems (plate/sinker, heart, and tap) in mature trees, and their importance for tree stability. Young trees normally possess a taproot and many horizontal roots, but as the tree matures, the taproot does not develop further, and plays a smaller role in the support of the tree. Ennos (1993) attributes this change to the expense of resources required in developing the taproot. As trees get larger, the efficiency of taproot systems will not increase, but that of plate systems will. The anchorage provided by the weight of the root–soil plate rises with the fourth power of linear dimensions rather than with their cube. Therefore plate systems should be favored more by trees than taprooted systems. Ennos (1993), however, does not acknowledge the cost of constructing heart systems, and presumably he classes them together with plate systems in his analysis. A large number of tree species possess heart root systems when mature, especially broadleaf species (Table 2). A tree will uproot if bending forces on the stem exceed root–soil strength, but are not strong

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183

Table 2 Compression, Bending, and Tensile Strength () of Woody Roots of Different Tree Species (mean values given in MPa)a Species

T

Pinus lambertiana Pinus ponderosa Abies concolor Aleurites mollucana Acacia confucia Pseudotsuga taxifolia (Rocky Mountain) (Coastal) (Coastal) Ficus microcarpa Pseudotsuga menziesii Pinus radiata Sambucus callicarpa Tsuga heterophylla Tilia parvifolia Alnus incana Uphill Downhill Fraxinus excelsior Tilia cordata Acer platanoides Populus deltoides (marsh) Populus deltoides (marah) Abies brachyphylla Picea abies Picea excelsa Pinus pinaster Populus  eur-americana (Clone 1–48) (Clone 1–78) Quercus rubra Kunzea ericoides Nothofagus sp. Picea sitchensis

10 10 11 6–19 11–40

Ziemer (1981) Ziemer (1981) Ziemer (1981) Greenaway (1987) Greenaway (1987)

15 45 63 16–24 13, 17 18 19 20 21 22 33 28 26 26 27 27 39 28 28 28 28

Burroughs and Thomas (1977) Burroughs and Thomas (1977) O’Loughlin (1974) Greenaway (1987) Ziemer (1981), Commandeur and Pyles (1990) O’Loughlin and Watson (1979) Ziemer (1981) Ziemer and Swanston (1977) Riedl (1937) Riedl (1937) Schiechtl (1980) Schiechtl (1980) Riedl (1937) Turmanina (1965) Riedl (1937) Hathaway and Penny (1975) Turmanina (1965) Riedl (1937) Turmanina (1965) Riedl (1937) Dupouy (1992)

Betula pendula Betula verrucosa Populus yunnanensis Salix matsudana Alnus japonica Uphill Downhill Quercus pedunculata Salix purpurea Pinus densiflora Uphill Downhill Horizontal

C

B

20

Reference

31 54 32 32 33 16, 35, 40 38 38 41 41

Hathaway and Penny (1975) Hathaway and Penny (1975) Turmanina (1965) Watson et al. (1999) O’Loughlin and Watson (1981) Ziemer and Swanston (1977), Coutts (1983), Lewis (1985) Turmanina (1965) Riedl (1937) Hathaway and Penny (1975) Hathaway and Penny (1975)

42 40 45 45

Schiechtl (1980) Schiechtl (1980) Riedl (1937) Hathaway and Penny (1975)

48 25 28

Schiechtl (1980) Schiechtl (1980) Schiechtl (1980) (continued)

184

Stokes Table 2 (continued) C

B

Species

T

Thuja plicata Pinus sylvestris (Paperpot) (Natural regeneration) Acer saccharum Populus nigra  P. italiensis Pinus sylvestris Castanea sativa Larix decidua Fraxinus excelsior Picea abies Fagus sylvatica

57

O’Loughlin (1974)

7 20

Reference

35 20

5.5

Lindstro¨m and Rune (1999) Lindstro¨m and Rune (1999) Niklas (1999) Stokes and Mattheck (1996)

23 24 25 26 27 34

3.5 10 5 12 6 15

Stokes Stokes Stokes Stokes Stokes Stokes

and and and and and and

Mattheck Mattheck Mattheck Mattheck Mattheck Mattheck

(1996) (1996) (1996) (1996) (1996) (1996)

a Arranged in order of increasing tensile, compression, and bending strength. Where two or more figures are available for the same species, they are grouped together.

enough to break the stem. Therefore, tree species with shallow rooting systems will be more likely to overturn compared to deeper-rooted species. In a study of the mode of failure of tropical hardwoods and wood quality, Putz et al. (1983) suggested that larger trees with dense, strong wood were more prone to uprooting than to stem snapping. The mass of large, heavy trees under dynamic stress increases the strain on the root–soil surface, and hence the likelihood of exceeding soil shear strength, which may contribute to slope failure. However, smaller trees generally have larger root systems and so will be more firmly anchored, thus increasing the possibility of stem breakage. Snapped broadleaf trees, especially most tropical species, are capable of resprouting from the stem, have larger root systems, and also have a positional advantage over smaller trees, hence occupying openings in the canopy resulting from tree fall. If a tree is healthy, resprouting from the tops of broken stems may compensate for stem failure and allow the tree to maintain its position in the canopy. Most gymnosperms are unable to produce fast-growing sprouts from a broken stem; therefore, whichever type of failure occurs, it will probably be fatal. As resprouting is one way in which an angiosperm can survive after mechanical failure, a relatively large investment in the root system will help prevent uprooting. Coniferous species, however, which are often found in exposed sites, e.g., on hillsides or near the tree line, may not always be able to develop a deep and wellanchored root system. A redistribution of resources to the most mechanically stressed parts of the root system will therefore be the most economical way for conifers

to compensate for a lack of stability, especially in shallow-plate systems (Stokes and Guitard, 1997). There are many areas of tree stability and root anchorage in which we are lacking knowledge, and clearly there is scope for much research in the future. Although the anchorage mechanics of trees are now begun to be examined and understood, several important questions remain unsolved. The role of the taproot and vertical roots in trees that grow on hard pans or waterlogged soils needs to be elucidated. Nicoll et al. (1995) showed that an increased allocation of biomass to the root system did not necessarily increase tree stability. Resources may have been allocated only to the stump region, and not to the lateral roots, which are more important for anchorage. A taproot that has not been able to develop fully may be considered as an extended stump, and serve only a minor role in anchoring the tree on both flat and sloping ground. However, this hypothesis would need to be verified through a series of winching tests similar to those of Coutts (1983, 1986) or by developing a model of the different components of anchorage of such a root system. Once the influence of root system architecture on tree stability has been clearly defined, this knowledge may be utilized in the selection and breeding of windresistant clones. It may also be possible to manipulate young trees by altering plantation methods, or using root pruning techniques. If a taproot system is considered desirable—for example, in the stabilization of a slope—taproot elongation should be encouraged in young trees by using deeper pots. However, for trees destined for planting on hard pans, it may be wiser to

Biomechanics

prune the taproot, thus encouraging greater lateral root development and ramification. Other factors that influence tree root anchorage and soil reinforcement, but which have been little studied, include plantation density and root grafting between trees; the strength of the root–soil bond; the relationship between root strength and root anatomy; and the factors that determine the development of a structural root as well as the death of certain roots.

ACKNOWLEDGMENTS This chapter was written with support from the European Community (project: Eco-slopes, QLRT2000-00289).

REFERENCES Barker DH, ed. 1995. Vegetation and Slopes. London; Thomas Telford. Burroughs ER, Thomas BR. 1977. Declining root strength in Douglas fir after felling as a factor in slope stability. Research paper INT-90, Intermountain Forest and Range Experiment Station, Ogden, UT: U.S. Forest Service. Bu¨sgen M, Munch E, Thomson T. 1929. The Structure and Life of Forest Trees. London; Chapman and Hall. Commandeur PR, Pyles MR. 1991. Modulus of elasticity and tensile strength of Douglas fir roots. Can J For Res 21:48–52. Coutts MP. 1983. Root architecture and tree stability. Plant Soil 71:171–188. Coutts MP. 1986. Components of tree stability in Sitka spruce on peaty gley soil. Forestry 59:173–197. Crook MJ, Ennos AR. 1996. The anchorage mechanics of deep-rooted larch Larix europea  L. japonica. J Exp Bot 47:1509–1517. Crook MJ, Ennos AR 1997. The increase in anchorage with tree size of the tropical tap rooted tree Mallotus wrayi, King (Euphorbiaceae). In: Jeronimidis G, Vincent JFV, eds. Plant Biomechanics. Reading, UK: Centre for Biomimetics, pp 31–36. Danjon F, Bert D, Godin C, Trichet P. 1999. Structural root architecture of 5-year-old Pinus pinaster measured by 3D digitising and analysed with AMAPmod. Plant Soil 217:49–63. Dupouy P. 1992. Conditions d’encastrement d’un arbre dans le sol. DEA de Me´canique, Universite´ Bordeaux I, France. Ennos AR. 1989. The mechanics of anchorage in seedlings of sunflower, Helianthus annuus L. New Phytol 113:85– 192.

185 Ennos AR. 1993. The scaling of root anchorage. J Theor Biol 161:61–75. Ennos AR. 1994. The biomechanics of root anchorage. Biomimetics 2:129–137. Eis S. 1978. Natural root forms of western conifers. Symposium on Root Form of Planted Trees, Victoria, BC, Canada, pp 23–27. Fourcaud T, Lac P. 1996. Mechanical analysis of the form and internal stresses of a growing tree by the finite element method. In: Engineering Systems Design and Analysis, ASME, Montpellier, France, Vol. 77, No. 5:213–220. Goodman AM, Ennos AR. 1999. The effects of soil bulk density on the morphology and anchorage mechanics of the root systems of sunflower and maize. Ann Bot 83:293–302. Hathaway RL, Penny D. 1975. Root strength in some Populus and Salix clones. NZ J Bot 13:333–344. Gray DH, Leiser AJ. 1982. Biotechnical Slope Protection and Erosion Control. New York; Van Nostrand Reinhold. Greenaway DR. 1987. Vegetation and slope stability. In: Anderson MG, Richards KS, eds. Slope Stability: Geotechnical Engineering and Geomorphology. Chichester, UK: John Wiley and Sons, pp 187–230. Hintikka V. 1972. Wind-induced movements in forest trees. Comm Inst For Fenn 76:1–56. Ko¨stler JN, Bru¨ckner E, Bibelriether H. 1968. Die Wurzeln der Waldba¨ume. Hamburg, Germany: Verlag Paul Parey. Lewis GJ. 1985. Root strength in relation to windblow. Forestry Commission Report on Forest Research, 1985. London; HMSO, pp 65–66. Lindstro¨m A, Rune G. 1999. Root deformation in containerised Scots pine plantations—effects on stability and stem straightness. Plant Soil 217:29–37. Mattheck C, Breloer H. 1994. The Body Language of Trees. London; Dept. of the Environment, HMSO. Nicoll BC, Easton EP, Milner AD, Walker C, Coutts MP. 1995. Wind stability factors in tree selection: distribution of biomass within root systems of Sitka spruce clones. In: Grace J, Coutts MP, ed. Wind and Trees. Cambridge, UK: Cambridge University Press, pp 276– 292. Niklas KJ. 1999. Variations of the mechanical properties of Acer saccharum roots. J Exp Bot 50:193–200. O’Loughlin CL. 1974. A study of tree root strength deterioration following clearfelling. Can J For Res 4:107– 113. O’Loughlin CL, Watson AJ. 1979. Root-wood strength deterioration in radiata pine after clearfelling. NZ J For Sci 9:284–293. Putz FE, Coley PD, Lu K, Montalvo A, Aiello A. 1983. Uprooting and snapping of trees: structural determinants and ecological consequences. Can J For Res 13:1011–1120.

186 Quine, CP, Coutts MP, Gardiner BA, Pyatt DG. 1995. Forests and wind: management to minimise damage. Forestry Commission Bulletin 114. London; HMSO. Riedl H. 1937. Bau und leistungen des wurzelholzes. Jahrbu¨cher fu¨r Wissenschaftliche Botanik. Leipzig, Germany: Verlag von Gebru¨der Borntrager, pp 1–75. Schiechtl HM. 1980. Bioengineering for Land Reclamation and Conservation. Edmonton, Alberta, Canada: University of Alberta Press. Stokes A. 1999. Strain distribution during anchorage failure in root systems of Maritime pine (Pinus pinaster Ait.) at different ages and tree growth response to windinduced root movement. Plant Soil 217:17–27. Stokes A, ed. 2000. The Supporting Roots of Trees and Woody Plants: Form, Function and Physiology. Developments in Plant and Soil Sciences No. 87. Dordrecht, Netherlands: Kluwer Academic Publishers. Stokes A, Guitard DG 1997. Tree root response to mechanical stress. In: Altman A, Waisel Y, eds. The Biology of Root Formation and Development. New York; Plenum Publishing, pp 227–236. Stokes A, Mattheck C. 1996. Variation of wood strength in roots of forest trees. J Exp Bot 47:693–699. Stokes A, Berthier S, Sacriste S, Martin F. 1998. Variations in maturation strains and root shape in root systems of Maritime pine (Pinus pinaster Ait.). Trees 12:334–339. Stokes A, Drexhage M, Guitard DG. 2000. A method for predicting the site of failure in trees under mechanical loading. In: Stokes A, ed. The Supporting Roots of

Stokes Trees and Woody Plants: Form, Function and Physiology. Developments in Plant and Soil Sciences No. 87. Dordrecht, Netherlands: Kluwer Academic Publishers, pp 279–285. Turmanina V. 1965. On the strength of tree roots. Bulletin of the Moscow Society of Naturalists, Biological Section 70:36–45 (in Russian with English summary). Waisel Y, Eshel A, Kafkaki U, eds. 1996. Plant Roots: The Hidden Half. 2nd ed. New York; Marcel Dekker. Watson A, Phillips C, Marden M. 1999. Root strength, growth, and rates of decay: root reinforcement changes of two tree species and their contribution to slope stability. Plant Soil 217:39–47. Wu TH, Watson A, El-Khouly MA. 1999. Soil–root interaction and slope stability. In: Proceedings of the First Asia-Pacific Conference on Ground and Water Bioengineering for Erosion Control and Slope Stabilization. April 19–21, 1999, Manila, Philippines, pp 514–521. Zhou Y, Watts D, Li Y, Cheng X. 1998. A case study of effect of lateral roots of Pinus yunnanensis on shallow root reinforcement. For Ecol Mgmt 103:107–120. Ziemer RR. 1981. Roots and the stability of forested slopes. In: Erosion and Sediment Transport in Pacific Rim Steeplands. Publication 132:343–361. London; International Association of Hydrological Sciences. Ziemer RR, Swanston DN. 1977. Root strength changes after logging in south-east Alaska. USDA Forest Service Research Note. PNW-306, pp 1–7.

11 Root Systems of Arboreal Plants Hans A˚. Persson Swedish University of Agricultural Sciences, Uppsala, Sweden

I.

INTRODUCTION

determine the competitive strength of plants are those expressed by the relative size of the absorbing root surface and by the disposition of root tips of competing root systems in relation to the soil available nutrients. Basically, roots within a single plant compete with each other for carbohydrates, and those occupying favorable soil regions tend to grow at the expense of the other roots. Most roots of trees range in diameter size from < 1 mm to several decimeters. There are several good reasons for classifying roots according to their diameters. While ‘‘fine roots’’ (often defined as < 1 mm in diameter) have their major function in absorbing water and nutrients from the surrounding soil, coarse roots may serve multiple functions, in which their size has an important role. There is no established conventional definition of the diameter of fine roots; thus, many forest biomass studies have defined fine roots as those with 5 mm as the upper limit in diameter (Vogt and Persson, 1991). Despite differences in morphology and function, fine and coarse roots often continue to be distinguished according to arbitrarily selected parameters. Different authors (cf. Wilson, 1964) classify roots without referring to diameters as nonwoody (fine) and woody (coarse) roots. It is advisable to realize that the described size classes of roots are not more than crudely related to function. The fine roots of forest trees are almost always infected by mycorrhizal fungi, which influences the diameter size and ramification pattern of the root tips.

Plant growth depends on water and nutrient supply from the soil and upon limitations for uptake imposed by the root system. Plant roots continuously exploit new soil regions where water and nutrients are available. The root systems of trees differ from those of annual plants by their woody framework of longlived structural roots supporting a mass of shortlived, nonwoody fine roots associated with mycorrhizal fungi. Investigations of tree root systems have been hampered since visual examination of the roots requires laborious methods whereby surrounding soil must be removed to expose the roots (Vogt and Persson, 1991). Additional impediments to the investigation of the distribution and function of root systems are their variable form and extensive branching that complicate their excavation. For example, exploration of the root system of one tree, which occupies an area of 10 m2 , requires the removal of up to 30 tons of soil. Once a root system is exposed, description of its threedimensional structure remains a major problem. A detailed morphological description of the root system of even a young tree is a formidable task. The excavation of complete tree root systems gives a good presentation of their morphology. Still it does not answer questions about the functional importance of each constituent with regards to water and nutrient uptake. Study of the differences between tree species and competition for soil resources between individual trees require functional analysis. Important factors that 187

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The white-colored, ‘‘unsuberized’’ area of the root tips, behind the region of cell elongation, is commonly held to be the most active in the uptake processes (cf. Caldwell, 1987). This ‘‘absorption zone,’’ often covered by root hairs, is short-lived in its effectiveness since suberization and loss of root hairs may often take place within a few weeks. Nonwoody roots have very little lignin in their cell walls. Woody roots have lignin along with cellulose and hemicellulose in their cell walls and an outer bark that contains suberin. As secondary xylem thickening takes place, the root tissue may change color from white to brown, caused by a layer of suberin. The suberin lamella develops in the passage cells opposite the protoxylem shortly after secondary growth has commenced. The change in root color may not be related to the development of suberized tissue, and the browning of roots may also be coincidental with the early division of phellogen (Atkinson, 1980). Suberization is strongly influenced by changes in soil moisture (Bartsch, 1987). Suberization of the endodermis may serve as a strategy to reduce water loss during a drought period. In rapidly growing root tips, the root hairs arise firstly several millimeters behind the apex. But, when growth slows down or ceases, root hair development occurs within some millimeters from the tip. Mycorrhizae and root hair are not necessarily exclusive of each other, although the occurrence of root hair seems to be more common in the absence of mycorrhizal infection (Laing, 1932; Cossmann, 1940). The degree of suberization varies during the growing period. Thus, the suberized area of Pinus taeda varied from 0.6% to 6.5% of the total surface area during a growth period (Kramer and Bullock, 1966). Although absorption occurs efficiently through the white apical root segments, significant absorption of nutrients may also occur through suberized and woody roots (Kramer and Bullock, 1966; Chung and Kramer, 1975; Van Rees and Comerford, 1990). The large proportion of woody roots that each tree preserves suggests that these roots are important for its water and nutrient acquisition. Root tips constantly change their position in relation to the embedding soil, and the rate of root penetration is an important factor controlling uptake processes. Thus, for immobile ions, such as phosphate, calcium, zinc, copper, molybdenum, and to some extent ammonium, movement to the root tips is a limiting factor and the activity of root tips and mycorrhizal fungi is crucial for ion uptake. Plant uptake of highly mobile ions such as nitrate, sulphate, and potassium are largely determined by the absorbing capacity of the

Persson

roots (see also Chapter 35 by Jungk in this volume). Maintenance of long-lived root elements seems to be beneficial to plants, at least with regard to energy conservation. However, long-lived fine root elements may be a poor evolutionary strategy, for many reasons. It may be less energetically expensive to continuously produce new roots (and mycorrhizae) than to systematically continue growth and explore microsites for immobile ions. The changes in the number of white root tips may be used as an indication of the periodicity of root growth (Olsthoorn and Titak, 1991). The fine roots, and/or the mycorrhizal hyphae, must constantly extend into undepleted volumes of soil, imposing a high carbon cost in fine root and fungal turnover. The necessity for continuous root penetration of the soil medium raises several questions: Is effective absorption of water and nutrients possible only by new root tissues? What is the adaptive significance of suberization and what processes are involved in it? How has suberization evolved? Is it an inherent property of some species and can it evolve rapidly? Are mycorrhizal fungi essential for uptake processes? Are viable root hairs also essential to uptake processes? What is the role of the dominating suberized part of the root system in the uptake processes? Roots are known to respond to changes in the soil medium, i.e., to irrigation, drought, fertilization, liming, and toxic levels of certain metals or soil acidity that may stimulate/reduce root growth and branching (Persson, 2000). Root branching varies with environmental conditions: the number of root tips is greater in nonfertilized plots, but is greater in irrigated than nonirrigated plots (Ahlstro¨m et al., 1988; Farrel and Leaf, 1974; Persson, 1980a,–c; Lloret and Casero, Chapter 8 in this volume). Production of new roots represents great carbon expenditure by the plant system that appears to be a significant phenomenon in a variety of ecosystems (Jackson et al., 1996). Together with litterfall, root production provides the primary input of organic carbon to forest soils (Vogt et al., 1986; Raich and Nadelhoffer, 1989). The high production and turnover of fine roots may be explained as an ‘‘adaptive strategy’’ (Persson, 2000). Trees have usually evolved in mixed ecosystems, in which survival in a competitive and varying environment (and not necessarily high productivity) has been the evolutionary stable strategy.

Root Systems of Arboreal Plants

The root environment in this context is a key factor, which determines whether a tree will survive. Roots comprise a substantial portion of the biomass of forest ecosystems. Generally, roots account for 15– 30% of the total tree biomass. In spite of their importance, root systems have received limited attention in ecological studies—in particular in the context of ecosystem functioning. Understanding and predicting ecosystem behavior (e.g., carbon and water fluxes) and the role of soils in carbon storage require an accurate knowledge of growth strategies of plant roots and their distribution (Jackson et al., 1996; Persson, 2000; see also Chapter 50 by Kottke and Chapter 49 by Sieber in this volume). The circulation of nutrients in forest ecosystems may follow specific pathways but may be altered at defined rates. Owing to the inherent structure of the forest ecosystems, the flow rates are to a great extent regulated by root functions. Accumulation and cycling of belowground carbon and other nutrient elements vary between the forest regions of the world. High amounts of root biomass (see Rodin and Bazilevich, 1967) accumulate in conifer forests (up to 85 tons/ha), up to 100 tons/ha in deciduous forests, and are particularly high in tropical and subtropical forests (up to 330 tons/ha). The tree constituents below- and aboveground form two different habitats. In the coniferous and deciduous forests the roots are almost exclusively to be found in the soil. In tropical forests, a considerable proportion of roots occur aboveground, and the soil root biomass cannot be regarded as the only root biomass (Jenik, 1971). Ecosystem-level studies in natural forests indicate that the latter probably allocate proportionally more carbon to root systems than agricultural crop systems (Dickmann and Pregitzer, 1992). However, although there are many observations of aboveground wood production, there are few corresponding belowground production rates. Few existing data can therefore contribute to a deeper analysis of belowground ecosystems.

II.

TREE ROOT CHARACTERISTICS

The root system of most trees consists initially of a taproot and its branches. From the development perspective, lateral roots are produced directly from the base of the taproot and may be referred to as axes or seminal roots (Fitter, 1982; Sutton and Tinus, 1983). As a tree grows and develops, multiple orders of lateral roots arise from the primary axes. Up to seven orders of lateral roots have been reported for trees (Sutton

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and Tinus, 1983). Important differences in phylogeny and ontogeny exist between the root systems of different tree species and between deciduous and coniferous trees. Nevertheless, compared with our understanding of the development of aboveground tree organs, our knowledge of the architecture of tree roots is limited. Complete descriptions of the branching patterns of roots of mature trees are generally lacking and should be sought. Deciduous tree root systems are often more extensively developed in the uppermost soil horizon (Table 1). In tropical and temperate regions, leaves of most deciduous trees decompose rapidly and the trees have evolved a superficial and extensively branched fineroot system to intercept and absorb nutrients from the litter layer. The litter layer comprises shoot material but also decaying root material. This humus layer is the basis for the complex biological cycles in the soil those include bacteria, fungi, and the soil fauna. As a rule, fine roots of deciduous species are smaller in diameter than those of coniferous trees, and they generally exhibit a higher turnover (Vogt and Persson, 1991; Strober et al., 2000). The thinner the diameter of a root, the shorter is its life span (Heikurainen, 1955; Dickmann and Pregitzer, 1992; Schoettle and Fahey, 1994). In deciduous forests most fine roots tend to be < 0:05 mm in diameter and therefore difficult to separate from the soil in a quantitative manner. An excavated root system of a tree consists of a mixture of roots of different diameters, root types, and ages (cf. Eshel and Waisel, 1996). Age determination in structural roots is difficult because they frequently form discontinuous rings. In most cases, the stem age cannot be used for determining the age of the roots, because the stem may be of sprout origin and younger than the root system, or because the roots are adventitous and younger than the stem (Wilson, 1964). Different types of root systems may be distinguished in different environments. Root characteristics are influenced by species, by competition, by soil characteristics, and by cultural practices (Ko¨stler et al., 1968; Kozlowski et al., 1991; Kutschera et al., 1997). Classification of root systems of different tree species is usually defined by their morphology (cf. Ko¨stler et al., 1968), but should also be based on direction and distribution of larger roots (Fig. 1). Four basic forms of root systems can be distinguished: heart root systems (Herzwurzelsystem), taproot systems (Pfahlwurzelsystem), flat root systems (Flachwurzelsystem) and sinker root systems (Senkerwurzelsystem) (see Lyr and Hoffmann, 1967). A central complex of many vertical roots characterizes

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Persson Table 1 Density of Live, Dead, and Total Fine Roots (< 1 mm in diameter) at Two Nearby Fagus sylvatica and Picea abies Stands in Northern France Species/depth (cm) Fagus sylvatica 0–2.5 2.5–5.0 5.0–10.0 10.0–20.0 20.0–40.0 Picea abies 0–2.5 2.5–5.0 5.0–10.0 10.0–20.0 20.0–40.0

Totals (live þ dead) (g cm1 m2 )

Live ðg cm1 m2 )

Dead ðg cm1 m2 )

12.1 aa 8.3 b 2.9 c 1.0 e 0.7 e

4.2 bc 5.7 a 4.7 a 3.6 c 2.5 b

16.3 14.0 7.7 4.7 3.3

5.0 cd 6.1 b 3.8 d 0.8 e 0.2 f

2.0 d 4.6 bc 3.9 bc 1.3 d 0.8 e

7.0 de 11.2 e 7.6 d 2.0 h 0.9 h

a a b b bc

Means (n ¼ 20) followed by the same letter are not significantly different by Duncan’s multiple range test. Source: Strober et al. (2000).

a

the heart root system. The taproot system originates from the taproot, i.e., the primary positively geotropic root that emerges from the seed. It is designated as the radicle and later as the taproot. The taproot may or may not be a prominent feature of the mature tree root system. Whereas most tree species retain a taproot for a long period, the initial taproot of some species is

replaced in early years by predominant lateral or by adventitious roots. Both heart and taproot systems attain pronounced depths. This is in contrast to the flat root systems, which mainly consist of horizontally distributed lateral roots. The sinker root system is usually more developed after the taproot is lost, bent, or stunted. Although the taproot may still persist, the

Figure 1 Schematic representation of a tree root system with nomenclature to be found in several European schools of classification.

Root Systems of Arboreal Plants

many sinker roots that descend vertically from the bases of lateral roots may hide it. Sinker roots are usually found within a few meters from the stem. The central part of the root system is at the base of the tree and may extend for some meters from the tree stem (Fig. 1). It consists of the taproot, main laterals, and several vertically and obliquely descending and ascending roots. The taproot is most strongly developed in both deciduous and coniferous tree species during their early stage of development. First-order (first degree of branching) lateral roots emerge from the taproot. Frequently, lateral roots, growing from the lower part of the taproot, tend to descend vertically almost as soon as they start to elongate. These roots, even in young trees, may penetrate into the soil as deeply as the taproot. In tropical forests, unusual aerial tree roots such as stilt roots and tabular roots (buttresses) make a description of their root systems even more difficult (Jenik, 1971; Sen, 1980). The most common coniferous forest trees in Northern Europe, e.g., Pinus sylvestris, Abies alba, sometimes Picea abies, and Larix decidua, are characterized by extensive taproot systems. Most deciduous tree species, except for the most common oak species (e.g. Quercus petrea and Q. robur) have developed heart root systems in their mature state. Typical heart root systems are to be found in Betula alba, Fagus sylvatica, and Tilia cordata. Sinker root systems are frequently a characteristic feature of the root system of Picea abies. Generally in tree roots, up to 10 first-order lateral roots at the base of the tree make the basic framework of the central root system and provide much of the support and anchoring system of the tree. First-order roots are eccentric and rapidly become cylindrical after extending a few meters from the tree. Root grafting between roots of the same tree is common within the central root system. The large-diameter roots in the central root system occupy so much space that it is easy to overlook the fact that many small firstto fourth-order laterals are also present. Most lateral roots are remarkably straight and appear to be oriented both with respect to the surface of the soil and with respect to the tree stems. The main drawback of these classifications lies in their disregard of fine root characterization. Roots continue to branch until the root tips reach a diameter of < 1 mm. The woody framework of horizontal and vertical root systems always bears many fine-root branches that have reduced cambial activity and may live for a few weeks. Approximately 90% of the total length of roots in Picea sitchensis and Pinus

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sylvestris plantations, excavated from soil cores, consisted of fragments < 0:5 mm in diameter (Ford and Deans, 1977; Roberts, 1976). Obviously, there is a large population of fine roots in forest soil. Many tree root systems have several kinds of roots and are therefore called ‘‘heterorhizic’’ (Tschirch, 1905; Noelle, 1910). Two broad root types can be frequently distinguished: long roots and short roots (Fig. 1). Anatomically they are very different and exhibit different elongation rates (Hatch and Doak, 1933). The long-roots are known as growth roots, or extension roots, and they are endowed with a potential capacity for extensive growth. Periodically new branches of short roots develop on new extending long roots (Orlov, 1980). Heterorhizic root systems, with contrasting long and short roots, are found among trees of the gymnosperms and angiosperms. Long roots exhibit secondary growth, are diarch or polyarchic, and have a root cap. Short roots are monoarchic, extend slowly in length, ramify extensively, and exhibit no radial growth. Long roots may continue growing for several years. The fairly thick and fast-growing long roots extend continuously into new areas of soil, or reoccupy areas that had been explored earlier. Although heterorhizic root systems occur in many Pinaceae, Betulaceae, Fagaceae, etc., they are absent from many other woody plants (Kozlowski, 1971a). Many plants of the Cupressaceae, for example, do not develop a heterorhizic root system. As a result of their frequent branching, short roots form dense networks of ramifications, often infected by fungi and form mycorrhizae (see Chapter 50 by Kottke in this volume). Such roots are continuously replaced as new areas of the soil are explored. Thus, the greatest natural loss of roots from trees occurs in short roots that reach a length of only a few millimeters. Mycorrhizal infections influence root form and the development of root tips by modifying fleshiness, elongation rate, branching patterns, root hair development, and the degree of suberization. The racemose form of mycorrhizae is most common in angiosperm and gymnosperm root systems, but pinnately branched and monopodial forms of mycorrhizae also occur (Harley, 1959). The most common mycorrhizae in Pinus are dichotomously branched (Hatch and Doak, 1933; Sutton, 1969). The short root tips are then often repeatedly dichotomized, forming branches of higher order in tuberlike masses (Ahlstro¨m et al., 1988). Short roots elongate more slowly than long roots, though in some cases they may reach considerable lengths (Lyford and Wilson, 1966). The growth rate of a short root of Pinus sylvestris may not exceed

192

Persson

0.10–0:15 mm day1 (Orlov, 1980). However, owing to their high number, their total length per unit area of the forest soil may be considerable. The root system of one 100-year-old Pinus sylvestris had 5 million root tips (Kalela, 1954). In a 15 to 20-year-old P. sylvesrtris stand, the number of root tips was
60,000 m2 and they grow about 2–3 mm2 day1 (Persson, 1978). In a 120-year-old stand the related number of root tips was 295,000 m2 and their mean growth rate was some 7 mm2 day1 (Persson, 1980a,b). Up to 95% of the root tips in Pinus sylvestris stands are short root tips. Although mycorrhizal fungi frequently infect long root tips, they rarely exhibit typical mycorrhizal ramifications. The growth rate of Pinus sylvestris long roots is in the range of 1–2 mm day1 (Orlov, 1980). Similarly to root hair formation, mycorrhizal development seems to be influenced by the interrelations between soil fertility and soil moisture. The initial size of a root tip determines whether the root becomes thickened or dies after a short while. Long roots and their branches facilitate an efficient system for exploration of the soil, and provide a longlasting structure from which the short roots can proliferate when local conditions are favorable. Long roots include the rapidly developing taproot. The latter enables seedlings to penetrate quickly into deep, moist horizons of the soil, thus enabling early establishment and survival of the seedlings. The total belowground biomass increases in importance in terms of dry weight with the age of the trees, whereas the opposite is some-

times observed for the taproot (Table 2). Few investigations in pure stands (even fewer in mixed stands) have centered on the relationship between mature trees and seedlings. In general, the increase in root weight parallels shoot development, and the living shoot to the living root ratio is fairly constant (Ovington, 1957; Ovington and Madgwick, 1959a,b). Generally root systems of individual trees do not function independently, since grafting between adjacent trees is a common feature (Bormann, 1966; Ku¨lla and Lo˜hmus, 1999). An increased number of root grafts with increased tree density in Picea abies and Fagus sylvatica stands was reported (HolstenerJoergensen, 1958–59). Translocation of water, minerals, and organic substances between trees through root grafts emphasizes the important role of grafted trees in developing tree stands. It is likely that when the aboveground parts of some trees are removed during thinning operations, their root systems may survive, adding to the root system of the remaining trees. Grafts between roots of the same species are quite common, while interspecific grafts do occur, but not so frequently (Woods and Brock, 1964). Nutrient transport by mycorrhizal links has also been demonstrated under experimental conditions. However, net gain of mineral nutrients by one living tree from another by mycorrhizal fungi is still questionable (Newman, 1988). Nevertheless, when roots die the transfer of important nutrients from the dead roots to the roots of surrounding trees occurs, most likely

Table 2 Belowground Dry Weight Proportions of Root Fractions in Relation to Total Tree Biomass Plantations of Pinus sylvestris in Great Britain on Sandy Subsoils Age 7 11 14 17 20 23 31 35 55 a

Total dry weight for the tree (kg)

Total dry weight for the root system (kg)

1.5 6.1 6.4 8.5 12.1 25.2 53.6 86.4 198.3

0.70 (47)a 2.51 (41) 2.00 (31) 2.26 (27) 2.60 (21) 7.71 (31) 11.67 (22) 23.49 (27) 44.84 (23)

Total dry weight for the taproot (kg) 0.06 0.45 0.55 0.85 1.05 3.92 4.65 11.17 19.53

(4) (7) (9) (10) (9) (16) (9) (13) (10)

The different root fractions are indicated as a percentage within parentheses of the total dry weight tree. Source: Ovington (1957).

Root Systems of Arboreal Plants

by mycorrhizal links. The total amount of nutrients removed from dead roots can be substantial. The absence of significant differences in N, P, K, and Mg concentrations between live and dead fine roots suggest that there is little direct translocation of nutrients from senescing fine-root tissue (Nambiar, 1987; Persson et al., 1995). To function efficiently, tree root systems must be both extensive and active enough to meet the needs of aboveground plant parts. Forest-thinning operations significantly affect root development and belowground living plant biomass recovers to preharvest levels more rapidly than aboveground biomass (Yin et al., 1989). Heavy thinning (60% reduction) in a Pinus radiata plantation has reduced the amount of live fine roots, while the amount of dead fine roots remained unchanged (Santantonio and Santantonio, 1987). Numerous observations have indicated that a healthy root system is necessary to secure vigorous growth and wood quality of the forest plantation (Sutton, 1980, 1983). Knowledge about root growth and root architecture during the seedling phase of root development will assist management decisions where intensive silviculture is practiced, and hence ensure survival and good plant performance. A newly planted tree has a root system, which provides access to a fairly limited volume of soil (cf. Scarratt et al., 1981). This makes the newly planted tree vulnerable to desiccation and nutrient deficiency. In the seedling stage new root growth is strongly dependent upon current photosynthates (Noland et al., 1997). Planting seedlings in forest sites with reduced light levels due to light competition from already established trees may result in reduced root growth and survival of the seedlings. Special tree seedling containers were developed to ensure good root growth and seedling establishment. The concept of ‘‘containerizing’’ tree seedlings is based on the premise that seedlings grown in the containers will have a protected root system, which will develop without restraint, after the seedling is outplanted. Enhancement of stem production is given high priority in most breeding programs. Breeding strategies until now seldom took genetic variation of root/shoot ratios into account. Allometric analysis of root/shoot ratios suggests that mechanisms exist that regulate the distribution of photosynthates between the shoot and root systems. Linear regression of log shoot weight against log root weight reported under experimental conditions is suggested to be related to normal changes in the root/shoot ratio with increasing plant size (Ledig et al., 1970). Results from various investigations support the idea that root

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system architecture is genetically determined (Santantonio et al., 1977; Drexhage et al., 1999). However, although developmental patterns may operate within a wide range of environments, to maintain a generally balanced growth of root and shoot, factors such as tree density, climatic differences, soil fertility etc., strongly influence the overall pattern.

III.

FACTORS INFLUENCING GROWTH RATES OF TREE ROOTS

One fundamental feature of the growth of plant populations is the resource restriction that limits their performance. From a functional point of view, forest trees consist of an aboveground part, which captures energy and carbon, and a belowground part, which captures water and mineral nutrients. It is widely believed that a functional equilibrium exists between the size and activity of the leaves/needles (which fix carbon) and the size and activity of the fine roots (which take up water and nutrients). Owing to the difficulty of accurately determining the root weights of large trees, most available measurements are from seedling material. It is not possible to use allometric equations for mature trees to predict the amount of fine roots from momentary measurements of the aboveground leave/needle biomass, since the fine-root biomass changes constantly at varying rates during the growth period (Vogt and Persson, 1991). Trees consist of extensive systems of surface areas both above- and belowground. The total area of a tree’s leaf/needle surface is, for example, several multiples of the ground covered by the tree’s crowns. Large amounts of plant tissue also accumulate in the root system (Stone and Kalisz, 1991). The horizontal roots may make up to 80% of the total biomass; more biomass is allocated to horizontal with increasing stem diameter, since the vertical root proportion is decreased (Drexhage et al., 1999). The amount of energy partitioned to the roots from aboveground photosynthesizing tissues depends on the demands of the roots as well as those of the shoots. As much as half of all photosynthates may be exported from the leaves to roots (Lambers, 1987). Between 8% and 52% of all carbohydrates produced per day by various species are respired in the roots during the same period (Lambers et al., 1996). Besides their functions in nutrient and water absorption, fine roots have a function as a reservoir of meristems, which are involved in the physiology of those plants.

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Unlike shoot and leaf growth, roots of mature trees can grow throughout the year. Since soil temperature and soil moisture levels fluctuate considerably in forests during a growing season, root initiation and growth must occur under a wide range of environmental conditions (Kuhns et al., 1985). High levels of starch are found in tree roots throughout the year and may be used up for root growth during periods of low supplies of photosynthates (Ericsson and Persson, 1980). Starch contents in roots may be looked upon as an indicator of tree vigor (Wargo, 1979). The functional role of starch reserves is to supply the active meristems with carbohydrates when sink demands on current assimilates are larger than the supplies (Cannell and Dewar, 1994). Starch is present before and after cessation of apical meristem activity of long root tips, indicating that the carbohydrate supply is not the limiting factor of their growth (Wilcox, 1954). All perennial organs of trees may serve as storage organs, but the highest concentration of carbohydrate reserves is usually found in root tissues. Root reserves respond rapidly to defoliation or to disruptions of photosynthesis, particularly late in season following the cessation of vegetative growth (Loescher et al., 1990). Marshall and Waring (1985) suggested that fineroot mortality follows closely the exhaustion of starch and sugar reserves. Starch is subsequently stored in growing roots to meet maintenance requirements. Marshall and Waring (1985) concluded that the initial starch concentrations in root tissues and soil temperature are the key variables in estimating fine-root turnover and the potential of fine-root biomass. Factors that can reduce plant growth, such as low temperatures and water deficit, favor carbohydrate investments in root growth (see Chapter 34 by Glass in this volume). The onset of root growth is generally controlled by soil warming in spring—the cost of root maintenance increases exponentially with temperature (Gill and Jackson, 2000). Root extension at low soil temperature in spring favors early water uptake and may be of competitive advantage. Soil temperature also affects rates of root maturation: low temperatures retard root growth and maturation, whereas high temperatures accelerate both processes. The fine roots occupy a key position in nutrient and water uptake, since, although they may represent only some 5% of the total dry weight, they can account for 95% of the root length and surface area (Persson, 1980a,b). While many large-diameter roots remain alive as long as the tree stays alive, fine roots have been demonstrated to be in a constant turnover,

Persson

often with a high seasonal rate of death and renewal (Lyr and Hoffmann, 1967; Kolesnikov, 1968). Studies of growth of structural organs of a forest tree—the main trunk, branches, stems, large-diameter roots etc.—may lead to a better understanding of how resources allocation of the tree change with age. Growth of leaves (or needles) and growth of fine roots have a more immediate functional importance. In response to the special features of the soil environment, extreme dilution and immobility of nutrients, evolution has given rise to root systems that by means of progressive penetration and branching bring an astonishingly large surface area in contact with the soil. The degree of root penetration may be regarded as a vitality criterion of the forest stand (Persson, 1996). The close contact between the soil and the penetrating root tips is probably one of the reasons why plants can remain stationary. The energy input to the soil from dead roots contributes significantly to sustain soil fertility. The rate of root elongation may vary for one individual root from a few millimeters to well over 25 mm a day during periods of active growth (Kozlowski, 1971b). In sandy sites lateral roots of some forest trees may extend up to 40 mm a day as they continually occupy new soil volumes (Lyr and Hoffmann, 1967). Often there seems to be a negative correlation between root/shoot ratio and fertility (Persson, 2000). High levels of nutrient supplies increase aboveground growth relative to root growth (Ledig, 1983; Persson, 1996). Aaltonen (1920) found that in poorer soil types the roots were more numerous, and that they extended farther both horizontally and vertically than on good sites. The number of roots increases with age and with increasing density of the crowns (Stevens, 1931). Considering the high cost of fine-root growth (A˚gren et al., 1980; Persson, 1995), greater attention should be given to the development of carbon and nitrogen budgets for different forest ecosystems. The effect of environmental factors on the intensity and course of plant investment in roots is a neglected but important area of plant ecophysiology. Water uptake, absorption of inorganic ions, and transport of the latter to the aboveground parts of the tree are closely related to the metabolic activity and growth rates of roots (Ericsson and Persson, 1980). Furthermore, certain hormones are synthesized at the root tips and later are moved to the aboveground parts of the tree and influence shoot development (Torrey, 1976; Bowen, 1985; see Chapter 25 by Emery and Atkins in this volume). The large photosynthate requirement for maintenance respiration of forests combined with high annual accumula-

Root Systems of Arboreal Plants

tion of organic matter in live and dead roots may limit forest growth in regions where photosynthate production is limited by the length of the growth season. Respiratory losses of roots can account for a large fraction of the fixed carbon and may be a more important determinant of yield than the photosynthetic rate (Cropper and Gholz, 1991). Most tree species require an adequate supply of oxygen in the soil atmosphere to meet their needs for high respiration and growth. Inadequacies in soil aeration influence root growth as well as top growth (Rickman et al., 1966). Pettersson et al. (1993) concluded that increased growth rate and net assimilation at elevated ambient CO2 were not associated with a shift in dry matter partitioning between roots and shoots. Other investigators challenged this. Two marked periods of root growth activity can be distinguished, one in spring and the other in autumn (Lyr and Hoffman, 1967). In regions that lack winter dormancy, for example in the Mediterranean region, root growth may take place in the whole soil profile during the winter months (Leshem, 1965). Under such conditions, growth may gradually cease during the dry summer, and the majority of roots normally enter then a rest stage (Leshem, 1970). Year-round continuation of root growth in glass houses or during mild winters supports the view that tree roots, unlike shoots, do not have an inherent period of dormancy (Morrow, 1950). Current views do not support the idea of autonomous growth rhythms of tree roots (Lyr and Hoffmann, 1967; Hermann, 1977). The frequently observed periodicity in root growth may instead reflect periodically recurring changes in environmental factors. Lyford and Wilson (1966) found that the optimum temperature for root growth for Acer rubrum was
12–15 C. In general, the minimum temperature for root growth for many tree species is considered to be between 0 and 5 C (Kuhns et al., 1985). Reduced root growth caused by low soil temperature is probably one of the factors that determine the natural tree line at high altitudes. Reduced root growth combined with high transpiration rate, limited water uptake by the roots, and desiccation, determines that line (Jackson et al., 1996). Frost damage to roots has become a primary cause of injury to the container-grown forest tree planting stock in high latitudes (Stattin, 1999). Under unfavorable conditions (drought, frost, lack of carbohydrates, etc.) many of the fine roots die. However, new ones rapidly form once conditions become more favorable. Santantonio and Hermann (1985), investigating the standing crop of live and

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dead fine roots on dry, moderate, and wet Pseudosuga menziesii stands, found that in the drier site the fine-root turnover rate was faster. During periods of prolonged heavy rainfall, and in poorly drained soils, the supply of oxygen is insufficient and root growth and other functional activities including water uptake are impaired. Only a freely drained soil contains sufficient air to supply the needs of the roots. One additional factor that controls root growth is resistance to root penetration—viz., the soil mechanical impedance (see Chapter 45 by Masle in this volume). Soil mechanical resistance reduces the rate of root penetration in dry or compacted soil. Frequent light showers, or frequent irrigation, encourage shallow rooting by keeping surface soil moist during the summer. In deeper soil horizons, the soil water level to a great extent influences the rooting depth. Fine roots grow toward more humid zones in the soil. The efficiency of root penetration depends on soil conditions as well as on the degree of suberization and mycorrhizal infection. In two nearby mature Pinus sylvestris stands on similar soil types, but with varying depths of the soil water table (Table 3), the fine roots were more deeply distributed in the stand with the highest soil water table. The fine roots in the latter case evidently penetrated more deeply in the soil profile owing to the available soil water and reduced soil resistance. Trees with deep root systems are considered more tolerant to drought stress than trees with shallow root systems because the upper soil layers are the ones that dry first.

Table 3 Vertical Distribution Down to 60 cm of the Mineral Soil of Living Fine Roots (< 2 mm in diameter) in Two Mature Pinus sylvestris Stands (IH0 and IH5) in Central Sweden with Different Groundwater Tables Soil horizons/depth (cm) LFH (6-7) Mineral soil 0–10 10–20 20–30 30–40 40–50 50–60 Total soil profile

Amount of living fine roots (g m2 ) IH0

IH5

79

99

59 37 18 20 6 1 221

72 15 2 1 1 1 189

The soil water table was at its highest 1–3 m in IH0 and 10 m level in IH5. Source: Persson (1982).

196

Persson

Tree fine roots are subjected to a variety of environmental conditions in the various soil horizons, and confront high variability in the availability of nutrients and soil water (Persson, 2000). Root growth responds to the presence of plant nutrients in the vicinity of the active root surface area (Marschner, 1988). The root surface area near the root tip is commonly held to be most active in the uptake processes. The uptake rate by a tree is therefore likely to be related to the number of white or unsuberized root tips (Caldwell, 1987). There is a greater proliferation of roots and of mycorrhizae in the uppermost layer of the soil horizon than in deeper ones, since the surface soil is richer in organic matter and in nutrients (Persson, 1980a,b). Tree roots grow most vigorously in these upper soil layers since the soil organic matter improves nutrient availability and reduces soil mechanical impedance. One point often neglected by root investigators is that roots are the principal source of organic matter in the deeper soil layers, and that the activity of soil microorganisms and of the soil fauna is to a large extent regulated by root activity. There is evidence that the success of tree roots in the uptake processes from organic matter depends on the activities of their mycorrhizae (see also Chapter 50 by Kottke and Chapter 49 by Sieber in this volume). Studies of nitrogen cycling suggest that inorganic nitrogen may be an insufficient source for boreal forests. Mineralization rates may underestimate the rates of nitrogen supply to plants, since the mycorrhizal plants short-circuit the mineralization step of decomposition by directly absorbing amino acids (Abuzinadah and Read, 1986, 1989). Among the factors that are known to influence root growth are water level, aeration, reducing properties of peat, concentration of the soil solution, nitrogen supply, iron starvation, the point of origin of roots, and mycorrhizae. Such factors that influence the life span of fine roots include also seasonal herbivore and pathogen pressure, competing sinks for carbohydrates in the plant, nutrient resorption, and recycling (Eissenstat and Yanai, 1987). Despite the basic knowledge of the importance of such factors for root system development, we know only little about the processes that have directed the evolution of roots in an adaptive fashion (Persson, 2000).

IV.

ROOT STRUCTURE

Tree roots may be regarded as an important plant organ that provides anchorage in the soil, prevents

wind damage and soil erosion, and builds up a major part of the organic soil horizon with regard to its biotic and abiotic components. The number and spatial distribution of structural roots are important traits for tree stability (Chapter 10 by Stokes in this volume). Data for coniferous and deciduous tree growth under widely differing environmental conditions show a remarkably consistent relationship between root system biomass and stem diameter at breast height (Santantonio et al., 1977). However, the proportion of tree biomass partitioned to the roots appears to decline during the first year of growth, and this decline may continue for a number of years thereafter (Dickmann and Pregitzer, 1992). A substantial amount of dry weight is accumulated in structural tree roots (Table 4). Thus, up to 150 tons/ ha of dry weight is accumulated in structural roots of mature trees. Roots comprise a substantial portion of forest ecosystems. Generally, roots account for up to a fourth of the total biomass of forests. Proportionally more structural roots are found in mature forest ecosystems, with mixed forest stands possessing deeper and more completely developed structural roots than monocultures of the same tree species (Ko¨stler et al., 1968; Parrotta, 1999). The competitive environment in mixed forest ecosystems may favor root investments as a survival strategy of trees. The ratio of structural roots/aboveground dry weight of woody tissues varies for different forest ecosystems from 4% to up to 48% (Table 4). The dry weight of woody roots of Pinus sylvestris seems to increase with age. But this is not a general phenomenon. Inverse trends are found for other species. When excavating structural root systems, emphasis has been placed on ‘‘typical’’ root systems. Root sampling involves the same principles as those for the aboveground parts of the tree. The major difference is that the root systems of trees intermingle far more than the crowns, and they are concealed in the soil. Depth of similar roots and spread of lateral root are the most frequently studied characteristics of tree roots. Often the three-dimensional distribution of the root systems were simplified into two-dimensional diagrams—either horizontally drawn from above, or vertically, from the side (Kutschera et al., 1997). Although soil properties affect root form, growth direction, and distribution, root architecture tends to be under genetic control (Ko¨stler et al., 1968). Explanations for increased aboveground biomass in trees in response to nutrient supply have been sought mainly in terms of increased foliage biomass and/or rate of photosynthesis. Ericsson (1995) reviewed the

Root Systems of Arboreal Plants

197

Table 4 Distribution of Various Above- and Belowground Structural Components (tons/ha) of Forest Trees and for Forest Ecosystems (fine roots and foliage not included) Species

Age

Woody roots

Abies amabilis Abies amabilis Betula verrucosa Betula verrucosa Fagus sylvatica Fagus sylvatica Fagus sylvatica Fagus crenata Fagus crenata Fagus crenata Larix gmelinii Larix leptolepis Leucaena leucocephala L. leucocephala (with C. equisetifolia) L. leucocephala (with E. robusta) Casuarina equisetifolia C. equisetifolia (with L. leucocephala) C equisetifolia (with E. robustus) Eucalyptus robusta Eucalyptus robusta (with L. leucocephala) Eucalyptus robusta (with C. equisitifolia) Picea abies Picea abies Picea abies Picea abies Picea abies Picea abies Picea abies Picea abies Picea abies Picea abies Picea abies Pinus densiflora Pinus sylvestris Pinus sylvestris Pinus sylvestris Pinus sylvestris Pinus sylvestris Pinus sylvestris

23 180 35 38 90 52 53 35 41 > 50 240–280 21 4 4

15.5 124.9 16.9 49.8 49 26.2 46.3 54.2 58.2 49.9 10.8 2.8 10.3 5.0

Pinus Pinus Pinus Pinus Pinus Pinus

sylvestris sylvestris sylvestris sylvestris sylvestris sylvestris

(30)a (23) (22) (24) (13) (17) (17) (21) (29) (17) (34) (15) (14) (14)

Stem

Total

27.7 356.1 48.0 134.5 221 131.1 232.7 168.0 165.5 202.2 18.1 7.9 63.9b 29.6b

50.9 548.9 76.7 211.3 375 157.3 279.0 258.4 202.2 296.5 32.1 19.3 74.2 34.6

References Grier et al., 1981 Griet et al., 1981 Ovington and Madgwick, 1959a,b Ovington and Madewick, 1959a,b Nihlga˚rd, 1972 Mo¨ller et al., 1954 Mo¨ller et al., 1954 Tadaki et al., 1969 Tadaki et al., 1969 Tadaki et al., 1969 Kajimoto et al., 1999 Satoo, 1960 Parrotta, 1999 Parrotta, 1999

4

7.6

(11)

63.3b

70.9

Parrotta, 1999

4 4

18.4 15.7

(19) (18)

77.9b 69.3b

96.3 85.0

Parrotta, 1999

4

12.1

(18)

53.4b

65.5

Parrotta, 1999

4 4

11.3 4.6

(20) (16)

45.6b 23.8b

56.9 28.4

Parrotta, 1999 Parrotta, 1999

4

4.4

(18)

20.4b

24.8

Parrotta, 1999

24 38 55 60 93 30–100c 30–100c 30–100c 30–100c 30–100c 30–100c 15 7 9 12 14 14 23

20.2 38.1 58 64.7 65.4 15.5 17.0 16.8 15.5 13.9 12.3 10.8 6.6 1.3 0.6 1.6 2.2 28.1

(23) (25) (16) (25) (21) (18) (20) (19) (18) (16) (15) (17) (7) (4) (4) (5) (6) (48)

69.1b 113.1b 262 195.6b 249.5 40.4 42.2 43.0 43.3 43.5 43.7 41.9 67.0 9.6 7.3 15.5 21.7 44.3

89.3 151.2 367 260.3 314.9 87.9 86.2 86.3 85.6 84.4 83.9 64.0 94.9 30.2 16.5 32.3 38.3 58.1

28 45 47 27 26 28

7.0 19.3 11.0 6.1 6.6 7.5

(28) (20) (21) (6) (5) (7)

11.6 60.9 30.4 74.1 84.3 75.8

17.9 75.9 41.9 104.7 121.0 112.6

Sonn, 1960 Sonn, 1960 Nihlga˚rd, 1972 Sonn, 1960 Sonn, 1960 Chibisov, 1995 Chibisov, 1995 Chibisov, 1995 Chibisov, 1995 Chibisov, 1995 Chibisov, 1995 Satoo, 1960 Albrektson, 1980 Albrektson, 1980 Albrektson, 1980 Albrektson, 1980 Albrektson, 1980 Ovington, 1957; 1959; Ovington and Madgwick, 1959a,b Ma¨lko¨nen, 1974 Ma¨lko¨nen, 1974 Ma¨lko¨nen, 1974 Albrektson, 1980 Albrektson, 1980 Albrektson, 1980 (continued)

198

Persson

Table 4 (continued) Species

Age

Pinus sylvestris Pinus sylvestris Pinus sylvestris Pinus sylvestris Pinbus sylvestris

29 33 34 50 55

Pinus sylvestris Pinus sylvestris Pinus sylvestris Pinus sylvestris Pinus sylvestris Populus deltoides Populus deltoides Populus deltoides Populus deltoides Quercus borealis Pseudosuga menziesii Pseudotsuga menziesii Equatorial forest Equatorial forest Intermediate savanna Open shrub savanna Dense shrub savanna Savanna woodland Mixed savanna Tropical evergreen Tropical deciduous Tropical deciduous Temperatate deciduous Northern hardwoods Northern hardwoods Pygmy cypress Subalpine spruce-fir

84 85 88 100 120–150 1d 2d 3d 4d 49 35–50 Uneven Uneven Uneven Uneven Uneven Uneven Uneven Uneven 18 Uneven 50 Uneven Uneven 79–106 Uneven 200–500

Woody roots

Stem

Total

5.7 32.7 8.6 7.6 34.1

(6) (18) (9) (8) (31)

63.2 118.8 66.7 64.6 96.7

94.3 185.9 98.5 90.8 109.0

10.0 17.9 21.1 12.5 14.2 13.8 16.0 16.5 16.7 15.0 67.4 141.3 2.8 2.6 3.6 9.2 14.3 26.6 18.6 31.3 34.3 24.7 39 28.3 20.6 7.1 18

(7) (34) (34) (11) (19) (19) (19) (19) (19) (9) (23) (16) (9) (11) (32) (29) (29) (31) (26) (21) (15) (9) (17) (18) (18) (32) (14)

108.6 41.0 48.2 81.3 45.2 48.2 49.4 53.6 55.1 111.9 263.282 704.5b 19.2 14.7 7.4b 21.9 32.6 54.3b 53.4b 116.4b 141.1 261.6b 231b 132.8 51.0 20.4 94

146.8 53.4 62.4 117.0 74.0 72.0 85.8 88.1 88.7 176.4 293.8 857.1 31.5 24.3 11.3 31.8 48.6 84.7 72.0 147.7 234.8 286.3 231 161.1 111.8 21.8 125

References Albrektson, 1980 Ovington and Madgwick, 1959a,b Albrektson, 1980 Albrektson, 1980 Ovington, 1957; 1959; Ovington and Madgwick, 1959a,b Albrektson, 1980 Fine´r, 1992 Fine´r, 1992 Albrektson, 1980 Bringmark, 1977 Lodihyal & Lodihyal, 1997 Lodihyal & Lodihyal, 1997 Lodihyal & Lodihyal, 1997 Lodihyal & Lodihyal, 1997 Ovington et al., 1962 Fogel and Hunt, 1992 Sollins et al., 1980 Bartholomev et al., 1953 Nye, 1961 Menat and Cesar, 1979 Menat and Cesar, 1979 Menat and Cesar, 1979 Menat and Cesar, 1979 Ogawa et al., 1961 Greenland and Kowal, 1960 Bandhu, 1970 Greenland and Kowal, 1960 Anderson, 1970 Gosz et al., 1976 Whittaker et al., 1974 Westman, 1978 Arthur and Fahey, 1991

a

Percentage share in woody root systems is indicated within parenthesis. Data are available only for stem þ branches. c Picea abies stands for natural origin that have undergone thinning of various intensity during the past 30 years (1000, 2000, 3000, 4000, 5000, and 6000 stems ha1 , respectively). d Plantations. b

influence of nutrient availability, light intensity and CO2 on growth and shoot/root ratio in structural components of young Betula pendula plants. He concluded that (1) root growth was favored when N, P, or S were major constraints for short roots; (2) shoot growth was favored when K, Mg, and Mn restricted shoot growth; (3) that shortage of Ca, Fe, and Zn regime had almost no effect on shoot:root ratio—that the light regime had no effect on dry matter allocation, and (4) that shortage of CO2 strongly decreased root development. The root biomass of forests is not static; it represents a varying proportion of the total tree biomass

during stand development (Table 4). Biomass accumulation in the woody roots does not occur at the same time as accumulation in aboveground structures. During stand development, root growth investments may even be higher than investments in aboveground structures (Lyr and Hoffman, 1967). Nevertheless growth of the above- and belowground structures are mutually interdependent. The ultimate rooting depth or the total lateral root spread in forest stands is usually not correlated with the aboveground size of the trees. While ‘‘living’’ fine roots persist more frequently in the upper part of the

Root Systems of Arboreal Plants

soil horizon, structural roots are generally spread more deeply in the soil profile (Persson, 1980b). The growth of the root tip determines the position, length, and orientation of the structural woody roots. The distribution of woody roots in forest stands, in relation to distance from trees, is dependent on the stand (Persson, 2000). Coarse roots are usually found, in dense forest stands, more frequently in the soil near the trees, while fine roots are distributed more regularly over the total stand (Persson, 1980a,b). Root biomass in the structural root system is usually minimal at maximum tree density (stem ha1 ) but increases proportionally to thinning intensity (Chibisov, 1995). However, the relationships between forest productivity and thinning operations are still not well understood. Tree roots are often spread as far as, or beyond, the projected crown (Fig. 2; Kutschera et al., 1997). In boreal forests, small and poorly developed trees frequently developed extensive root systems extending far beyond the tree crowns. Conservation of essential nutrients and their effective utilization is required to sustain forest production, especially in nutrient-poor sites. Trees on poor sandy soils allocate considerably more biomass to the structural roots than trees growing on fertile clay soil (Nielson and Dencker, 1998). At high elevations or in permafrost areas, the root/shoot ratio of forest trees is higher than in neighboring, climatically more favourable areas (Van Cleve et al., 1981). Roots of woody plants are stiffened and thickened by secondary xylem/wood production. Variations in growth rates around the root circumference are a general feature, especially for horizontal roots. Therefore root cross sections tend to be very eccentric, though their structure on the wind side differs from that on the lee side. Young roots are generally circular in shape. However, later they change toward greater xylem production on the upper side near the root base or in the strained side than on the stressed side. Thickening of

199

roots started first in more distal parts of the roots (Head, 1968). During some years the roots do not thicken at all. Thus, the number of rings of the secondary wood cannot be safely used for estimation of the age of roots. Moreover, radial growth tends to differ in root sections of different-diameter classes. The largest contribution to root biomass and the largest relative radial growth was found in thinner-diameter classes (Deans, 1981). The uprooting of trees by wind (i.e., windthrow) occurs very irregularly in forests, mainly on sites with shallow structural roots. The increased root/stem ratio with increasing thinning intensity of forest stands is presumably caused by the increase in water and nutrient availability, but also by enhanced mechanical stress in stems and structural roots (Chibisov, 1995; Nielson and Dencker, 1998; see also Chapter 10 by Stokes in this volume). The tree root systems subjected to wind movements lose a high proportion of fine roots, which must continuously be replaced. The impression of stability and permanence of the trees mask the essentially dynamic nature of forest ecosystems. In forest ecosystems, large-diameter tree roots survive and function for many seasons. Many large diameter roots remain alive as long as the trees stay alive, and in temperate regions may extend to > 100 years. As the forest stand ages, a considerable weight fraction accumulates in large-diameter roots (Ovington, 1962; Ovington and Madgwick, 1959a,b; Persson et al., 1995). However, carbon budgets suggest that the annual investment in the maintenance of the fine roots may even be higher (A˚gren et al., 1980; Axelsson and Axelsson, 1986). There are no good methods for determination of mortality and decomposition of large roots (Fogel, 1985, 1990). Many large-diameter roots of forest trees survive for a long time, though generally they exhibit a varying rate of mortality and high masses of dead root tissue have been reported for various forest ecosystems (Puhe, 1994).

Figure 2 Distribution of the structural root system in a 13-year-old Pinus sylvestris stand on a sandy podsol in Central Sweden. The soil water table was at 10 m. No taproot was found under those circumstances. (From Kutschera et al., 1997.)

200

V. CONCLUSIONS Structural roots of arboreal trees play important roles in the mechanical support of the trees, and protecting the soil against erosion. Fine roots derive their greatest functional importance in supplying the needs of the aboveground parts of trees with water and nutrients. Roots play an important role as producers of growthregulating compounds that affect the shoots. The amount of fine roots is for functional reasons in constant flux; fine roots repeatedly penetrate the soil horizons, often at the expense of high rates of death and renewal. Production and replacement of fine roots accounts for the high rate of primary production of forests. Extensive growth of fine root requires an adequate supply of carbohydrates and hormones and sufficient soil water and nutrient supply, adequate temperature and oxygen, low soil impedance, and limited competition with neighboring roots. Water and some nutrients move through the soil to the roots, but this is not enough. Tree roots extend into new areas by means of fast-growing lateral roots originally extending from the taproot. By penetrating the soil substrate, the roots improve soil structure, and the nutrient and soil water holding capacity. Roots compete with each other for carbohydrates, and those occupying favorable soil sites tend to grow at the expense of other components of the same root system. Most roots of the common tree species of the world grow within the upper 1–2 m of the soil. The most active fine roots are concentrated in the top 5 cm. Roots of most trees are usually spread far beyond the tree crowns. Biomass studies of tree roots at the ecosystem level indicate that the belowground investment is high and that root systems are expensive to maintain. Compared to our knowledge of the aboveground organs of trees, our knowledge about root function and behavior still needs boosting.

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Persson A˚gren G, Axelsson B, Flower-Ellis JGK, Linder S, Persson H, Staaf H, Troeng E. 1980. Annual carbon budget for a young Scots pine. Ecol Bull (Stockh) 32:307–313. Ahlstro¨m K, Persson H, Bo¨rjesson I. 1988. Fertilization in a mature Scots pine (Pinus sylvestris L.) stand—effects on fine roots. Plant Soil 106:179–190. Albrektson A. 1980. Relationships between tree biomass fractions and conventional silvicultural measurements. Ecol Bull (Stockh) 32:315–327. Andersson F. 1970. An ecosystem approach to vegetation, environment and organic matter of a mixed woodland and meadow area. Summary of doctoral Thesis, Univ. Lund, Sweden. Arthur MA, Fahey TJ. 1991. Biomass and nutrients in an Engelmann spruce—subalpine fir forest in north central Colorado: pools, annual production, and internal cycling. Can J For Res 22:315–325. Atkinson D. 1980. The growth and activity of fruit tree root systems under simulated orchard conditions. In: Sen DN, ed. Environment and Root Behaviour. Jodhpur, India: Geobios International, pp 171–185. Axelsson E, Axelsson B. 1986. Changes in carbon allocation patterns in spruce and pine trees following irrigation and fertilization. Tree Physiol 2:189–204. Bandhu D. 1970. A study of the productive structure of northern tropical dry deciduous forests near Varanasi. I. Stand structure and non-photosynthetic biomass. Trop Ecol 11:92–106. Bartholomew WY, Meyer J, Laudeleaut H. 1953. Mineral nutrient immobilization under forest and grass fallow in the Jangambi (Congos region) with some preliminary results. Publ Inst Natn Etude Agron Congo Belge, Ser Sci 57:1–40. Bartsch N. 1987. Response of root systems of young Pinus sylvestris and Picea abies plants to water deficits and soil acidity. Can J For Res 17:805–812. Bormann FH. 1966. The structure, function, and ecological significance of root grafts in Pinus strobus L. Ecol Monogr 36:2–26. Bowen GD. 1985. Roots as a component of tree productivity. In: Cannel MGR, Jackson JE, eds. Attributes of Trees as Crop Plants. Edinburgh; Institute of Terrestrial Ecology, Natural Environment Research Council, pp 303–315. Bringmark L. 1977. A bioelement budget of an old Scots pine forest in Central Sweden. Silva Fenn 11:201–257. Caldwell MM. 1987. Competition between roots in natural communities. In: Gregory PJ, Lake JV, Rose DA, eds. Root Development and Function. New York; Cambridge University Press, pp 167–185. Cannell MGR, Dewar RC. 1994. Carbon allocation in trees: a review of concepts for modelling. Adv Ecol Res 25:59–104. Chibisov GA. 1995. Bioproductivity of spruce stands in northern European Russia. Water Air Soil Pollut 82:87–96.

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201 lengths, weights and carbohydrate concentrations of fine roots. Plant Soil 47:463–485. Gill RA, Jackson RB. 2000. Global patterns of root turnover for terrestrial ecosystems. New Phytol 147:13–31. Gosz JR, Likens GE, Bormann FH. 1976. Organic matter and nutrient dynamics of the forest and forest floor in the Hubbard Brook Forest. Oecologia 22:305–320. Greenland DJ, Kowal JML. 1960. Nutrient content of the moist tropical forest of Ghana. Plant Soil 12:154–174. Grier CC, Vogt KA, Keyes MR, Edmonds RL. 1981. Biomass distribution and below and above-ground production in young and mature Abies amabilis zone ecosystems of the Washington Cascades. Can J For Res 11:155–167. Harley JL. 1959. The Biology of Mycorrhiza. London; Leonard Hill. Hatch AB, Doak KD. 1933. Mycorrhizal and other features of the root systems of pine. J Arnold Arbor 14:85–99. Head GC. 1968. Seasonal changes in the diameter of secondarily thickened roots of fruit trees in relation to growth of other parts of the tree. J Hort Sci 43:275– 282. Heikurainen L. 1955. U¨ber Vera¨nderungen in den Wurzelverha¨ltmissen der Kiefernbesta¨nde auf Moorbo¨den im Laufe des Jahres. Acta For Fenn 65:1–70. Hermann RK. 1977. Growth and production of tree roots. In: Marshall JK, ed. The Belowground Ecosystem: A Synthesis of Plant-Associated Processes. Fort Collins, CO: Colorado State University, Range Science Department Science Series 26:7–28. Holstener-Joergensen H. 1958–59. Investigations of root systems of oak, beech and Norway spruce on groundwater-affected moraine soils with a contribution to elucidation of evapotranspiration of stands. Forstl Forsoeksv Danm 15:227–289. Jackson RB, Canadell J, Ehleringer JR, Mooney HA, Sala OE, Schulze ED. 1996. A global analysis of root distributions for terrestrial biomes. Oecologia 108:389– 411. Jenik J. 1971. Root structure and underground biomass in equatorial forests. In: Duvigneau P, ed. Productivity of Forest Ecosystems. Paris; UNESCO, pp 323–331. Kalela E. 1954. U¨ber die Wurzelverha¨ltnisse der Kiefernsamenba¨ume und -Baumbesta¨nde. Acta For Fenn 61:1–17. Kajimoto T, Matsuura Y, Sofronov MA, Volokitina AV, Mori S, Osawa A, Abaimov AP. 1999. Above and belowground biomass and net primary productivity of a Larix gmelinii stand near Tura, Central Sibiria. Tree Physiol 19:815–822. Kolesnikov VA. 1968. Cyclic renewal of roots in fruit plants. In: Ghilarov MS, Kovda A, Novichkova-Ivanova LN, Rodin LE, Sveshnikova VM, eds. Methods of Productivity Studies in Root Systems and

202 Rhizosphere Organisms. St. Petersburg, Russia: Publishing House NAUKA, pp 102–106. Kozlowski TT. 1971a. Growth and Development of Trees. I. Seed Germination, Ontogeny, and Shoot Growth. New York; Academic Press. Kozlowski TT. 1971b. Growth and Development of Trees. II. Cambial Growth, Root Growth, and Reproductive Growth. New York; Academic Press. Kozlowski TT, Kramer PJ, Pallardy SG. 1991. The Physiological Ecology of Woody Plants. New York; Academic Press. Ko¨stler JN, Bru¨ckner E, Bibelriether H. 1968. Die Wurzeln der Waldba¨ume. Berlin; Verlag Paul Parey. Kramer PJ, Bullock HC. 1966. Seasonal variations in the proportions of suberized and unsuberized roots of trees in relation to the absorption of water. Am J Bot 53:200–204. Kuhns MR, Garret HE, Teskey RO, Hinckley TM. 1985. Root growth of black walnut trees related to soil temperature, soil water potential, and leaf water potential. Forest Sci 31:617–629. Kutschera L, Lichtenegger E, Sobotik M, Haas D. 1997. Bewurzelung von Pflanzen in der verschiedenen Lebensra¨umen. 5. Band der Wurzelatlas-Reihe. Stapfia 49:1–331. Ku¨lla T, Lo˜hmus K. 1999. Influence of cultivation method on root grafting in Norway spruce (Picea abies [L.] Karst.). Plant Soil 217:91–100. Laing EV. 1932. Studies in Tree Roots. Forestry Com Bull 13, 81 pp. Lambers H. 1987. Growth, respiration, exudation and symbiotic associations: the fate of carbon translocated to roots. In: Gregory PJ, Lake JV, Rose DA, eds. Root Development and Function. London; Cambridge University Press, pp 24–35. Lambers H, Scheurwater I, Atkin OK. 1996. Respiratory pattern in roots in relation to their functioning. In: Waisel Y, Eshel, A, Kafkafi U, eds. Plant Roots. The Hidden Half. 2nd ed. New York; Marcel Dekker, pp 323–362. Ledig FT. 1983. The influence of genotype and environment on dry matter distribution in plants. In: Huxley PA, ed. Plant Research and Agroforestry. Nairobi, Kenya: Int. Council Res. Agroforestry, pp 427–454. Ledig FT, Bormabb FH, Wenger KF. 1970. The distribution of dry matter between shoot and roots in loblolly pine. Bot Gaz 131:349–359. Leshem B. 1965. The annual activity of intermediary roots of the Aleppo pine. Forest Sci 11:291–298. Leshem B. 1970. Resting roots of Pinus halepensis: structure, function, and reaction to water stress. Bot Gaz 131:99– 104. Loescher WH, McCamant T, Keller JD. 1990. Carbohydrate reserves, translocation, and storage in woody plants. HortScience 25:274–281. Lodhiyal LS, Lodhiyal N. 1997. Variation in biomass and net primary productivity in short rotation high density

Persson central Himalayan popular plantations. For Ecol Manag 98:167–179. Lyford WH, Wilson BF. 1966. Controlled growth of forest tree roots: techniques and application. Harvard For Pap 16:1–12. Lyr H, Hoffmann G. 1967. Growth rates and growth periodicity of tree roots. Int Rev For Res 2:181–236. Ma¨lko¨nen E. 1974. Annual primary production and nutrient cycle in some Scots pine stands. Comm Inst For Fenn 84:1–87. Marschner H. 1988. Mineral Nutrients of Higher Plants. London: Academic Press. Marshall JD, Waring RH. 1985. Predicting fine root production and turnover by monitoring root starch and soil temperature. Can J For Res 15:791–800. Menaut JC, Cesar J. 1979. Structure and primary productivity of Lamto savannas, Ivory Coast. Ecology 60:1197– 1210. Mo¨ller CM, Mu¨ller D, Nielsen J. 1954. Respiration in stem and branches of beech. Forstl Sorsoeksv Danm 21:273–301. Morrow RR. 1950. Periodicity and growth of sugar maple surface layer roots. J For 48:875–881. Nambiar EK. 1987. Do nutrient retranslocate from fine roots? Can J For Res 17:913–918. Newman EI. 1988. Mycorrhizal links between plants: their functioning and ecological significance. Adv Ecol Res 18:243–270. Nielson CN, Dencker I. 1998. Root architecture and root/ shoot-ratios of Norway spruce as affected by thinning intensity and soil type in Denmark. In: Box JE, ed. Root Demographics and Their Efficiencies in Sustainable Agriculture, Grasslands and Forest Ecosystems. Dev Plant Soil Sci 82:721–735. Nihlga˚rd B. 1972. Plant biomass, primary production and distribution of chemical elements in a beech and a planted spruce forest in South Sweden. Oikos 23:69– 81. Noelle W. 1910. Studien zur vergleichenden Anatomie und Morphologie der Koniferenwurzeln mit Ru¨cksicht auf Systematik. Bot Zeit 68:169–266. Noland TL, Mohammed GH, Scott M. 1997. The dependence of root growth potential on light level, photosynthetic rate, and root starch content in jack pine seedlings. New For 13:105–119. Nye PH. 1961. Organic material and nutrient cycles under moist tropical forest. Plant Soil 13:250–263. Ogawa H, Yoda K, Kira T. 1961. A preliminary survey on the vegetation of Thailand. Nat Life SE Asia 1:21–157. Olsthoorn AFM, Titak A. 1991. Fine root density and root biomass of two Douglas-fir stands on sandy soils in the Netherlands. Neth J Agric Sci 39:67–77. Orlov AY. 1980. Cyclic development of roots of conifer and their relation to environmental factors. In: Sen DN, ed. Environment and Root Behaviour. Jodhpur, India; Geobio International, pp 43–61.

Root Systems of Arboreal Plants Ovington JD. 1957. Dry-matter production by plantations of Pinus sylvestris L. Ann Bot NS 21:287–314. Ovington JD. 1962. Quantitative ecology and the woodland ecosystem concept. Adv Ecol Res 1:103–192. Ovington JD, Madgwick HAI. 1959a. The growth and composition of natural stands of birch. 1. Dry-matter production. Plant Soil 10:271–283. Ovington JD, Madgwick HAI. 1959b. The growth and composition of natural stands of birch. 2. The uptake of mineral nutrients. Plant Soil 10:389–400. Parrotta JA. 1999. Productivity, nutrient cycling, and succession in single- and mixed-species plantations of Casuarina equisetifolia, Eucalyptus robusta, and Leucaena leucocephala in Pourto Rico. For Ecol Manag 124:45–77. Persson H. 1978. Root dynamics in a young Scots pine stand in Central Sweden. Oikos 30:508–519. Persson H. 1980a. Spatial distribution of fine root growth, mortality and decomposition in a young Scots pine stand in Central Sweden. Oikos 34:77–87. Persson H. 1980b. Death and replacement of fine roots in a mature Scots pine stand. Ecol Bull (Stockh) 32:251– 260. Persson H. 1980c. Fine-root dynamics in a Scots pine stand, with and without near optimum nutrient and water regimes. Acta Phytogeogr Suec 68:101–110. Persson H. 1982. Changes in the tree and dwarf shrub fineroots after clear cutting in a mature Scots pine stand. Swed Con For Proj, Techn Rep 31, 18 pp. Persson H. 1995. The role of roots in carbon cycling in forests. In: Helmisaari H-S, Smolander A, Suokas A, eds. The Role of Roots, Mycorrhizas and Rhizosphere Microbes in Carbon Cycling in Forest Soil. Fin For Res Inst Res Pap 537:119–126. Persson H. 1996. Fine-root dynamics in forest trees. Acta Phytogeogr Suec 81:17–23. Persson H. 2000. Adaptive tactics and characteristics of tree fine roots. Dev Plant Soil Sci 33:337–346. Persson H, Majdi H, Clemensson-Lindell A. 1995. Effects of acid deposition on tree roots. Ecol Bull (Stockh) 44:158–167. Pettersson R, McDonald AJS, Stadenberg I. 1993. Response of small birch plants (Betula pendula Roth.) to elevated CO2 and nitrogen supply. Plant Cell Environ 16:1115– 1121. Puhe J. 1994. Die Wurzelentwicklung der Fichte (Picea abies [L.] Karst.) bei unterschiedlichen chemischen Bodenbedingungen. Ber. Forschungszentrums Waldo¨kosysteme Univ Go¨ttingen 198:1–129. Raich JW, Nadelhoffer KJ. 1989. Belowground carbon allocation in forest ecosystems: global trends. Ecology 70:1346–1354. Rickman RW, Letey J, Stolzy LH. 1966. Plant response to oxygen supply and physical resistance in the root environment. Proc Soil Sci Soc Am 30:304–307.

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12 Root–Shoot Relations: Optimality in Acclimation and Adaptation or the ‘‘Emperor’s New Clothes’’? Peter B. Reich University of Minnesota, St. Paul, Minnesota

I.

INTRODUCTION

also focused on the range of components of root and shoot systems (including differences in morphology, chemistry, phenology, metabolism, and longevity), although data on root properties under contrasting environmental conditions are still scarce. In this chapter I revisit the issues raised above, focusing on the existing literature. I will not consider the actual internal communication devices used by plants to signal differential rates of growth or senescence of aboveground versus belowground tissues. Instead I will ask whether root/shoot relationships (1) maintain a so-called functional balance over time; (2) differ phenotypically in relation to differences in supply of light, nutrients, CO2, or water, or merely differ due to variation in plant size resulting from variation in these factors; (3) differ among plant species differing in habitat affinity; and (4) of aggregations of plants in stands are similar in their behavior to individual plants.

Differences in the relative sizes and/or function of root and shoot systems can arise due to differences in allocation of biomass, in morphology and chemistry of absorptive root and shoot tissues, and in the turnover rates of those tissues. For as long as the topic has been considered by plant biologists, a consistent theme has prevailed—that, given the relatively opposite roles of shoots and roots in uptake and use of key resources, there should be some kind of balance in size (e.g., biomass) and surface area (or related metric) between the root and shoot systems of individual plants (and perhaps stands). Such a balance should vary in relation to factors such as resource supply and species habitat affinity. For example, this would lead to a proportionally greater root than shoot system when nutrients were in short supply. Although such ideas have been taken as near-paradigm by several generations of physiologists and ecologists, we need to ask: How firmly does the evidence support these ideas? Historically, the majority of discussion of root– shoot relationships has focused on the relative biomass fractions in roots versus shoots. Scientists have

A.

Conceptual Considerations

The close coordination of growth of root and shoot systems has long intrigued plant biologists. It led to the functional equilibrium model of coordinated shoot and root growth (Brouwer, 1962a, 1983). It also led to the optimality theory (Wilson, 1988; Bloom et al., 1985; Thornley, 1972, 1998) that argues for preferential partitioning to the part of the plant

Abbreviations: LAR, leaf area ratio; LMF, leaf mass fraction; NAR, net assimilation rate (mol m2 h1); RGR, relative growth rate (d1); RMF, root mass fraction; SLA, specific leaf area (cm2 g1); SRL, specific root length (cm g1).

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which acquires the currently most limiting resource. Such partitioning hypothetically increases the ability of the plant to acquire that most limiting resource. Consistent with the optimality theory is the idea that species will show marked differences in biomass distribution in a fashion related to their differences in habitat affinities. According to such hypotheses, for example, plants adapted to growth in deep shade should tend to display high biomass allocation to leaves (Givnish , 1988), and plants adapted to growth in conditions with limited soil resources (nutrients or water) should tend to display high biomass allocation to roots (Chapin, 1980). Thus, theory suggests that there should be functional balance among root and shoot systems, that this balance should vary with resource supply, and that this balance should vary among species in relation to variation in life history traits and/or habitat affinities. As demonstrated below, the first point has been shown to be true so far as it has been tested. In fact, the evidence suggests that a tendency toward functional balance may be so pronounced that the second and third points, often taken as paradigm and nearparadigm, respectively, are in fact weak trends compared to the tendency toward individual functional balance. They have been overemphasized in studies of young plants due to failure to account for ontogenetic drift (e.g., Rice and Bazzaz, 1989; Coleman et al., 1994; Reich et al., 1998a). Moreover, differences in morphology, chemistry, and metabolism of roots and leaves are so profound in terms of impact on uptake and use of resources (e.g., Fitter, 1985; Poorter and Remkes, 1990; Poorter et al., 1990; Garnier, 1992, 1998; Lambers and Poorter, 1992; Ryser and Lambers, 1995; Reich et al., 1998b; Poorter and Nagel, 2000) that they exert greater influence on growth than differences in root–shoot biomass fraction in young plants. B.

Terminology

1. Allocation, Partitioning, and Distribution Although root/shoot ratio has often been criticized for being a static ratio, at least it is defined similarly by most authors, as the ratio of dry mass of belowground [i.e., roots] vs. aboveground plant parts at a given point in time. In contrast, the terms biomass allocation and partitioning (often used similarly) have had diverse uses. These range from narrow definitions in which allocation or partitioning refers only to the shortterm transfer and placement of new increments of biomass or specific substances (including carbohydrates,

proteins, lipids, etc.) to broad definitions in which allocation or partitioning refers specifically to the relative distribution of biomass pools among plant parts at a point in time (e.g., Poorter and Nagel, 2000). For purposes of this chapter, I will follow the definitions used by Lutze and Gifford (1998) and refer to allocation and partitioning as synonyms for the transfer and placement of new increments of biomass. I will refer to the amount of biomass present in any organ system at any comparable time or plant size, relative to the total plant biomass, as the biomass distribution. Hereafter I will use the term carbon roughly synonymously with biomass, since carbon represents roughly half of plant biomass. Biomass distribution then is a function of biomass allocation and biomass turnover rate, as shown in this example for root mass fraction (RMF): RMF = (Biomass allocated to roots – root biomass turnover)/(Biomass allocated to roots – root biomass turnover) + (Biomass allocated to stems – stem biomass turnover) + (Biomass allocated to foliage – foliage biomass turnover) In such a definition, turnover would include biomass lost via respiration as well as by tissue senescence or herbivory. It is clear that biomass distribution (which is frequently measured) is equally a result of turnover as of allocation (both of which are rarely measured), although this important and simple point is often overlooked. Hence, since turnover rates of roots are still very poorly quantified for the vast majority of species and situations (Eissenstat and Yanai, 1997), we have little reliable data for biomass allocation for whole plants, except during very early rapid growth phases when fine-root turnover is likely to be very low. This lack of empirical data on allocation is further highlighted when we consider our failure to account for carbon allocated to root symbionts such as mycorrhiza in most species and to root nodule bacteria in others, and carbon exuded from roots. Thus, the vast majority of usable, interpretable data are for biomass distribution. Again, for young seedlings, this might largely reflect biomass allocation since turnover has not yet begun in a major fashion. However, for older plants (including older ‘‘seedlings’’), any conclusions reached in the literature about biomass allocation per se (usually based on biomass distribution) are likely suspect, since the turnover and allocations parts of the equation are generally unknown.

Root–Shoot Relations

2.

Root–Shoot Ratios, Biomass Fractions, and Allometric Analyses

Given historic use of the root–shoot ratio I will repeatedly refer to it in this chapter, although I prefer the term biomass fraction to refer to the fraction of total plant mass in a given plant component. The term biomass fraction is preferred for statistical reasons over root/shoot ratio, although they convey the same information. In addition to evaluating patterns of biomass fractions, I shall also discuss the use and value of allometric relationships in which the log-transformed biomass of the shoot is plotted against the logtransformed biomass of the roots or of the whole plant (Pearsall, 1927; Evans, 1972). This enables the separation of differences in biomass distribution due to differences in size from those due to true shifts in partitioning. Although allometry is not perfect, it is ‘‘the only routine method of showing an effect of treatment on net partitioning’’ (Farrar and Gunn, 1998). When allometry is performed to relate roots to shoots, the allometric slope constant k is the ratio of the relative growth rate (RGR) of the shoot versus the RGR of the root, and hence a precise measure of relative net change in biomass for the shoot vs. the root. It is useful to evaluate whether k is less than or greater than unity, since this directly defines the direction of the ontogenetic drift in partitioning. Although k is useful as typically defined, it does not distinguish among stems, leaves, and roots, which may limit its usefulness (Poorter and Nagel, 2000). However, one can determine a k for any specific pair of tissues. Additionally, nonlinearities in the allometric relationships are problematic and the slopes of allometric relationships do not fully account for differences in the overall elevation of the allometric lines. Apparently, plants might allocate proportionally less to one component (roots, for example) as they grow larger under a given treatment A as compared to another treatment B (i.e., have a lower slope under treatment A if roots are the Y-axis variable). Yet they still might have a greater proportion of total biomass in that tissue component at any plant size because of differences in intercept. Hence, allometric relationships should consider the slopes and intercepts to adequately interpret potential differences in allocation and distribution. Finally, allometry provides some information on the outcome of partitioning, not the process itself. Despite these limitations it is far more appropriate as a means of addressing questions of partitioning than comparing plants in different treatments at the same time (Evans, 1972; Coleman et al., 1994; Lutze

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and Gifford, 1998). A number of authors have also compared leaf mass fraction (LMF) or RMF to plant size (Peace and Grubb, 1982; McConnaughay and Coleman, 1999; Poorter and Nagel, 2000), which accomplishes roughly the same purpose as the direct allometric approach. C.

Evidence

In evaluating the published data in support of or against the idea of optimality in acclimation and adaptation of biomass allocation or distribution, I used all my reprints plus 675 articles gleaned from an electronic search for ‘‘root’’ and ‘‘shoot’’ using the database Agricola. Roughly 200 citations met the criteria needed to be relevant to the topic at hand. Although not an exhaustive search, it is likely relatively comprehensive. Moreover, when I refer to papers that did not account for ontogenetic drift and yet arrived at conclusions regarding shifts in biomass allocation, there is no attempt to denigrate these authors. To demonstrate this, I point out here that my own work (e.g., Reich et al., 1987; Walters and Reich, 1989) has suffered from the failure to account for ontogenetic drift. II.

FUNCTIONAL BALANCE AND RESPONSES OF ROOT VERSUS SHOOT SYSTEMS TO VARIATION IN RESOURCES

A.

Functional Balance, Optimality Theories, and Ontogenetic Drift

The relative differences in response of root versus shoot systems to variation in resources such as light, water, nutrients, or CO2 has long been a central question in plant biology. Brouwer (1962a,b) performed several pioneering investigations that showed that leaf- or root-pruned seedlings rapidly regained their original root–shoot biomass balance and that the pruned plants were then on the same line in the allometric analysis as the control plants; i.e., when compared to a (younger) control plot with a similar dry mass, they had the same proportion of that mass in roots and shoots. Surprisingly few studies were ever made to validate this finding, although work by Caloin et al. (1991), Farrar and Gunn (1998), and Poorter and Nagel (2000) demonstrate the same result for herbaceous plants. Indirect evidence in this same vein has been seen for woody plants by Eissenstat and Duncan (1992), Reich et al. (1993), Kruger and Reich (1993), and Vanderklein and Reich (1999) for lightly to

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moderately shoot-pruned or defoliated woody plants. Hence I will presume that this is a general process until proven otherwise. If true, this is remarkable, in that the control and conservation of biomass distribution occurs only due to plastic variation in the allocation or partitioning of new carbon (Farrar and Gunn, 1998). The recovery of root–shoot balance in partially defoliated plants comes at a cost, such as of fruit production in citrus (Eissenstat and Duncan, 1992). Even if we accept that all plants behave in such a fashion, many possible mechanisms may be involved, and understanding of this process is surprisingly limited to this date (Farrar and Gunn, 1998; Poorter and Nagel, 2000). Building conceptually on earlier studies that showed greater shoot fraction in low light or root fraction under low soil resource supply (which one should note were typically based on root–shoot ratios of plants of different sizes), Brouwer (1983) synthesized his own work to develop the ‘‘functional equilibrium’’ theory. In a simplified framework this can be stated as (Poorter and Nagel, 2000): plants shift their allocation toward shoots if the carbon gain of the shoot is limited by a low level of aboveground resources, such as light or CO2. Allocation is shifted toward roots if belowground resources, such as nutrients or water, are at low levels. Optimal partitioning theory further suggests that plants respond to variation in the environment by partitioning biomass among various plant organs to optimize the acquisition of nutrients, light, water, and carbon in a manner that maximizes plant growth (Thornley, 1969; Bloom et al., 1985). For example, plants experiencing low levels of nutrient supply would be predicted to shift resources toward root growth and to processes associated with nutrient capture rather than those associated with carbon uptake. In this sense we can think of the optimal partitioning theory as an extension of the ‘‘functional equilibrium’’ model. Both functional equilibrium and optimal partitioning models explain the recovery of a stable root–shoot biomass distribution following pruning or nutrient starvation. They also explain shifts toward higher root biomass fraction under low soil resources and higher shoot biomass fraction under high soil resources believed to be a common whole-plant behavior. Moreover, these shifts could be considered adaptive, since they might lead toward enhanced capture of the resources most limiting to plant growth. However, optimality may not be a good explanation. Instead, one could argue that the plant is reestablishing a functional equilibrium selected for over time for reasons unrelated to optimality and determined genetically.

Reich

Since the mechanisms controlling functional equilibrium are still at best partially known, and other aspects of the ‘‘optimal allocation vis-a`-vis resource supply’’ theory are only weakly supported, I propose that functional equilibrium may be as much or more a case of plants maintaining homogeneity rather than responding optimally to variation in resource supply. Optimal partitioning theory is generally accepted— in fact, it is loosely used as a paradigm in plant ecology, based on concept and empirical evidence (Bloom et al., 1985; Chapin, 1991; Reynolds and D’Antonio, 1996), although there is no consensus about the mechanisms involved. This idea still merits partial support, but a variety of studies have indicated that some significant fraction of the evidence taken as support for such theory is actually due to failure to account for ontogenetic drift (Evans, 1972; Rice and Bazzaaz, 1989; Walters et al., 1993a,b; Coleman et al., 1994; Reich et al., 1998a; Farrar and Gunn, 1998; McConnaughay and Coleman, 1999). Such allometric evidence suggests that the plasticity of allocation in response to resource variation is often relatively muted (and differentially dependent on the resource involved). The large differences reported in the literature and attributed to plasticity sometimes reflect large differences in plant size, rather than true differences in allocation. In fact, failure to account for ontogenetic drift can cause authors to misjudge the magnitude, duration, and even direction of many of the adjustments in biomass allocation patterns found for many species (McConnaughay and Coleman, 1999). Those investigators noted that a surprisingly small number of studies have explicitly distinguished between biomass distribution differences that result as a natural consequence of ontogenetic drift (i.e., plant growth and development) and ‘‘true’’ adjustments in biomass distribution (i.e., those that require an adjustment in biomass allocation). The numbers of such publications are small as compared to the much larger number of papers on plant responses to nutrients, light, water, or CO2 published in the past several decades without considering ontogenetic drift. Poorter and Nagel (2000) have argued that root– shoot differences that occur among treatments at a point in time due only to differences in prior growth rate, and hence in plant size at that time, are nonetheless meaningful. This is because the plants actually co-occur in time and differences in biomass fractions may influence their response to current conditions. Although this latter possibility may be true, the hypothetical plants are also likely to differ in many other ways (leaf or root chemistry, metabolism, plus

Root–Shoot Relations

overall plant size) that may influence their functions. Hence, Evans (1972), Coleman et al. (1994), and Lutze and Gifford (1998) have argued, and I agree, that the comparison of biomass distribution in plants, that are of widely differing sizes because of microenvironmental differences, sheds no light on the actual process of allocation, or of plasticity in allocation. If plants of a given species under low light conditions actually shifted their proportional allocation of new biomass toward leaves (which I define as a plastic response), they would have a higher k and likely a higher LMF at the same size as high light plants. If the plants make absolutely no change in allocation, high light will cause faster overall growth and hence over time the plants diverge along the same developmental trajectory and with exactly the same partitioning patterns, and yet have divergent LMF at the same time. B.

Responses to Resource Gradients

1.

Light

Optimal partitioning theory hypothesizes increased allocation to leaf production should be a phenotypic response of plants to lower light environments (e.g., Givnish, 1988). The vast majority (in fact, every test I could find) of studies that compared plants under varying light conditions reported higher leaf mass fraction in low-light-grown plants when plants were harvested at a common time (cf. Olff et al., 1990; Callaway, 1992; Latham, 1992; Lei and Lechowicz, 1998) and a meta-analysis of such studies also reports a significant shift toward leaves and stems and away from roots under low light (Poorter and Nagel, 2000). This evidence of differences in biomass distribution of plants of widely different size that grew under differing light conditions led to the common paradigm that plants shift allocation toward leaves when grown at low light. However, most of the studies that addressed this issue examined plants only at a common time. In contrast, the vast majority of the smaller set of studies that explicitly tested for allocation by accounting for ontogenetic drift, have not found increasing allocation to leaf mass in low light. This is the case for 23 species (of 26 tested) as disparate as herbs (Evans and Hughes, 1961; Hughes and Evans, 1962; Peace and Grubb, 1982; Rice and Bazzaz, 1989; Philippot et al., 1991; Casper et al., 1998; McConnaughay and Coleman, 1999), crop plants (Terry, 1968; Corre, 1983), and woody plant seedlings (Ledig et al., 1970; Steinbrenner and Rediske, 1964; Walters et al., 1993a; Stoneman and Bell, 1993; Chen, 1997; Chen and Klinka, 1998; Reich et al., 1998a). Thus, most

209

studies that accounted for ontogenetic drift found no evidence of allocation shift toward leaf production at low light. Using an alternative approach, Ingestad and Agren (1991) also concluded that there is no effect of irradiance on allocation if a plant is at steady-state nutritional conditions. Thus the preponderance of evidence suggests that plastic allocational shifts in biomass in response to light gradients predicted by optimality theory are the exception rather than the rule. As an example of the evidence regarding biomass allocation patterns under contrasting light environments, I present data for seedlings of nine woody species that were summarized in a condensed fashion in Reich et al. (1998a). It is evident (Fig. 1; Table 1) that there is no consistent difference in the allometric coefficient k (here defined as the change in leaf RGR versus root RGR, ignoring stem mass) in contrasting light treatments. Only one of the nine species had a significant shift in k, and many species even had lower k in lower light. The intercepts were generally not significantly different either. Only Larix had a significantly different intercept, showing for this species that even for a given root size, low-light plants had greater leaf mass despite a similar k. Using these data, we also see no significant differences between light treatments if LMF is regressed against plant size. One alternative way of examining allocational shifts bears mention. Some studies which reported an effect of light on biomass allocation and attempted to adjust for plant size, did so using analysis of covariance (ANCOVA), wherein the relationship between leaf fraction and light environment was statistically adjusted for plant size (cf. Veneklass and Poorter, 1998; Poorter et al., 1999). For our data set (Fig. 1; Table 1), ANCOVA results were often substantially different from those from a strictly allometric analysis, and the range of plant size occupied by each treatment was quite distinct, so that the statistical adjustment using ANCOVA may not adequately account for ontogenetic drift. Therefore, until such data are examined using both allometry and ANCOVA, studies that use only ANCOVA should be considered weak evidence as a test of plasticity. When analyzed allometrically, for woody plants there was no shift toward higher LMF at lower light for 12 species (Walters et al., 1993a; Reich et al., 1998a), but there was a consistent shift toward stems and away from roots. This was also seen earlier for etiolated peas (Pearsall, 1927) and for the woodland herb Impatiens in studies by Evans and Hughes (1961), Huges and Evans (1962), and Peace and

210

Reich

Figure 1 Variation in leaf mass versus root mass, shown on a logarithmic scale, for young seedlings of nine woody plant species grown at low and high light (5% and 25% of full sunlight, respectively) (n ¼ 59 plants on average per light level per species). Statistical summaries of slope and intercept differences across light treatments are provided in Table 1. Individual data points are individual plants harvested at nine different times over the course of the growing season. (Expanded from data summarized in Reich et al., 1998a.)

Table 1 Summary of Values of the Allometric Constant (k) for the Relationship Between Leaf Mass and Root Mass for Each of Nine Woody Species in Two Contrasting Light Environments—Low (5% of full sunlight) and High (25% of full sunlight). Species Populus tremuloides Betula papyrifera Larix laricina Pinus banksiana Betula allegheniensis Pinus strobus Picea glauca Picea mariana Thuja occidentalis

Shade tolerance classification Intolerant Intolerant Intolerant Intolerant Intermediate Intermediate Tolerant Tolerant Tolerant

Low light 0.92 1.07 0.91 0.76 0.96 0.73 0.89 0.90 0.89

        

0.05 0.03 0.04 0.07 0.07 0.04 0.08 0.08 0.08

High light

P ðkÞ

P (intercept)

        

ns * ns ns ns ns ns ns ns

ns — * ns ns ns ns ns ns

0.84 0.89 0.93 0.87 0.97 0.73 0.94 0.96 0.81

0.02 0.02 0.02 0.03 0.02 0.02 0.03 0.03 0.03

Mean is followed by 1 SE. Species are arrayed from least to most shade tolerant. Probabilities (P) contrast the two light levels and are those for the slope (the allometric constant k) and for the intercept of the allometric relationship (not testable for Betula papyrifera since there were significant slope differences). Source: Expanded from data summarized in Reich et al. (1998a).

Root–Shoot Relations

Grubb (1982). These patterns could be construed as indirect support for the optimal allocation model, since stems and leaves collectively are needed to harvest light and fix carbon, while roots acquire water and nutrients. If both leaf fraction and stem fraction increase under low-light conditions then one could argue that these changes supported the optimality theory, since immediate resource gain would occur. However, if only stem fraction increases, as commonly observed, it suggests a strategy to improve the likelihood of future potential resource gain and perhaps to increase the likelihood of survival. Moreover, since the plants in these experiments were growing as isolated individuals, with shade coming from high above the plant, increased carbon gain would have come directly from more leaves or leaf area but not from more stem mass or stem length. Hence, the observed shift toward stems rather than leaves must represent an acclimation that has been selected for because of its value in natural environments, rather than a plastic response to increase carbon gain in the near term. In contrast to the weak evidence for optimal biomass partitioning with respect to light—or, in fact, any consistent shift in LMF—dramatic shifts in leaf morphology were routinely shown, e.g., with leaves in low light having lower density, lower thickness, or both. This was demonstrated by higher SLA values, even when accounting for ontogenetic drift in SLA with plant size (Hughes, 1965; Peace and Grubb, 1982; Walters et al., 1993a; Reich et al., 1998a). There are few data available examining shifts in root properties (such as specific root length) under differing light conditions, but the data are consistent with leaf responses. Plants of nine species grown under low-light conditions had lower specific root lengths and higher SLA than plants of the same size grown under higher light conditions (Fig. 2; Reich et al., 1998a). Although all nine species showed shifts in SRL with light environment, these are not easily explained simply by differences in the slope of the change in root length with root dry mass or whole plant mass. The initial differences between light levels (in the smallest, youngest plants measured) were sometimes so large that SRL was higher in high-light than in low-light plants across their common range of plant sizes. This is despite a steeper slope of root length versus root mass for low light plants. Acclimation of tissue morphology appears to represent a greater means of plasticity than shifts in biomass allocation in response to variation in light (Evans, 1972; Walters et al., 1993a; Reich et al., 1998a). Moreover, greater SLA and lesser SRL at low light

211

Figure 2 Specific leaf area (cm2 foliage per g foliage) and specific root length (m root length per g root) for seedlings of nine species grown at low and high light (5% and 25% of full sunlight, respectively). See Fig. 1 or Table 1 for species names. (From Reich et al., 1998a.)

are consistent with the idea of improving the uptake of the most limiting resource (Givnish, 1988), assuming that uptake of resources is related to high surface area or length of the tissue in question (Reich et al., 1998b). Hence, optimality theory might be better supported if it were considered using models that incorporate the full constellation of plant traits and responses, including morphological ones that result in changes in the relative area or length of absorptive tissues. 2.

Nutrients and Water

The evidence for phenotypic shifts in partitioning is stronger for nutrients than any other resource, but still is not universal. The predominant response noted in studies that did not account for ontogenetic drift was a large shift toward roots and away from shoots at low nutrient supply (Poorter and Nagel, 2000). Most studies that adjusted for plant size also found shifts in allocation across varied nitrogen supply, as reported by Ryser and Lambers (1995), Volin and Reich (1996), Gedroc et al. (1996), Lutze and Gifford (1998), Baxter et al. (1997) and McConnaughay and Coleman (1999) (Fig. 3). The relative magnitude of the shift was typically smaller when examined ontogenetically rather than at a single

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Figure 3 Variation in root mass versus shoot mass, shown on a logarithmic scale, for young seedlings of the annual herbaceous plant species Abutilon theophrasti (left) and Chenopodium album (right) grown at low (open circles) and high (closed circles) nutrient regimes (n=22 plants per nutrient regime per species). Individual data points are individual plants harvested at different times over the course of the growing season. (From Gedroc et al., 1996.)

point in time. Evidence in response to supply of P and other nutrients is less clear. Eissenstat et al. (1993) found shifts toward decreasing RMF with increasing P supply in sour orange, even when comparing plants at a common size. However, using data from a study of common bean plants (Nielsen et al., 1998), there is no evidence of decreasing RMF with increasing P supply, and in fact, the evidence suggests the opposite (Fig. 4). Ryser and Lambers (1995) found declining RMF with increasing P supply in one grass species (Brachypodium pinnatum) but not in another (Dactylis glomerata). Using a different experimental approach, several authors concluded that there is a systematic shift in allocation for variable P and N supply, but not for K (Ingestad and Agren, 1991; see also Van der Werf et al., 1993a,b). Similar to the limited database on root traits across light gradients, there are few data for root traits across nutrient gradients. Ryser and Lambers (1995) found that in the grass species Dactylis glomerata, SRL decreased with increasing N supply at various levels of P supply, but in the grass species Brachypodium pinnatum there was no decline in SRL with increasing N supply. In neither species was there a consistent shift in SRL with variation in P supply. In contrast, Eissenstat et al. (1993) found a decrease in SRL with increasing P supply in sour orange. Only scant data on plastic allocation responses to water availability are available in experiments where ontogenetic drift was accounted for in the design. Of those data, increased allocation toward roots, when water was in low supply, have been reported for some woody plants (Ledig et al., 1970; Tomlinson and Anderson, 1998) and during certain phenotypical phases in different woody seedlings (McMillan and

Reich

Wagner, 1995). However, it was not observed for annual herbs (McConnaughay and Coleman, 1999). This database is too scant and inconsistent to provide a basis for reaching firm conclusions. Moreover, with regard to water acquisition, optimality theory may not apply in cases of uncertainty (Lerdau, 1992; Lerdau and Gershenzon, 1997). If plants were to develop roots after water shortage occurred, it would be too late. Therefore they may have been selected to invest in excessive amounts of roots in habitats that may be subjected to periodic and unpredictable drought.

Figure 4 Variation in root mass versus total plant mass, shown on a logarithmic scale, for young common bean plant seedling grown (lower panel) at low and high phosphorus supply rates and (upper panel) with or without mycorrhizal infection. Individual data points are average values for plants harvested at four dates over the time course of the experiment, pooled across the other treatment. (From Nielsen et al., 1998.)

Root–Shoot Relations

3.

213

Elevated Atmospheric CO2

C.

Consistent with optimality theory, it was hypothesized (e.g., Reynolds and Thornley, 1982) and often empirically observed (e.g., Eamus and Jarvis, 1989; Ceulemanns and Mousseau, 1994; among many such publications) that growth in elevated CO2 results in increased dry-mass partitioning to roots. However, few if any of these reports accounted for ontogenetic drift in biomass fraction with plant size. In contrast, opposite results were reported by researchers who made explicit tests with woody and herbaceous species of the role of ontogenetic drift vis-a`-vis biomass allocation under elevated CO2 (Tjoelker et al., 1998; Gunn et al., 1999; Lutze and Gifford 1998). They found that the strong size dependence of partitioning of dry mass among plant parts resulted in ‘‘apparent’’ differences in root and shoot dry mass partitioning, that in fact were the result of differences in plant size and not a functional adjustment to CO2 environment (e.g., Table 2, Fig. 5). Recent meta-analyses that did not account for plant size (Curtis and Wang, 1998; Poorter and Nagel, 2000) also indicated that these earlier suggestions of optimality in root–shoot partitioning in response to elevated CO2 are not supported by critical evidence.

Conclusions Regarding Experimental Tests of Allocation Across Resource Gradients

A better understanding of acclimation of biomass distribution under variable environmental conditions is critical to our ability to comprehend plant growth, or the responses of plants to multiple resource scenarios (McConnaughay and Coleman, 1999). Optimal partitioning theory has often been the basis for models which attempt to predict plant responses to global environmental changes. If the assumption that plants shift biomass partitioning in response to environmental cues is untrue, or much more limited in degree than previously believed, the predictions of such models must be questioned. Some studies show pronounced shifts in allocation as hypothesized, but others do not. What can we therefore conclude? If optimality allocation theory holds for all plant resources, we should see predicted shifts in response to variation in light, CO2, water, and nutrients. There is strong evidence against such claims for light and CO2, strong evidence in favor of these claims for N, and variable evidence for P or water.

Table 2 Allocation of Dry Mass to Roots Among Five Boreal Species (Populus tremuloides, Betula papyrifera, Larix laricina, Pinus banksiana, and Picea mariana) Grown as Seedlings Under Ambient (370
mol mol1) and Elevated (580
mol mol1) Concentrations of CO2 in Combination with Five (day/night) Growth Temperatures Growth temperature (8C) Genus

CO2

18/12

21/15

24/18

27/21

30/24

Populus

370 580 P > Fa 370 580 P>F 370 580 P>F 370 580 P>F 370 580 P>F

1.19  0.04 1.18  0.05 ns 1.07  0.02 1.07  0.01 ns 0.93  0.01 0.96  0.01 0.09 1.05  0.02 1.07  0.02 ns 1.01  0.01 1.01  0.01 ns

1.10  0.02 1.08  0.02 ns 1.05  0.01 1.05  0.01 ns 1.03  0.02 0.99  0.02 ns 1.01  0.02 1.05  0.02 ns 0.97  0.02 0.94  0.01 ns

1.11  0.02 1.21  0.02 .001 1.15  0.03 1.09  0.02 ns 1.02  0.01 1.01  0.01 ns 0.98  0.02 1.02  0.01 0.05 1.00  0.01 1.00  0.01 ns

1.04  0.02 1.08  0.02 ns 1.05  0.01 1.05  0.01 ns 0.99  0.01 1.01  0.01 ns 0.99  0.01 0.99  0.01 ns 0.97  0.01 0.98  0.01 ns

1.11  0.02 1.12  0.01 ns 1.08  0.02 1.04  0.01 ns 1.04  0.01 1.05  0.01 ns 1.00  0.02 0.99  0.01 ns 0.99  0.02 1.04  0.02 0.08

Betula

Larix

Pinus

Picea

Mean (SE) allometric coefficients relating change in root mass to change in plant mass are shown. Slope values determined from individual plant dry mass data using the linear regression equation: ln (root) = slope  ln (plant) + b, all R2  0.97. Values >1.0 indicate an increased partitioning of dry mass to roots for a given change in plant mass. a P values for test of separate slopes for CO2 treatment in analysis of covariance; ns indicates P > :1. Source: Tjoelker et al. (1998).

214

Reich

Figure 5 The effect of atmospheric CO2 concentration on (A) shoot–root ratio at a number of harvest dates, and (B) the allometric relationship between shoot and root, for Bellis perennis and Trifolim repens. Plants were grown hydroponically in controlled environments of either 350 or 700
mol CO2 mmol1. Open bars of each pair (above) represent elevated CO2 levels. Open squares (below) represent elevated CO2 levels. (From Gunn et al., 1999.)

Collectively, the body of evidence suggests that the optimal allocation theory is not a general theory. Why is this so? An idea central to this theory is that plants plastically shift biomass allocation to have a higher resource gain under a new situation than would otherwise occur without such a shift. However, several important issues make this idea untenable: 1. A variety of traits influence plant resource balance, including morphology, metabolism, and chemistry, that are often more biologically significant and influential than biomass allocation (Evans, 1972; Lambers and Poorter, 1992; Reich et al., 1998a,b). For example, a plant can change carbon gain by either changing LAR or by changing the photosynthetic rate

of that leaf area (NAR). A plant can change leaf area by changing either SLA or LMF. In short, there are numerous ways to enhance C gain and RGR, and only few of them require shifts in biomass allocation. Thus, even if plants did routinely phenotypically change to maximize resource gain, they would not necessarily need to shift biomass allocation to do so. Apparently it is a less effective means of doing so as compared to other alternatives. 2. Shifts in allocation, morphology, architecture, chemistry, and metabolism have likely been selected for to simultaneously improve C and N balance and survival, not just to improve C and N balance. For instance, responses to an increase from a very low to a low level of a limiting resource are often inconsistent with optimality arguments. Almost all shifts, such as

Root–Shoot Relations

decreased SLA (and hence decreased LAR) in higher but still limiting light or CO2 conditions, should in theory reduce C gain and RGR (Lambers and Poorter, 1992) when compared to a plant that had not shifted SLA or LAR. 3. Traits that enhance resource gain do not necessarily enhance survival and vice versa, especially in resource-poor and/or disturbed habitats (Walters and Reich, 1996, 2000a,b). Thus, shifts that have been selected for to increase survival may in fact work against a shift toward enhanced resource status. Only a whole-plant model that simultaneously examines shifts in allocation, morphology, chemistry, metabolism, turnover, and architecture, and their interaction, can characterize such changes. Unfortunately, it is easier to propose such a model than to develop it. III.

VARIATION IN ROOT–SHOOT RELATIONSHIPS AMONG SPECIES

Species of differing resource environments have been hypothesized to differ in their root–shoot relations (Grime, 1979; Chapin, 1980; Tilman, 1988; Gleeson and Tilman, 1990), especially for resources that either are soil based, such as nutrients and water, or aboveground based, such as light and CO2. In essence, if it is advantageous to be rootier as an individual when soil resources are poor, all species should phenotypically act this way, and species adapted to poor soils should be intrinsically rootier than those adapted to richer soils. But what do the data show? Species found in soil resource-poor habitats have been hypothesized to be inherently ‘‘rootier’’ than species from richer habitats (Chapin, 1980). A test of this idea requires comparison of individuals in comparable habitats. Several studies designed to address this question have had conflicting results (Poorter and Remkes, 1990; Garnier, 1991; Fichtner and Schultze, 1992; Van der Werf et al., 1993a,b), and no conclusion can be made in support of the hypothesis. Moreover, the best objects for such test would be of species in natural field settings. However, there are few if any such published data available. Moreover, no clear distinction in root morphological or chemical traits was apparent among species varying in fertility of habitat (cf. Ryser, 1996; Craine et al., 2001). Optimality theory also suggests that if it is advantageous to allocate proportionally more biomass to leaves when an individual plant is shaded, shadeadapted species should tend to be leafier than shade-

215

intolerant ones (Givnish, 1988). However, in comparing shade-tolerant versus intolerant species, recent results show opposite patterns vis-a`-vis biomass distribution, allocation and tissue traits as long-held hypotheses. Shade-tolerant species actually appear to have equal or higher RMF than intolerant species. This was true in a comparison of tropical woody species considered shade tolerant versus intolerant, (Veneklass and Poorter, 1998) and for winter deciduous temperate broadleaf species (Walters and Reich, 1999). A neutral pattern was observed for evergreen broad-leaved species (Walters and Reich 1999) and for our study species (Table 1; note that k is not different for tolerant vs. intolerant species). Hence, comparisons among species whose distribution is related to fertility or light gradients do not lend strong support for the optimal allocation or distribution theory. In contrast to the weak evidence for differences in biomass distribution among species in relation to habitat affinity, there is much stronger evidence of differences in tissue morphology, chemistry, metabolism, and turnover rates, increasingly of roots as well as shoots (Walters et al., 1993a,b; Ryser, 1996; Reich et al., 1998a,b; Walters and Reich, 2000b). Plants adapted to low fertility and/or low light may in fact be selected for low turnover of leaf and root tissues, rather than to high allocation to roots or shoots (Aerts, 1990; Reich et al., 1992, 1998a). They are also selected for traits associated with slow tissue turnover that minimize resource losses, such as dense tissues with low respiration rate. These include extended nutrient residence time via long tissue life span, for plants adapted to infertile habitats (Aerts, 1990). They also maintain low carbon losses via the combination of low respiration rates of all tissue types, and low tissue turnover rates, for plants adapted to deep shade (Walters and Reich, 1999, 2000a,b). Generally, species from resource-rich microhabitats have higher concentrations of nutrients, faster photosynthetic and respiration rates, higher SRL and SLA, and faster turnover of leaves than those from resource-poor environments (Chapin, 1980; Reich et al., 1992, 1998a,b, 1999). However, the comparative multispecies database for roots is still weak. Thus, it is uncertain whether root turnover rates are also higher for species from resource-rich microhabitats (Eissenstat and Yanai, 1997). The importance of leaf and root morphology and metabolism, rather than biomass allocation, was shown in contrasts of diverse species (Poorter and Remkes, 1990; Walters et al., 1993a; Reich et al., 1998a; Cornellissen et al., 1996) and among populations within a species (Ryser and Aeschlimann, 1999).

216

Reich

V. PLANT COMMUNITIES AND PLANTS IN COMMUNITIES Field studies that include whole-plant biomass data are rare. The root–shoot distributions of five conifer species planted in the field did not differ in trenched plots with higher nitrogen and water supply than in untrenched plots (Machado et al., 2001), even for plants of common size. Similarly, naturally grown sugar maple seedlings growing across a soil fertility and soil moisture gradient did not differ in biomass fraction distributed to roots, even when accounting for plant size and light environment (Walters and Reich, 1997), but did differ in other important plant traits such at tissue %N. It is possible that modest natural gradients in soil resource supply have different, or inconsequential, effects, compared to the large gradients observed in experimental studies with very young plants. It is also likely that biomass fractions in older seedlings are markedly influenced by turnover rates of leaves and roots. Do stands or patches of plants behave similarly vis-a`-vis root–shoot relationships as individual plants? Answering this question is difficult since most accounts of individual plants involve small chamber-grown plants, whereas most studies of plant stands involve larger, older, field-grown plants. For forests, both observational and experimental studies show that plants growing under lower nutrient and water availability tend to have a greater RMF and a greater shift toward fine-root than toward foliage production, as compared with plants of high nutrient and water availability (Linder and Axelsson, 1982; Gower et al., 1992). These patterns could result either from shifts toward greater allocation to roots at low resource supply, from lower fineroot turnover rates, or from both. The relative contribution of each is practically impossible to calculate, given the paucity of root turnover data. Across a N gradient that resulted from 32 years of variable experimental fire frequency regimes, a strong gradient in root:foliage fraction was found (Fig. 6; Reich et al., 2001). Oak savanna stands under chronically low N supply have a greater fraction of fine biomass in fine roots rather than in foliage. This pattern is consistent with optimality theory, but it is impossible to know whether it results from variation in dominance of one vegetation type over another, since the low-fertility, frequently burned end of the continuum was dominated more by grasses than trees. Moreover, again we cannot separate allocation from turnover rates in this instance.

Figure 6 The fraction of total fine root plus leaf mass distributed to fine roots in relation to net nitrogen mineralization rate for 14 oak savanna stands in a fire frequency experiment in Minnesota. Along with the variation in nitrogen mineralization rate are parallel gradients in fire frequency and relative tree versus grass dominance, with frequently burned stands being dominated more by grasses than trees and having low mineralization rates, and vice versa. (From Reich et al., 2001.)

At a very coarse, but very large scale, Jackson et al. (1996) summarized existing data regarding root and shoot biomass for different biomes of the world. Even segregating woody from nonwoody systems (because these two classes vary in root–shoot ratio primarily due to the differences in aboveground woody biomass), there were substantial differences among biomes. For forests, there were differences among boreal (0.32), temperate coniferous (0.18), temperate deciduous (0.23), tropical deciduous (0.34), and tropical evergreen (0.19) forests, but these do not provide any discernible pattern. This could be a result of the confounding of the myriad of differing environmental factors across such broad gradients. One way to separate out genetic from environmental effects would be to compare different species or populations in common gardens, or the same genotype in plantations across environmental gradients. Some such data are available for Scots pine populations, wherein those populations from colder environments show greater biomass fractions in roots, both as stands of trees (Oleksyn et al., 1999) and as individual seedlings. Across this climate of origin gradient, there are strong gradients in temperature, nutrient supply, length of growing season, and day length, so discerning the most responsible factors for such patterns is difficult.

Root–Shoot Relations

VI.

SUMMARY

It is not clear why young plants phenotypically shift biomass allocation in response to gradients in N supply in a manner consistent with optimality theory, fail to do so consistently for light or CO2, and show mixed patterns for P and water. This may not even be an important question to consider in isolation. It may well be that plants benefit more from shifting morphology, phenology, metabolism, chemistry, tissue longevity, or architecture in response to such factors than from shifting allocation. Only holistic consideration at the whole-plant level can answer such questions. Knowledge of ontogenetic drift in root–shoot biomass distribution with plant size and age is not at all new. There are now a considerable number of publications calling attention to the possible misinterpretation of root–shoot ‘‘snapshots’’ in time. Nonetheless, despite systematic tests of the role of ontogenetic drift, these have failed to alert plant scientists sufficiently to these patterns. Instead, many authors continue to refer to differences in root–shoot relations as differences in plasticity of allocation that may well be largely the result of ontogenetic drift (e.g., Walters and Reich, 1989; Conroy et al., 1992; Latham, 1992; Graves, 1994; Lusk et al., 1997; Lei and Lechowicz, 1998; Messier et al., 1999; Valladares et al., 2000). For instance, root–shoot ratio was reported to be significantly higher in bean plants under low than moderate P supply and higher in nonmycorrhizal than mycorrhizal plants, based on comparisons of plants at common harvest times (Nielsen et al., 1998). It is difficult to reconcile results of such ‘‘snapshots’’ with those obtained by allometric relationships (Fig. 4) showing that at a common plant size, low P plants had fewer roots, not more. The point of such a specific example for P (Fig. 4), plus an example for CO2 (Fig. 5), is to provide graphic examples of a persistent problem of inadequate evaluation of whether shifts in biomass allocation are real, or whether we often ‘‘see’’ what we have been trained to ‘‘see.’’ Similarly, many elegant studies have used sophisticated techniques to study the mechanisms involved in the control of allocation, as influenced by resource supply. However, their results may teach us about how plants developmentally adjust biomass allocation as they grow larger and older, rather than teaching us about how resource supply influences allocational processes. Only careful scrutiny of all such studies can help us decide when the right interpretation was made. Changes in allocation patterns are relatively strong when nutrient supply is varied, but changes in other

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aspects of plant morphology or physiology more generally dominate the responses of plants to variation in a wide set of resources, including light, nutrients, water, and CO2. In essence, rather than adjust allocation of biomass, plants change tissue morphology, chemistry, metabolism, and turnover to alter their capability for resource interception, and to vary the rate of resource capture or resource loss. To the best of our knowledge, evidence for optimality in biomass allocation is even scarcer when examining differences among species that vary in habitat association, along either fertility or light gradients. Again, shifts in other attributes that may positively or negatively affect rates of resource capture or resource conservation appear to be far more important. Hence, it seems timely to suggest the removal of the significance of biomass allocation from its historically premier position, when discussing both how plants respond phenotypically to their environment and how plants have arrived through natural selection at their intrinsic genotypic traits. REFERENCES Aerts R. 1990. Nutrient use efficiency in evergreen and deciduous species from heathlands. Oecologia 84:391–397. Baxter R, Ashenden TA, Farrar JF. 1997. Effect of elevated CO2 and nutrient status on growth, dry matter partitioning and nutrient content of Poa alpina var. vivipara L. J Exp Bot 48:1477–1486. Bloom AJ, Chapin FS, Mooney HA. 1985. Resource limitations in plants—an economic analogy. Annu Rev Ecol Syst 16:363–392. Brouwer R. 1962a. Distribution of dry matter in the plant. Neth J Agric Sci 10:361–376. Brouwer R. 1962b. Nutritive influences on the distribution of dry matter in the plant. Neth J Agric Sci 10:399–408. Brouwer R. 1983. Functional equilibrium: sense or nonsense? Neth J Agric Sci 31:335–348. Callaway RM. 1992. Morphological and physiological responses of three California oak species to shade. Int J Plant Sci 153:434–441. Caloin M, Cle´ment B, Herrmann S. 1991. Regrowth kinetics of Dactylis glomerata following root excision. Ann Bot 68:435–440. Casper BB, Cahill JF Jr, Hyatt LA. 1998. Above-ground competition does not alter biomass allocated to roots in Abutilon theophrasti. New Phytol 140:231–238. Ceulemans R, Mousseau M. 1994. Effects of elevated atmospheric CO2 on woody plants. New Phytol 127:425– 446. Chapin FS III. 1980. The mineral nutrition of wild plants. Annu Rev Ecol Syst 11:233–260. Chapin FS III. 1991. Effects of multiple stresses on nutrient availability and use. In: Mooney HA, Winner WE, Pell

218 EJ, eds. Response of Plans to Multiple Stresses. San Diego, CA: Academic Press, pp 67–88. Chen HYH. 1997. Interspecific responses of planted seedlings to light availability in interior British Columbia: survival, growth, allometric, and specific leaf area. Can J For Res 27:1383–1393. Chen HYH, Klinka K. 1998. Survival, growth, and allometry of planted Larix occidentalis seedlings in relation to light availability. For Ecol Manag 106:169–179. Coleman JS, McConnaughay KDM, Ackerly DD. 1994. Interpreting phenotypic variation in plants. Trends Ecol Evol 9:187–191. Conroy JP, Milham PJ, Barlow EWR. 1992. Effect of nitrogen and phosphorus availability on the growth of Eucalyptus gradis to high CO2. Plant Cell Environ 15:843–847. Cornelissen JHC, Castro-Diez P, Hunt R. 1996. Seedling growth, allocation and leaf attributes in a wide range of woody plant species and types. J Ecol 84:755–765. Corre WJ. 1983. Growth and morphogenesis of sun and shade plants. I. The influence of light intensity. Acta Bot Neerl 32:49–62. Craine JM, Tilman DG, Wedin DA, Reich PB, Tjoelker MJ, Knops JMH. 2001. The relationship between plant functional strategies and growth in low-nutrient habitats. (Submitted to J Veg Sci.) Curtis PS, Wang X. 1998. A meta-analysis of elevated CO2 effects on woody plant mass, form, and physiology. Oecologia 113:299–313. Eamus D, Jarvis PG. 1989. Direct effects of CO2 increases on trees and forests (natural and commercial) in the UK. Adv Ecol Res 19:1–55. Eissenstat DM, Duncan LW. 1992. Root growth and carbohydrate responses in bearing citrus trees following partial canopy removal. Tree Physiol 10:245–257. Eissenstat DM, Yanai RD. 1997. The ecology of root lifespan. Adv Ecol Res 27:1–60. Eissenstat DM, Graham JH, Syvertsen JP, Drouillard DI. 1993. Carbon economy of sour orange in relation to mycorrhizal colonization and phosphorus status. Ann Bot 71:1–10. Evans GC. 1972. The Quantitative Analysis of Plant Growth. Berkeley, CA: University of California Press. Evans GC, Hughes AP. 1961. Plant growth and the aerial environment. I. Effect of artificial shading on Impatiens parviflora. New Phytol 60:150–180. Farrar JF, Gunn S. 1998. Allocation: allometry acclimation—and alchemy? In: Lambers H, Poorter H, Van Vuuren MMI, eds. Inherent Variation in Plant Growth. Physiological Mechanisms and Ecological Consequences. Leiden, Netherlands: Backhuys Publishers, pp 183–198. Fichtner K, Schulze E-D. 1992. The effect of nitrogen nutrition on growth and biomass partitioning of annual plants originating from habitats of different nitrogen availability. Oecologia 92:236–241.

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Root–Shoot Relations Lei TT, Lechowicz MJ. 1998. Diverse responses of maple saplings to forest light regimes. Ann Bot 82:9–19. Lerdau M. 1992. Future discounts and resource allocation in plants. Funct Ecol 6:371–375. Lerdau M, Gershenzon J. 1997. Allocation theory and the costs of chemical defenses in plants. In: Bazzaz F, Grace J, eds. Resource Allocation in Plants and Animals. San Diego, CA: Academic Press, pp 265– 277. Linder S, Axelsson B. 1982. Changes in carbon uptake and allocation patterns as a result of irrigation and fertilization in a young Pinus sylvestris stand. In: Waring RH, ed. Carbon Uptake and Allocations: Key to Management of Subalpine Forest Ecosystems, International Union Forest Research Organization (IUFRO) Workshop. Corvallis, OR: Forest Research Laboratory, Oregon State University. Lusk CH, Contreras O, Figueroa J. 1997. Growth, biomass allocation and plant nitrogen concentration in Chilean temperate rainforest tree seedlings: effects of nutrient availability. Oecologia 109:49–58. Lutze JL, Gifford RM. 1998. Acquisition and allocation of carbon and nitrogen by Danthonia richardsonii in response to restricted nitrogen supply and CO2 enrichment. Plant Cell Environ 21:1133–1141. Machado J-L M, Walters MB, Reich PB. 2001. In deeply shaded forest understories, belowground resources limit seedling growth but do not alter biomass distribution patterns or survival. (Submitted to Forest Ecology and Management.) McConnaughay KDM, Coleman JS. 1999. Biomass allocation in plants: ontogeny or optimality? A test along three resource gradients. Ecology 80:2581–2593. McMillan JD, Wagner MR. 1995. Effects of water stress on biomass partitioning of ponderosa pine seedlings during primary root growth and shoot growth periods. For Sci 41:594–610. Messier C, Doucet R, Ruel J-C, Claveau Y, Kelly C, Lechowicz MJ. 1999. Functional ecology of advance regeneration in relation to light in boreal forests. Can J For Res 29:812–823. Nielsen KL, Bouma TJ, Lynch JP, Eissenstat DM. 1998. Effects of phosphorus availability and vesicular-arbuscular mycorrhizas on the carbon budget of common bean (Phseolus vulgaris). New Phytol 139:647–656. Oleksyn J, Reich PB, Chalupka W, Tjoelker MG. 1999. Differential above- and belowground biomass accumulation of European Pinus sylvestris populations in a 12year-old provenance experiment. Scand J For Res 14:7–17. Olff H, Van Andel J, Bakker JP. 1990. Biomass and shoot/ root allocation of five species from a grassland succession series at different combinations of light and nutrient supply. Funct Ecol 4:193–200.

219 Peace WJH, Grubb PJ. 1982. Interaction of light and mineral nutrient supply in the growth of Impatiens parviflora. New Phytol 90:127–150. Pearsall WH. 1927. Growth studies VI. On the relative sizes of growing plant organs. Am J Bot 41:549–556. Philippot S, Allirand JM, Chartier M, Gosse G. 1991. The role of different daily irradiations on shoot growth and root/shoot ratio in Lucerne (Medicago sativa L.). Ann Bot 68:329–335. Poorter L. 1999. Growth responses of 15 rain-forest tree species to a light gradient: the relative importance of morphological and physiological traits. Funct Ecol 13:396–410. Poorter H, Nagel O. 2000. The role of biomass allocation in the growth response to plants to different levels of light, CO2, nutrients and water: a quantitative review. Aust J Plant Physiol 27:595–607. Poorter H, Remkes C. 1990. Leaf area ratio and net assimilation rate of 24 wild species differing in relative growth rate. Oecologia 83:553–559. Poorter H, Remkes C, Lambers H. 1990. Carbon and nitrogen economy of 24 wild species differing in relative growth rate. Plant Physiol 94:621–627. Reich, PB, Schoettle AW, Stroo HF, Troiano J, Amundson RG. 1987. Influence of O3 and acid rain on white pine seedlings grown in five soils. I. Net photosynthesis and growth. Can J Bot 65:977–987. Reich PB, Walters MB, Ellsworth DS. 1992. Leaf lifespan in relation to leaf, plant and stand characteristics among diverse ecosystems. Ecol Monogr 62:365–392. Reich PB, Walters MB, Krause SC, Vanderklein D, Raffa KF. 1993. Growth, nutrition and gas exchange of Pinus resinosa following artificial defoliation. Trees 7:67–77. Reich PB, Tjoelker MG, Walters MB, Vanderklein D, Buschena C. 1998a. Close association of RGR, leaf and root morphology, seed mass and shade tolerance in seedlings of nine boreal tree species grown in high and low light. Funct Ecol 12:327–338. Reich PB, Walters MB, Tjoelker MG, Vanderklein D, Buschena C. 1998b. Photosynthesis and respiration rates depend on leaf and root morphology and nitrogen concentration in nine boreal tree species differing in relative growth rate. Funct Ecol 12:395–405. Reich PB, Ellsworth DS, Walters MB, Vose J, Gresham C, Volin J, Bowman W. 1999. Generality of leaf traits relationships: a test across six biomes. Ecology 80:1955–1969. Reich PB, Peterson DA, Wrage K, Wedin D. 2001. Fire and vegetation effects on productivity and nitrogen cycling across a forest-grassland continuum. Ecology (in press). Reynolds HL, D’Antonio C. 1996. The ecological significance of plasticity in root weight ratio in response to nitrogen: opinion. Plant Soil 185:75–97.

220 Reynolds JF, Thornley JHM. 1982. A shoot:root partitioning model. Ann Bot 49:585–597. Rice SA, Bazzaz FA. 1989. Quantification of plasticity of plant traits in response to light intensity: comparing phenotypes at a common weight. Oecologia 78:502– 507. Robinson D. 1986. Compensatory changes in the partitioning of dry matter in relation to nitrogen uptake and optimal variations in growth. Ann Bot 86:841–848. Robinson D, Rorison IH. 1988. Plasticity in grass species in relation to nitrogen supply. Funct Ecol 2:249–257. Ryser P. 1996. The importance of tissue density for growth and life span of leaves and roots: a comparison of five ecologically contrasting grasses. Funct Ecol 10:717– 723. Ryser P, Aeschlimann U. 1999. Proportional dry-mass content as an underlying trait for the variation in relative growth rate among 22 Eurasian populations of Dactylis glomerata s.l. Funct Ecol 13:473–482. Ryser P, Lambers H. 1995. Root and leaf attributes accounting for the performance of fast- and slow-growing grasses at different nutrient supply. Plant Soil 170:251–265. Steinbrenner EC, Rediske JH. 1964. Growth of ponderosa pine and Douglas-fir in a controlled environment. Weyerhauser Forest Paper No. 1. Centralia, WA: Weyerhauser Forestry Research Center. Stoneman GL, Dell B. 1993. Growth of Eucalyptus marginata (Jarrah) seedlings in a greenhouse in response to shade and soil temperature. Tree Physiol 13:239–252. Terry N. 1968. Developmental physiology of sugar beet. I. The influence of light and temperature on growth. J Exp Bot 61:795–811. Thornley JM. 1969. A model to describe the partitioning of photosynthate during vegetative plant growth. Ann Bot 33:419–430. Thornley JM. 1972. A balanced quantitative model for root:shoot ratios in vegetative plants. Ann Bot 36:431–441. Thornley JHM. 1998. Modelling shoot:root relations: the only way forward? Ann Bot 81:165–171. Tilman D. 1988. Plant Strategies and the Dynamics and Structure of Plant Communities. Princeton, NJ: Princeton University Press. Tjoelker MG, Oleksyn J, Reich PB. 1998. Temperature and ontogeny mediate growth response to elevated CO2 in seedlings of five boreal tree species. New Phytol 140:197–210. Tomlinson PT, Anderson PD. 1998. Ontogeny affects response of northern red oak seedlings to elevated CO2 and water stress. New Phytol 140:493–504. Valladares F, Wright SJ, Lasso E, Kitajima K, Pearcy RW. 2000. Plastic phenotypic response to light of 16 congeneric shrubs from a Panamanian rainforest. Ecology 81:1925–1936.

Reich Van der Werf A, Van Nuenen M, Visser AJ, Lambers H. 1993a. Contribution of physiological and morphological plant traits to species’ competitive ability at high and low nitrogen supply. Oecologia 94:434–440. Van der Werf A, Visser AJ, Schieving F, Lambers H. 1993b. Evidence for optimal partitioning of biomass and nitrogen at a range of nitrogen availabilities for a fast- and slow-growing species. Funct Ecol 7:63–74. Vanderklein DW, Reich PB. 1999. The effect of defoliation intensity and history on photosynthesis, growth and carbon reserves of two conifers with contrasting leaf lifespans and growth habits. New Phytol 144:121–132. Veneklaas EJ, Poorter L. 1998. Growth and carbon partitioning of tropical tree seedlings in contrasting light environments. In: Lambers H, Poorter H, Van Vuuren MMI, eds. Inherent Variation in Plant Growth. Leiden, Netherlands: Backhuys Publishers, pp 337–361. Volin JC, Reich PB. 1996. Interaction of carbon dioxide and ozone on C3 and C4 grasses and trees under contrasting nutrient supply. Physiol Plant 97:674–684. Walters MB, Reich PB. 1989. Response of Ulmus americana seedlings to varying nitrogen and water status. I. Photosynthesis and growth. Tree Physiol 5:159–172. Walters MB, Reich PB. 1996. Are shade tolerance, survival, and growth linked? Low light and nitrogen effects on hardwood seedlings. Ecology 77:841–853. Walters MB, Reich PB. 1997. Growth of Acer saccharum seedlings in deeply shaded understories of northern Wisconsin: effects of nitrogen and water availability. Can J For Res 27:237–247. Walters MB, Reich PB. 1999. Low light carbon balance and shade tolerance in the seedlings of woody plants: do winter deciduous and broad-leaved evergreen species differ? New Phytol 143:143–154. Walters MB, Reich PB. 2000a. Seed size, nitrogen supply and growth rate affect tree seedling survival in deep shade. Ecology 81:1887–1901. Walters MB, Reich PB. 2000b.Trade-offs in low-light CO2 exchange: a component of variation in shade tolerance among cold temperate tree seedlings. Funct Ecol 14:155–165. Walters MB, Kruger EL, Reich PB. 1993a. Growth, biomass distribution and CO2 exchange of northern hardwood seedlings in high and low light: relationships with successional status and shade tolerance. Oecologia 94:7– 16. Walters MB, Kruger EL, Reich PB. 1993b. Relative growth rate in relation to physiological and morphological traits for northern hardwood seedlings: species, light environment and ontogenetic considerations. Oecologia 96:219–231. Wilson JB. 1988. A review of evidence on the control of shoot:root ratio, in relation to models. Ann Bot 61:433–449.

13 Root Life Span, Efficiency, and Turnover David M. Eissenstat The Pennsylvania State University, University Park, Pennsylvania

Ruth D. Yanai State University of New York, Syracuse, New York

I.

INTRODUCTION

than shoot competition (Wilson, 1988). Just as perennial structures aboveground can give plants a competitive advantage for light capture, there may be advantages to long-lived roots in the capture of limited soil resources. Resource preemption can be an important component of competitive success. For example, in climates with winter precipitation, perennial grasses with an established root system are much more effective than seedlings of perennial grasses at competing with annual grass species during the spring and summer (Harris, 1967). Clearly, root demography can have important consequences on species distribution and abundance. The demography of roots also influences ecosystem processes associated with material and energy flows. Approximately 33% of global net primary production is used for fine-root production, based on fine-root biomass in 253 field studies in a wide range of ecosystems and assuming roots have a life span of 1 year, possibly a conservative estimate (Jackson et al., 1997). In other studies, belowground net primary productivity (BNPP) has been estimated to be at least as great as aboveground net primary productivity (Vogt et al., 1986; Caldwell, 1987). Clearly, the production and death of fine roots can have a substantial influence on ecosystem carbon and mineral nutrient cycling. Many ecologists have been concerned with understanding how BNPP varies among ecosystems and pre-

Like other plant organs, roots have a life history in which they pass from birth to death. The size and population structure of the root system is determined by the birth rate and death rate of the individual roots. The study of root demography is of interest to many disciplines, including crop science, physiology, ecology, and soil science. For example, a better understanding of root demography could enable agronomists and horticulturalists to increase yields while reducing agrochemical inputs. Severe root losses, such as those caused by drought or pathogens, clearly are not conducive to crop production. Growing too many roots, however, may also be undesirable, since large amounts of carbohydrates and mineral nutrients are needed for root growth and maintenance that otherwise might be allocated to photosynthetic organs or harvested parts. An optimization approach suggests that, other things being equal, total plant growth should be greatest when a root system maximizes water and nutrient acquisition per unit resource supplied from the shoot (e.g., Thornley, 1998). If roots are produced in the most favorable soil patches and shed when they are no longer efficient in water and nutrient absorption, then production, theoretically, should be maximized. The birth and death of roots also influence plant competition. Root competition can be more intense 221

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dicting how it may change in response to tropospheric ozone concentrations (Coleman et al., 1996), nitrogen deposition (Nadelhoffer, 2000), temperature (Gill and Jackson, 2000; Pregitzer et al., 2000a), drought (Joslin et al., 2000) and elevated CO2 (Arnone et al., 2000; Tingey et al., 2000). We need a more mechanistic understanding of factors controlling root longevity if BNPP is to be incorporated into models of ecosystem response to global climate change (Norby and Jackson, 2000; Jackson et al., 2000). The first roots of plants developing from seed are indeterminate, typically extending greatly in length as the taproot or other seminal roots develop. The major laterals that first emerge from these primary roots and the adventitious or nodal roots that emerge from the stem base are also typically indeterminate, often extending decimeters or more in length. These indeterminate roots form the basic framework of the root system and may live as long as the plant lives. This chapter focuses on the more ephemeral portion of the root system. Ephemeral roots are the fine laterals that may be replaced several times during a growing season and may have only a few orders of branching. In at least some woody species, these roots never undergo secondary development of the stelar tissue or the development of a periderm (Brundrett and Kendrick, 1988; Eissenstat and Achor, 1999). In this chapter, we examine variations in root life span and causes for this variation. We discuss different methods of assessing root life span and root turnover. We describe a cost-benefit model of root deployment, which defines the root life span that maximizes the efficiency of resource acquisition. We review studies that have examined biotic and abiotic factors that influence root life span in the context of our hypothesis that plants modulate root life span to maximize root efficiency. Finally, we extend the model of individual root efficiency to describe a cohort of roots with a median life span, and we include allocation to defense in defining the optimal root life span. II.

VARIATION IN ROOT LIFE SPAN

A.

Sources of Variation

Estimates of root life span vary widely. The median life span of the finest roots can range from <20 days in fast-growing trees and deciduous fruit crops to >1 year in slow-growing forest trees, according to studies using transparent windows in the soil (Eissenstat and Yanai, 1997). In a data set containing 190 studies in nonagricultural ecosystems, based mainly on changes

in biomass from sampling soil monoliths, soil cores or ingrowth cores, average root life spans ranged from
290 d in tropical ecosystems to
3 years in highlatitude ecosystems, with considerable variation within each ecosystem type (Gill and Jackson, 2000). Recent studies based on tracer approaches have indicated that fine roots may live considerably longer—averaging 4–8 yr in some temperate forests (Matamala et al., 2000; Gaudinski et al., 2000). Although differences in methods contribute substantially to differences in estimates of life span, as we will discuss, undoubtedly much of the variation in reported root life span is caused by differences in environmental conditions and plant species. B.

Patterns of Variation Among Species

It is difficult to assess the relative importance of genetic and environmental variation on root life span. Few studies have tracked individual roots of more than one species under the same environmental conditions. In a greenhouse study of seedlings of four tree species, root life span varied from 26 d in Prunus avium to 86 d in Picea sitchensis (Black et al., 1998). In a Valencia orange citrus rootstock trial in central Florida, we measured a median root life span of 90 d in Poncirus trifoliata and 152 d in Citrus volkameriana. Weaver and Zink (1946) banded individual nodal roots of perennial range and pasture grasses. After 3 years, root survival ranged from 45% in Bouteloua gracilis to 10% in Stipa spartea. The fine laterals of the nodal roots presumably had shorter life spans, but they could not be followed with this approach. The same theories that attempt to explain variation in leaf life span have been applied to roots (Grime, 1977; Chapin, 1980; Aerts, 1995). Plants that have slow growth rates and are adapted to chronically low-nutrient sites, for example, should have long life span of the absorptive organs compared to more fertile sites. Tissue retention in nutrient-poor sites allows nutrients to be retained as well, which is important if root and shoot growth rates are restricted by nutrient limitations. There is considerable evidence that leaf longevity is consistent with this hypothesis (Reich et al., 1997), but roots have been less well studied. In the pot study of Black et al. (1998), the species with the shortest root life span, Prunus avium, had considerably faster growing root and shoot systems than the species with longest root life span, Picea sitchensis. In a study comparing grasses from nutrient-poor and nutrient-rich habitats in pots in the field, the grasses from the nutrient-rich habitat had lost a greater

Life Span, Efficiency, and Turnover

percentage of their leaves and roots by the end of the second growing season (Ryser, 1996). Roots also lived longer in species adapted to more infertile soils among trees in mixed hardwood forests in Wisconsin (estimated by nitrogen budgeting; Aber et al., 1985; Nadelhoffer et al., 1985) and among heathland shrubs and grasses (estimated by minirhizotron and soil core sampling; Aerts et al., 1989, 1992). These results suggest that roots and leaves do have similar adaptations of longevity to resource availability. There are notable exceptions to this generalization, however. Desert succulents have long-lived leaves but short-lived ‘‘rain’’ roots (Huang and Nobel, 1992; North et al., 1993; see also Chapter 53 by Nobel in this volume). In seasonal dry climates, cluster roots of evergreen woody plants (Lamont, 1995) and ericoid mycorrhizal root hairs of plants in the Epacridaceae (Smith and Read, 1997) are shed during extended dry periods. Generalizations about the relationship of tissue longevity to resource availability may apply better to nutrients than water. Long leaf life span has been associated with other leaf traits, including low specific leaf area (area/mass ratio), N concentration, maximum assimilation rate, high leaf thickness, toughness, lignin content, and tissue density (Reich et al., 1997). Similar suites of correlated traits may also occur in roots (Eissenstat, 1992; Reich et al., 1998), but the scarcity of observations makes patterns more difficult to detect. One such study by Ryser (1996) found higher tissue density in grasses with longer-lived roots. Similarly, in a comparison of apple and citrus, long root life span was associated with coarse root diameter and high tissue density (Eissenstat et al., 2000), low maintenance respiration, and a low P uptake capacity (Bouma et al., 2001). III.

METHODOLOGICAL CONSIDERATIONS

A.

Difficulties and Definitions

The single greatest impediment to the study of root life span is the difficulty of studying roots in their natural environment. Many approaches have been taken with varying degrees of success. Often studies are not long enough to establish clear year-to-year variation or to have allowed the plants to fully adjust to installation of root measuring devices (e.g., minirhizotrons) or treatments. For example, fertilization studies are often conducted for only a few years, so they may not characterize steady-state responses to a new level of fertility. The interpretation of estimates of root life span is hindered by inconsistencies in methods of reporting

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root dynamics. In many ecosystem studies, the main objective is to estimate BNPP (kg ha1 yr1). The term ‘‘root turnover’’ has often been used synonymously with annual root production or annual root mortality and thus has units such as kg ha1 yr1. Alternatively, root turnover may be used to describe the specific rate of root mortality, in units of yr1. One way to report the specific rate of root mortality is the rate constant in exponential decay (described in Section VII, below). Root turnover rate is also commonly reported as annual root production or annual root mortality divided by root standing crop. Studies differ in whether minimum (Hendrick and Pregitzer, 1993), average (Aber et al., 1985; Aerts et al., 1992), or maximum (Dahlman and Kucera, 1965; Gill and Jackson, 2000) root biomass are used to estimate standing crop. Gill and Jackson (2000) found that about onethird of root turnover studies report only the mean standing crop. An important disadvantage of using minimum or maximum standing crop is that the minimum or maximum value in any distribution is dependent on the number of samples collected and the sampling error associated with sample measurement. Nonetheless, Gill and Jackson (2000) found that maximum standing crop could be accurately estimated by mean standing crop by a regression approach ðr2 ¼ :90Þ, based on 20 data sets that included both maximum and mean root biomass. Root life span is inversely proportional to root turnover rate, with the constant of proportionality dependent on the definitions of turnover rate and life span. Many recent studies that follow the fate of individual roots with minirhizotrons report only median life span (or similarly half-life of the cohort), partly because many of the roots in the study have not died by the end of the study and partly because the median is a better estimator of the central location of a highly skewed distribution—a condition common to survivorship curves. Clearly, average life span may be considerably longer than median life span if an appreciable fraction of the population lives a very long time. Studies that follow individual roots typically report median life spans of specific cohorts (roots born at the same point in time), because different cohorts may exhibit very different median life spans (Kosola et al., 1995). B.

Methods of Estimating Root Life Span

Early techniques estimated root turnover at ecosystem scales by measuring average standing crop and seasonal root production using sequential coring, root

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ingrowth cores, and elemental budgeting (see reviews by Caldwell and Eissenstat, 1987; Vogt and Persson, 1991; Fahey et al., 1999). More recently, studies have used minirhizotrons (Cheng et al., 1990; Hendrick and Pregitzer, 1993) and other direct observational techniques (Fahey and Hughes, 1994), which focus on the fate of individual roots. In addition, tracer techniques hold considerable promise as an independent estimator of root longevity (Gaudinski et al., 2000; Matamala et al., 2000). No method of estimating root turnover has emerged as the best for all conditions. In the 1970s and ’80s, the most popular approach to estimating ecosystem BNPP was sequential coring. This approach involves collecting soil cores over the growing season (often monthly) and estimating BNPP based on changes in the mass of live and dead roots (e.g., Vogt et al., 1981). The advantages of sequential coring are that the roots being measured have not been altered in any way prior to coring, estimates can be scaled up to the ecosystem, and equipment costs are low. The labor required to separate roots from the soil core and to separate live from dead roots, however, is considerable (Bloomfield et al., 1996). The very finest roots, which may be very fragile, are probably never completely separated from the soil. Another limitation of this method is a lack of information on turnover of deeper roots; cores are commonly collected only to 20 cm depth. There are also several sources of error in the calculations, which involve the differences between cores collected over time. Simultaneous birth and death of roots during a single sampling interval is not detected (Rytter, 1999). The very finest roots probably die within weeks, not months (Wells and Eissenstat, 2001). It is also difficult to separate spatial and sampling variation in root mass from the parameter of interest, temporal variation (Singh et al., 1984; Sala et al., 1988). Typically, soil-coring or soil monolith methods are used to estimate annual root production, and a steady-state assumption is required to equate annual root mortality with annual root production. Root turnover (yr1) is obtained by dividing production (kg ha1 yr1) by some estimate of standing crop (kg ha1), which can introduce further errors. Various approaches have been used to improve biomass-based estimates using compartment-flow models (Santantonio and Grace, 1987; Ma¨kela¨ and Vanninen, 2000), but these methods require accurate information on fine-root decomposition, which is difficult to acquire (Fahey et al., 1999), especially for the very finest roots (Comas et al., 2000; Wells and Eissenstat, 2001).

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The ingrowth-core technique for estimating root production and turnover assumes that root production in a soil volume initially devoid of roots reflects root production in the undisturbed soil (Fabia˜o et al., 1985; Fine´r et al., 1997). It also assumes that no root mortality (or if dead roots are followed, no root decomposition) has occurred during a sampling interval. Like sequential coring, this method of estimating root mortality assumes steady-state conditions and requires an estimate of root standing crop. Ingrowth-core approaches are relatively inexpensive, requiring considerably less labor than either minirhizotron or sequential coring methods, because they do not involve distinguishing live roots from dead ones or removing them from soil organic matter. The biggest drawback is their artificiality. Soil disturbance can increase water and nutrient availability by increasing decomposition and reducing root competition (Eissenstat, 1991). Soil disturbance may also favor root growth by decreasing soil bulk density and impedance. For these reasons, root growth may be considerably higher in small volumes of disturbed soil than in surrounding undisturbed soil, biasing estimates of production and turnover. Various elemental budgeting techniques have been used to assess root turnover (Nadelhoffer, 2000). The nitrogen budgeting approach estimates fine-root production as the difference between annual net mineralization and net N uptake into aboveground production (Aber et al., 1985; Nadelhoffer et al., 1985). This method depends on the accuracy of the estimate of net N mineralization, which is usually based on in situ soil incubations, and assumes that there is no change in ecosystem storage of mineralized N. To estimate root turnover or root longevity further requires estimating the standing crop of roots and assuming a steady state, as for the other methods described above. The budgeting techniques are also subject to errors in other fluxes such as N deposition, denitrification, and leaching. Another budgeting approach uses soil carbon fluxes to estimate root turnover. Soil respiration less root respiration and aboveground litterfall should equal fine root production (Raich and Nadelhoffer, 1989; Nadelhoffer and Raich, 1992; Haynes and Gower, 1995). This method depends on the accuracy of the estimates of soil and root respiration. It also assumes that soil organic matter is at steady state, unless the rate of change can be estimated (see Chapter 40 by Cramer in this volume). Tracers of C and N have been used to estimate root turnover, typically by calculating the dilution of the

Life Span, Efficiency, and Turnover

tracer in the structural tissues at various intervals after labeling (Caldwell and Camp, 1974; Milchunas and Lauenroth, 1992; Hendricks et al., 1997). The chief problems with this approach have been achieving uniform labeling of the structural tissue of the fine roots, estimating turnover rates of the very finest roots, which may be more rapid than the sampling intervals, and, for C, labeling whole trees. Recently, 13C in free-air CO2 exposure (FACE) experiments (Matamala et al., 2000) and the spike in atmospheric 14C caused by bomb-testing in the 1950s (Gaudinski et al., 2000) have been used to provide estimates of root longevity for large trees. There are some additional techniques that allow for the examination of factors influencing root demography. Tagging roots (Weaver and Zink, 1946) and following tillers of known age and root number (Shaver and Billings, 1975; Brundrett and Kendrick, 1988) permit estimation of the life span only of the major nodal roots, not the fine laterals. Root screens (Fahey and Hughes, 1994) can be useful for estimating the longevity of fine roots that form a readily accessible root mat. The most versatile technique for the direct observation of root demography is to track roots growing against transparent windows. Large root observation windows, referred to as rhizotrons, were initially used to study root phenology (seasonal patterns of root growth), including root mortality, in relation to shoot phenology (Head, 1973). Rapid progress in our understanding of root demography has occurred with the development of minirhizotrons (transparent tubes typically 2–6 cm in diameter), which allow roots to be observed in diverse ecosystems with minimal disturbance and a reasonable degree of replication (Taylor, 1987; Fahey et al., 1999; see Chapter 18 by Polomski and Kuhn in this volume). This technique suffered in its early years from limitations in the quality of images and the amount of labor required to process thousands of root images. In the late 1990s, improvements in miniature cameras or borescopes, direct digital capture of images, fast low-cost computers with greater storage capacity, and more sophisticated statistical approaches have made this technique more powerful and accessible. Minirhizotron studies have provided detailed information about root life span, such as age-specific mortality rates, mortality rates of roots born at different times of year or at different depths in the soil, mortality rates among roots of different orders or diameters, and effects of localized soil conditions on root mortality (Hendrick and Pregitzer, 1993; Ruess et al., 1998; Arnone et al., 2000; Wells and Eissenstat, 2001).

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Despite the widespread use of transparent wall techniques, they, too, have disadvantages. Transparent walls create an unnatural environment that may affect root production and longevity (Samson and Sinclair, 1994; Joslin and Wolfe, 1999). They can be difficult to use in rocky soils, shrink-swell soils, and clays that smear the tube surface, although various modifications have been devised (Gijsman et al., 1991; Meyer and Barrs, 1991; Lopez et al., 1996; Phillips et al., 2000). The biggest limitations are the cost of the camera equipment and the still considerable labor required to process the large numbers of root images. C.

Influence of Root Diameter and Root Order

The reported variation in root life span is partly due to the imprecise definition of the classes of roots under study. ‘‘Fine’’ roots are typically defined by an arbitrary diameter limit. The diameter limits for tree roots are generally large (1–5 mm) relative to the very finest roots. These finest roots can have much shorter life spans than the larger-diameter roots, which are still considered part of the fine-root system. For example, the median life span of apple roots 0.1–0.2 mm in diameter was only
40 d, while the median life span of roots 0.5–1.1 mm in diameter was longer than the observation period (211–240 d, depending on the year; Wells and Eissenstat, 2001). In peach, roots

0.25 mm in diameter had a median life span of 77 d while not a single root in the 0.5–1.7-mm class (n = 45) had died by the end of the study (369 d; Wells et al., submitted). Root order, which describes the position of a root in the branching pattern, is also important to root life span. In sugar maple, among roots <0.25 mm in diameter, roots with dependent laterals lived
400 d longer than those with no laterals, which had a median life span of 319 d (Wells, 1999; Eissenstat et al., 2000). Similar effects of root order have also been found in peach (Wells et al., 2002). Differences in longevity of roots of different diameter and order affect estimates of root turnover. For example, sugar maple roots <0.25 mm in diameter have a median life span of 319 d, with coarser roots (0.25–1.0 mm) living 694 d (Wells, 1999). Sugar maple has
50% of its fibrous root length, but only
20% of the mass, in the <0.25-mm-diameter class (Pregitzer et al., 1997). As a result, the average median life span is 503 d on a length-weighted basis but 616 d on a massweighted basis. Clearly, some of the variation in reported root life spans is associated with the size

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and order of the roots being studied and the methods of reporting the data. IV.

MODELING ROOT LIFE SPAN

A.

Value of Cost-Benefit Models Applied to Roots

The factors controlling root life span are not well understood. One way to explore hypotheses concerning the observed patterns in root life span is by constructing simulation models. The science of root life span is not so far advanced as to allow predictive modeling. The main value of our modeling efforts is heuristic, as we will illustrate: we compare predictions of our model to observations of root life span, and when inconsistencies occur we analyze the possible explanations. Fine roots provide a service, which, from the point of view of the whole plant, comes at a cost. The service is the uptake of nutrients and water. While roots may have other important functions such as the supply of hormones, these are not included in an efficiency model based on resource acquisition. The cost is the material and energy required to build and maintain the roots. If a plant were deploying roots to maximize return on investment, then the ratio of benefit (defined by the uptake of the limiting resource, be it water or a nutrient element) to cost (defined by the carbon or nutrient expended) should be maximized. This cost-benefit approach is not limited to exploring the optimal life span of roots. It can be used to show the value of roots of small diameter (Yanai et al., 1995), proliferation of roots in new soil (Caldwell, 1979; Eissenstat et al., 2002), and mycorrhizal association (Eissenstat et al., 1993; Peng et al., 1993). B.

An Optimization Model for Root Life Span

We have used a cost-benefit analysis to describe the theoretically optimal life span that maximizes the efficiency of nutrient capture. We define the efficiency, E, as the ratio of nutrient uptake to carbon cost: E ¼ Uptake=Cost The instantaneous efficiency of the root, E, can be calculated at a single point in time, using appropriate rates of Uptake (e.g., mmol P/g root/day) and Cost (mol C/g root/day). Alternatively, the costs and benefits can be summed over time to find the cumulative efficiency. Cumulative Uptake has units, in our examples, of mmol P/g root, while cumulative Cost has units

of mol C/g root. The units of both instantaneous and cumulative E are therefore mmol P/mol C. The optimal life span of a single root is defined as that with the highest cumulative efficiency. Plant carbon allocated to the root system will produce the highest possible rate of nutrient return if the roots live to the age with the highest cumulative Uptake/Cost ratio. The instantaneous efficiency always peaks at a younger age than the cumulative efficiency, which does not begin to decline until the instantaneous efficiency falls below the cumulative efficiency. The carbon cost of the root includes the carbon in the root and the carbon respired in constructing and maintaining the root: Cost ¼ carbon content + respiration The initial carbon investment in roots is high, because of the cost of constructing them. In our examples, the C content (42.5%) of a citrus root is 35.3 mmol C/g root (dry weight). The C content of the root is much higher than the daily maintenance respiration costs; in citrus,
3 weeks of maintenance respiration costs (
2 mmol/g/d in young roots) equal the C content of the root (Eissenstat and Yanai, 1997; see also Chapter 32 by Lambers et al., in this volume). Respiration costs of roots are not constant; they are initially high because of the metabolic energy used during root construction, and they decline over time, because of minimal growth respiration in fully formed roots and because of the decreasing metabolic activity of living cells in the root (Comas et al., 2000). This decline in respiration with age is evident in the daily carbon cost of citrus roots (Fig. 1c) (Bouma et al., 2001). A decline in C cost would tend to make a root more efficient over time, if uptake rates were constant. Cumulative efficiency always increases early in the life of a root, as the initial construction cost is amortized over a longer period (Fig. 1d). If the capacity of roots for nutrient uptake were not affected by age, then the theoretical optimal life span in a constant soil environment would be infinite (Yanai et al., 1995). Simply put, it would always be more costly to rebuild roots than to keep the old ones, if new ones were no better. The factors that make new roots better than old ones include the declining uptake capacity of older roots and the depletion of the soil around active roots. Comparisons of uptake capacity between woody and nonwoody roots and along different regions of the new roots of seedlings (Clarkson, 1991; Van Rees and Comerford, 1990) have shown declines related to root age. Our previous simulations (Yanai et

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Figure 1 Daily P uptake (a), cumulative P uptake (b), daily C cost (c), cumulative C cost (d), daily efficiency (e), and cumulative efficiency (f) of citrus roots. Dashed line represents simulated uptake with no soil depletion. Solid line represents uptake with soil P depletion based on soil parameters of Chandler fine sand. (From Bouma et al., 2001.)

al., 1995; Eissenstat and Yanai, 1997; Bouma et al., 2001) assumed that uptake capacity declined with root age, although measurements were not yet available to parameterize that relationship. We now have measurements of nutrient uptake capacity in fine lateral roots of varying ages for citrus (Fig. 1a) and apple (Bouma et al., 2001). We used a model for uptake that described the effect of various root and soil properties on nutrient uptake (Yanai, 1994). Either root or soil properties, or both, may limit uptake (Williams and Yanai, 1996). C.

Optimization Model Applied to Individual Roots

The optimization model has been applied to individual roots of citrus and apple, using observed patterns of uptake capacity and respiration as a function of root age. Citrus groves are fertilized with P and other nutrients and are commonly planted on sandy soils with low inherent fertility. We simulated P uptake and carbon costs for citrus roots growing in Chandler soil in Florida (Fig. 1). We used age-dependent P uptake kinetics and C respiration measured on excised roots

of known ages. The cumulative efficiency of P uptake increased initially, as P uptake increases and C costs decrease as the root develops. If the availability of P in the soil is assumed to remain high, as in a fertilized grove, then the efficiency remains high, and the optimal life span is infinite. Citrus roots are quite coarse and long-lived, but they do not live forever. Alternatively, if the P in the soil is assumed to be depleted by the root over time, then the efficiency of the root declines after
35 days and the cumulative root efficiency peaks at
50 days. This is much shorter than the observed life span of citrus roots (
300 days under low biotic pressure; Eissenstat et al., 2000), suggesting that the roots remain effective at nutrient capture for longer than predicted by the optimization model. The nutrient concentration in the soils is probably intermediate between the two cases illustrated; some depletion occurs, but not as much as if there were no nutrient supply to the soil. Mycorrhizal fungi may contribute significantly to the success of citrus in obtaining P from the soil. The effects of mycorrhizae on root longevity are discussed in a later section. The most distal lateral roots of apple, in contrast to those of citrus, are very fine and widely spaced. They are

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also more ephemeral than citrus roots, with a median life span of only
30–60 days (Eissenstat et al., 2000; Wells and Eissenstat, 2001). A simulation of the costs and benefits of apple roots failed to produce an optimal life span, even when root uptake was assumed to deplete the soil of P (Bouma et al., 2001). In this case, we suspect that P is not the important benefit to model. The optimal life span of the root is that which maximizes the ability of the plant to acquire the limiting resource. Since apple trees in the study orchards respond more to additions of N than to P, a better test of the optimization model would be conducted using N acquisition as the benefit, but the age-dependent kinetics of N uptake by apple roots have not been measured. This cost-benefit model of root life span has been applied to explore the effects of environmental factors such as drought and soil fertility on the costs and benefits of root deployment. These applications, which will be described below, reveal some of the needs for future research as well as illustrating concepts affecting optimal root life spans. V. ABIOTIC FACTORS INFLUENCING ROOT LIFE SPAN A.

Soil Moisture

Changes in the soil environment may change the nutrient uptake efficiency of the root and hence the optimal longevity. For example, in citrus, carbon allocation to the roots and root respiration were slowed substantially when roots were in dry soil for more than a couple of weeks (Kosola and Eissenstat, 1994; Espeleta and Eissenstat, 1998; Espeleta et al., 1999; Bryla et al., 1997). Root respiration was only 10– 20% of that in wet soil. Drought also affects the supply of nutrients from the soil to the roots, because of its effects on diffusion and transpiration rates. Phosphorus uptake by citrus in dry surface soil was reduced by 95–98% (Whaley, 1995); after the soil was rewetted, the roots recovered almost immediately, as indicated by water and P uptake rates. Sour orange seedlings whose surface roots were exposed to dry soil for > 40 d fully recovered their ability to take up water and P within the time interval of the first measurement (1–24 h; Eissenstat et al., 1999). Thus, while citrus roots have greatly diminished uptake in dry soil, they also have greatly diminished costs and essentially complete recovery, causing root efficiency to be only moderately affected by drought, according to model simulations (Eissenstat and Yanai, 1997). Not surprisingly, drought reduces citrus root life span in field stu-

dies by only a very modest amount (Akritas et al., unpublished data). Fine lateral roots of citrus have a well-developed exodermis that likely helps prevent desiccation in dry soil (Eissenstat and Achor, 1999; Huang and Eissenstat, 2000). Dry soil can greatly increase root mortality in grasses that lack an exodermis, such as big bluestem (Andropogon gerardii) (Hayes and Seastedt, 1987) and many agronomic plants (Huck et al., 1987; Smucker and Aiken, 1992) and turf grasses (Huang et al., 1997; Huang and Fry, 1998). Species that have very fine lateral roots of high hydraulic conductivity tend to shed their roots in dry soil and regrow them quickly when soil is rewetted. The costs and benefits of this plant strategy have been described for the rain roots of desert succulents (Nobel et al., 1992; see Chapter 53 by Nobel in this volume). B.

Soil Temperature

The importance of soil temperature as a factor influencing root life span is difficult to assess. Experimental manipulations of soil temperature have shown either no effect or a decrease in root longevity with increased temperature (see Chapter 41 by McMichael and Burke in this volume). In a study of trembling aspen (Populus tremuloides) in temperature-controlled containers in the field, cooling soil temperature to about 138C (28C) from roughly 208C (108C, 3.5-d average) decreased cumulative root production and mortality, but had no clear effect on root longevity (King et al., 1999). Elevating soil temperature by 2:88C at a 2 cm depth caused no clear change in root longevity in upland grassland in the United Kingdom (Fitter et al., 1999). In contrast, a growth chamber experiment with perennial ryegrass (Lolium perenne) found that grasses grown at 158C exhibited 30% root mortality after 35 d, while grasses grown at 278C had 84% root mortality (Forbes et al., 1997). Plants grown at 218C exhibited intermediate root mortality. Seasonal patterns and cross-site comparisons provide indirect evidence that high soil temperatures diminish root life span. Several studies have noted longer life spans of tree roots produced in the fall than those produced in the spring (Head, 1969; Hendrick and Pregitzer, 1993; Johnson et al., 2000; Wells and Eissenstat, 2001). Life spans of sugar maple roots were 75 d longer at the more northerly of two sites in Michigan, which corresponded with 2– 48C cooler soil temperatures at a 15-cm soil depth during the spring and summer months (Hendrick and Pregitzer, 1993). Life spans of fine roots of Lolium

Life Span, Efficiency, and Turnover

perenne and Trifolium repens were
30 d shorter in Italy (44 N latitude) than in the United Kingdom (57 N), which the investigators attributed primarily to differences in temperature (Watson et al., 2000). In a comparison of grassland sites along an altitudinal gradient in the United Kingdom where mean soil temperature at 2 cm ranged from 9:18C to 4:58C, root life spans were generally longer at the higher altitude sites except for roots produced in May, when they were shorter (Fitter et al., 1998). No differences were detected, however, in root life spans of aspen (Populus), jack pine (Pinus banksiana), and black spruce (Picea mariana) forests between a southern (54 N latitude) and northern (56 N) site in Saskatchewan and Manitoba, Canada (Steele et al., 1997). Global data sets can be used to suggest the effect of temperature on root turnover (Gill and Jackson, 2000). Mean annual temperature described more variation in fine-root turnover than any other variable, with an increase in mean annual temperature of 108C causing a 40–90% decrease in root life span. One explanation is that soil temperature increases root respiration more than nutrient uptake and accelerates the rate at which root efficiency decreases with age, causing a decrease in optimal life span. Simulations of the effects of temperature on root costs (using Q10 = 2 for maintenance respiration and no change in root benefit), however, indicate only about a 15-d decrease in root life span with a 108C increase in soil temperature (Eissenstat and Yanai, 1997). This clearly does not account for the approximately 0.5-year shift in root life span observed in the global data set of Gill and Jackson (2000). Unfortunately, studies of latitudinal and altitudinal variation in temperature and life span are readily confounded by covarying factors such as soil fertility, moisture, growing-season length, and herbivore and pathogen activity, making it nearly impossible to distinguish direct effects of temperature. Other factors, such as reduced root herbivory and parasitism in climates where soil freezes, may be a better explanation for the apparent effects of temperature on root longevity. In summary, higher temperatures have occasionally, but not always, been associated with shorter root life span. It is difficult, however, to distinguish the direct effects of temperature on root life span from the numerous indirect effects that temperature can have on the abiotic and biotic factors that influence root longevity. C.

Soil Nutrients

Root life span is responsive to fertility, but the results have been inconsistent. Among studies that have

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tracked the fate of individual roots, increased N availability has been associated with decreased root life span in Populus (Pregitzer et al., 1995, 2000b) and Picea abies (Majdi and Kangas, 1997). However, greater root life span has been observed in surface roots of forests dominated by sugar maple (Acer saccharum) (Burton et al., 2000); in localized fertile patches created with water and N in a forest stand dominated by Populus grandidentata, Prunus pennsylvanica, and other second-growth hardwoods (Pregitzer et al., 1993); in a forest dominated by Acer saccharum, Fagus grandifolia, and Betula alleghaniensis (Fahey and Hughes, 1994); and in a nearly pure 60-yr-old Acer saccharum stand (Wells, 1999). Information on variation in root life span among species along fertility gradients is sparse. Root life span is negatively correlated with plant potential growth rate in heathland species. Molina, which tends to dominate wet, moderately fertile heathlands, has median root life spans of
160–220 d, whereas Caluna, which dominates low-nutrient heathlands, has a median root life span of
570 d (Aerts et al., 1989, 1992). A comparison of 14 forest stands showed a strong inverse relationship between N availability and root life span using an N-budgeting approach, with average life spans ranging from 167 d in oakcherry-maple forest on fertile soil to 1223 d in pine forest on very infertile soil (Aber et al., 1985; Nadelhoffer et al., 1985). Average root life span in coniferous forests ranged from
80–580 d with no relationship with forest floor N (mean live root mass/ root turnover; recalculated from Fig. 14 in Vogt et al., 1986). Comparisons between species adapted to low and high fertility may differ from plastic responses to nutrient availability within species (Burton et al., 2000). In studies that examined the same species under different fertility regimes, sugar maple (using minirhizotrons; Burton et al., 2000) and Sitka spruce (Picea sitchensis) (using sequential coring; Alexander and Fairley 1983) exhibited increased root longevity with increased soil fertility. However, Populus (Pregitzer et al., 1995, 2000b) and Picea abies (Majdi and Kangas, 1997) exhibited the reverse response. Douglas fir (Pseudotsuga menziesii) exhibited similar mortality of root tips in fertile and infertile sites in Washington (determined by root observation windows; Keyes and Grier, 1981). In an efficiency context, plants should optimize carbon expenditure for uptake of nutrients that limit growth. To predict how increased nutrient availability might affect optimal root life span requires informa-

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tion on root respiration and uptake capacity as a function of root age. In some plants, roots of high metabolic activity associated with high root N concentrations might be expected to exhibit rapid declines in uptake capacity with age and therefore earlier mortality than roots of lower metabolic activity (cf. Pregitzer et al., 1998). Indirect effects may complicate the interpretation of N-gradient studies. For example, plants in more fertile soils may exhibit higher water

Figure 2 Daily root efficiency of nitrate acquisition and median life span of apple roots grown in split pots (Wang, Eissenstat, Flores-Alva, unpublished data). Plants received either high (H; 0.4 mmol) or low (L; 0.16 mmol) nitrate-N twice weekly in each pot separately. Treatments were: high N to both pots (HH), high N to one pot and low N to the other pot (HL), and low N to both pots (LL). The asterisk indicates the pot being measured (i.e., HL* indicates the low side of the high-low treatment is being measured). Root efficiency was determined by determining daily nitrate uptake at 75 d after transplanting using 15N-nitrate and carbon costs by determining root construction cost (elemental analysis), root growth rates (minirhizotrons), and respiration (continuous gas exchange over 48-h period of the pot head space). Median life span was determined for two root cohorts using minirhizotrons and a rigid borescope.

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use, causing their roots to be periodically exposed to drier soil. Spatially localized nutrient enrichment can have different effects on root efficiency and root longevity than variation in site fertility, in which the whole root system is affected. Studies with trees in the field have demonstrated enhanced fine-root persistence in fertile patches (Pregitzer et al., 1993; Fahey and Hughes, 1994; Wells, 1999). Studies of potted herbaceous species have indicated both increases and decreases in root longevity in fertile patches (Gross et al., 1994; Hodge et al., 1999a,b). To maximize root efficiency, life span should be greater in fertile soil patches because root efficiency is higher where nutrients are more available. For example, in a split-root study using apple seedlings, root efficiency was considerably higher for roots receiving greater N additions, because root benefits were increased more than root costs in the fertile soil (Fig. 2). Consistent with the increased efficiency, median root life span was also increased in the highnutrient side of the split-pot system (Fig. 2). In summary, there are conflicting results on the effects of soil nutrients on root life span. Inconsistencies may be partially related to indirect effects of fertility and to differences in methodology. More direct observations of the survival of individual roots along fertility gradients, in long-term fertilization trials, and in response to nutrient patches are needed before we can generalize about the effect of soil fertility on life span. VI.

BIOTIC FACTORS INFLUENCING ROOT LIFE SPAN

A.

Available Photosynthate and Competition with Other Sinks

Root mortality can be strongly affected by available photosynthate (Eissenstat and Yanai, 1997). Factors that reduce shoot carbon acquisition, such as canopy loss (Head, 1969; Eissenstat and Duncan, 1992) or shading (Marshall, 1986), can strongly diminish root longevity. For example, removal of the top third of the canopy of Valencia orange trees caused at least a 20% reduction in fine root length (Eissenstat and Duncan, 1992). Strong carbon demands during reproduction have also been associated with high root mortality. Declines in total root length during and after flowering are common in annual crops (Eissenstat and Yanai, 1997). Farmers are concerned when their trees produce too many fruit, thereby ‘‘weakening’’ the root system. For

Life Span, Efficiency, and Turnover

example, high root mortality has been associated with very heavy fruit crops of Prunus (Chandler, 1923) and Citrus (Smith, 1976; Graham et al., 1985). B.

Mycorrhizal Fungi

Approximately 90% of plants form mycorrhizal associations (Smith and Read, 1997). The primary benefit associated with vesicular arbuscular (VA) mycorrhizas is improved plant acquisition of P. Because plants may be more resistant to pathogens if not P deficient, many putative mycorrhizal benefits against pathogens may simply be an indirect result of improved P nutrition (Graham, 1988). There is, however, some evidence that mycorrhizae may enhance root longevity independent of P nutrition. Compared to nonmycorrhizal roots, root life span was extended in VA mycorrhizal roots exposed to dry surface soil (Espeleta et al., 1999), fungal pathogens (Benhamou et al., 1994; Newsham et al., 1995), and insect herbivores (Gange et al., 1994). Hooker et al. (1995), in contrast, found mycorrhizal colonization to diminish root longevity in Populus. In ectomycorrhizal associations, the fungal sheath that surrounds the roots probably protects the root from many forms of herbivory (Smith and Read, 1997). The effects of mycorrhizal fungi on root life span can also be examined in the context of root efficiency (Eissenstat and Yanai, 1997). If the mycorrhizal roots of a plant are acquiring more nutrients for less carbon (including costs of construction and maintenance of extramatrical hyphae) than those that have not been colonized, then the plant may more actively maintain and defend the mycorrhizal roots. A root system differentially colonized by mycorrhizal fungi may behave similarly to one in patchy soil fertility, as described above. C.

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in the rhizosphere. For example, Fusarium solani, a fungus whose inoculum is ubiquitous in root tissues of citrus, is able to develop only when starch reserves in the citrus roots are depleted, as may occur following canopy loss or during heavy fruit set (Graham et al., 1985). Wells (1999) examined the effects of selective pesticides on root life span by drenching pesticide monthly around minirhizotron tubes. In a 60-yr-old sugar maple stand, the fungicide metalaxyl (an inhibitor of protein synthesis in Oomycetes like Phytophthora and Pithium) caused an increase in median life spans from 270 d in water-drenched sites to 690 d in the fungicide treatment (Fig. 3). When both the fungicide and the insecticide chlorpyrifos (a broad-specturm cholinesterase inhibitor) were applied, median life spans were extended beyond the duration of the experiment (after 714 d, only 38% of the initial root population had died). The insecticide increased sugar maple root life span only when used in combination with the fungicide. In peach, chlorpyrifos also increased root life span, although the magnitude of the effect depended on the age of the roots when the insecticide was applied (Wells et al., submitted). For roots <50 days old, drenching with insecticide increased median life span by >250 d. For roots >50 d old, median life span was

Herbivores and Pathogens

Roots are constantly influenced by the myriad organisms that reside in the rhizosphere. Some soil organisms feed on roots directly, with obvious impact on plant communities (Weste, 1986). Others affect roots indirectly through root efficiency. Rhizosphere organisms may feed upon or compete with beneficial organisms such as mycorrhizal fungi and bacteria. They can also immobilize nutrients that would otherwise be available to the roots. The extent to which root herbivory and parasitism influence root life span is poorly understood. In most cases roots probably are not actively shed but simply succumb to weak parasites and herbivores that reside

Figure 3 Survivorship of sugar maple roots following monthly drenches of metalaxyl fungicide (open circles), chlorpyrifos insecticide (closed circles), both pesticides (closed triangles), or the water control (open triangles). Survivorship was determined using the minirhizotron technique. Both the fungicide and the fungicide+insecticide roots were significantly different from the control roots (P < :05; Cox proportional hazards regression). (From Eissenstat et al., 2000.)

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increased by only 44 d (from 56 d in control trees to
100 d in treated) by the insecticide treatment. In both the peach orchard and the sugar maple stand, the trees looked healthy and exhibited no root-related problems. Yet the influence of soil insects and fungi on patterns of root survivorship was clearly substantial. While we cannot say whether the pesticide treatments influenced root survivorship by directly removing root herbivores or fungal pathogens, we can conclude that root life span in many communities is likely to be strongly influenced by complex interactions with soil organisms. Factors controlling root life span likely include both biotic and abiotic factors. VII. A.

MODELING THE DEFENSE OF A COHORT OF ROOTS Optimization Model Applied to a Cohort of Roots

The mechanisms controlling the shedding of fine roots are not clear. An optimization model does not specify how roots should be shed, but only when they should be shed. Roots in minirhizotron studies are commonly observed to disappear, rather than to die in place and decompose over time. If herbivory or parasitism cause the death of roots, then optimal life spans from the cost-benefit point of view might not be achieved. Another observation in conflict with the optimization model described above is the wide distribution of life spans in a single root cohort (Fig. 3). If roots are produced in similar environments, with similar patterns of uptake capacity and respiration over time, they should reach maximal efficiencies at the same time. The observed range of life spans suggests that the control over root life span is very inexact. An alternative to a single-root optimization model is a cohort model, which describes a population of roots born at the same time. One method for describing a distribution of life spans in a population is a decay function, or a proportional-hazards model. Exponential decay is the simplest example of a hazard model, in which the chance of failure is constant over time. In this idealized case, the number of roots dying is a constant proportion of roots living, and every root has an equal chance of failure at every point in time. Empirical root survivorship curves look roughly exponential, with seasonal variation in mortality causing important deviations (Wells and Eissenstat, 2001). We use a simple exponential model to illustrate our approach to analyzing the efficiency of a cohort, but more complex models could be developed to include

factors such as climate, phenology, herbivore pressure, and the like. In simple exponential decay, only one parameter describes the mortality rate: A ¼ A0 expðk  tÞ where A0 is the original mass, length, or number of roots in the cohort; t is the age of the root (d), and k is the decay constant (day1), which equals the fraction of the roots that die each day. This equation can also be described by the median life span, or half-life, which is the age at which half the roots have died, or A ¼ 1=2A0 . At this point age = lnð1=2Þ=k. The cumulative efficiency of a cohort of roots is defined as the total cumulative uptake by all the roots divided by the total cumulative cost. This efficiency can be calculated by summing the costs and benefits over finite time intervals as the mass, length, or number of the roots decline. If the expressions for cost and benefit could be integrated, numeric integration would not be necessary. To illustrate the efficiency of a cohort of roots, we use equations for uptake and respiration that have the convenient property of exhibiting an optimal life span even when soil depletion is not simulated. We used uptake rates from apple and respiration rates from citrus (Fig. 1c). The resulting behavior cannot be attributed to any species, but it is computationally simple, and it illustrates the value of an efficiency approach applied to a cohort of roots. Because of the change in the number of roots present over time, the median life span of an optimal distribution does not coincide with the optimal individual life span. This point is illustrated in Fig. 4, using the equations for respiration and uptake described in the legend. In this illustration, the optimal life span for an individual root to maximize E is 86 days; the optimal half-life of the cohort using the same parameters is
60 d, assuming exponential decline in number of roots. From the point of view of the individual root, the exponential model means that there is a constant risk of root death from all sources. The plant could be imagined, however, to have some control over the magnitude of this risk. Root browning, for example, has been associated with reduced mortality in apple (Wells and Eissenstat, 2001) and peach (Wells et al., submitted). In addition, reduced insect herbivory was associated with delayed root browning (Wells et al., submitted). The optimization approach can explore what magnitude of investment in root defense is justified by an increase in root efficiency.

Life Span, Efficiency, and Turnover

Figure 4 The cumulative efficiency of a single root as a function of age and the efficiency of cohort of roots as a function of the median life span, or ln(.5)/k, assuming that the distribution of life spans in the cohort follows first-order kinetics, where the death rate is k times the pool of living roots. This illustration is based on Uptake (mmol P/g root/ day) = 21.7 * age/(age2 + 6.96 * age + 83.6) and Respiration (mol C/g root/day) = 14.3 + 12.6 * age3.98/ (age3.98 + 2560), where age is in days.

B.

Including Plant Defense and Herbivory in the Optimization Model

We can suggest an approach for determining whether allocation to defense is advantageous in terms of maximizing root efficiency. We should acknowledge at the outset, however, that to our knowledge, the data are not available to parameterize such a model for any species. We will illustrate the concept using respiration rates measured for citrus and uptake rates measured for apple, consistent with the demonstration of cohort efficiency, above. We wish to describe how the allocation of C to defense (Cdef) reduces the chance of mortality. A hypothetical relationship between Cdef and the halflife of a cohort is illustrated in Fig. 5 (inset). This relationship will vary with the intensity of pressure from herbivores and pathogens. For simplicity, we assume a linear relationship between Cdef and the median life span of roots. This life span is shorter in the case of higher pressures, for the same investment in Cdef. In our example, in the case of lower pressure, an investment of 10 mmol C/g root decreases the chance of mortality by 29%, which corresponds to an increase in the median life span of 40%. In the case of higher pressure, the same investment in defense results in a 50% decrease in the risk of mortality or a

233

doubling of the median life span. This sounds impressive, but still leaves the cohort with higher mortality and shorter life spans than the cohort under low pressure without any defense. We use these numbers for illustration, since we do not know specifically how much C associated with root construction is used for defense. Indirect evidence in support of C investment in defense is provided by the observation that coarser roots of longer longevity have higher tissue density and more lignified secondary walls (Eissenstat and Achor, 1999). In addition, as mentioned previously, older peach roots that have more condensed tannins and thicker secondary walls benefit less from insecticide application than young peach roots (Wells et al., submitted). We can now compare the efficiency of cohorts of roots under different degrees of herbivore and pathogen pressure with different investments in defense (Fig. 5). The optimal allocation to defense in the scenario with lower herbivore and pathogen pressure is 5 mmol C/g root in this illustration; under higher pathogen pressure, a greater C allocation to defense is desirable to increase median life spans toward the optimum predicted in the absence of herbivory (Fig. 4). The efficiencies and life spans achieved are less than in the case without herbivory, where the optimal median life span was
60 d, and the cohort efficiency was > 23 mmol

Figure 5 The efficiency of a cohort of roots as a function of C expended for defense (CDef). The efficiency of the cohort is based on respiration and uptake rates of individual roots (see Fig. 1) and exponential decay at rates determined by the defensive C investment. The inset shows the assumed relationship between C expended for defense of the root and the resulting half-life of the cohort of roots, for scenarios of higher (High) and lower (Low) pressure from herbivores and pathogens.

234

P/mol C (Fig. 4). Under low pressure, where we assumed the median life span would be reduced to 35 d without an additional C investment, the optimal median life span was increased to 42 d by an additional C investment that resulted in 94% of the ideal maximum efficiency. With higher pressure, where we assumed the median life span would be only 14 d without the expenditure of C for defense, the optimal C investment was 25 mmol/g root, compared to only 5 mmol C/g root in the low-pressure case. This investment, according to our guess at the return on investment shown in Fig. 5 (inset), resulted in a median life span of 50 days, and a 10% loss in cohort efficiency, but a vast improvement over the efficiency achieved by the unprotected cohort. We do not expect the quantitative relationships illustrated here to apply to any real situations in nature. The relationship between defensive C investment and median life span (Fig. 5, inset), upon which the optimal investment in defense depends (Fig. 5), was not based on data from any specific case. This illustration, however, serves to highlight some useful concepts and some needs for future research, as follows. Applying a cost-benefit analysis to a cohort of roots with a distribution of life spans reveals that the optimal individual life span is not the same as the optimal median life span of the population. The relationship between the individual and population optima depends on the form of the survivorship curve or on its hazard function. Plant control over root life span may be indirect, as the direct causes of mortality, such as herbivory and disease, are largely external to the root. One indirect influence of the plant over root life span is investment in defense of the root. The optimal investment in defense of the root will depend on the magnitude of the imposed risk of mortality. The optimal investment in defense will be larger when the herbivore or pathogen pressure is high. Inducible defenses may help finetune the relationship between allocation to defense and herbivore or pathogen pressure. The maximal efficiency of the cohort will be reduced by the C expenditure for defense, compared to a situation without herbivore or pathogen pressure. Beyond the optimal expenditure, allocating additional C to root defense would increase life spans but reduce efficiencies, such that nutrient acquisition would be better served by constructing new roots than by defending old ones. The absence of data required to better quantify or test these relationships is due to the difficulty of collecting information on root demography, C allocation, and age-dependent C expenditures

Eissenstat and Yanai

and uptake rates of ephemeral roots under field conditions

VIII.

SUMMARY

The ability of a root system to forage efficiently for water and nutrients depends on the production and loss of individual roots in soil of spatially and temporally heterogeneous moisture and fertility and on the physiological activity of these roots, which changes with age. There is enormous variation in root life span, and sources for the variation are not well understood. We hypothesize that plant variation in root life span often relates to plant potential growth rate and the nutrient availability where the plant has evolved. More and wider species comparisons under common garden conditions are needed to test this hypothesis. One of the difficulties in generalizing about factors controlling root life span is the lack of agreement among methods. No method has emerged as best for all conditions, although the minirhizotron approach seems to hold the most promise for developing a better understanding of root demography under a wide range of conditions. Both abiotic and biotic factors affect root life span, and often these factors interact. Higher temperature, for example, may diminish root life span more by allowing for more root herbivores and pathogens than by directly affecting root maintenance costs. Reductions in available photosynthate for root maintenance, such as caused by grazing or pruning of the shoot or by high fruit production, often leads to greater root mortality. We previously approached the cost-benefit analysis of root life span with the implicit assumption that plants controlled the life span of roots. Our current approach acknowledges the role of exogenous factors in root mortality, with the plant having indirect control through allocation to defense. A cohort analysis of root efficiency allows a distribution of life spans to be optimized. Additional studies examining factors influencing root life span combined with optimization modeling are needed to unravel the numerous controls and constraints on the life span of plant roots.

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Eissenstat and Yanai Thornley JHM. 1998. Modelling shoot:root relations: the only way forward? Ann Bot 81:165–171. Van Rees KCJ, Comerford NB. 1990. The role of woody roots of slash pine seedlings in water and potassium absorption. Can J For Res 20:1183–1191. Vogt KA, Edmonds RL, Grier CC. 1981. Seasonal changes in biomass and vertical distribution of mycorrhizal and fibrous-textured conifer fine roots in 23- and 180-yearold subalpine Abies amabilis stands. Can J For Res 11:223–229. Vogt KA, Grier CC, Vogt DJ. 1986. Production, turnover, and nutrient dynamics of above- and belowground detritus of world forests. Adv Ecol Res 15:303–377. Vogt KA, Persson H. 1991. Root methods. In: Lassoie JP, Hinckley TM. Ecophysiology of Forest Trees. Vol. 1, Techniques and Methodologies. Boca Raton, FL: CRC Press, pp 477–501. Watson CA, Ross JM, Bagnaresi U, Minotta GF, Roffi F, Atkinson D, Black KE, Hooker JE. 2000. Environment-induced modifications to root longevity in Lolium perenne and Trifolium repens. Ann Bot 85:397–401. Weaver JE, Zink E. 1946. Length of life of roots of ten species of perennial range and pasture grasses. Plant Physiol 21:201–217. Wells CE. 1999. Advances in the fine root demography of woody species. PhD thesis. Pennsylvania State University, University Park, PA. Wells CE, Eissenstat DM. 2001. Marked differences in survivorship among apple roots of different diameters. Ecology 82:882–892. Wells CE, Eissenstat DM, Glenn DM. Submitted. Soil insects alter fine root demography in peach (Prunus persica). Submitted to Plant Cell Environ. Wells CE, Eissenstat DM, Glenn DM. 2002 Submitted 2 Changes in the risk of fine root mortality with age: a case study in peach (Prunus persica). Am J Bot. Weste G. 1986. Vegetation changes associated with invasion by Phytophthora cinnamomi of defined plots in the Brisbane Ranges, Victoria, 1975–1985. Aust J Bot 34:633–648. Whaley EL. 1995. Uptake of phosphorus by citrus roots in dry surface soil. MSc thesis, University of Florida, Gainesville, FL. Williams M, Yanai RD. 1996. Multi-dimensional sensitivity analysis and ecological implications of a nutrient uptake model. Plant Soil 180:311–324. Wilson JB. 1988. Shoot competition and root competition. J Appl Ecol 25:279–296. Yanai RD. 1994. A steady-state model of nutrient uptake improved to account for newly-grown roots. Soil Sci Soc Am J 58:1562–1571. Yanai RD, Fahey TJ, Miller SL. 1995. Efficiency of nutrient acquisition by fine roots and mycorrhizae. In: Smith WK, Hinckley TM, eds. Resource Physiology of Conifers. New York; Academic Press, pp 75–103.

14 Maize Root System and Genetic Analysis of Its Formation Gu¨nter Feix, Frank Hochholdinger, and Woong June Park University of Freiburg, Freiburg, Germany

I.

INTRODUCTION

tion and morphogenesis of various root types and root hairs. Identification and correct phenotypic assessment of such mutants were essential for detailed analysis of the structure and development of the root system (cf. Feldman, 1994; Kisselbach, 1999). As this aspect remains important for further mutant work, a short structural account of the root system of maize is given below.

The genetic analysis of root formation in maize has long been neglected, although maize has been a favored plant object for genetic analysis (cf. Coe et al., 1988; Sheridan and Clark, 1988). The lack of genetic research of root structure and its formation in monocotyledonous plants was largely influenced by the difficulty of observing the complex underground root systems. Furthermore, the large environmental influence on root formation makes it difficult to identify mutants in large plant populations. Thus, only one maize root mutant had been characterized until the mid-1990s, rt1 (Jenkins, 1930), a mutant that shows a reduced lodging resistance caused by defects in shootborne root formation. The deficiency of available root mutants was recently overcome in the model plant Arabidopsis by applying new methods and concepts, and this led to the isolation of an impressive number of mutants allowing new insights into the general mechanisms of root formation and morphogenesis (cf. Scheres and Wolkenfelt, 1998). However, as the root system of maize is more complex (Feldman, 1994) than that of Arabidopsis, and because a detailed knowledge of root structure and formation of food plants like maize is still needed, a concerted effort was recently undertaken in search for root mutants in maize (Wen and Schnable, 1994; Hetz et al., 1996; Hochholdinger and Feix, 1998a). This effort has concentrated so far on the isolation and characterization of monogenic mutants with an influence on the forma-

II.

ROOT SYSTEM OF MAIZE

A.

Various Root Types and Their Formation During Development

The primary root system of maize consists of the embryonic primary and seminal roots, the postembryonic shootborne crown and brace roots, and lateral roots emerging from all root types. The term adventitious root has often been associated with the crown and brace roots (Esau, 1977; Fahn, 1990; Kisselbach, 1999), but adventitious root formation occurs, strictly spoken, only after an external influence like injury or hormone treatment and is not developmentally programmed (Feldman, 1994). The primary root is an endogenous part of the developing embryo (Yamashita, 1991). It grows out of the basal meristem and appears to be essential for the growing at the seedling stage only. At later stages of development the growth of the primary root usually stops (Larson and Hanway, 1977; Feldman, 1994). However, it may resume growth and support the 239

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plant to maturity if necessary (Kausch, 1967; McCully and Canny, 1988; Hetz et al., 1996; Kisselbach, 1999). The seminal roots are also of embryonic origin and their primordia emerge from the scutellar node during the later phase of embryogenesis. In more recent work they have also been called lateral-seminal roots (Hetz et al., 1996). The number of the seminal roots is a polygenic trait and highly variable (0–20) between different maize lines (Sass, 1977; Feldman, 1994; Kisselbach, 1999). The ontogenic origin of the seminal roots is still unclear especially when relating to the issue of allorhizie and homorhizie (Tillich, 1977). It is still unclear which role the seminal roots play for a good performance of the seedling. The shootborne crown roots are formed in variable numbers at consecutive underground stem nodes and initiate at the inner cell layer of the nodes (Martin and Harris, 1976; Varney and McCully, 1991; Varney et al., 1991; Wang et al., 1991, 1994). Numbers and sizes of the crown roots increase on higher nodes of the stem. When formed from the aboveground nodes, such roots are designated as brace roots (Feldman, 1994). Crown roots represent the backbone of the overall root architecture of maize endowing the plant its lodging resistance and are essential for the stability of the growing plant (McCully and Canny, 1988). Lateral roots initiate in the pericycle cell layer (Esau, 1977) or sometimes from some endodermal cells of the root differentiation zone (Bell and McCully, 1970). The primordia of such roots are formed by a multistep mechanism (Laskowski et al., 1995; Malamy and Benfey, 1997). They are much thinner than crown

roots (Peterson, 1991) and can lead to the formation of secondary and higher-order lateral roots which have a great influence on the architecture of the root system (Lynch, 1995). Lateral roots increase the absorbing surface of the root system and are essential for water and nutrient uptake. Root hairs are epidermal extensions that are formed on both embryonic and postembryonic roots and have an essential function for nutrient and water uptake by further increasing the absorbing surface of the root (Gilroy and Jones, 2000; see also Chapter 5 by Ridge and Katsumi in this volume). However, the importance of these root hair functions in maize is unclear, because some mutants lacking root hairs showed normal growth and development (Wen and Schnable, 1994). A summary of some important properties of the various root types is given in Table 1. B.

Structure of Individual Roots

The basic anatomical structure of maize roots (Avery, 1930; Sass, 1977; Feldman, 1994; Ishikawa and Evans, 1995) is comparable to that of Arabidopsis roots (Dolan et al., 1994; Benfey and Schiefelbein, 1994; Scheres and Wolkenfelt, 1998), but maize roots display some additional features. The quiescent center is much larger (it contains >1000 cells; Feldman, 1994), the cortical cell files are more numerous (up to 10–15; Feldman, 1998) and border cells are continuously secreted at the tip region (Vermeer and McCully, 1982). The mesocotyl connecting the scutellar and coleoptilar node has structural features similar to

Table 1 The Root System of Maize Root type Primary root Nodal roots Seminal roots Crown roots Brace roots Lateral roots Adventitious roots

Site and time of root outgrowth

Time of primordia formation

Number of roots

Influence on lodging resistance

From root apical meristem in germinating embryo

Early in embryogenesis

1

—

From scutellar node in first days of seedlings growth From underground nodes at developing stem From aboveground nodes at stem (later growing stage) From pericycle of differentiated roots From stem at random sites after exogenous induction

Late in embryogenesis

—

Late in plant development

0–12(variety dependent) Major root stock of plant Several

+

Not defined

Many

(+)

After dedifferentiation and reprogramming of stem regions

Stimulus dependent

(+)

During growth of plant

+++

Maize Root System

roots. This is demonstrated by the extensive growth of fine roots from the mesocotyl in some maize lines. Field-grown roots normally have a well-developed rhizosphere that influences the structural features of the root; e.g., hypoxic or anaerobic soil condition can lead to arenchyma formation (He et al., 1996).

241

tance was undertaken with the option for a later isolation of the genes impaired by the mutation. An account of these attempts will be given in the following paragraphs.

A. C.

Transition from Early to Late Root Architectures

The overall architecture of the root system is determined by the transition from the temporary embryonic to the persisting postembryonic root structures. The early root system consists of the primary and other seminal roots together with their lateral roots. It is only essential for the development of the young seedling. The shape of this structure is comparable to the root system of the dicot plants (e.g., Arabidopsis) except for the additional presence of the seminal roots. At about 2 weeks after germination, the postembryonic shootborne roots start to develop and gradually determine the form of the root system architecture. The final shape of the root system is governed by the number and growth direction of the postembryonic crown roots together with their branching laterals (Lynch, 1995). The development of the root system is strongly influenced by environmental factors like water and nutrient availability. Those can determine the rate of root initiation and growth and are the major causes for the plasticity of the root architecture (Aiken and Smucker, 1996; McCully, 1999).

III.

GENETIC ANALYSIS OF ROOT FORMATION

The formation of the complex root structure of maize is controlled by many interacting genes, leading to polygenic inheritance patterns of many root-related traits. Breeders succeeded in manipulating and modifying the root system in line with agronomic requirements leading to the isolation of varieties with a more shallow or deep root system responsive to changes of soil conditions. More recently, quantitative trait loci (QTL) analyses were performed with rootconnected traits associating genetic determinants of root formation to chromosome regions (Lebreton et al., 1995; Zheng et al., 2000). The dissection, however, of such QTLs into their monogenic components has not been achieved. In a first step to identify genes involved in root formation and morphogenesis, a search for mutants with a strictly monogenic inheri-

Variability of Root Formation and Induced Mutagenesis

The variability of root architecture of maize varieties is mostly of polygenic nature and could only rarely be used as starting material for the isolation of monogenic mutants (Schiefelbein and Benfey, 1991). Depending on the crosses performed with lines displaying particular structural features of their root system, a monogenic trait of interest may be identified by segregation analysis and be used for the isolation of a mutant. One such rare case was apparently present in the progenitor line used for the isolation of the rtcs mutant (described in Section III.C). However, such an approach is very unpredictable in its outcome and not suitable for a more systematic search for root mutants. Systematic searches for monogenic recessive mutants were successfully performed with maize lines mutagenized by an ethylmethanesulfonate (EMS) treatment of pollen (Neuffer, 1994). The search for mutants was then undertaken in segregating F2 families generated from the mutagenized line by selfings. The identification of monogenic recessive mutants required the availability of large numbers of F2 families. For example, in the case of the screening for the lateral root mutant lrt1 (described in Section III.C), 9811 segregating F2 families were used. The second mutagenesis procedure used depends on the insertion of transposons by working with lines containing active transposons in large amounts. After the initial use of the Ac-Ds and the En-Spm transposon systems, the Mutator Mu system is now preferred because of its high mutation rate and the apparent random insertion of Mutator into chromosomal sites (Gierl and Saedler, 1992). It should be remembered that the Mu system does generate germinal reversions only at a very low rate that have been proven advantageous for the identification of mutants (Bennetzen et al., 1993; Bennetzen, 1996). For example, the transposon mutagenesis was applied in the case of the mutants slr1 and slr2 (1969 segregating families were used in this screening; described in Section III.D) or in the screening of root hair mutants (2892 segregating families revealed the mutants rth2 and rth3; described in Section III.E).

242

B.

Feix et al.

Isolation of Mutants

Two major test systems were applied for the screening of large numbers of segregating F2 families for recessive monogenic mutants. The search for mutants with aberrations in the formation of the early root system was performed in a paper roll test (Hetz et al., 1996). This is a system which allows the screening of large numbers of seedlings under controlled conditions, in limited space. In brief, surface sterilized kernels were placed on properly arranged filter paper sheets, which were rolled up and put with their lower part into beakers containing water only. The kernels were then left to germinate for up to 4 weeks. The developing root system of the seedlings was visually analyzed after opening the rolled-up sheets. Such examinations could be repeated several times if needed. This test system is very reliable and could, of course, be extended by supplying further screening criteria to the germinating kernels. The search for mutants with defects at later stages of root formation was performed in field tests of the segregating F2 population. This test is more demanding because only in rare cases a direct correlation of the observed changes to the root system can be obtained. This was the case with the laterdescribed root lodging mutant rtcs. Isolates, with changes in root phenotypes and with wild-type siblings showing the expected Mendelian segregation pattern, should in the beginning be considered as tentative mutants. After further selfings and outcrossings to other genetic backgrounds, the mutant candidates may then prove to be monogenic recessive, stable, and true mutants. The screening conditions,

under which the isolation of the mutants was done, need also to be strictly reproducible to exclude environmental influences on the observed phenotypes. The major classes of ‘‘root’’ mutants isolated so far showed defects in the formation or morphogenesis of the primary or of lateral roots. They displayed a different architecture or were agravitropic. The mutant classes described below in more detail were selected for further analysis because of their highly root-specific phenotype with relevance to the mechanism of root formation and development. C.

Root Initiation Mutants

The two mutations rtcs (deficient in the formation of nodal roots; Hetz et al., 1996) and lrt1 (deficient in the formation of lateral roots; Hochholdinger and Feix, 1998a) are of particular interest. In both mutants primordia cannot be detected by microscopic examination, indicating that the defects of these loss-offunction mutants act at an early stage of root initiation. The monogenic recessive mutant rtcs (rootless for crown and seminal roots; Hetz et al., 1996) was isolated from a cross of a dent line with the local variety DK 105 from Germany and is apparently not caused by any of the known active transposon lines. A graphic illustration of a 14-day-old seedling of rtcs is depicted in part B of Fig. 1. Although the aboveground phenotype (complete loss of root lodging resistance) is very dramatic, no mutant of this phenotype has been reported before. The mutation affects all crown roots and the embryonic seminal roots by inhibiting primor-

Figure 1 Graphic illustrations of the mutants described. The root systems of 14-day-old seedlings of wild-type (A), rtcs (B), lrt1 (C) and slr1 (D) are shown. Drawings by Miwa Kojima, Agronomy Department, Iowa State University, Ames, IA.

Maize Root System

dia formation. The effect of the mutation seems to be highly specific, since no pleiotropic effects on other parts of the plant have been observed. Normal seed set can be achieved if the impaired lodging mutant plants are supported by a stick. The absence of cyclines as shown by in situ hybridization experiments (Hochholdinger and Feix, 1998b) indicates that this mutation prevents all cell divisions in the region of primordia formation. The deficiency of the mutant is confined to the root-initiating part of the nodes with no influence on their tiller-forming part tested by introgressing the rtcs locus into the heavily tillered gaspe flint line (Hochholdinger and Feix, 1998c). The RTCS locus was mapped with the help of a cosegregating RAPD marker (Hetz et al., 1996) and by a B-A translocation test (Hochholdinger and Feix, unpublished) to the small arm of chromosome 1. The monogenic recessive mutant lrt1 (lateral rootless 1; Hochholdinger and Feix, 1998a) was isolated from EMS-mutagenized B73 seeds. It lacks postembryonic root formation at the early seedling stage; i.e., no lateral roots emerge from the primary root or from the crown roots that grow out of the coleoptilar node (see part C of Fig. 1). Root growth of these plants resumes later leading to normally developed root systems of the mature plants. The defect of lrt1 operates very early in root initiation before the primordia can be seen. Furthermore, the wild-type phenotype cannot be rescued from mutants by the application of auxin to germinating kernels. The mutant does not form an elongated mesocotyl in the dark, although its photomorphogenic response appears to be normal in the mutant. Double mutants prepared from lrt1 and rtcs show a strict additive behavior. The LRT1 locus was mapped to chromosome 2S by B-A translocation analysis (Hochholdinger and Feix, 1998a). D.

Root Elongation Mutants

In the mutants slr1 and slr2 (short lateral root), the mechanism of cell elongation of lateral roots is disturbed leading to lateral roots with impaired morphogenesis (Hochholdinger et al., 2001). The two recessive and nonallelic mutants isolated from selfed Mutator stocks both displayed short lateral roots of about one-fourth the length of wild-type roots (see part D of Fig. 1) and very limited root hair formation because of their incomplete cell elongation zone. In the case of slr1, the mutation was mapped by B-A translocation analysis to the short arm of chromosome 3. A particular feature of these mutants is the strict root type specificity, since only lateral roots of the embryonic

243

primary and of other seminal roots are affected. Furthermore, the deficiency of the mutants acts transiently during the first 4 weeks, and the wild-type phenotype is restored afterward. Confocal laser scanning microscopy demonstrated that the short lateral roots are caused by the presence of smaller cells compared to wild-type lateral roots. In the double mutant slr1 slr2, the defect does not only influence lateral root-specific cell elongation, but also leads to disarranged longitudinal cellular patterns in the primary and in the other seminal roots. The transient nature of the single mutants is, however, retained in the double mutant, indicating that the two loci affected in slr1 and slr2 are cooperating in the establishment of the lateral root specificity during early root development. E.

Root Maturation Mutants

Root hair formation is a typical trait of the root maturation zone. So far, the three monogenic recessive mutants rth1-rth3 (root hairless), which are defective in the morphology of root hairs of the primary root and the seminal roots, have been isolated from selfed Mutator stocks (Wen and Schnable, 1994). These mutants are of particular interest, since root hairs play an important role in plant nutrition by facilitating the uptake of water and nutrients (Gilroy and Jones, 2000). The rth1 and rth2 mutants form normal root hair primordia. However, the root hairs of the mutant rth1 hardly elongate and reach only 1/20 to 1/10 of the length of the root hairs of the wild-type plants. The root hairs of the mutant rth2 show elongation to about one-fifth to one-fourth of those of the wild type. In contrast to rth1 and rth2, the mutant rth3 displays abnormal root hair primordia already at a very early stage of development, as can be seen by scanning electron microscopy. Such an early expression eventually leads to very short root hairs similar to the case of rth1. This implies that different genes regulate root hair elongation at different stages of development. The mutant rth1 shows pleiotropic nutritional defects leading to stunted plants with purplish leaves that never form ears and only rarely produce tassels. In contrast to rth1, the plants of the mutants rth2 and rth3 show a normal growth performance during their entire development despite their drastically impaired root hair morphology. This might suggest that under some conditions root hairs are less important for plant growth than had been previously thought. rth1, rth2, and rth3 were mapped by B-A translocation tests to chromosome arms 1L, 5L, and 1S, respectively (Wen and Schnable, 1994).

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F. Conclusions from Mutants Analysis

phenotype that persists until about the third week after germination cannot be rescued by auxin and is not under the influence of a phytochrome signal transduction pathway. Similarly, a development-specific signal can also be implied in the case of the slr mutants which are affected only in the lateral root formation of the embryonic but not of the postembryonic shootborne roots. Another aspect that can be learned from the chromosomal map positions of the mutant loci indicates that a larger root locus combining root relevant genes is not evident from the available data. A summary of these findings of the mutant analysis is given in Table 3. Although root hairs immensely increase the absorbing surface and are known to play an important role in nutrient and water uptake (Gilroy and Jones, 2000), they should, depending on growth conditions, be considered as less important in maize growth than previously thought. This is indicated by the root hair mutants rth2 and rth3 which are drastically impaired in root hair elongation but do not show any other defects during the development of the whole plant (Wen and Schnable, 1994).

The use of mutants revealed several properties of the mechanism of root formation not easily accessible otherwise. For example, the analysis of rtcs demonstrated that the initiation of embryonic and postembryonic nodal roots depends on an intact RTCS gene, while the initiation of crown roots from the scutellar node depends also on the LRT1 gene. The two genes act independently of each other as shown by the additive nature of the double mutant from rtcs and lrt1. This implies that at least two independent mechanisms are effective in the formation of root primordia. The LRT1 gene is also essential for the initiation of lateral roots emerging from the primary root showing that the formation of roots initiated either from preformed roots or from nodal tissue shares expression features of LRT1. Interestingly, gene functions with an influence on several root types had been seen by most of the genes impaired in the available mutants (see Table 2). A case of extreme root type specificity is demonstrated by the transient deficiency in lateral root elongation effective only on lateral roots emerging from the primary root. This root type, tissue, and stage specificity is governed by at least two independent genes (SLR1 and SLR2). The transient occurrence of the deficiency of several mutants together with further findings indicates that various steps of root formation depend on sets of signals operating for defined periods of time in the development of the plant. In the case of lrt1, the mutant

IV.

OUTLOOK

The mutant work, although in its early stage and still very fragmentary, has already proven very valuable for the advancement of the knowledge on root formation and morphogenesis. A schematic summary of the mutants isolated so far, with indications of where in

Table 2 Root Typesa Affected in the Mutants Are Indicated by Shaded Areas Mutant

PR

SR

CR

rtcs rt1 lrt1 rth1-3 slr1 slr2 brt1 des21 agt Asr1 a

PR, primary root; SR, seminal root; CR, crown root; BR, brace root. See references for des21 (Gavazzi et al., 1993), agt (Pilet, 1983), and Asr1 (De Miranda et al., 1980).

BR

Maize Root System

245

Table 3 Characteristics of the Mutants

Name

Gene

Chromosomal map position

rootless for crown and seminal root lateral rootless

rtcs

1S

lrt1

2S

short lateral root 1

slr1

3S

short lateral root 2

slr2

N/A

root hairless 1

rth1

1L

root hairless 2

rth2

5L

root hairless 3

rth3

1S

Root type affected All crown and brace roots Lateral roots and crown roots from coleoptilar node Lateral roots from primary and seminal roots Lateral roots from primary root and seminal roots Root hairs from primary and seminal roots (others N/A) Root hairs from primary and seminal roots (others N/A) Root hairs from primary and seminal roots (others N/A)

the pathways of root formation they can be placed, is shown in Fig. 2. It is evident that mutants for only a few steps in the mechanism of root formation have been found, and it is hoped that great efforts will be made to fill in the many gaps of lacking knowledge. So far, only visual inspection has been used for mutant isolation using the paper roll test. Screening procedures with better information are expected to be used in the future. For example, the monogenic recessive mutant brt1 (for brown root), displaying a brown-looking central cylinder of the primary root starting 7 days after germination and leading to a drying out of the seedling, was identified under water stress conditions in a paper roll screening experiment (Hochholdinger and Feix, 1998d). It was found that this mutant shows deficiencies in a function relevant to survival under anaerobic conditions (Chang et al., 2000). The search for mutants in late-appearing traits of the root system of maize will continue to depend on their detection in field tests. This is because of the difficulties to test thousands of segregating families under hydroponic or aeroponic conditions or in root boxes. The need for additional efforts for the identification of mutants in field experiments is evident from

Observed defects Primordia formation Primordia formation Cell elongation

Plant growth period affected after germination

References

Permanent

Hetz et al. (1996) First 4 weeks Hochholdinger and Feix (1998a) First 4 weeks Hochholdinger et al. (2001)

Cell elongation

First 4 weeks Hochholdinger et al. (2001)

Root hair elongation

Permanent

Root hair elongation

Not affected

Abnormal primordia, root hair elongation

Not affected

Wen and Schnable (1994) Wen and Schnable (1994) Wen and Schnable (1994)

the isolation and investigation of the recessive mutant rtd (root degradation). This mutant is characterized by the drying of lower leaves of approximately 4-week-old plants (Krebs and Feix, unpublished). It was then found, in an experimental setup allowing the direct observation of the roots, that the drying of the leaves was the consequence of a premature degradation of the root system. The screening for specific mutants with defects at later stages of development is important for the recognition of genes acting at later phases of postembryonic development. It is hoped that aboveground traits cosegregating with root deficiencies and relevant markers will become increasingly available for the detection of such mutants in field experiments. Further insights into the genetic network operating in the formation and function of roots can be expected from double-mutant analyses and the generation of second-site mutations. Such studies have proven powerful in many gene systems for the analysis of gene interactions. Double mutants will not only be informative if generated from different single-root mutants as shown in the case of slr1 and slr2, but also in combinations with single mutants with defects in other parts of the plants. For example, it will be interesting to check, by using the appropriate double

246

Feix et al.

Figure 2 Genetic control of root formation in maize. All mutants are described in the text.

mutants, whether the root apical meristem shares important gene functions and characteristics with the other meristems of the maize plant. Furthermore, double-mutant analyses may yield important information on common features between developmentally controlled root initiation and root formation induced by external signals. Regarding the identification of target genes of the mutated gene structures and of the influence of such genes on cell functions, the use of newly established profiling techniques like proteomics (Touzet et al., 1996; Thiellement et al., 1999; Chang et al., 2000) or

EST microarray technology (Richmond and Sommerville, 2000; Van Hal et al., 2000) will be very rewarding. Assistance in the identification of gene activities, with relevance to root formation and function, can also be expected from the use of reverse genetic methods by identifying cDNAs and ESTs with root-specific expression. Using this approach it was possible to isolate a cDNA coding for a glycine rich protein (Goddemeier et al., 1998) which was shown by in situ hybridization to be highly specifically expressed in the exodermis of the root tip of several root types

Maize Root System

(Hochholdinger and Feix, unpublished). Furthermore, the isolated DNA elements can be used in the Trait Utility System of Corn (TUSC) with the aim to search for mutants caused by genes coding for the cDNA of interest. Isolation of the mutated genes, which is in progress in the case of some of the mutants, will allow the understanding of the genes involved in root development. Sequence elements identified that way might reveal cases of root-specific genes as part of larger gene families. They will also allow scientists to search for homologous genes in other plants, like rice. Furthermore, sequence elements will be very helpful in the identification of more alleles of the genes affected by the mutation as well as the phenotypes caused by these alleles. This could be achieved by using the TUSC system. It is of particular interest to identify the specific promoters and potential root-specific regulatory elements of the isolated genes. Root-specific promoters are very much in demand for the construction of transgenes, as for instance in expression cassettes of transgenes intended for the tissue- and stage-specific in planta synthesis of Bt-type insecticides (de Maagd et al., 1999). The information gained by the analysis of the cloned genes will be very useful for selecting the bestsuited alleles for the construction of transgenic plants for application purposes.

REFERENCES Aiken RM, Smucker AJM. 1996. Root system regulation of whole plant growth. Annu Rev Phytopathol 34:325– 346. Avery GS. 1930. Comperative anatomy and morphology of embryos and seedlings of maize, oats and wheat. Bot Gaz 89:1–39. Bell JK, McCully ME. 1970. A histological study of lateral root initiation and development in Zea mays. Protoplasma 70:179–205. Benfey PN, Schiefelbein JW. 1994. Getting to the root of plant development: the genetics of Arabidopsis root formation. TIG 10:84–88. Bennetzen JL. 1996. The mutator transposable element system of maize. Curr Top Microbiol Immunol 204:195–229. Bennetzen JL, Springer PS, Cresse AD, Hemdrickx M. 1993. Specificity and regulation of the mutator transposable element system of maize. Crit Rev Plant Sci 12:57–95. Chang WWP, Huang L, Shen M, Webster C, Burlingame AL, Roberts JKM. 2000. Patterns of protein synthesis and tolerance of anoxia in root tips of maize seedlings acclimated to a low-oxygen environment, and identification of proteins by mass spectrometry. Plant Physiol 122:295–317.

247 Coe EH Jr, Neuffer MG, Hoisington DA. 1988. The genetics of corn. In: Sprague GF, ed. Corn and Corn Improvement. Madison, WI: American Society of Agronomy. de Maagd RA, Bosch D, Stiekema W. 1999. Bacillus thuringiensis toxin-mediated insect resistance in plants. TIPS 4:913. De Miranda LT. 1980. Inheritance and linkage of root characteristic from Puebla maize. Maize Genet Newslett 54:18–19. Dolan L, Duckett CM, Grierson C, Linstead P, Schneider K, Lawson E, Dean C, Poethig S, Roberts K. 1994. Clonal relationships and cell patterning in the root epidermis of Arabidopsis. Development 120:2465– 2474. Esau K. 1977. Plant Anatomy. 2nd ed. New York; John Wiley and Sons. Fahn A. 1990. Plant Anatomy. 4th ed. Oxford, UK: Pergamon Press. Feldman L. 1994. The maize root. In: Freeling M, Walbot V, eds. The Maize Handbook. New York; SpringerVerlag, pp 29–37. Feldman LJ. 1998. Not so quiet quiescent centers. TIPS 3:80–81. Gierl A, Saedler H. 1992. Plant transposable elements and gene tagging. Plant Mol Biol 19:39–49. Gavazzi G, Dolfini M, Galbiati M, Helentjari T, Landon M, Pelucci N, Todesco G. 1993. Mutants affecting germination and early seedling development in maize. Maydica 38:265–274. Gilroy S, Jones DL. 2000. Through form to function: root hair development and nutrient uptake. TIPS 5:56–60. Goddemeier M, Wulff D, Feix G. 1998. Root-specific expression of a Zea mays gene encoding a novel glycine-rich protein, zmGRP3. Plant Mol Biol 36:799–802. He C-J, Morgan PW, Drew MC. 1996. Transduction of an Ethylene signal is required for cell death and lysis in the root cortex of maize during aerenchyma formation induced by hypoxia. Plant Physiol 112:463–472. Hetz W, Hochholdinger F, Schwall M, Feix G. 1996. Isolation and characterisation of rtcs, a mutant deficient in the formation of nodal roots. Plant J 10:845– 857. Hochholdinger F, Feix G. 1998a. Early post-embryonic root formation is specifically affected in the maize mutant lrt1. Plant J 16:247–255. Hochholdinger F, Feix G. 1998b. Cyclin expression is completely suppressed at the site of crown root formation in the nodal region of the maize root mutant rtcs. J Plant Physiol 153:425–429. Hochholdinger F, Feix G. 1998c. Tiller formation in Gaspe Flint is not affected by the rtcs mutation. Maize Genet Newslett 72:30–31. Hochholdinger F, Feix G. 1998d. Isolation of the new necrotic mutant brt1. Maize Genet Newslett 72:29–30.

248 Hochholdinger F, Park WJ, Feix G. 2001. Cooperative action of SLR1 and SLR2 is required for lateral root-specific cell elongation in Maize. Plant Physiology 125:1529–1539. Ishikawa H, Evans ML. 1995. Specilized zones of development in roots. Plant Physiol 109:725–727. Jenkins MT. 1930. Heritable characters of maize XXXIVrootless. J Hered 21:79–80. Kausch W. 1967. Lebensdauer der Primaerwurzel von Monokotylen. Naturwissenschaften 54:475. Kisselbach TA. 1999. The Structure and Reproduction of Corn. 50th Anniversary ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Laskowski MJ, Williams ME, Nusbaum C, Sussex IA. 1995. Formation of lateral root meristems is a two-stage process. Development 121:3303–3310. Larson WE, Hanway JJ. 1977. Corn production. In: Spargue GF, ed. Corn and Corn Improvement. Madison, WI: American Society of Agronomy, pp 625–669. Lebreton C, Lazic-Jancic V, Steed A, Pekic S, Quarrie SA. 1995. Identification of QTL for drought responses in maize and their use in testing causal relationships between traits. J Exp Bot 46:853–865. Lynch J. 1995. Root architecture and plant productivity. Plant Physiol 109:7–13. Malamy JE, Benfey PN. 1997. Organization and cell differentiation in lateral roots of Arabidopsis thaliana. Development 124:33–44. Martin EM, Harris WM. 1976. Adventitious root development from the coleoptilar node in Zea mays L. Am J Bot 63:890–897. McCully ME. 1999. Roots in soil: unearthing the complexities of roots and their rhizospheres. Annu Rev Plant Physiol Plant Mol Biol 50:695–718. McCully ME, Canny MJ. 1988. Pathways and processes of water and nutrient movements in roots. Plant Soil 111:159–170. Neuffer MG. 1994. Mutagenesis. In: Freeling M, Walbot V, eds. The Maize Handbook. New York; SpringerVerlag, pp 212–219. Peterson RL. 1991. Adaptations of root structure in relation to biotic and abiotic factors. Can J Bot 70:661–675. Pilet PE. 1983. Elongation and gravireactivity of roots from an agravitropic maize mutant—implications of growth inhibitors. Plant Cell Physiol. 24:333–336. Richmond T, Sommerville S. 2000. Chasing the dream: plant EST microarrays. Curr Opin Plant Biol 3:108–116. Sass JE. 1977. Morphology. In: Spargue GF, ed. Corn and Corn Improvement. Madison, WI: American Society of Agronomy, pp 89–110.

Feix et al. Scheres B, Wolkenfelt H. 1998. The Arabidopsis root as a model to study plant development. Plant Physiol Biochem 36:21–32. Schiefelbein JW, Benfey PN. 1991. The development of plant roots: new approaches to underground problems. Plant Cell 3:1147–1154. Sheridan WF, Clark JK. 1988. Maize developmental genetics: genes of morphogenesis. Annu Rev Genet 22:353– 358. Thiellement H, Bahrman N, Damerval C, Plomion C, Rossignol M, Santoni V, de Vienne D, Zivy M. 1999. Proteomics for genetic and physiological studies in plants. Electrophoresis 20:2013–2026. Tillich HJ. 1977. Vergleichend morphologische Untersuchungen zur Identitaet der GramineenPrimaerwurzel. Flora 166:415–421. Touzet P, Riccardi F, Morin C, Damerval C, Huet J-C, Pernollet J-C, Zivy M, de Vienne D. 1996. The maize two-dimensional gel protein database: towards an integrated genome analysis program. Theor Appl Genet 93:997–1005. Van Hal NL, Vorst O, Van Houwelingen AM, Kok EJ, Peinenburg A, Aharoni A, Van Tunen AJ, Keijer J. 2000. The application of DNA microarrays in gene expression analysis. J Biotechnol 78:271–280. Varney GT, McCully ME. 1991. The branch roots of Zea. II. Developmental loss of the apical meristem in fieldgrown roots. New Phytol 118:535–546. Varney GT, Canny MJ, Wang XL, McCully ME. 1991. The branch roots of Zea. First order branches, their number, sizes and division into classes. Ann Bot 67:357– 364. Vermeer J, McCully ME. 1982. The rhizosphere in Zea: new insight into its structure and development. Planta 156:45–61. Wang XL, Canny MJ, McCully ME. 1991. The water status of the roots of soil-grown maize in relation to the maturity of their xylem. Physiol Plant 82:157–162. Wang XL, McCully ME, Canny MJ. 1994. The branch roots of Zea. IV. The maturation and openness of xylem conduits in first-order branches of soil-grown roots. New Phytol 126:21–29. Wen T-J, Schnable PS. 1994. Analyses of mutants of three genes that influence root hair development in Zea mays (Gramineae) suggest that root hairs are dispensable. Am J Bot 81:833–843. Yamashita T. 1991. Ist die Primaerwurzel bei Samenpflanzen exogen oder endogen? Beitr Bio Pflanzen 66:371–391. Zheng HG, Babu RC, Pathan MS, Ali L, Huang N, Courtois B, Nguyen HT. 2000. Quantitative trait loci for rootpenetration ability and root thickness in rice: comparison of genetic backgrounds. Genomics 43:53–61.

15 Root Architecture—Wheat as a Model Plant Gu¨nther G. B. Manske and Paul L. G. Vlek Center for Development Research, University of Bonn, Bonn, Germany

I.

INTRODUCTION

However, not all primordia always develop. In wheat, three to six seminal roots normally emerge from the seed, but genetic variations exist among the various cultivars (Robertson et al., 1979). Seed size and seminal root number are positively correlated, although in some genotypes this is not the case (O’Toole and Bland, 1987). The seminal roots constitute 1–14% of the entire root system. They grow and function throughout the whole vegetative period, and penetrate the soil earlier and deeper than the adventitious roots. The wheat crown root system develops on the lower shoot internodes, typically 1–2 cm beneath the soil surface. The seminal root system is very important for the establishment of wheat seedlings. Crown roots begin to develop only at the first foliar node, when the fourth mainstem leaf appears. Root axes (generally, two to four per node) then emerge from successive higher nodes (Klepper et al., 1984). The unique relationships between stem nodes and crown roots permits the establishment of correlations between shoot and root development (Klepper, 1991). The stage of development of the crown root system can be calculated from the phyllochron, which is the unit of time between equivalent growth stages of successive leaves. The phyllochron depends on the air temperature. In wheat, each phyllochron requires
100 growing degree days (Rickman et al., 1984). Adventitious roots mostly occupy the upper soil layers and their number depends mainly on the tillering ability of the plant. Root number and tillering thus are

Root characteristics play an important role in the development of new wheat germplasm with improved drought tolerance, nutrient and water uptake efficiency, lodging resistance, and tolerance to mineral toxicity. These traits are especially relevant for the adaptation of wheat to marginal environments, with increasing demand for wheat production at a time when water and phosphorus are becoming scarce commodities worldwide. This chapter describes the morphology, physiology, ecology, and function of wheat roots, and reviews the current status of knowledge regarding heritability and genetic diversity of root traits of wheat.

II.

MORPHOLOGY AND PHYSIOLOGY OF WHEAT ROOTS

Two root types are distinguished in cereals—the seminal roots (also called primary roots), which develop at the scutellar and epiblast nodes of the embryonic hypocotyl of the germinating caryopsis, and adventitious roots (also called shootborne, nodal, secondary, or crown roots), which subsequently emerge from the coleoptilar nodes at the base of the apical culm and tillers. These two categories of roots function in a complementary manner, and thus the root system must be considered as a whole. The number of seminal roots in cereals is usually five to seven but may reach 10. 249

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positively correlated (Hockett, 1986). The ratio of seminal to adventitious roots is also altered by the degree of tillering, and consequently by interplant competition. A high-input ideotype of wheat is characterized by a low number of tillers, by a high harvest index, and by seminal root dependence. In contrast, low-input genotypes develop a larger root system, essentially based on adventitious roots exploring a greater soil volume. Indeed, research results at CIMMYT in Mexico showed that the number of tillers was positively correlated with root length density and grain yield of semidwarf bread wheat cultivars (BWs) grown under P deficiency in acid soils (Manske et al., 2000). Tiller numbers did not affect grain yield and root length density when P was amply available. The average root radius of wheat plants ranges between 0.07 and 0.15 mm. The root length density (RLD) varies between 2 and 10 cm cm3 soil depending on the stage of plant development, soil depth, and environmental factors. The horizontal spread of the roots of a wheat plant is usually between 30 and 60 cm. However, under deficient moisture conditions, they invade lower horizons and have a greater vertical distribution (Mishra et al., 1999). The roots can be abundant at soil depths >100 cm with some roots even reaching <200 cm (Gregory et al., 1978). Root hairs and extramatrical hyphae of vesiculararbuscular and arbuscular mycorrhizal fungi (AMF) enlarge the effective absorbing surface area considerably. Root hairs have a diameter of 0.003–0.007 mm, a length of 3–13 mm, and a normal life-span of a few days. They emerge just behind the root tip, at the zone of root elongation. The root surface of a single wheat plant thus varies in terms of number, size, length, and life span of root hairs. Mycorrhiza is the mutualistic association between soilborne fungi and roots of higher plants. The AMF derive their carbon compounds from the host plants and in return for a delivery of mineral nutrients to the host plant. Intercellular arbuscels are the main sites of nutrient and carbon exchange between the fungi and the host cytoplasm. Wheat roots are usually infected by soilborne vesicular-arbuscular and AMF (Young et al., 1985). The AMF extramatrical hyphae, which also extend several centimeters away outside of the roots into the soil rhizosphere, are 5–10 times thinner than the root hairs and explore an area around the root, which exceeds the zone of nutrient depletion around uninfected roots (Smith and Smith, 1990). The finer hyphae enter pores of soil aggregates not accessible to roots and root hairs. These hyphae absorb relatively immobile elements (P, Zn, Cu) in such inaccessible

Manske and Vlek

fractions of the soil. This enables these fungi to benefit plants. Another advantage of mycorrhizae is that the extramatrical hyphae remain functional for a long time, and continue to provide an uptake pathway even when roots become suberized (Bowen et al., 1975). In field surveys conducted in India, Mexico, and Germany, with >200 wheat genotypes grown in different soils and under different water and fertilizer regimes, the roots were always infected by AMF (authors’ unpublished information). In one trial in India, where sewage water was used for irrigation, AMF was absent (Lu¨ttger, 1996). AMF spores are sensitive to the accumulation of heavy metals.

III.

ECOLOGY AND FUNCTION OF WHEAT ROOTS

Extensive root growth is often required for improved growth of shoots and for higher yields in marginal environments. Complex feedback systems between roots and soil take place in the rhizosphere, which commonly comprises 20–30% of the topsoil volume. In compacted soils, roots are shorter, thicker, and more irregularly shaped than the thinner, fibrous roots that develop under noncompacted conditions (Dexter, 1987; Chapter 45 by Masle in this volume). Wheat produces finer roots per unit soil volume under conditions of low nutrient and water supply. Moreover, it exhibits a remarkable plasticity in root growth, adjusting to the soil nutrient and water status (Cholick et al., 1977; Vlek et al., 1996; Chapter 34 by Glass in this volume). Wheat plants can respond to nutrient and water stress via various mechanisms— alternations of root branching and root extension rates (Horst et al., 1996), increased rate of uptake per unit root length or weight (Egle et al., 1999), higher root:shoot ratios (Manske, 1989; Hamblin et al., 1990), more and longer root hairs (Foehse et al., 1991), enhancement of root exudates (Neumann and Romheld, 1999), and AMF infection (Manske et al., 1995)—and by lowering demand for nutrients and water for growth. Each of these parameters can be altered by selection and breeding. Roots tend to proliferate in the soil zones of more favorable nutrient and water supply, but avoid sites of hypoxia or toxic levels of minerals. As a result, wheat roots are more abundant in the upper soil layers which are better aerated and richer in nutrients than deeper horizons. Usually, 70% of the total root length are found in the 0–30 cm soil layer, where the nutrients are concentrated in most agricultural soils. The five

Root Architecture

principal functions of roots are listed below and discussed in relation to wheat. A.

Nutrient Uptake Efficiency

Wheat roots in the upper soil layers have an outstanding capability to absorb nutrients, and most of the nutrient requirements can be satisfied by these roots. Only a fraction of the nutrients are absorbed from the deeper soil (Bole, 1977). However, under P stress, roots will proliferate in the deeper soil profile, allowing access to residual, native P in the deeper soil layer in an Andisol in Mexico (cf. Manske et al., 2000). The geometry of the root system is a key to improvement of nutrient uptake as it may be manipulated to maximize RLD in those places where nutrients and water are more readily available. Wheat plants can only extract immobile nutrients such as P from soil close to the root surface. The quantities extracted are limited by the concentration at the root–soil interface. The soil solution concentrations of phosphate are small (<0.05
g g1) compared to that of nitrate-N (100
g g1). Very little of the phosphate is moved to the root by capillary water movement (Fo¨hse et al., 1991). The wheat roots have to grow until they come in contact with unexplored soil from which they can extract phosphate. Therefore, root length is the major determinant of the absorbing surface area. Wheat genotypes with higher root length density are able to take up more P (Manske et al., 2000). This is most important in soils of low P availability. At low-P supply, the linear correlation between RLD and P uptake or grain yield is usually 0.50–0.60, but with adequate P supply this correlation is weaker (Manske et al., 1995, 2000). Root diameter and root hair abundance are other important determinants of P uptake efficiency in wheat (Jones et al., 1989). Genotypes with thinner roots showed improved P uptake (Manske et al., 1996a). At high P and ample water supply, root hairs are rudimentary, whereas under deficient conditions long root hairs are abundant (Fo¨hse et al., 1991). Root hair length and density play a significant role in P acquisition, because they modify the P depletion profiles in the rhizosphere (Gahoonia et al., 1997). Root hair density, scored in plants grown in pot cultures with quartz sand, varied considerably among 19 BWs, and were positively correlated ðr ¼ :77Þ with P uptake at anthesis when grown on a P-deficient calcareous Aridisol (Manske, 1997). Vesicular-arbuscular and arbuscular mycorrhizal fungi (AMF) are also of importance for the uptake of nutrients that are relatively immobile—e.g., P

251

(Hayman and Mosse, 1971), Cu (Gildon and Tinker, 1983), and Zn (Swaminathan and Verma, 1979). The AMF infection improves P influx (P uptake per unit root length). Increased fertilizer doses reduce the infection by native AMF. With reduced fertilizer application in an irrigated field trial, improved AMF infection at the stage of tillering resulted in higher grain yields (Manske et al., 1995). In a P-fixing, acid Andisol, AMF infection rate was positively correlated with P uptake into wheat shoots ðr ¼ :47Þ (Manske et al., 2000). However, this explains only 25% of the variance of P uptake. Other traits like RLD and phosphatases excretion by the roots were also important. Roots induce changes in the pH of their rhizosphere. Carboxylic acids, especially citric and malic acid, are the major compounds in exudates of wheat roots that are responsible for this process. Compared with other monocots, wheat is highly resistant to limeinduced chlorosis caused by Fe deficiency. Under conditions of Fe deficiency, the roots release high amounts of chelating exudates (phytosiderophores), which form plant available Fe-phytosiderophore complexes and transport Fe to the roots (Marschner et al., 1986). Exudation promotes the dissolution of poorly available nutrients such as P (Neumann and Romheld, 1999) and Mn (Godo and Reisenhauer, 1980). Poorly available organic P in the soil, which commonly accounts for 40–50% of the total P supply to plants, can be utilized with the help of root phosphatases (Helal, 1990). Genotypic differences in root phosphatase, excreted or bound at the root surface, exist (McLachlan, 1980). The acid phosphatase activity at the surface of intact wheat roots was determined from 4-week-old plantlets of 42 bread wheats from the CIMMYT collection grown in P-free nutrient solution by the p-nitrophenyl assay (Tabatabai and Bremner, 1969). Results were positively correlated ðr ¼ :49Þ with P uptake of those genotypes grown in the field on a Pdeficient Andisol (Manske, 1997; Portilla et al., 1998), which indicates that the wheat plants could utilize organic P from the soil. The measurement of phosphate activity in nutrient solution is a nonsophisticated but reliable method to screen wheat germplasm for root phosphate activity. B.

Drought Tolerance and Water Accessibility

The traits associated with high-uptake efficiency of water are similar to those for nutrient uptake efficiency. However, the soil water status is more dynamic in nature. Thus, a well-developed root system com-

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bines the ability to reach residual moisture deep in the soil profile with a high degree of plasticity to adjust to rapid changes in topsoil water status. In rain-fed wheat, root length densities are much higher in drier years than in humid ones (Hamblin et al., 1990). Wheat grown on residual moisture depends on roots that reach the deep soil layers (Mian et al., 1993; Bai et al., 1997). Varietal differences in rooting depth of wheat were demonstrated (cf. Hurd, 1968). Droughttolerant semidwarf bread wheats formed more roots in the deeper layers (by the cultivars Pastor, Sujata Dharwar, Synthetic 2), whereas the nontolerant checks (Sonalika, Tevee2, Baviacora, and Pavon) had fewer roots in the deep soil (Table 1). Improved total root volume in the whole soil profile (0–100 cm) was not responsible for improved water use efficiency. RLD in the upper soil was even negatively correlated ðr ¼ :70 Þ with grain yield, but not in the deeper soil. Scores for green stay were positively correlated ðr ¼ :69 Þ with RLD in deep soil layer (80–100 cm). Especially the high-yielding genotypes Suajat and Synthetic 1 stayed long green. Grain yield was not significantly related to RLD in the deep soil layer, because Check 3 and Synthetic 2 had high RLD in the deep soil, but low yield potential (Table 1). Therefore, breeding for improved drought tolerance

has to combine both high-yield potential and improved root systems. Genetic factors also influence the spatial distribution of seminal and adventitious roots in wheat. The plagiogravitropic growth of the axes, from which the root emerges, determines the shape of the root system. The plagiogravitropic response of a nodal root depends on its diameter and on the position of the internode from which it emerges. The rooting depth is closely related to the growth angle of the seminal roots (Oyanagi et al., 1993). C.

Lodging Resistance

Lodging is often caused by strong winds that affect plants with a poorly developed anchoring root system. The plants lean at an angle, though each of the stems remains rigid and straight. Lodging-resistant wheat is positively correlated with the spreading angle of roots (Pinthus, 1967). D.

Tolerance to Waterlogging

In considerably large areas of wheat cropping, yields are reduced due to waterlogging. Prolonged periods of rainfall combined with poor soil drainage often causes

Table 1 Grain Yield, Scores for Stay Green, and Root Length Density (RLD) at Different Soil Layers (0–100 cm) of 12 Wheat Genotypes with Different Drought Resistance Grown on Residual Moisture (one single irrigation at sowing time) in an Arid Environment in Northwest Mexico (means of six replication) RLD in diff. soil layers Genotypes

Grain yield t ha1

Scorea stay green

0–100 cm cm cm3

0–20 cm cm cm3

80–100 cm cm cm3

Sonalika Baviacora Pavon Nesser Tevee Seri Check 3 Synthetic 1 Synthetic 2 Dharwar Sujata Pastor Mean LSD 5%

2.29 2.51 2.15 2.07 2.29 1.76 0.71 2.44 2.15 2.20 2.65 2.52 2.15 0.38

1 3 1 2 3 2 5 4 5 3 5 3 3 0.93

0.95 0.98 1.11 1.00 0.88 1.08 1.14 0.92 1.04 1.09 0.88 1.01 1.01 0.18

1.92 1.85 2.45 1.82 1.92 2.09 2.71 1.76 1.89 2.25 1.42 1.62 1.97 0.72

0.40 0.43 0.45 0.45 0.49 0.56 0.61 0.65 0.68 0.70 0.71 0.72 0.57 0.24

a

Score stay green after anthesis; 1<5 for increasing green.

Root Architecture

low pO2 in the soil. Decreases in soil O2 content, under flooded conditions, are often accompanied by increase in soil CO2 and ethylene content. Such changes have detrimental effects on root and shoot growth of wheat. Waterlogging-tolerant wheat genotypes are less responsive to increased CO2 and decreased O2. In part this is because roots of wheat, especially those of the waterlogging-tolerant genotypes, produce aerenchyma under conditions of hypoxia, a feature that helps them cope with such conditions (Huang et al., 1997).

IV.

HERITABILITY AND GENETIC DIVERSITY OF ROOT TRAITS IN WHEAT

Information on the genetic background of a plant character is a prerequisite for effective breeding. Knowledge on heritability and inheritance pattern of wheat root characters is still limited, but indicates that those traits are controlled by a polygenetic system. The control of root systems is largely influenced by additive genetic systems, which allow progress to be made by selection on root quantity and depth of penetration (Monyo and Whittington, 1970). The partitioning of assimilates between roots and shoots appears highly dependent on the wheat genotype (Sadhu and Bhaduri, 1984). Over 32% of the total phenotypic variability for root:shoot ratio is conditioned by additive gene effects (Kazemi et al., 1979). Although this additive portion of the total variance is not especially high, the authors suggested that lines with favorable root:shoot ratios may be obtained from direct selection in early generations of wheat crossings. Moderate heritability values for the total root length (0.62) and root branching (0.42) have been reported (Monyo and Whittington, 1970). Hexaploid wheat has >90% genetic duplication. High levels of genetic duplication reduce the likelihood of natural or spontaneous singlegene recessive variation of any given rooting characteristic. This reduces also the narrow-sense heritability for multigenic characterstics. Narrow-sense heritability for root length ranged between 0.38 and 0.46. Zhang et al. (1999a) analyzed the genetic behavior of apparent tolerance to aluminum toxicity in triticale crosses as measured by root regrowth of seedlings after exposure to Al stress in nutrient solutions. Their results suggested that Al tolerance is of polygenetic inheritance. The moderate high value of estimates of heritability and genetic advance showed that this method could be used in a breeding program aimed at greater

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Al tolerance. There exist no association between Al and Mn tolerance measured in nutrient solution, and selective breeding for tolerance to acid soils has to focus on both stress tolerances (Zhang et al., 1999b). Screening experiments with wheat have shown considerable genotypic variability in AMF colonization and efficiency (Bertheau et al., 1980; Manske, 1990; Kapulnik and Kushnir, 1991; Hetrick et al., 1992). Old wheat cultivars rely more on symbiosis than modern wheats (Manske, 1990). In comparison between landraces from Asia and the United States, the Asian ones showed higher root colonization levels of AMF (Hetrick et al., 1992). Both the extent of AMF infection and the degree of benefit from AMF are plant heritable traits (Manske, 1990; Vlek et al., 1996). High AMF efficiency of a wheat variety can be transferred by crossing to a nonefficient one (Manske, 1990; Diederichs and Manske, 1991). Depending on the crossing combination and environment, recessive, intermediate, dominant inheritance modi, plasmatic effects, effects of heterosis, and even a change of dominance within the same genotypes in different environments were found (Manske, 1989, 1990; Diederichs and Manske, 1991). A.

Differences Among Wheat Genotypes of Different Ploidy Level

Bread wheat Triticum aestivum is an alloploid species with three different genomes (A, B, D) and 42 chromosomes (2n = 6x = 42). In a pot experiment with 57 wheat genotypes of different ploidy level grown on an acid, P-deficient soil, almost all wheat genotypes benefited from the AMF inoculation. However, AMF inoculation effects varied between and within the three ploidy levels of wheats (Table 2). The di- and tetraploid genotypes were more dependent upon AMF than the hexaploid ones. When grown in a sterilized soil, without AMF, the di- and tetraploid genotypes formed little biomass and almost no kernels. Inoculation with the AMF fungus Glomus manihot drastically improved growth. There was a strong enhancement in total root length with AMF, especially with spelt, tetra- and diploid wheats. Within the hexaploid group, the landraces, spelt wheats, and their wild relatives were most responsive to AMF inoculation (Table 2). Some of the inoculated high-yielding varieties formed also very large root systems. Kapulnik and Kushnir (1991) showed that the response of hexaploid wheat to AMF is controlled by factors of the A and B genomes which are epistatic over those located in the D genome. They also found

254

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Table 2 Aboveground Biomass at Maturity and Total Root Length at Anthesis of 34 Hexaploid Wheat Genotypesa Total root length (cm/pot) FePO4

Biomass dry weight (g/pot)

MCP

FePO4

MCP

Groups

n

AMF

þAMF

AMF

þAMF

AMF

þAMF

AMF

þAMF

Hexaploid High-yielding varieties Landraces Spelt wheats Tetrapoid Diploid

25 10 9 16 6

195 236 183 196 158

293 346 349 482 345

531 685 767 631 982

990 1196 1153 892 1246

581 660 538 299 365

624 778 808 579 670

3673 4531 7393 2889 3991

5359 5909 4674 3313 3349

ab bc ab ab a

cd d d e d

a ab bc a d

d de de cd e

c c bc a a

cd d e c d

bc d bc a c

e f d b b

a
a high AMF colonization and response in T. timopheevii var. araraticum, which has an AAGG genome. The latter could not be confirmed by Hetrick et al. (1992) when using a different AMF inoculum.

B.

Old, Tall vs. Modern, Semidwarf Cultivars

Genetic diversity in root characteristics is well documented for bread wheat (MacKey, 1973; O’Brien, 1979) and for durum wheat (Motzo et al., 1993). Many wild forms and landraces of wheat possess large root systems, but tend to lodge because of their tall culms (Manske, 1989; Vlek et al., 1996). There is concern that breeding for improved yield potential under high-input conditions would result in germplasm with smaller root systems. This was checked with 44 historically important lines released by CIMMYT during the last 45 years grown under conditions of adequate nutrient and water supply. Grain yield was increased on the average by 50.4 kg ha1 y1 over the years, but the absolute values of root length density were not significantly changed (Fig. 1) (Manske, 1997). Selection for adaptation to acid soils, low-P conditions, and tolerance to Al toxicity have resulted into superior semidwarf bread wheats. The modern semidwarf lines had improved grain yield, P uptake, and root length density at low and high P input conditions (Table 3) (Manske et al., 1996b). This demonstrates that yield potential can be increased by wheat breeding without adverse effects on root length.

C.

Influence of Dwarfing Genes

The dwarfing genes Rht-B1b and Rht-D1b (with the former designations Rht1 and Rht2, respectively), incorporated singly or together, are in the background of most of the successful wheat cultivars in use today. Rht-B1c (former Rht3) reduces culm length more than Rht-B1b and/or Rht-D1b. It is often used in physiological studies, but has not been incorporated into com-

Figure 1 The effect by the year of release on root length density (RLD) in the upper soil layer (0–20 cm) of 44 important CIMMYT lines released since 1942, grown on an alkaline soil with nutrients and water amply supplied. Means of three replicated plots.

Root Architecture

255

Table 3 Grain Yield and Root Length Density (at the 0–20 cm soil depth) of Eight Semidwarfs and Eight Tall Wheat Lines Grown on a P-exhausted Alkaline Soil with Irrigation High-P regime Semidwarf Grain yield Root length density

kg/ha cm/cm3

Low-P regime Tall

6240 b 7.7 b

4598 a 6.6 a

Semidwarf 4539 b 10.4 b

Tall 3169 a 8.7 a

a
mercial cultivars. The extent of rooting can be modified by selection during breeding, irrespective of the dwarfing genes (MacKey, 1973; Gale and Yousefian, 1985). McCaig and Morgan (1993) found no significant relationships between dwarfing genes and root dry matter. Similar results were obtained in our own experiments with 28 semi- and double-dwarf hexaploid wheat genotypes containing Rht-B1b and/or Rht-D1b or Rht-B1c. The dwarfing genes showed no effect on total root length and AMF infection, although the variation in total root length was large. The few root studies with near-isogenic lines showed contradicting results (Siddique et al., 1990; Miralles et al., 1997), because root growth varies with genetic background, growing conditions, and stage of development (Bush and Evans, 1988). In a field trial with near-isogenic lines carrying different combinations of the genes Rht-B1b, Rht-D1b, and Rht-B1c in two varietal backgrounds, the analysis of root length density in the upper soil layer (0–20 cm depth) revealed some effects of the Rht alleles (Table 4). The introduction of RhtB1b or Rht-B1c was associated with lower RLD at the stage of tillering in both cultivars. In addition, RhtD1b reduced root length density in the Mexican cultivar Nainari 60. The double dwarfs had RLD similar to

the tall controls in both cultivars at tillering. Between tillering and anthesis, Rht-B1b and Rht-B1c genotypes enhanced their RLD, so that at anthesis no differences between Rht alleles were present (Table 4). The RhtB1c genotypes had lower grain yields but thick roots (data not shown), which is related to a surplus of assimilates during stem elongation. The assimilates were translocated to the roots due to the lack of alternative sinks (Miralles et al., 1997). D.

Effect of 1B/1R Translocation

Many high-yielding wheat cultivars carry the 1B/1R translocated segment from rye. Substantial yield increases occurred in germplasm containing the 1B/ 1R translocation (Villareal et al., 1995). Rye has a larger root system than wheat, and a segment of rye chromosome may enlarge the wheat root system. Genotypes with the 1B/1R translocation performed better on an acid soil, having more grains per spike. They also had thinner roots and higher RLD, two traits that enhance root surface area (Table 5). However, the same germplasm showed no advantage of the 1B/1R translocation under better soil conditions or irrigation. In the latter location, this was confirmed

Table 4 Effect of Dwarfing Genes (Rht-B1b, Rht-D1b, Double Dwarfs of the Two Genes; Rht-B1c) and Tall Control on Root Length Density (cm cm3 soil) of the Bread Wheat Cultivars Nainari and Maringa Nainari Tall control Rht-B1b Rht-D1b Double dwarf Rht-B1c Mean

3.7 c 2.9 b 2.7 ab 3.3 bc 2.7 ab 3.1 b

Maringa 3.1 2.0 3.0 2.6 2.1 2.6

b a b ab a a

Mean(dwarfing genes) 3.4 2.5 2.9 3.0 2.4

b a ab ab a

a
256

Manske and Vlek

Table 5 Effect of 1B/1R Translocation (26 lines), 1B/1B, Without Translocation (14 lines) on Grain Yield, Root Surface Area, and Root Diameter (0–20 cm soil layer), CIMMYT Wheat Lines Grown on Acid Andisol in Mexico Under Rainfed Condition, with 35 kg P ha1 Applied as Superphosphate

1B1B 1B1R

n

Grain yield (kg/ha)

14 26

2391 a 2622 b

Root surface area (cm2/sample) Tillering Anthesis Postanthesis 1959 a 2159 a

11867 a 14769 b

Root diameter (mm) Tillering

9354 a 11446 b

173 b 164 a

a
by studies with near-isogenic lines carrying the 1B/1R in different varietal backgrounds (Manske and Vlek, unpublished data). Further studies, especially in marginal environments, are necessary to elucidate the effect of the 1B/1R translocation.

V. COST-BENEFIT ANALYSIS A cost-benefit analysis of the wheat root system is essential for the elucidation of its function. The partition of the assimilated carbohydrates among shoots, roots, and grains is determined by the plant genotype and by environmental factors such as climate, soil conditions, and the availability of mineral nutrients and water. It is also determined by the demand for carbohydrates by microbial associations in the roots of the host plants. Carbon allocated to the roots and used for construction and maintenance is considered the currency for such an analysis. Wheat root dry matter deposited in the soil was estimated as being equal to the dry weight of grain produced (Samtsevich, 1965). Wheat uses 52–67% of the carbohydrates translocated to the roots for root respiration (Lambers et al., 1996). The benefits of large RLD generally diminish in irrigated wheat with high fertilizer input. Root length density of wheat germplasm grown with irrigation in

India, Bangladesh, and Mexico was significantly and positively correlated with grain yield in only 50% of the cases (Table 6). A positive correlation occurred in most cases when the fertilizer was reduced. However, in Trial 2 in India and Trial 4 in Mexico, with the recommended rate of fertilizer applied, RLD and grain yield were negatively correlated. If the demand for carbohydrates by large root systems is not compensated by improved P and water acquisition, the roots may become yield limiting. Plant growth response to VAM infection also depends on the balance between the costs and benefits of symbiosis. The energy required to build and maintain an extensive AMF-infected root system needs to be offset by the improved nutrient uptake of the plant, a condition that may apply in marginal soils. From the plant’s side, AMF fungi are strong competitors for assimilates, which is especially relevant if C availability for growth and grain filling is limited. Depending on the host plant genotype or on P supply, 4–14% of the photosynthates are allocated to AMF-infected roots (Harris and Paul, 1987). Response of plant growth to AMF usually declines as stress conditions are relaxed, e.g., as sufficient nutrients are supplied. The enhanced uptake of P is gained at the cost of host photosynthate utilized by AMF. However, if P becomes nonlimiting and photosynthate utilization by AMF is not curtailed,

Table 6 Phenotypic Correlation Between Grain Yield and Root Length Density (RLD) in the Upper Soil Layer (0–20 cm) at Tillering Time and Early Anthesis India

Mexico

Fertilizer rate

Trial 1 (n ¼ 20) half full

Trial 2 ðn ¼ 9Þ half full

RLD at tillering RLD at early anthesis

0.28 0:17

0:16 0.11

0.04 0.00

0:80b 0.16

Trial 3 (n ¼ 30) P þP 0:51b 0.50b

0:05 0.03

Significant by t-test: aP < :05, bP < :001; — data not assessed; n = number of genotypes.

Bangladesh

Trial 4 (n ¼ 20) P þP 0:52b 0.41a

0:54 0:16

Trial 6 (n ¼ 12) P þP — 0.45*

— 0:10

Root Architecture

the AMF plant may become carbon limited, a situation that leads to parasitic effects of AMF (Vlek et al., 1996). VI.

OUTLOOK

The root system of wheat has received far less attention by plant biologists and plant breeders than the aboveground shoot. This is particularly true for field experiments. Nevertheless, a considerable part of the research to date has dealt with the analysis of the genotypic variation of root traits. Wheat breeders have only rarely selected for root properties, mainly for resistance to root diseases. However, ‘‘roots’’ mean far beyond the mere tissues of that organ. The importance of AMF in regulating the water and nutrient uptake by plants under stress, and improving their resource acquisition efficiency as well as yields, has been documented. In spite of that, no practical means exist to produce a viable inoculum of the obligate symbiotic AMF in order to introduce effective strains on a field scale (Heinzemann, 1994). The lack of suitable nondestructive methods to observe growth and development of intact root systems in situ is one of the reasons for an inadequate knowledge of root system phenology and inheritance. Many of the methods are too expensive to allow for routine genetic studies. The advances in our knowledge of root characteristics have largely been made with relatively simple, but time-consuming, methods. Techniques like minirhizotron, image analysis systems, and root washing machines have been developed, and although they are often more precise and faster, they are expensive and/or unsuitable for field studies with many genotypes (cf. Box, 1996; Chapter 18 by Polomski and Kuhn in this volume). However, those techniques combined with biotechnological methods may lead to new knowledge about the mode of inheritance of root traits in wheat, and the identification of genes to genetically engineer specific root characteristics (see also Chapter 17 by Bucher in this volume), in the near future. REFERENCES Bai QA, Sinclair TR, Ray JD. 1997. Variation in transpirational water-use efficiency and root length development among wheat cultivars. Soil Crop Sci Soc Florida Proc 56:14–19. Bertheau Y, Gianinazi-Pearson V, Gianinazi S. 1980. Development and expression of endomycorrhizal asso-

257 ciations in wheat. I. Evidence of varietal effects. Ann de Amelioration Plantes 30:67–78. Bole JB. 1977. Uptake of tritiated water and phosphorus32 by roots of wheat and rape. Plant Soil 46:297– 307. Bowen GD, Bevege DI, Mosse B. 1975. Phosphate physiology of vesicular-arbuscular mycorrhizas. In: Sanders FE, Mosse B, Tinker PB, eds. Endomycorrhizas. London: Academic Press, pp 242–260. Bush MG, Evans LT. 1988. Growth and development in tall and dwarf isogenic lines of spring wheat. Field Crops Res 18:243–270. Cholick FA, Welsh JR, Cole CV. 1977. Rooting patterns of semidwarf and tall winter wheat cultivars under dryland field conditions. Crop Sci 17:637–639. Dexter AR. 1987. Compression of soil around roots. Plant Soil 97:401–406. Diederichs C, Manske GGB. 1991. The role of VA mycorrhiza for crop nutrition in warmer regions. In: Saunders DA, ed. Wheat for the Nontraditional Warm Areas. Mexico City: CIMMYT, pp 352–371. Egle K, Manske GGB, Ro¨mer W, Vlek PLG. 1999. Improved phosphorus efficiency of three new wheat genotypes from CIMMYT in comparison with an older Mexican variety. J Plant Nutr Soil Sci 162:353– 358. Fo¨hse D, Claassen N, Jungk A. 1991. Phosphorus efficiency of plants. II. Significance of root radius, root hairs and cation-anion balance for phosphorus influx in seven plant species. Plant Soil 132:261–272. Gahoonia TS, Care D, Nielsen NE. 1997. Root hairs and phosphorus acquisition of wheat and barley cultivars. Plant Soil 191:181–188. Gale MD, Yousefian S. 1985. Dwarfing genes in wheat. In: Russel GE, ed. Progress in Plant Breeding. London: Butterworth Scientific, Vol 1, pp 1–35. Gildon A, Tinker PB. 1983. Interactions of vesicular-arbuscular mycorrhizal infection and heavy metals in plants. I. The effects of heavy metals on the development of vesicular-arbuscular mycorrhizas. New Phytol 95:247– 261. Godo GH, Reisenhauer HM. 1980. Plant effects on soil manganese availability. Soil Sci Soc Am J 44:993–995. Gregory PJ, McGowan M, Biscoe PV, Hunter B. 1978. Water relations of winter wheat. I. Growth of the root system. J Agric Sci Camb 91:91–102. Hamblin A, Tennant D, Perry MW. 1990. The cost of stress: dry matter partitioning changes with seasonal supply of water and nitrogen to dryland wheat. Plant Soil 122:47–58. Harris D, Paul EA. 1987. Carbon requirements of vesiculararbuscular mycorrhiza. I. Growth of Endogone inoculated plants in phosphate deficient soils. New Phytol 70:19–27. Hayman DS, Mosse B. 1971. Plant growth response to vesicular-arbuscular mycorrhiza. I. Growth of Endogone

258 inoculated plants in phosphate deficient soils. New Phytol 70:19–27. Heinzemann J. 1994. Production of glass-wool inoculum of arbuscular mycorrhizal fungi. PhD Dissertation in German, abstract in English, University of Go¨ttingen, Germany. Helal HM. 1990. Varietal differences in root phosphatase activity as related to the utilization of organic phosphates. Plant Soil 123:161–163. Hetrick BDA, Wilson GWT, Cox TS. 1992. Mycorrhizal dependence of modern wheat varieties, landraces and ancestors. Can J Bot 70:2032–2040. Hockett EA. 1986. Relationship of adventitious roots and agronomic characteristics in barley. Can J Plant Sci 66:257–266. Horst WJ, Abdou M, Wiesler F. 1996. Differences between wheat cultivars in acquisition and utilization of phosphorus. Z Pflanzenerna¨hrung Bodenkunde 159:155– 161. Huang B, Johnson JW, NeSmith DS. 1997. Response to root-zone CO2 enrichment and hypoxia of wheat genotypes differing in waterlogging tolerance. Crop Sci 37:464–468. Hurd EA. 1968. Growth of roots of seven varieties of spring wheat at high and low moisture levels. Agron J 60:201– 205. Jones GPD, Blair GJ, Jessop RS. 1989. Phosphorus efficiency in wheat—a useful selection criteria. Field Crops Res 21:257–264. Kapulnik Y, Kushnir U. 1991. Growth dependency of wild, primitive and modern cultivated wheat lines on vesicular-arbuscular mycorrhizal fungi. Euphytica 56:27–36. Kazemi H, Chapman SR, McNeal FH. 1979. Components of genetic variance for root/shoot ratio in spring wheat. Proc 5th Int Wheat Genet Symp, New Delhi, pp 597– 605. Klepper B. 1991. Root–shoot relationships. In: Waisel Y, Eshel A, Kafkafi U, eds. Plant Roots: The Hidden Half. New York; Marcel Dekker, pp 265–286. Klepper B, Belford RK, Rickman RW. 1984. Root and shoot development in winter wheat. Agron J 76:117–122. Lambers H, Atkin OW, Scheurwater I. 1996. Respiratory patters in roots in relation to their function. In: Waisel Y, Eshel A, Kafkafi U, eds. Plant Roots: The Hidden Half. 2nd ed. New York; Marcel Dekker, pp 323–362. Lu¨ttger A. 1996. The importance of root length and indigenous arbuscular mycorrhizae in P-fixing soils in northern India for the nutrient and transpiration efficiency in spring wheat (Triticum aestivum L.). PhD dissertation in German, abstract in English, University of Go¨ttingen, Germany. MacKey J. 1973. The wheat root. Proc 4th Int Wheat Genet Symp, Columbia, MO, pp 827–842.

Manske and Vlek Manske GGB. 1990. Genetical analysis of the efficiency of VA Mycorrhiza with spring wheat. I. Genotypical differences and reciprocal cross between an efficient and non-efficient variety. In: El Bassam N, Dambroth, M., Loughman, B.C., eds. Genetic Aspects of Plant Mineral Nutrition. Dordrecht, Netherlands: Kluwer Academic Publisher, pp 397–405. Manske GGB. 1989. The efficiency of the inoculation by the VA mycorrhizal fungi Glomus manihotis in spring wheat genotypes and its inheritance in F1- and R1generations at different phosphate forms applied and different weather conditions. PhD dissertation in German, abstract in English, University of Go¨ttingen, Germany. Manske GGB. 1997. Utilization of the genotypic variability of VAM-symbiosis and root length density in breeding phosphorus efficient wheat cultivars at CIMMYT. Final report of a special project. Mexico City: CIMMYT. Manske GGB, Lu¨ttger AB, Behl RK, Vlek PLG. 1995. Nutrient efficiency based on VA mycorrhizae and total root length of wheat cultivars grown in India. Angew Bot 69:108–110. Manske GGB, Ortiz-Monasterio JI, Van Ginkel M, Gonza´lez R, Vlek PLG. 1996a. Phosphorus uptake, utilization efficiency and grain yield of semidwarf wheat grown in acid or alkaline, P deficient soils. 5th International Wheat Conference, June 10–14, 1996, Ankara, Turkey. Manske GGB, Ortiz-Monasterio JI, Rajaram S, Vlek PLG. 1996b. Improved phosphorus use efficiency in semidwarf over tall wheats with and without P fertilization. Second International Crop Science Congress, November 17-24, 1996, New Delhi, India. Manske GGB, Ortiz-Monasterio JI, Van Ginkel M, Gonza´lez, R, Rajaram S, Molina E, Vlek PLG. 2000. Traits associated with improved P-uptake efficiency in CIMMYT’s semidwarf spring bread wheat grown on an acid Andisol in Mexico. Plant Soil 221: 189–204. Marschner H, Kissel M, Ro¨mheld V. 1986. Different strategies in higher plants in mobilization and uptake of iron. J Plant Nutr 9:695–713. McCaig TN, Morgan JA. 1993. Root and shoot dry matter partitioning in near-isogenic wheat lines differing in height. Can J Plant Sci 73:679–689. McLachlan KD. 1980. Acid phosphatase activity of intact roots and phosphorus nutrition of plants. I. Assay conditions and phosphatse activity. Aust J Agric Res 31:429–440. Mian MAR, Nafziger ED, Kolb FL, Teyker RH. 1993. Root growth of wheat genotypes in hydroponic culture and in the greenhouse under different soil moisture regimes. Crop Sci 33:129–138.

Root Architecture Miralles DJ, Slafer GA, Lynch V. 1997. Rooting patterns in near-isogenic lines of spring wheat for dwarfism. Plant Soil 197:79–86. Mishra HS, Rathore TR, Tomar VS. 1999. Root growth, water potential and yield of irrigated wheat. Irrig Sci 18(3):117–123. Monyo JH, Whittington WJ. 1970. Genetic analysis of root growth in wheat. Agric Sci Camb 74:329–338. Motzo R, Atenne G, Deidda M. 1993. Genotypic variation in durum wheat root systems at different stages of development in a Mediterranean environment. Euphytica 66:197–206. Neumann G, Ro¨mheld V. 1999. Root excretion of carboxylic acids and protons in phosphorus-deficient plants. Plant Soil 211:121–130. O’Brien L. 1979. Genetic variability of root growth in wheat (T. aestivum L.). Aust J Agric Res 30:587–595. O’Toole JC, Bland WL. 1987. Genotypic variation in crop plant root systems. Adv Agr 41:91–145. Oyanagi A, Nakamoto T, Wada M. 1993. Relationship between root growth angle of seedlings and vertical distribution of roots in the field in wheat cultivars. Jpn J Crop Sci 62:565–570. Pinthus MJ. 1967. Spread of the root system as indicator for evaluating lodging resistance of wheat. Crop Sci 7:107– 110. Portilla CI, Molina GE, Cruz-Flores G, Ortiz-Monasterio I, Manske GGB. 1998. Colonizacion micorrizica arbuscular, actividad fosfatasica y longitud radical como respuesta a estres de fosforo en trigo y triticale cultivados en un andisol. Terra 16:55–60. Rickman RW, Klepper B, Pumphrey FV, Rickman RW. 1984. Developmental relationships among roots, leaves and tillers in winter wheat. In: Day W, Atkin RK, eds. Wheat Growth and Modeling. New York; Plenum Press, pp 83–98. Robertson BM, Waines JG, Gill BS. 1979. Genetic variability for seedling root numbers in wild and domesticated wheat. Crop Sci 19:843–847.

259 Sadhu D, Bhaduri PN. 1984. Variable traits of roots and shoots of wheat. Z Acker- Pflanzenbau 153:216. Samtsevich SA.1965. Active secretions of plant root and their significance. Fiziol Rast 12:837–846. Siddique KHM, Belford RK, Tennant D. 1990. Root:shoot ratios of old and modern, tall and semi-dwarf wheats in a Mediterranean environment. Plant Soil 121:89–98. Smith SE, Smith FA. 1990. Structure and formation of the interfaces in biotrophic symbioses as they relate to nutrient transport. New Phytol 114:1–38. Swaminathan K, Verma BC. 1979. Reponse of three crop species to vesicular-arbuscular mycorrhizal infection on zinc-deficient Indian soils. New Phytol 82:481–487. Tabatabai MA, Bremner JM. 1969. Use of p-nitrophenylphosphate for assay of soil phosphatase activity. Soil Biol Biochem 1:301–307. Villareal RL, Del Toro E, Mujeeb-Kazi A. 1995. The 1BL/ 1RS chromosome translocation effect on yield characteristics in a Triticum aestivum L. cross. Plant Breeding 114:497–500. Vlek PLG, Lu¨ttger AB, Manske GGB. 1996. The potential contribution of arbuscular mycorrhiza to the development of nutrient and water efficient wheat. In: Tanner DG, Payne TS, Abdalla OS, eds. The Ninth Regional Wheat Workshop for Eastern, Central and Southern Africa. Addis Ababa, Ethiopia: CIMMYT, pp. 28–46. Young JL, Davis EA, Rose SL. 1985. Endomycorrhizal fungi in breeder wheats and Triticale cultivars field-grown on fertile soil. Agron J 77:219–224. Zhang XG, Jessop RS, Ellison F. 1999a. Inheritance of root regrowth as an indicator of apparent aluminum tolerance in triticale. Euphytica 108:97–103. Zhang XG, Jessop RS, Ellison F. 1999b. Differential genotypic tolerance response to manganese stress in triticale. Commun Soil Sci Plant Anal 30(17–18):2399– 2408.

16 Banana Roots: Architecture and Genetics Xavier Draye Universite´ catholique de Louvain, Louvain-la-Neuve, Belgium

I.

INTRODUCTION

when crossed with selected fertile diploids, which paves the way for the genetic analysis of important traits and for the improvement of the narrow cultivated germplasm (Vuylsteke et al., 1993a,b; Ortiz and Vuylsteke, 1994, 1995; Swennen et al., 1995). Root research in Musa is now slowly entering the genetics age, instigated by the growing need for improved cultivars that are adapted to local conditions (Swennen et al., 1986; Draye et al., 1999; Blomme, 2000; Stoffelen, 2000).

Bananas and plantains (Musa spp.) are among the most important fruit crops of the tropics (FAO, 1999). These giant perennial herbs grow as clumps with a number of side shoots or suckers arising from the main rhizome. In commercial plantations, suckers are regularly pruned, leaving one shoot to replace the parent plant (Gowen, 1988). The need for a suitable root architecture in Musa arises from the considerable resource uptake and anchorage demands of banana plants, combined with the importance of biotic and abiotic interactions taking place in the rhizosphere. It is a case for applied and multidisciplinary research. Since the first report on Musa root architecture at the beginning of the last century (Fawcett, 1921), much has been accomplished in various aspects of the morphologic, physiologic, and agronomic aspects of root structure and development. Cultivated Musa plants are mostly triploids. They are classified as AAA dessert banana, AAA East African highland cooking and beer banana, AAB plantains, AAB dessert banana, and AAB, ABB, and BBB cooking bananas. All were derived from the diploid wild species M. acuminata Colla and M. balbisiana Colla which contributed the A and B genomes (Simmonds, 1966; Jones, 1994). Being almost sterile, bananas are vegetatively propagated from rhizomes, suckers, or from in vitro plants. Nonetheless, some of the cultivated triploids display very low levels of fertility and produce viable diploid and tetraploid progenies

II.

THE BANANA ROOT SYSTEM

The functional root system of Musa is based entirely on adventitious root axes, also known as cord roots. Those arise from a subterranean rhizome, and produce primary and higher-order laterals. The topology of the Musa root system resembles that of a herringbone pattern (Fitter, 1996) repeated at each order of branching. The descriptive, dynamic, anatomical, and functional features of the root system of a reference Musa type are reviewed in the following section. The genus Musa lends itself to the selection of a reference type, with considerably more research performed on the dessert banana (AAA genome) and especially on cultivars from the Cavendish subgroup (cv’s Gros Michel, Williams, Grande Naine, Giant Cavendish, Valery, Poyo). In view of the large genetic variability of the genus Musa, it seems reasonable to adopt these genotypes as reference types, without any intention to hide differences that exist between them. 261

262

A.

Draye

Constituents

1. Rhizome It is difficult to discuss the root system of banana without referring to the rhizome, from which the adventitious root system arises. The rhizome constitutes the true stem of the banana plant and attains a diameter of >30 cm and a height of 35 cm. It is dominated by a primary apical meristem surrounded by a rosette of leaves whose elongated sheaths overlap closely to form a tall pseudo-stem. In the early reproductive phase, the apical meristem elongates considerably through the center of the pseudostem and later emerges from the top of the plant as the inflorescence (cf. Skutch, 1932). Behind the primary meristem, a massive central cylinder exists surrounded by a thick cortex. A cambiumlike layer, from which adventitious roots are differentiated, is situated at the outer surface of the central cylinder and extends from
5 to 35 mm from the growing point (Mangin zone, after Mangin, 1882). This special meristem produces large longitudinal bundles that prolong the leaf bundles and large horizontal bundles, to which the root vascular bundles are connected. These horizontal bundles girdle the central cylinder and anastomose with the longitudinal bundles. Thus, water or nutrients entering the rhizome through any root are distributed among the leaf bundles on any side of the rhizome. During the vegetative phase, the rhizome produces lateral buds, some of which develop into lateral shoots or suckers. While attached to the parent plant, the rhizome of these suckers may produce a number of roots axes exceeding that of the parent plant itself (Turner, 1972). It is thus essential, when considering root architecture, to distinguish between roots that have originated from different rhizomes.

2. Seminal Root In the germinating seed of Musa balbisiana, the seminal root is short-lived and its growth is overtaken by that of adventitious roots within a week (Simmonds, 1959). By the time the seedling is 4–6 weeks old, the adventitious root system is by far the dominant one (McGahan, 1961). As the cultivated bananas are reproduced vegetatively, seeds are rarely formed and the entire root system is adventitious (Simmonds, 1966).

3.

Adventitious Root Axes

Adventitious root axes originate in the Mangin zone, in longitudinal groups of up to four roots. In their early development, they penetrate the parenchyma of the cortex and the leaf bases. Their advance in these tissues is facilitated by softening of the surrounding cells. When they reach a few millimeters from the surface of the rhizome, the root tips, which are
1.5 mm in diameter, broaden abruptly (Skutch, 1932). Because the Mangin zone is restricted to a 3-cm-long arc near the growing point of the rhizome, new roots are produced higher up (acropetally) as the rhizome grows (Summerville, 1939; Kwa, 1993). This leads ultimately to the production of roots above the soil surface, which may be detrimental in soils of low fertility, in windy areas, and under conditions of high parasite infestation (Moreau and Le Bourdelle`s, 1963). This phenomenon may be exaggerated by the tendency of rhizomes of successive ratoons to rise above the soil surface (‘‘high mat’’) (Stover, 1972; Braide and Wilson, 1980). The root axes are white and ropelike in the young portions, and somewhat corky and brown in the mature portions. The roots are relatively straight and cylindrical, up to 5.2 m long, and
4–10 mm in diameter. Root axes have reached lengths of 6 m in the ‘‘Sarah Racine’’ aeroponic root lab of Tel Aviv University. Roots growing from mature plants have larger diameters than those of young plants. The root tips of the main roots are very soft and covered by a 0.8- to 1.2-mm-thick and 3- to 4-mm-long root cap. Root elongation occurs in a region extending from 3 to 15 mm behind the root tip, with a zone of maximal extension between 4 and 6 mm (Fawcett, 1921; Acquarone, 1930; Riopel, 1960; Riopel and Steeves, 1964; Monnet and Charpentier, 1965; Simmonds, 1966; Blomme, 2000). Root axes develop a dense cover of large root hairs often exceeding 2 mm in length. These appear immediately behind the elongation zone and reach full size 8–12 cm proximal to their origin. They persist for
3 weeks, viz. 60 cm behind the root tip of actively growing roots (2.5–3 cm/day), and much less during periods of arrested root growth. The maintenance of a root hair zone thus depends on a continuous root growth (Acquarone, 1930; Summerville, 1939; Riopel, 1960; Riopel and Steeves, 1964; Weckx, 1982; Robinson, 1987, 1996). In hydroponics, root hairs live only for a few days and are sloughed off 7–8 cm behind the root tip (Swennen et al., 1986).

Banana Roots

Two types of axes have been distinguished—feeder and pioneer axes—which differ by their high and low density of primary laterals. Compared with the large conical pioneer roots, feeder axes are cylindrical and have a diameter similar to that of large primary lateral roots (Winderickx, 1985; Swennen et al., 1986, 1988). Feeder roots were only observed on suckers and in vitro plantlets undergoing a transfer to hydroponics but not on plants already grown in hydroponic. Further attempts to distinguish feeder and pioneer root types in the field were rather difficult (Blomme, 2000). Feeder roots might be replacement roots arising from portions of root axes situated in the cortex of the rhizome. The first reports on banana roots gave the impression that there are horizontal and vertical roots (Fawcett, 1921; Hartman et al., 1928). The difference, however, is one of age and length. The oldest roots produced at the base of the young rhizome tend to grow downward, and ultimately 70% of the total root mass is confined to the upper 20–40 cm of the soil (Acquarone, 1930; Summerville, 1939; Champion and Sioussaram, 1970; Lassoudie`re, 1978; Gousseland, 1983; Robinson, 1985; Araya et al., 1998; Blomme, 2000). This general scheme, however, has to be reconsidered by taking account of soil heterogeneity and/or suboptimal soil conditions. The horizontal and vertical extension of the banana root system would be primarily determined by variations of bulk density and mechanical impedance in the soil profile, and affected by soil type, texture, and structure, and by land preparation, climate, previous crop, and plantation age (Godefroy, 1969; Delvaux and Guyot, 1989). 4.

Lateral Roots

Lateral roots are initiated in the vicinity of root tips. Lateral root primordia are differentiated by tangential divisions from a circular group of pericycle cells opposite the protoxylem strand (Acquarone, 1930; Charlton, 1982). In the case of primary laterals, the first cell divisions may be detected
450
m from the root/cap junction, although the initial events leading to the differentiation of lateral root primordia probably occur even closer to the tip (Charlton, 1996; Draye et al., 1999). The cells of the endodermis lying against the dividing pericycle cells give rise to a digestive pocket and to the new cap. The growth of the primordium is symmetrical so that each primordium is centered on the protoxylem and clear protoxylem-based longitudinal ranks of primordia result.

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The initiation of lateral roots is apparently acropetal within each rank. Primordia tend to arise at a roughly constant proportion of the distance between the root tip and the previous primordium of the same rank. However, the initiation occurs independently in different ranks and is therefore not strictly acropetal at the root level (Charlton, 1987). The pattern of lateral root initiation thus comprises two elements: restriction to protoxylem-based ranks, and a rather regular spacing within ranks. It has recently been shown that this pattern is conserved among genotypes (Draye et al., 1999). This overall rigidity in lateral root initiation contrasts with the variability of root branching that is generally observed. The latter probably reflects the space–time variability of the rhizosphere, yet the conditions in which initiated primordia develop into lateral roots or enter a dormant phase remain unexplored. Primary laterals, 0.5–4.0 mm in diameter, protrude 10–30 cm behind the tip of the leader root axis. Their growth is determinate: they extend at a rate of 0.7–1.5 cm/day and reach a final length of 3–15 cm (Laville, 1964; Riopel and Steeves, 1964; Lassoudie`re, 1978; Blomme, 2000). Swennen et al. (1986) observed a succession, along water-grown root axes, of segments covered with, alternately, shorter and longer primary laterals. Given cyclical variations of pH and nutrients, the length of primary laterals was probably determined by rhizospheric conditions prevailing near the tip of the axis at the time when each lateral root primordium was initiated. The life span of primary laterals ranges from as short as 6–10 days to as long as 8 weeks depending on biotic and abiotic constraints (Robinson, 1987). Primary laterals can reach a density of 8–10/cm of root axis in hydroponics and in the field (Blomme, 2000; Stoffelen, 2000). The density and length of lateral roots may be slightly depressed in the proximal decimeter of root axes, probably under the influence and proximity of the rhizome. Secondary laterals, 1 mm or less in diameter and 0.3–4 cm long, are produced on primary laterals. They remain alive for up to 5 weeks and reach densities of 8.7/cm of primary lateral (Robinson, 1987; Blomme, 2000). In nutrient solution, 95% of primary laterals are covered with secondaries, with a density of 3.0/cm (Stoffelen, 2000). Tertiary laterals are rare and <200
m in diameter (Acquarone, 1930; Riopel and Steeves, 1964). Root hairs are produced on all lateral roots, starting at 4 mm from the root tip. They persist for 3 weeks or until the root dies (Robinson and Bower, 1988).

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In conditions favoring uniform root growth, primary laterals protrude at a remarkably constant distance behind the tip of the axes. Conversely, on roots whose apex has become less active, lateral roots protrude closer to the tip (Lassoudie`re, 1978), indicating that the progress of lateral root emergence is less affected by adverse conditions than the growth of the root axis. That progress might even be promoted when the sink strength of the root apex is reduced. The determinate nature of the lateral roots in Musa is being questioned for the cv Grande Naine. In hydroponics and sand culture, primary lateral roots of this cv grow continuously and commence rotting before they reach a putative determinate length. This suggests that these laterals are of indeterminate growth type. Such a growth type may be valuable for maintaining the quantity of root hairs and higher-order laterals. 5. Replacement Roots Musa root axes that meet a hard obstacle at right angle cannot grow around it. The apex dies and several lateral roots, almost as large as the parent root, develop behind the dead tip. These replacement roots may be lateral roots that change order and become root axes: they have an indeterminate growth and the same internal organization as primary roots (Acquarone, 1930; Riopel, 1960; Laville, 1964). Considering the abundance of lateral root primordia behind Musa root tips and since the density of replacement roots does not exceed that of primary laterals, such roots probably develop from primordia of the normal sequence of primary laterals. Root axes experiencing mechanical constraints, such as abrupt direction changes, also produce large lateral roots with an apparent indeterminate growth. This reaction seems to be proportional to the intensity of the constraint (Lassoudie`re, 1971). B.

Production, Growth, and Survival of the Root Axes

One of the most striking features of the Musa root system is the fact that root hairs and lateral roots, which best enhance the uptake of water and nutrients, are only transient structures. Consequently, the maintenance of sufficient root hairs and laterals depends on the continued production and growth of root axes. 1. Number of Root Axes The production of root axes by young Musa plants depends on the type of planting material, e.g., suckers,

bits (section of rhizome containing a lateral bud), or in vitro plantlets. On suckers, 10-34 roots emerge during the first fortnight after planting. These are preformed roots that were developing in the cortex of the rhizome at the time the sucker was uprooted. The formation of new roots is then interrupted until 3 months after planting (Champion and Olivier, 1961; Moreau and Le Bourdelle`s, 1963; Beugnon and Champion, 1966; Gousseland, 1983; Lavigne, 1987). In the case of bits, one to 10 small fibrous roots are initially produced, which do not extend beyond the planting hole. New root formation on the new plant starts 3–5 weeks after planting and continues thereafter (Turner, 1972). Finally, the roots produced by in vitro plants in the regeneration medium do not grow further in soil or hydroponics and new root formation starts 4–5 weeks after planting, at the end of the acclimation phase (Blomme, 2000; Stoffelen, 2000). Except for one report of a second period of root emission in aeroponics, root formation continues steadily until flowering. It is, however, highly variable with age and date, promoted by high planting densities and can be arrested during periods of low temperatures. The number of visible roots increases and reaches >600 roots at flowering. The plant loses its capacity to form new roots at the end of the vegetative phase, and subsequent emission is confined to the suckers. Most of the root axes present at flowering remain alive during the reproductive period, and approximately half of them may still be present at harvest of the follower (Moreau and Le Bourdelle`s, 1963; Beugnon and Champion, 1966; Turner, 1970, 1972; Lassoudie`re, 1980; Gousseland, 1983; Mohan and Madhava Rao, 1984; Lavigne, 1987; Robinson, 1987; Blomme, 2000). A sucker attached to the parent plant may produce 200–300 roots before developing the first active, nonlanceolate leaf. In addition, counts >400 roots have been recorded on the subsistent rhizome of pruned suckers (Champion and Olivier, 1961; Robin and Champion, 1962; Turner, 1972). Glass rhizotron experiments, as well as comparisons between numbers of visible roots and total numbers of roots produced (inferred from root scars in the cortex), indicate that the life span of root axes is
2–6 months (Moreau and Le Bourdelle`s, 1963; Robinson, 1987). However, Lassoudie`re (1980) found live roots, presumably 10 months old, on the remains of the parent rhizome, 8 months after harvest. The level of activity of the latter roots is unknown. Interestingly, they were less affected by nematodes than the roots of the follower rhizome.

Banana Roots

Consistent with the lack of new roots to replace dying roots, the proportion of healthy roots is lower during the reproductive (6–17%) than at the end of the vegetative phase (16–50%) (Champion and Olivier, 1961; Robin and Champion, 1962; Blomme, 2000). Besides, root length decreased by 40% during the reproductive period, as compared with only 8% for root number (Valsamma et al., 1987; Blomme, 2000). This indicates that long roots would be more prone to root decay than short ones. This may also be attributed to their being older and to the fact that they explore larger volumes of soil and have higher chances of encountering hazards. The number of roots of the parent plant increases with the leaf area, suggesting fixed allometric ratios. The root number is also correlated with size of the plant such as bunch size (both number of hands and fingers) and pseudo-stem diameter. In addition, the fruit weight of the parent plant is correlated with the number of roots of the tallest sucker and with the pseudo-stem circumference. Many of these correlations are dependent on place and season (Champion and Olivier, 1961; Beugnon and Champion, 1966; Lassoudie`re, 1980; Gousseland, 1983; Lavigne, 1987; Blomme, 2000). 2.

Length of Root Axes

The length of root axes depends primarily on their elongation rate and the time over which elongation proceeds. In Musa, the elongation rate ranges between 1.2 and 4.0 cm/day and is independent of root length. It follows the diurnal and seasonal variations of the leaf emergence rate and is adversely affected by mechanical impedance, low pH, elevated water tables, suboptimal soil temperatures, and oxygen deficiency (Riopel and Steeves, 1964; Beugnon and Champion, 1966; Lassoudie`re, 1978; Gousseland, 1983; Lavigne, 1987; Robinson, 1987; Robinson and Alberts, 1989; Aguilar et al., 1998; Rufyikiri, 2000). Based on an average 5-month life span, a single axis may thus reach a length of 2.6–7.5 m, a range which encompasses most observations of maximum root length in good conditions. The most informative representation of root length with respect to root system architecture and soil exploration is the distribution of the number of roots Ni among successive length classes. Ni equals the number of roots of lengths between Xi and Xi+w, where w is a constant length interval. In 10-week-old healthy plants (propagated in vitro) growing in nutrient solution, Ni appears to be constant, which would be

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expected if both new root formation and elongation were constant and roots had not been exposed to severe hazards, or at least not long enough. In such conditions, the average root length of young plants sometimes exceeds 75 cm (Swennen et al., 1986; Stoffelen, 2000). In mature field-grown plants, Ni decreased exponentially and the number of roots in any length class (w=10 cm) was
65% that of the preceding class: the 0- to 10-cm length class included 32–42% of the roots and only 1% were in the 91- to 100-cm class (Moreau and Le Bourdelle`s, 1963). The average root length of field-grown plants during the vegetative phase is thus usually very short and cannot indicate the volume of soil explored. In principle, the exponential distribution corresponds to a situation where new roots are produced continuously, grow at a constant rate, and die only of hazards that must occur at a constant rate and be independent of root age. In conditions of constant root production and growth, the rate of hazards would thus provide a measure of the global impedance on the root system. The higher the death rate, the lower the proportion of roots growing to the next length class. In the study by Moreau and Le Bourdelle`s, the proportions of roots growing to the next length class were 64%, 65%, and 66% in three different soils, suggesting, under the conditions stated above, that the biotic and abiotic pressures in these soils were rather similar. In this respect, crude estimates of root weight data as a function of distance from the rhizome may be inappropriate to address issues of root length and root dynamics in soil, as long as the various orders of roots are not considered separately. C.

Anatomical Features of the Various Root Types

As thorough descriptions of the anatomy of mature Musa roots have been given elsewhere (Acquarone, 1930; Riopel and Steeves, 1964), the following account is limited to those features that are meaningful for root architecture, uptake, and transport. The epidermis and hairs of the leader root axes are sloughed off at 50–60 cm from the tip as the underlying cells of the cortex become slightly suberized. It is still unclear whether this occurs when the epidermis cells reach a given age or a given distance from the tip. This does not happen on lateral roots, however. The cortex is organized in discrete inner and outer regions which are composed of fewer layers of cells in lateral roots than in axes. Root axes constitutively develop radially widened lysigenous lacunae between

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the inner and outer regions of the cortex. In aerated nutrient medium, this aerenchyma is formed within 10 cm from the tip, increases in extent up to
30 cm from the tip, then gradually decreases toward the base of the root. Under poor aeration, root axes increase the potential oxygen flow toward the tip by increasing the aerenchyma and by speeding up its production to within 5 cm of the root tip (Aguilar et al., 1999). Casparian strips are formed in the endodermis, 12– 18 mm from the tip. In primary laterals and in large secondary laterals, prominent secondary thickening appears on the inner walls and is completed 60–110 cm from the apex. The stele of the root axes is polyarchous, with 28 or more xylem strands (proto- and early metaxylem) disposed in radii at its periphery, alternating with phloem strands (proto- and early metaphloem). Primary laterals are polyarchous with 10–20 xylem strands, secondary laterals are tri- or tetrarchous, and tertiary laterals are diarchous. Whether the number of strands is correlated with the stelar diameter remains unclear (Riopel and Steeves, 1964; Draye et al., 1999). Unusually large individual vessels of late metaxylem, 300–350
m in diameter, and strands of late metaphloem are dispersed in the central region of the stele. Maturation of the xylem occurs 1– 55 cm from the tip, although some data indicate that it may not be completed until 2 m (McCully, unpublished data). D.

Mycorrhizal Symbiosis

The root cortex of bananas is commonly invaded by arbuscular mycorrhizal fungi, with
68% colonization of banana roots (Stover and Simmonds, 1987; Iyer et al., 1988). Under artificial inoculation, frequencies of colonization range from 17% to 85% depending on fungal species, plant cultivar, age, substrate, and conditions (Lin and Chang, 1987; Knight, 1988; Jaizme Vega et al., 1991; Declerck et al., 1994, 1995). Mycorrhizal infections cause an increase of shoot and root weight, pseudo-stem diameter, plant height, leaf area, water content, transpiration, and photosynthesis and appear as effective as optimum P fertilization. Mycorrhization may also reduce the number of roots, independently of their length. Attempts to characterize the effects of mycorrhizae on nutrient uptake (shoot content) were inconclusive, which is probably due to a lack of precise knowledge of the root conditions and of the symbiotic partner (Lin and Chang, 1987; Lin and Fox, 1987; Knight, 1988; Umesh et al., 1989; Jaizme Vega et al., 1991, 1997; Declerck et al., 1994, 1995; Yano Melo et al., 1999).

The benefits of the symbiosis are especially pronounced for cultivars developing a smaller root system with shorter and fewer root hairs, and for young plants propagated in vitro (Declerck et al., 1995). Mycorrhizae also reduce nematode-induced lesions on banana roots and improve the resistance to aluminum stress (Umesh et al., 1989; Jaizme Vega et al., 1997; Rufyikiri et al., 2000a). The conditions for successful inoculation during the in vitro propagation of banana plantlets was recently established (Declerck, personnal communication), which will allow the systematic inoculation of this planting material before its distribution. E.

Root Function

1.

Uptake

Under banana cropping in the subtropics,
40% of total water loss occurs from the upper 10 cm of soil and 80% from the upper 30 cm (Robinson and Alberts, 1989). This seems consistent with the vertical distribution of roots. The crop water use coefficient (Et/Eo) appears to be primarily determined by the leaf area index (LAI) and potential evaporation (Eo, evaporation from a Class A pan; Et, evaporation from the plant and the soil surface) (Robinson and Bower, 1988). However, root activity may limit water uptake during the winter, even in conditions of wellwatered soil and high LAI. This was ascribed to the progressive loss of root hairs following cessation of root growth under low soil temperature. This interpretation was based on the traditional assumption that water uptake in Musa occurs mainly in the root hair zone of roots (Hartman et al., 1928; Robinson, 1987). It will have to be reconsidered, as it appears that old roots may still be active in uptake (Clarkson, 1996). Nutrient uptake has been mainly considered with regard to fertilization. The first experiments involved localized applications of 32P followed by the determination of 32P activity in the leaves. These revealed that most of the P was taken up in the upper 15 cm of the soil, in agreement with the vertical distribution of root axes, and within 60 cm from the rhizome (Walmsley and Twyford, 1968; Ssali, 1977; Mohan and Madhava Rao, 1985). However, these were in contrast with the observations of increasing root weight with increasing distance from the rhizome. The rationale of these experiments merits some comment. Firstly, they take no account of root phenotypic plasticity expressed by the enhanced branching in response to localized nutrient supply (Robinson, 1994).

Banana Roots

Ideally, the localized 32P supply should be accompanied by an equivalent supply of the stable isotope on the remaining areas. Secondly, the fertilizer was supplied in fixed amounts irrespective of the distance, i.e., with a subsequent reduction of the 32P concentration in the soil solution with increasing distance from the rhizome. Finally, the effectiveness of different root segments in absorbing nutrient was not considered. It is indeed hard to admit that P uptake is maximum near the rhizome, where lateral roots and root hairs are the less abundant (Summerville, 1939). The effect of varying temperature and nutrient supply on the whole root system uptake (N, P, K, Ca, Mg, Na, Mn, Cu, Zn) in nonstressful conditions was addressed by Turner and colleagues (Turner and Barkus, 1981; Turner and Lahav, 1985). Their results suggest that root system size was not a limiting factor for uptake: nutrient absorption was influenced by the additional growth more than by the direct effects of temperature or supply. The effect of temperature on the root uptake rates reflected changes in the distribution of dry matter and in the accumulation of the elements in the different organs. The efficiency of root uptake may thus be expected to vary both seasonally and with the developmental stage. In particular, soil and air temperatures may affect uptake in a different way, given their respective effects on root and shoot growth (Turner and Lahav, 1983; Ramcharan et al., 1995). Uptake effectiveness and its response to the various treatments were dependent on the nutrient element considered and also on the element varied among treatments, with K causing large changes in growth. This illustrates further the risk of basing fertilizer placement recommendations on observations of P uptake only. Given the many connections between vascular bundles within a rhizome and between parent and follower rhizomes (Skutch, 1932), it is likely that the various components of a banana stool share a common pool of water and nutrients. The two-way translocation of water and nutrients between the parent plant and its followers have been demonstrated with 32P and 45Ca (Walmsley and Twyford, 1968; Teisson, 1970). The parent plant benefits from the root activity of young suckers with narrow leaves, especially in view of their possessing similar numbers of roots. The continued production of roots by the suckers certainly compensates for the lack of new root formation on the parent rhizome after floral initiation. However, suckers can also withdraw nutrients from the parent plant, with predictable effects on crop yield. The final balance would largely depend on architectural and functional

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aspects of the parent’s and followers’ root systems, and ultimately on internal and external influences on plant growth. The complexities of this issue, however, remain to be addressed. 2.

Biomechanics and Anchorage

Little attention has been given to the mechanical aspects of root architecture in Musa. The stature of the plant and, in many cultivars, large bunch weight (20–50 kg) place a considerable mechanical demand on the root system which must resist bending moments set up by wind or plant weight (Fitter and Ennos, 1989). Part of these moments are converted to tensile forces applied to oblique root axes. These tensile forces are transferred to the soil along a length of root which depends on the nature and extent of soil–root contact and on their elasticities. The remainder of the bending moments tend to pull out the root–soil plate, causing section strains on the horizontal axes. The higher the cohesion of the root-soil plate, the higher the moments that can be resisted. Anchorage thus comprises four components, all of which may change in time and space: the rhizome size, the number and direction of root axes, the tensile and section strength of root axes, and the soil mechanical properties (see also Chapter 10 by Stokes in this volume). The stability of the plant and the tensile forces experienced by the oblique root axes depend on the number of rhizomes forming the banana stool and on the size of the rhizomes. The latter, however, offers limited scope for improving plant stability because large rhizomes correlate with tall pseudo-stems (and high bending moments) (Swennen and De Langhe, 1985). Unfortunately, the cultivation practice of selecting a single sucker for the following ratoon crop also prevents the establishment of a wide base, and has a negative impact on anchorage capability of the plants. One may expect that the increasing number of live root axes with the age of the plant meets the increasing demand set up by the elongating pseudo-stem. At flowering, however, new root formation terminates while the growing bunch keeps increasing the anchorage demand. Root decay at that stage can be minimized by healthy soil conditions (Turner, 1972), yet it remains necessary that an excess of roots be present at the end of the vegetative phase. Again, the role of roots attached to the suckers should not be underestimated. The tensile and section strength of roots determines the maximum strain that can be absorbed by

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individual roots. It is not only a function of root diameter, but also of the mechanical properties of the root tissues—e.g., the relative thickness of the cortex and of the stele, the extent of aerenchyma, and the architecture of cell walls. While the former has been discussed above, data regarding the latter are essentially lacking.

3. Regulation of Sucker Growth Lateral shoot development in Musa consists of three different stages: peeper (young and apparently dormant sucker bearing scale leaves), sword sucker (sucker bearing narrow swordlike leaves), and maiden sucker (large sucker with foliage leaves) (Simmonds, 1966). On the one hand, the production of lateral buds and the transition from the sword to the maiden sucker is stimulated by high cytokinin/auxin ratios. On the other hand, peeper development would be regulated by an interplay of stimulating gibberellins and inhibiting factors manifesting apical dominance of the parent’s primary meristem (De Langhe et al., 1983; Swennen and Wilson, 1983; Swennen et al., 1984). It was recently suggested that high apical dominance, which inhibits suckering of plantains (AAB), is under the influence of a recessive allele ad at a single genetic locus (Ortiz and Vuylsteke, 1994). In the early 1980s, it was suggested that densely branched root systems with many active tips would synthesize high amounts of gibberellins and cytokinins, thereby inducing the development of peepers into sword suckers and maiden suckers (De Langhe et al., 1983). The idea was further supported by observations that mulch application to one side of a plant promoted root branching and sucker development in the mulched sector (Swennen, 1984). However, this depends on the cultivar: positive relationships between root system size (number of axes, weight, and total axis length) and the height of the tallest sucker were reported for cv Obino l’Ewai (AAB genome) but not for other cv’s (Blomme, 2000). The lack of such relationship among different cv’s indicate that genetic factors may influence root system size without affecting sucker development and vice versa. As of today, conclusive evidence of GA synthesis in the roots is still lacking and it appears that gibberellins are principally synthesized in the meristematic shoot tissues and in green leaves (see also Chapter 24 by Tanimoto in this volume). There is thus a need to reconsider the role of the root system in mediating the suckering behavior.

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III.

ARCHITECTURE UNDER STRESS

Unfavorable biotic and abiotic soil conditions impede the growth and development of the Musa root system and are held responsible for important yield losses. Root parasites, drought, anoxia, soil acidity, mechanical impedance, and suboptimal temperatures constitute the leading sources of root stress. The following section comments on the effect of these conditions and on the possible sources for adaptation and resistance. A.

Root Parasites

Root parasites are among the major constraints for banana and plantain production worldwide. They are mainly composed of root lesion nematodes (Radopholus similis and Pratylenchus coffeae), root knot nematodes (Meloidogyne spp.), the banana weevil Cosmopolites sordidus, and the soilborne fungus Fusarium oxysporum f. sp. cubense. Root lesion nematodes feed, multiply, and migrate in the rhizome and roots and cause a necrotic and reduced root system, while root knot nematodes are sedentary endoparasites which cause root galling. The vertical distributions of roots and of nematodes in the soil are very similar, and it was suggested that the occurrence of fewer nematodes at greater depth was only caused by the smaller quantity of roots (Araya et al., 1998, 1999). Conversely, the number of R. similis nematodes increases faster than the weight of roots with decreasing horizontal distance from the plant base. Apparently, nematode infection intensifies in the vicinity of the plant base, where the distance between nearby roots is extremely small. This extensive colonization might contribute to some inhibition of development of the sedentary root knot nematodes (Pinochet, 1977; Araya et al., 1999). R. similis is found equally on root axes and laterals, and the distribution over the root types seems independent of the banana variety and inoculum density (Mateille, 1994). There are, however, little relationships between the number of R. similis per gram of roots and the decrease of root weight (Hahn et al., 1996). Resistance to R. similis is unknown in any of the commonly grown banana or plantain cultivars, but has been identified in Yangambi Km5 (AAA) and in the Pisang Jari Buaya group (AA). The mechanisms of resistance to R. similis reveal some constitutive anatomical adaptations—i.e., preformed phenolic cells in Yangambi, lignified cell walls in Pisang Jari Buaya, and inducible production of a phytoalexin in Pisang Jari Buaya (Fogain and Gowen, 1996; Binks et al., 1997).

Banana Roots

Resistant varieties, grown in hydroponics, have long secondary laterals (Stoffelen, 2000). In Yangambi, lateral roots are very tiny, in both water and sand culture. The significance of such architecture for nematode resistance remains unexplored (see also Chapter 51 by Koltai et al. in this volume). Adult banana weevils (Cosmopolites sordidus) deposit eggs in holes made in the rhizome at ground level between the basal leaf scars (Gowen, 1995). The damage done by the weevil is primarily the result of destruction of tissue of the rhizome by larvae. Although the beetle does not attack banana roots, root initiation and/or root growth through the cortex may be hampered due to the tunneling larvae. Little is known of the sources of resistance to the weevil borer. Mechanical resistance in rhizome tissue may be an important factor (Jones, 1994). F. oxysporum f. sp. cubense is the pathogen responsible for the banana wilt which devastated the commercial banana plantations of the Americas during the first half of the 20th century. The problem was managed by the use of resistant Cavendish cultivars instead of the widely grown susceptible Gros Michel (Simmonds, 1966). Although symptoms develop only on the aerial parts of the plant, infection occurs mostly on lateral roots. Growth in the roots is slowed down and only few of the lateral root infections reach the stele of the main roots and the vascular tissues of the rhizome. The development of the pathogen is encouraged by actively growing root systems, light soils, and badly drained soils. The increase of aerenchyma under anaerobic conditions may favor the spread of the pathogen inside the roots (Aguilar et al., 1999). Resistant clones seem to limit the growth of the fungus in the roots, and this is probably determined by root metabolism. Vascular gel plugs, which form in infected xylem vessels, contribute to the localization process. Nevertheless, no differences were found between resistant and susceptible cultivars (Vandermolen et al., 1986). The role of timing, location, and extent of gel production has still to be considered. B.

Water Deficits

Musa plants are extremely sensitive to depletion of available soil water. The banana maintains its internal water status during drought by leaf folding and by closing stomata (Turner and Thomas, 1998; Thomas and Turner, 1998). Predictably, growth declines considerably as soil water decreases below two-thirds of the total available (Ke, 1979; Robinson and Bower, 1987). Root elongation of field-grown plants decreases

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considerably during the dry season (Lassoudie`re, 1978). A diminution of root hairs and lateral roots was further inferred from a reduction of P uptake during the dry season (Ssali, 1977). Controlled irrigation experiments indicate that at given depletion levels, water tended to be extracted deeper with increasing soil water depletion:
30% of water uptake occurred in the 30- to 50-cm layer with soil water deficits >50%, against 13% with water deficits of 16% (Robinson and Alberts, 1989). This suggests that increased water deficits and a high water loss in the superficial layers may have promoted root growth in the subsoil. Finally, root axes were found to increase slightly in number, length, and both horizontal and vertical spread with increasing soil water depletion (Krishnan and Shanmugavelu, 1980). Unfortunately, no further data were given to allow a clear analysis of this observation. Among the Musa genome groups, the ABB cooking banana have been said to be drought tolerant (Hahn et al., 1990). Failure to relate this tolerance to a deeper rooting with cv’s Cardaba and Fougamou suggests that the drought tolerance of the ABB cooking bananas would be the result of some physiological adaptation (Blomme, 2000). C.

Oxygen Deficiency

Anoxia is the most frequent chemical constraint acting on banana roots (Gousseland, 1983). Under anoxic conditions, roots present a soft consistency and a pale bluish-gray color (Delvaux, 1995). Oxygen deficiency is favored by soil compaction and waterlogging. The first condition, soil compaction, induces higher water saturation in the soil spaces and symptoms on roots (Champion and Sioussaram, 1970; Delvaux, 1995). The effects of soil compaction can also be attributed to mechanical impedance, and this should be exacerbated with the large roots of Musa. The second condition, waterlogging, restricts root growth and the number of axes, causes shallow root systems, and ultimately reduces plant size and yield (Stover, 1972; Lassoudie`re, 1978; Irizzary et al., 1980; Holder and Gumbs, 1983). The oxygen profile across banana roots growing in aerated solution was determined using microelectrodes. Very low oxygen concentrations were measured within the stele. Thus, a small reduction in external oxygen supply would cause the stele to become anoxic. Reduced transport of nutrients into the stele is one of the earliest responses of roots exposed to low oxygen supply. Root tips, which have high metabolic rates and oxygen needs, were very sensitive to oxygen deficiency (Aguilar et al., 1998). The

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presence of aerenchyma contributes to the adaptation of banana to waterlogging. Significant differences of root porosity were found among four genotypes, with the porosity of Williams (AAA) and Gros Michel (AAA) being about twice that of Sugar (AAB) and Goldfinger (AAAB) (Aguilar et al., 1999). Yet, the estimated physical resistance to gaseous diffusion along the root was similar for these cultivars under stagnant conditions, due to differences in root thickness among the cultivars examined. Further work is thus needed to explore the physiological significance of increased root porosity and its impact on the growth of Musa roots. D.

Low Soil pH

There is little available information on the effect of soil acidity on Musa. Crop response to liming varies widely with soil type. Classical effects of low pH such as Ca and Mg deficiencies and manganese toxicity seem to occur in banana as in many other crops, although to a much lower extent. Indeed, banana is able to produce high yields over a pH range of 4.5–8.5. Depending on the soil, acidity also leads to the release of exchangeable Al. In this respect, plantain seems highly tolerant as it survives aluminum saturation exceeding 70% of the cation exchange capacity (Rodrı´ guez-Garcı´ a et al., 1985; Delvaux et al., 1986; Lahav and Turner, 1989; Delvaux, 1995). The long-term effects of high soluble Al on root architecture were recently addressed by Rufyikiri and colleagues (2000b, 2001) for five different genotypes: the dessert banana Grande Naine (AAA), the East African beer bananas Igitsiri (AAA-EA) and Kayinja (ABB), and the plantains Agbagba and Obino l’Ewai (AAB). The plants were grown with or without aluminum in hydroponics, at temperature regimes that suited their respective cultivation areas. None of the genotypes displayed the impairment of root elongation most commonly observed under Al stress (see also Chapter 46 by Matsumoto in this volume). Effect of Al on root elongation involves also calcium deficiency that eventually occurs in acid soils, but which may have been prevented in the Ca-rich nutrient solution. Depending on the genotype, axis diameter and lateral root dry weight were severely reduced. The direct effect of Al on root architecture was thus a dramatic reduction of total root surface. The lower root system conductance contributed to the reduction of water transport and was largely responsible for the reduction of nutrient uptake. The water deficit caused a reduction of leaf area and ultimately of new root production

(Turner, 1995). The latter, indeed, can hardly be attributed to direct effects of Al, as root formation occurs in the upper part of the rhizome and translocation of Al from the roots to the shoot was very limited. The effect of Al on the synthesis of growth substances in root apices and its consequences on root formation have not been investigated in Musa. In most considered aspects (root and shoot growth, water, and nutrient uptake), the two plantains (AAB) were significantly less affected by Al than the other genotypes, and Kayinja (ABB) was the most sensitive genotype. Aluminum tolerance is thus not confined to either of the A or B genomes. Compared to the other genotypes, Kayinja had the highest root cation exchange capacity (CECR), and this was correlated with higher Al content in the root tissues. It will be interesting to investigate whether CECR could be a suitable marker for aluminum tolerance in Musa. E.

Mechanical Impedance

Mechanical impedance affects the root system size and root distribution of bananas (Hartman et al., 1928; Godefroy, 1969; Dorel, 1993; Robinson, 1996; Blomme, 2000). The soft root tips are very sensitive to penetration resistance and especially to soil structural discontinuities occurring at soil layer interfaces or caused by land cultivation (Delvaux and Guyot, 1989). Such conditions, often exacerbated by reduced soil macroporosity, restrict the volume of soil accessible to the large Musa roots and reduce the uptake capacity of the plant. The effect of reduced macroporosity on soil aeration also influences the availability and chemical form of the nutrients and makes it difficult to distinguish between the mechanical and the induced chemical effects of soil compaction. The lower uptake capacity of the root system also mediates a cascade of growth responses which ultimately loops back and further influences root development. Thus, long-term effects of soil compaction are difficult to interpret (see also Chapter 45 by Masle in this volume). The first direct effect of mechanical impedance is a reduction of the rate of root elongation, inferred from a reduction of the mean axis length and of the volume of soil explored (Irizarry et al., 1981; Dorel, 1993; Robinson, 1996; Blomme, 2000). The sensitivity of root elongation to mechanical impedance varied among six Musa genotypes but did not reveal consistent effect of genome constitution. Root length was reduced by as much as 56% in a tetraploid plantain hybrid but was barely influenced in the plantain Obino l’Ewai (AAB) and cooking banana Fougamou (ABB).

Banana Roots

The second direct effect of mechanical impedance is the induction of thicker root axes and lateral roots (Lassoudie`re, 1978; Gousseland, 1983; Dorel, 1993), which is likely to involve mechanical and physiological responses (Bennie, 1996; Chapter 45 by Masle in this volume). One may expect the uptake efficiency of roots to decrease with increasing diameter, especially in the case of tiny lateral roots. Indeed, if we assume that nutrients can be extracted within given distances from the root surface and that root volume reflects the amount of resources allocated to the root, then the ratio of useful soil volume to root volume decreases with increasing root radius. Both direct effects of mechanical impedance on root architecture tend therefore to decrease the uptake capacity and hydraulic conductance of the root system. The effects of mechanical impedance in Musa are reflected by many aspects of plant growth—e.g., root number, weight, and density; pseudo-stem circumference; leaf area; number of leaves; time to flowering; and bunch traits (Dorel, 1993; Blomme, 2000). It is most likely that the reduction of axis number is a consequence of the diminution of leaf area. It is hardly conceivable how soil pressure may exert a significant direct effect on root formation which occurs in the upper part of the rhizome. This may also explain why such effects are not always observed in short-term experiments or with all genotypes (Gousseland, 1983; Blomme, 2000). It is tempting to assume that roots of some genotypes maintained an even elongation rate under compacted conditions by exerting higher axial pressures on the facing soil at the cost of lower resource allocation to other parts of the plant. Both lateral root elongation and development were reduced by mechanical impedance (unpublished data). In particular, 67.6% of sand-grown root axes lacked laterals as compared with 11.1% for water-grown axes. This was consistent for five genotypes of the AA, AAA, and AAB genomes. However, lateral root density (number of primary laterals per centimeter of axis) was fairly similar. It has been postulated that interactions might exist between adjacent sites of lateral root initiation, which may prevent primordia from developing, as long as the distance separating them is less than a given threshold (Charlton, 1982; Draye et al., 1999). If the elongation of lateral-free sand root axes was inferior to that of sand roots bearing laterals, such interactions may explain the lower proportion of sand root axes bearing laterals. This, however, remains to be shown.

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F.

Suboptimal Temperatures

Suboptimal temperatures for Musa occur mainly in the subtropics. Available data on the effect of suboptimal temperature on the Musa root system consist of measurements of root extension rate, root dry weight, and nutrient uptake (Turner and Lahav, 1983; Robinson, 1987). Root extension has been observed in situ (root laboratory), where soil and air temperatures vary simultaneously. Extension was inhibited at soil temperatures < 11:58C and increased with increasing temperature up to 218C. Above 218C, root extension exhibited large variations, and its tendency to increase with increasing temperature was much less pronounced, suggesting that root systems were experiencing a heat stress (Robinson and Alberts, 1989). Soil temperatures > 388C cause severe damage to the root apex. Such temperatures would disrupt water uptake and subsequently favor oxygen deficiency in roots under high irrigation volumes (Ramcharan et al., 1995). Root dry weight has been measured in growth chambers, with similar soil and air average temperatures (Turner and Lahav, 1983). The maximum root dry weight was observed at a 25=188C air temperature regime, corresponding to a soil temperature of approximately
21:58C. Root dry weight decreased progressively at suboptimal temperatures. Compared with the 25=188C regime, growth was reduced by 75% at 17=108C and at 37=308C. In root heating tubes, root dry weight decreases with increasing soil temperatures from 28 to 438C (lower temperatures were not tested) (Ramcharan et al., 1995). The correspondance between the temperatures of maximum root extension and dry weight suggests that suboptimal root temperatures primarily affects root extension. This, however, should not preclude the possibility of additional effects of root temperature on root formation.

IV.

ARCHITECTURE AND GENETICS

Our understanding of the genetic background of root architecture in Musa has been hampered by the difficulties in root system assessment and by the difficulty of obtaining appropriate segregating material for genetic analysis. Recently, the International Network for the Improvement of Bananas and Plantains (http:// www.inibap.fr) started to coordinate international efforts toward creating segregated populations tagged with genetic marker data. Some of these populations will reveal genetic polymorphism for root architecture

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Draye

and allow detailed genetic analysis. As of today, the root polymorphism within the Musa germplasm remains virtually unexplored. The number of assessed genotypes within genome groups is often insufficient to tackle even the simplest issue of ploidy and genomespecific effects. Beyond such a depressing picture, the available data can be used to draw a preliminary list of genetically variable components of root architecture, along with the amplitude of this variation. A.

Controlled-Conditions Studies of Root Architecture

Precise and detailed data regarding the genetics of root architecture of Musa were obtained in hydroponics and in sand culture experiments under greenhouses or in growth chambers (Swennen et al., 1986; Stoffelen, 2000; Rufyikiri et al., 2001; Draye, unpublished data). The recourse to controlled conditions addresses the main limitations of Musa root system assessment in the field, viz., the limited number of traits which can be reasonably measured and the unrealistic sample size needed to compensate for the considerable variability of architectural data. It is also felt that the limited pertinence of controlled conditions for predicting field performance may be overcome by the characterization of genotypes by their phenotypic responses to major environmental factors. Hopefully, such response profiles may provide a basis for predicting the expression of root architecture in a broad range of environmental conditions. Until now, however, experiments have been performed in a single, at most two, environments. Most of the root traits studied under controlled conditions display an appreciable amount of genetic variability (given hereafter as ratios). Among eight cultivars of the AA, AAA, AAB, ABB, and AAAB genomes, the average length of axes and primary and secondary laterals varied respectively from 1 to 2, 3.6, and 3.3 (Stoffelen, 2000). The number of axes varied from 1 to 1.9, the density of primary and secondary laterals from 1 to 2.4 and 3, and the proportion of primary laterals bearing secondaries from 1 to 1.5 (see also Swennen et al., 1986; Rufyikiri et al., 2001). In sand and nutrient solution, the axis external link length (distance between the apex of an axis and the most distal lateral) varied from 1 to respectively 1.6 and 2.7. In addition, the lateral roots of Grande Naine (AAA) were of indeterminate growth. The nature of such variability (discrete/quantitative, degree of dominance, epistasis, dosage effect, pleiotropy) has not been investigated. A few traits seem to

be attributable to the genome constitution, although a clear conclusion would be premature in view of the low number of genotypes considered within genome groups. The percentage of primary laterals covered with secondary laterals was 68.5% for three AAB plantains, against 98.7% for four M. acuminata cultivars (Swennen et al., 1986). Similar values of 77% and 90% were observed for another AAB and for three M. acuminata genotypes (Stoffelen, 2000). The total root length of bananas seems to be twice that of plantains (Swennen et al., 1986). The root axes of two AAB plantains were fewer, shorter, and slightly thinner and produced higher dry weight of laterals per centimeter of axis than AAA and ABB cultivars (Rufyikiri et al., 2001). Sand and hydroponics experiments confirmed this result and suggested further that the higher dry weight of laterals of the two plantains result from larger lateral root diameters and higher densities. When comparing size-related characters, attention must be paid to the large dissimilarities between the growth of the different cultivars, which may obscure the comparison of their root architecture. In fact, the low number of axes of the AAB plantains may simply be a consequence of the low leaf area of these cultivars which grew less vigourously than the others (Rufyikiri et al., 2001; Stoffelen, 2000). Differences in root architecture between Musa genotype are the outcome of increase and/or reduction of the length of root axes, the density and length of primary and secondary laterals, and the proportions of axes and primary laterals bearing respectively primary and secondary laterals (Swennen et al., 1986; Stoffelen, 2000). It is therefore unlikely that synthetic architectural parameters lead to a better understanding of the genetic variability in the Musa germplasm. Whenever possible, parameters with established functional, physiological, or morphogenetic significance should be preferred. B.

Field Studies

Although field experiments provide direct insights of the root system in cropping conditions, their usefulness to predict root behavior in other soil and climatic conditions remains limited. The greatest advantage of fieldwork could be the possibility to conduct experiments over complete crop cycles. This may be of importance because root characteristics of mature plants cannot always be adequately estimated from observations on young plants (Blomme, 2000). The range of genetic variation for root traits under field conditions appears to be comparable with, or

Banana Roots

slightly smaller than, the one reported for controlled conditions. Twelve weeks after planting, the number of axes, total axes length, and root dry weight varied from 1 to respectively 1.6, 2, and 2.2 among eight cultivars (AAA, AAB, ABB, AAAB), while the density and length of primary and secondary laterals varied from 1 to 2.3 and 1.9 (Blomme, 2000). At shooting, the number, average, and maximum lengths of root axes varied from 1 to 1.3, 1.6, and 1.9 among nine cultivars (AA, AAB, AB, ABB) (Valsamma et al., 1987). Similar ranges were found with another three cultivars (AAA, AAB, ABB) (Chakrabatty, 1977; Mohan and Madhava Rao, 1984). The reduction of axes number, mean, and maximum length from shooting to harvest varied considerably more among cultivars. This variability reflected different strategies of root decay during the reproductive phase: the AA cultivar lost many axes, irrespective of their length, while one of the ABB lost few axes, preferentially the longest. The importance of dynamic studies of root architecture is hereby stressed. The effects of ploidy level and genome group have also been investigated. The axis diameter was found to be 5.35 mm for M. acuminata diploids, 6.45 mm for AAA, 7.46 for AAB, but only 7.35 for AAAA (Monnet and Charpentier, 1965; see also Blomme, 2000). The variability among the triploids was at least three times higher than that among the diploids. Root:shoot ratio for parent plants at flower emergence varied significantly among 27 genotypes (Blomme, 2000). However, the variations of the ratio for the whole plant were much lower than for the parent plant, indicating that genotypes differ most by the partitioning of the dry matter between the different plant parts. Nonetheless, multiple regression revealed that shoot growth parameters could account for 90% or more of the variability for important root traits among the genotypes. If the regression model and parameters would turn out to be applicable to a broader range of genotypes, such results may allow indirect assessments of the root system based on simple shoot observations.

V.

FUTURE RESEARCH

The genus Musa is far from being suited to address leading topics of genetics and root architecture. However, having a single type of root axis, unlike other important cultivated monocots, bananas and plantains lend themselves to a dynamic modeling of their root architecture, a step toward an integrated

273

and rational understanding of crop growth and performance. The experience accumulated for over a century of research provides a framework for a developmental model of Musa root architecture, comprising root formation, branching, elongation, and senescence. Each of those should be expressed as a function of genotype, plant status, root age, and rhizospheric conditions. In principle, a model could be extended to take account of soil space–time heterogeneity and genetic correlations among the various root parameters, and could be ultimately connected to data on root function. While observing architectural differences among genotypes or treatments, one should always attempt to connect these differences to root function. In several instances, there appears to be an excess of roots, the significance of which remains unexplored. In addition, root construction costs have been assumed to be proportional to root weight, while it is likely that the amount of photosynthetic energy required to form a unit of root length increases in stressful conditions. With regard to crop performance, the assurance of a desired root behavior depends on our ability to account for root plasticity. The most significant causes of root stress in Musa have been identified and the nature of their architectural consequences are being clarified. However, the effects of these factors on the late development of lateral roots remain unknown. In the context of Musa improvement, it may be wise to establish standard root system descriptors accounting for root plasticity and to be evaluated in defined and rigorously controlled conditions. Given the huge variability of root development under field conditions, this may be the only way to bring together the outcome of different research projects. With the exception of a few traits such as the determinate/indeterminate type of growth of lateral roots, the absence of a particular type of roots or the abnormal characteristics of root mutants, most of the natural genetic variation for root traits is of a quantitative nature. Unraveling these variations in Musa should start by screening germplasm accessions, especially fertile diploids which lend themselves to the constitution of segregating populations. Care should be taken when interpreting the correlation between root traits among accessions, as such correlations reveal the combined effects of correlated selection, genetic correlations (pleiotropy and tight linkage), and correlations with aboveground or sucker characters. Segregating populations should be used to estimate true genetic parameters such as genetic correlation between various components of root architecture, and to initiate gene/ QTL mapping where it seems reasonable to do so.

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Many questions should be answered before we can embark on a rational improvement of root architecture by backcrossing of interesting alleles into elite germplasm. A few recommendations can, however, be formulated to answer the call of the breeders (Jones, 1994). A root system is a population of roots of different types and age that evolve dynamically according to root formation and root senescence. The overall capacity of the root system to keep pace with the various demands depends on the ability of the plant to maintain an appropriate root population. Whenever possible, this should be achieved by the use of appropriate cultivation practices (Delvaux and Guyot, 1989). The yield decline of plantains, for example, does not occur on backyard or compound gardens (Braide and Wilson, 1980). The genetic improvement of the root system should come second in order and may proceed by increasing root formation or decreasing root senescence. In this regard, Blomme (2000) proposed that a regulated suckering would be the most reasonable breeding objective for the time being. ACKNOWLEDGMENTS We are especially grateful to Dr. David Turner for his thorough reading and invaluable advices on the manuscript, and to Ste´phane Declerk who kindly reviewed the section on mycorrhizae. This work was supported by postdoctoral research associate fellowships from the Universite´ catholique de Louvain (Belgium) and from the Fonds National de la Recherche Scientifique (Belgium). REFERENCES Acquarone P. 1930. The roots of Musa sapientium L. United Fruits Co Res Bull 26:831–868. Aguilar EA, Turner DW, Gibbs DJ, Sivasithamparam K, Amstrong W. 1998. Response of banana (Musa sp.) roots to oxygen deficiency and its implication for Fusarium wilt. Acta Hort 490:223–228. Aguilar EA, Turner DW, Sivasithamparam K. 1999. Aerenchyma formation in roots of four banana (Musa spp.) cultivars. Sci Hort 80:57–72. Araya M, Vargas A, Cheves A. 1998. Changes in distribution of roots of banana (Musa AAA cv. Valery) with plant height, distance from the pseudostem, and soil depth. J Hort Sci Biotech 73:437–440. Araya M, Vargas A, Cheves A. 1999. Nematode distribution in roots of banana (Musa AAA cv. Valery) in relation to plant height, distance from the pseudostem and soil depth. Nematology 1:711–716.

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275 Meloidogyne incognita and Glomus mosseae in banana. Plant Soil 196:27–35. Jones DR. 1994. Report on the meeting. Banana and Plantain Breeding: Priorities and Strategies. Proceedings of the first Meeting of the Musa Breeders’ Network, 2–3 May 1994, La Lima, Honduras. Ke LS. 1979. Studies on the physiological characteristics of bananas in Taiwan. II. Effects of soil moisture on some physiological functions and yield of the banana plant. J Agric Assoc China 108:11–23. Knight S. 1988. The phosphorus nutrition of bananas with special emphasis on V.A. mycorrhizal fungi and the effect of nitrogen. Banana Newslett 11:18. Krishnan BM, Shanmugavelu KG. 1980. Effect of different soil moisture depletion levels on the root distribution of banana cv. Robusta. South Indian Hort 28:24–25. Kwa M. 1993. Architecture, morphogene`se et anatomie de quelques cultivars de bananiers. PhD dissertation, Universite´ de Montpellier II, Montpellier, France. Lahav E, Turner DW. 1989. Banana nutrition (IPI-Bulletin 7). Berne, Switzerland: International Potash Institute. Lassoudie`re A. 1971. La croissance des racines du bananier. Fruits 26:501–512. Lassoudie`re A. 1978. Quelques aspects de la croissance et du de´veloppement du bananier ‘Poyo’ en Coˆte d’Ivoire. Le syste`me radical. Fruits 33:314–338. Lassoudie`re A. 1980. Matie`re ve´ge´tale e´labore´e par le bananier Poyo depuis la plantation jusqu’a` la re´colte du deuxie`me cycle. Fruits 35:405–446. Lavigne C. 1987. Contribution a` l’e´tude du syste`me racinaire du bananier. Mise au point de rhizotrons et premiers re´sultats. Fruits 42:265–271. Laville E. 1964. Etude de la mycoflore des racines du bananier ‘Poyo’. Fruits 19:435–449. Lin C-H, Chang DCN. 1987. Effect of three Glomus endomycorrhizal fungi on the growth of micropropagated banana plantlets. T Mycol Soc R O C 2:37–45. Lin M-L, Fox RL. 1987. External and internal P requirements of mycorrhizal and non-mycorrhizal banana plants. 10th International Plant Nutrition Colloquium, College Park, MD, pp 1341–1348. Mangin L. 1882. Origine et insertion des racines adventives. Ann Sci Nat Bot Ser 6, 14:216–363. Mateille T. 1994. Comparative host tissue reactions of Musa acuminata (AAA group) cvs Poyo and Gros Michel roots to three banana-parasitic nematodes. Ann Appl Biol 124:65–73. McGahan MW. 1961. Studies on the seed of banana. II. The anatomy and morphology of the seedling of Musa balbisiana. Am J Bot 48:630–637. Mohan NK, Madhava Rao VN. 1984. The effect of plant density on the banana root system. South Indian Hort 32:254–257.

276 Mohan NK, Madhava Rao VN. 1985. Determination of active zone in banana using radioactive phosphorus. South Indian Hort 33:217–220. Monnet J, Charpentier JM. 1965. Le diame`tre des racines adventives primaires des bananiers en fonction de leur degre´ de polyploı¨ die. Fruits 20:171–173. Moreau B, Le Bourdelle`s J. 1963. Etude du syste`me racinaire du bananier ‘Gros Michel’ en Equateur. Fruits 18:71– 74. Ortiz R, Vuylsteke D. 1994. Genetics of apical dominance in plantain (Musa spp., AAB group) and improvement of suckering behavior. J Am Soc Hortic Sci 119:1050– 1053. Ortiz R, Vuylsteke D. 1995. Inheritance of dwarfism in plantain (Musa spp, AAB group). J Plant Breeding 114:466–468. Pinochet J. 1977. Occurrence and spatial distribution of rootknot nematodes on bananas and plantains in Honduras. Plant Dis Rep 61:518–520. Ramcharan C, Ingram DL, Nell TA, Barrett JE. 1995. Interactive effects of root-zone temperature and irrigation volume on banana vegetative growth in two environments. Fruits 50:225–232. Riopel JL. 1960. Studies on development and wound responses of the roots of Musa ‘Gros Michel’ in relation to Panama disease. PhD dissertation, Harvard University, Cambridge, MA. Riopel JL. 1966. The distribution of lateral roots in Musa acuminata ‘Gros Michel’. Am J Bot 53:403–407. Riopel JL, Steeves TA. 1964. Studies on the roots of Musa acuminata ‘Gros Michel’ 1. The anatomy and development of main roots. Ann Bot 28:475–494. Robin J, Champion J. 1962. Etude des e´missions des racines de la varie´te´ du bananier Poyo. Fruits 17:93–94. Robinson D. 1994. The responses of plants to non-uniform supplies of nutrients. New Phytol 127:635–674. Robinson JC. 1985. Root depth in bananas. CSFRI Information B 155:6–8. Robinson JC. 1987. Root growth characteristics in banana. CSFRI Information B 183:7–9. Robinson JC. 1996. Bananas and Plantains. Wallingford, Oxon, UK: CAB International. Robinson JC, Alberts AJ. 1989. Seasonal variations in the crop water-use coefficient of banana (cv. ‘Williams’) in the subtropics. Sci Hort 40:215–225. Robinson JC, Bower JP. 1987. Transpiration characteristics of banana leaves (cultivar ‘Williams’) in response to progressive depletion of available soil moisture. Sci Hort 30:289–300. Robinson JC, Bower JP. 1988. Transpiration from banana leaves in the subtropics in response to diurnal and seasonal factors and high evaporative demand. Sci Hort 37:129–143. Rodrı´ guez-Garcı´ a J, Rivera E, Abrun˜a F. 1985. Crop response to soil acidity factors in ultisols and oxisols

Draye in Puerto Rico. XIV. Plantains and bananas. J Agric Univ Puerto Rico 69:377–383. Rufyikiri G, Declerck S, Dufey JE, Delvaux B. 2000a. Arbuscular mycorrhizal fungi might alleviate aluminium toxicity in banana plants. New Phytol 148:343– 352. Rufyikiri G, Nootens D, Dufey JE, Delvaux B. 2000b. Effect of aluminium on growth and uptake of bananas (Musa spp.) cultivated in acid solutions. I. Growth and chemical composition. Fruits 55:367–379. Rufyikiri G, Dufey JE, Nootens D, Delvaux B. 2001. Effect of aluminium on growth and uptake of bananas (Musa spp.) cultivated in acid solutions. II. Water and nutrient uptake. Fruits 56:5–16. Simmonds NW. 1959. Experiments on the germination of banana seeds. Trop Agric 36:259–273. Simmonds NW. 1966. Bananas. 2nd ed. London: Longman. Skutch A. 1932. Anatomy of the axis of the banana. Bot Gaz 93:233–258. Ssali H. 1977. Root activity of bananas during wet and dry seasons as measured by 32P uptake. East Afr Agr For J 42:304–308. Stoffelen R. 2000. Early screening of Eumusa and Australimusa bananas against root-lesion and rootknot nematodes. PhD dissertation, Katholieke Universiteit Leuven, Leuven, Belgium. Stover RH. 1972. Banana, Plantain and Abaca Diseases. Kew, Surrey, UK: Commonwealth Mycological Institute. Stover RH, Simmonds NW. 1987. Bananas. 3rd ed. Essex, UK: Longman Scientific and Technical. Summerville WAT. 1939. Root distribution of the banana. Queensland Agric J 52:376–392. Swennen R. 1984. A physiological study of the suckering behavior in plantain (Musa cv. AAB). PhD dissertation, Faculty of Agriculture, Katholieke Universiteit Leuven, Leuven, Belgium. Swennen R, De Langhe EA. 1985. Growth parameters of yield of plantain (Musa cv. AAB). Ann Bot 56:197– 204. Swennen R, Wilson GF. 1983. La stimulation du developpement du rejet baı¨ onnette du bananier plantain (Musa spp. groupe AAB) par application de giberrelline (GA3). Fruits 38:261–265. Swennen R, Wilson GF, De Langue EA. 1984. Preliminary investigation of the effect of giberrellic acid (GA3) on sucker development in plantain (Musa cv. AAB) under field conditions. Trop Agric 61:253–256. Swennen R, De Langhe E, Janssen J, Decoene D. 1986. Study of the root development of some Musa cultivars in hydroponics. Fruits 41:515–524. Swennen R, Wilson GF, Decoene D. 1988. Priorities for future research on the root system and rhizome in plantains and bananas in relation with nematodes and the banana weevil. Proceedings of the Workshop on Nematodes and the Borer Weevil in Bananas:

Banana Roots Present Status of Research and Outlook, Bujumbura, Burundi, pp 91–96. Swennen R, Vuylsteke D, Ortiz R. 1995. Phenotypic diversity and patterns of variation in West and Central African plantains (Musa spp., AAB group Musaceae). Econ Bot 49:320–327. Teisson C. 1970. Condution vers le bananier d’e´le´ments mine´raux absorbe´s par son rejet. Fruits 25:451–454. Thomas DS, Turner DW. 1998. Leaf gas exchange of droughted and irrigated banana cv. ‘Williams’ (Musa spp.) growing in hot, arid conditions. J Hort Sci Biotech 73:419–429. Turner DW. 1970. Banana roots. Agric Gaz N S W 81:472– 473. Turner DW. 1972. Banana plant growth. 1. Gross morphology. Aust J Exp Agr 12:209–215. Turner DW. 1995. The response of the plant to the environment. In: Gowen S, ed. Bananas and Plantains. London; Chapman and Hall. Turner DW, Barkus B. 1981. Some factors affecting the apparent root transfer coefficient of banana plants (cv. ‘Williams’). Fruits 36:607–613. Turner DW, Lahav E. 1983. The growth of banana plants in relation to temperature. Aust J Plant Physiol 10:43–53. Turner DW, Lahav E. 1985. Temperature influences nutrient absorption and uptake rates of bananas grown in controlled environments. Sci Hort 26:311–322. Turner DW, Thomas DS. 1998. Measurements of plant and soil water status and their association with leaf gas exchange in banana (Musa spp.): a laticiferous plant. Sci Hort 77:177–193. Umesh KC, Krishnappa K, Bagyaraj DJ. 1989. Interaction of Radopholus similis with Glomus fasciculatum in

277 banana. Twenty-Eighth Annual Meeting of the Society of Nematologists, University of California, Davis, CA, pp 592–593. Valsamma M, Arvindakshan M, Valsalakumari PK, Parameswaran NK. 1987. Root distribution of banana cultivars under rainfed condition. South Indian Hort 35:334–338. Vandermolen GE, Labavitch JM, Devayje. 1986. Fusariuminduced vascular gels from banana roots—a partial chemical characterization. Physiol Plant 66:298–302. Vuylsteke D, Ortiz R, Ferris S. 1993a. Genetic and agronomic improvement for sustainable production of plantain and banana in sub-Saharan Africa. Afr Crop Sci J 1:1–8. Vuylsteke D, Swennen R, Ortiz R. 1993b. Registration of 14 improved tropical Musa plantain hybrids with black sigatoka resistance. HortScience 28:957–959. Walmsley D, Twyford IT. 1968. The translocation of phosphorus within a stool of Robusta bananas. Trop Agric 45:229–233. Walmsley D, Twyford IT. 1968. The zone of nutrient uptake by the Robusta banana. Trop Agric 45:113–117. Weckx G. 1982. Invloed van mulching en minerale bemesting op het wortelstelsel van plantaan en banaan. Dissertation, Katholieke Universiteit Leuven, Leuven, Belgium. Winderickx D. 1985. Invloed van pH en temperatuur op het wortelstelsel van banaan en plantaan. Dissertation, Katholieke Universiteit Leuven, Leuven, Belgium. Yano Melo AM, Saggin OJ, Lima JM, Melo NF, Maia LC. 1999. Effect of arbuscular mycorrhizal fungi on the acclimatization of micropropagated banana plantlets. Mycorrhiza 9:119–123.

17 Molecular Root Bioengineering Marcel Bucher Institute of Plant Sciences, Plant Biochemistry and Physiology, Zurich, Switzerland

I.

INTRODUCTION

tal factors such as nutrient availability, interacting symbiontic organisms, or pathogens will be given. Finally, recently published promising applications in molecular root bioengineering will be presented.

The root system serves many tasks; it anchors the plant and absorbs water and minerals from the soil and delivers certain growth regulators. Roots also have the remarkable ability to secrete a vast array of low- and high-molecular-weight molecules into the rhizosphere in response to biotic and abiotic stresses. The underground growth habit and the lack of suitable experimental systems to approach roots directly made root research problematic in the past. In the past 10 years, progress in root biology has been made by using molecular-genetic tools, thus offering novel perspectives for both the understanding and exploitation of root processes. In molecular root bioengineering genetic engineering principles are applied to problems involving root cells, tissues, or molecules that exert biological activity at the level of the plant root. A prerequisite for successful application of molecular-genetic tools toward modification of root functions is the characterization of tissue-specific gene expression, the isolation of suitable promoters, and the characterization of gene function. The expression of endogenous or heterologous genes can then be directed in a precise spatial and temporal manner to modify and improve root properties. This chapter attempts to give insight into molecular approaches in root research and to point out opportunities for root bioengineering. It includes an overall view of standard and modern methods suitable to investigate root-specific gene expression. Examples for the control of root gene expression by environmen-

II.

APPROACHES TO CLONE ROOTSPECIFIC GENES

Differential and subtractive hybridization techniques are two classical methods in identifying mRNAs in comparative studies. Differential screening routinely allows the detection and cloning of abundant mRNAs. For the identification of genes preferentially expressed in roots, cDNA probes originating from leaf and root mRNA are used to differentially screen a root cDNA library from the same species. This approach has led to the cloning of the root-specific tobacco gene TobRB7 (Conkling et al., 1990). This gene shares sequence homology with genes encoding major intrinsic proteins (MIPs) that function as water or ion channels across membranes. In another approach, a tomato cDNA library from distinct root cells, i.e., the root hairs, was synthesized. A differential screening resulted in the cloning of several cDNAs the corresponding transcripts of which were found to be highly abundant in root hairs and to encode extensinlike proteins (Bucher et al., 1997, 2001). Extensin genes abundantly expressed in root hairs were also cloned from other species, such as Vigna unguiculata (ArsenijevicMaksimovic et al., 1997), suggesting a structural role 279

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for extensins in root hair elongation. In contrast to differential hybridization, subtractive hybridization is more sensitive and can thus detect fairly rare mRNAs (Rodriguez and Chader, 1992). This technique has allowed the cloning of genes that are differentially regulated during nodule development in legume roots (Kouchi and Hata, 1993; Cook et al., 1995). A more recent method, differential display (Liang and Pardee, 1992), is a more elaborate technique which allows cloning of rare mRNAs and mRNAs that are expressed in just a small set of cells or in a temporal manner. It was used, for example, to clone marker genes that are induced within 1–3 h after the application of the nodulation factor to Vicia sativa (Heidstra et al., 1997). Microarrays prepared by high-speed robotic printing of DNAs on glass grids now enable researchers to monitor the expression of up to several thousand genes in parallel in different organs, cell types, or under different environmental conditions (Schena et al., 1995; Schenk et al., 2000). With the imminent completion of the sequencing of both Arabidopsis thaliana and rice genomes, DNA microarray analysis will allow us to obtain a comprehensive view of the genes that define a root and will thus broaden our understanding of root biology. Two-dimensional gel electrophoresis allows the comparison of protein populations from several sources and the subsequent isolation of abundant and differentially expressed products. The use of oligonucleotides based on partial amino acid sequence data of tryptic digests of such a protein enables the cloning of its respective cDNA via RT-PCR or library screening. This approach led to the cloning of a developmentally regulated pathogenesis-related gene which was expressed in the rhizodermis of pea and during embryo development (Mylona et al., 1994). The improvement of two-dimensional electrophoresis now permits the reproducible separation of up to 2000 proteins on a single gel. Such gel-separated proteins can be rapidly identified by mass spectrometric (MS) methods and N-terminal Edman sequencing. If genomic information is also available, such analyses permit the systematic identification of the protein complement of a genome, the proteome. Proteomics tools therefore bridge the gap between genomic sequence information and the actual protein population in a specific tissue, cell, or cellular compartment (Shevchenko et al., 1996; Peltier et al., 2000). Thus, it could assist the targeted search for tissue-specific genes and their promoters. A hypothesis-driven approach toward the identification of root-specific genes includes the cloning of

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genes involved in typical root processes, such as nutrient and water uptake, or root development. The expression of such genes may even be restricted to specific root tissues. The tomato root hair-specific cDNA library (Bucher et al., 1997) has been screened by several research teams to identify nutrient channels or transporter proteins, such as the potassium channel LKT1 (Hartje et al., 2000), several nitrate and ammonium transporters (Lauter et al., 1996; Von Wire´n et al., 2000), and a phosphate transporter (Daram et al., 1998). The transcripts corresponding to the respective genes were found to be highly abundant in root hair cells, but occasionally also in other tissues of the plant, thus corroborating the general function of root hairs in nutrient acquisition. Root development in Arabidopsis is amenable to genetic analysis, because of the many advantages of this organism (Benfey and Schiefelbein, 1994). In the past 10 years, a rich collection of transfer DNA (TDNA)-tagged mutants has been described which have defects in genes governing organ formation, meristem activity, cell differentiation, and responses to environmental conditions (Schiefelbein and Benfey, 1991; Benfey and Schiefelbein, 1994; Azpiroz-Leehan and Feldmann, 1997). The mutated genes carrying the inserted chimeric T-DNA construct as a result of Agrobacterium-mediated TDNA transfer during transformation can then be cloned using molecular-genetic tools (Dilkes and Feldmann, 1998). The genes regulating root development and cell fate are expected to be expressed in young tissues of the root. However, most of such genes identified so far are not expressed in a strictly root-specific manner. Transcripts from the ROOT HAIRLESS 1 and ROOT HAIR DEFECTIVE 3 genes, which are required for root hair initiation and expansion, respectively (Wang et al., 1997; Schneider et al., 1998), are both detectable throughout the plant. Similarly, expression of the SCARECROW gene, which is involved in establishing radial patterning in the root (Di Laurenzio et al., 1996), is not restricted to the root. The WEREWOLF (WER) gene determines the fate of epidermal cell differentiation in the root and hypocotyl of Arabidopsis (Lee and Schiefelbein, 1999). The highest steady-state concentration of WER RNA was found in root tips, with a lower abundance in hypocotyl/cotyledon RNA. In summary, numerous molecular-genetic methods are now available to clone root-specific genes. It is the complexity of the scientific problem to be solved that determines the approach to be taken.

Molecular Root Bioengineering

III.

REGULATION OF ROOT-PREFERRED GENE EXPRESSION

Plants are sessile organisms and as such their survival is crucially dependent on rapid adaptation to environmental changes. In the soil, these changes may relate to soil humidity, oxygen, or CO2 stresses, to nutrient availability, and to the presence of microorganisms. During soil flooding, oxygen levels in the rhizosphere decrease rapidly and hypoxic or even anoxic conditions occur in the submerged tissues (see Chapter 42 by Armstrong and Drew in this volume). Such changes normally result in a rapid modification of gene expression including the upregulation of anaerobiosis-specific proteins (Sachs et al., 1980). Hypoxic induction of the genes is often not unique to roots and also occurs in aerial parts of the plant. To understand the plant’s response to environmental stress and to develop strategies for the genetic improvement of plant species which are normally sensitive to stress, such as flooding, it seems advisable to carefully study the physiological and molecular responses of resistant species under both defined laboratory conditions and the more complex situation in the natural habitat (Bucher and Kuhlemeier, 1993; Bucher et al., 1996; Drew, 1997). This approach allows an assessment of the role of proposed adaptation mechanisms involved in stress tolerance under natural conditions and identification of the genes playing a key role in the genetic network determining the stress response. The key regulatory genes can then be transferred to other, stress-sensitive species by genetic engineering leading to the generation of transgenic plants expected to exhibit increased stress resistance or serving as a model system for further improvement (Bucher et al., 1994; Tadege et al., 1998; Saijo et al., 2000). A.

Regulation of Root Gene Expression by Nutrients

Plant roots are constantly mining the soil for nutrients to sustain plant growth, development, and reproduction. Efficient nutrient acquisition is determined by an interplay between the intrinsic developmental and metabolic programs and external biotic and abiotic stimuli (Schiefelbein and Somerville, 1990; Lynch, 1995; see also Chapter 34 by Glass in this volume). A well-studied external stimulus is nitrate concentration. The first committed step in nitrate assimilation is its uptake into the root. Genes encoding nitrate uptake transporters in tomato are induced in root hairs upon exposure to nitrate (Lauter et al., 1996). In

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Nicotiana plumbaginifolia, the genes for the three successive steps in nitrate assimilation, i.e., transport into root cells, nitrate, and nitrite reduction, were shown to be coordinately induced in roots by nitrate after exposure to nitrogen starvation (Krapp et al., 1998). Nitrate transporter transcript concentrations were highest in roots. Similar results were obtained with genes involved in nitrate reduction in plant roots (Miyazaki et al., 1991; Aoki and Ida, 1994; Ritchie et al., 1994). It is assumed that plant cells are capable of sensing external and internal nitrate concentrations. Root branching is probably modulated by opposing signals reflecting the plant’s internal N status and the external supply of nitrate (Zhang et al., 1999). In this scenario, the product of the nitrate-inducible and root-specific ANR1 gene for a putative transcription factor acts as a putative component of the signal transduction pathway linking external nitrate to increased lateral root proliferation (Zhang and Forde, 1998). Transport of sulfate and phosphate clearly responds to the sulfur and phosphorus status of the plant. Expression of the sulfate and phosphate transporter genes is repressed by a sufficient S or P supply. Upon S or P starvation, mRNAs of the respective transporter genes rapidly accumulate due to derepression of the genes, which is consistent with a role of the encoded proteins in nutrient uptake by the roots (Smith et al., 1995; Takahashi et al., 1997; Raghothama, 1999; Smith et al., 2000). Other structural genes for sulfate assimilation in Arabidopsis are inducible by sulfate starvation (Takahashi et al., 1997). Plant genes serving functions in phosphate mobilization in the soil environment are induced by phosphate starvation of roots, such as ribonucleases (Kock et al., 1995; Dodds et al., 1996; Bariola et al., 1999). However, their expression is not confined to the root tissue. Similar to S or P transporters, an Arabiodpsis zinc transporter is strongly induced under zinc deficiency in a root-preferred manner (Grotz et al., 1998). Iron uptake by dicotyledonous plants, involves an initial stage where Fe3+ is reduced to Fe2+ by the action of a ferric-chelate reductase. The gene for such a reductase is induced in iron-deficient roots of Arabidopsis (Robinson et al., 1999). These examples demonstrate that genes involved in nutrient transport are preferentially expressed in roots and may respond to changes in nutrient availability. B.

Gene Regulation in the Mycorrhizal Symbiosis

Approximately 80% of the terrestrial plants undergo a mutually beneficial interaction with arbuscular-mycor-

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rhizal (AM) fungi. This symbiosis is advantageous for the plant mainly during P or Zn nutrient limitation. Nutrients are taken up by AM fungal hyphae in soil areas distant from the root surface and are transported to symbiotic structures within the root cortex, where they are taken up by the vascular plant in exchange for carbohydrate delivery to the fungus (Smith and Read, 1997). The molecular basis for this symbiosis is largely not understood. Recent investigations, however, begin to shed light on molecular mechanisms governing mycorrhizae formation (Varma and Hock, 1999), and the first plant genes, either up- or downregulated in the symbiotic interaction, have been identified. Three genes are not or are only weakly expressed in Medicago truncatula roots prior to formation of the symbiosis but are significantly induced following colonization by Glomus versiforme (Van Buuren et al., 1999). One gene shares homology to human translation initiation factor 3 (eIF3). The two others encode a xyloglucan endotransglycosylase-related protein and a putative arabinogalactan protein (AGP). Apparently, both proteins contribute to cell wall structure and are involved in alterations in the extracellular matrix of the cortical cells following colonization by mycorrhizal fungi. Two mycorrhiza-specific class III chitinase genes are induced in mycorrhizal M. truncatula roots and are considered to play an important role during the establishment of arbuscular mycorrhiza in this plant (Salzer et al., 2000). Chitinases possibly degrade fungal elicitors and may thus contribute to a suppression of the hypersensitive response (HR). HR normally occurs during interaction with incompatible pathogens and leads to rapid cell death in the infected tissue to reduce the spreading of the pathogen. A more complex regulatory mechanism governs expression of Mt4, a M. truncatula gene whose expression is enhanced during P deprivation, but strongly reduced upon colonization of the root with AM fungi (Burleigh and Harrison, 1997). Furthermore, expression is only observed when plants are grown under nitrogen-sufficient conditions. The function of Mt4 is unknown. Thus, several putative components of the genetic network governing the formation of the AM symbiosis have been identified. Many more genes that are involved in this interaction will hopefully be cloned in the future, providing opportunities for further fruitful investigations into the genetic components of AM symbiosis development. The understanding of the molecular mechanisms underlying the AM symbiotic interaction could finally lead to strategies resulting in improved colonization of crop roots by AM fungi and hence an increased nutrient transfer into crop plants.

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C.

Gene Regulation During Nodule Formation

Leguminous species are capable of forming symbiotic associations with rhizobia (see Chapter 47 by Vance in this volume). This interaction leads to the formation of nodules, specialized root structures in which nitrogen fixation occurs. Nodules are formed through a series of unique developmental processes which are the result of Rhizobium species-dependent alteration of plant gene expression within root cells. During infection and nodule formation, several plant genes are activated. The study of nodulin gene expression may provide insight into root-nodule development and the mechanism of communication between bacteria and host plant. The pea cDNA clone PsENOD12 represents a gene involved in the infection process early during the establishment of the symbiosis (Scheres et al., 1990). ENOD12 expression is induced by infection with Rhizobium leguminosarum bv. viciae or exposure to the respective nodulation (Nod) factor. Nod factors are lipochitooligosaccharide signals of rhizobial origin that elicit key symbiotic developmental responses in the host legume root. In situ hybridization experiments with the M. truncatula ortholog of ENOD12 have shown that, within the indeterminate nodule, transcription of the MtENOD12 gene begins in cell layers of meristematic origin situated more central than the periferal infection zone, suggesting that these cells are undergoing preparation for bacterial infection (Pichon et al., 1992). Thus, the expression pattern of ENOD12 may prove to be a useful marker for early plant responses to Nod factors. Many of the nodulin genes seem to be nodule specific, but several of them are also expressed in plant organs other than the nodule (Govers et al., 1991). Numerous novel proteins have been identified, expressed both early and late during nodulation, among them proteins of unknown function (Kouchi and Hata, 1993), proline-rich proteins (Long, 1996), glycine-rich proteins (Schroeder et al., 1997), or a polygalacturonase (Munoz et al., 1998). A thorough understanding of the genetic control governing nodule formation and dinitrogen fixation may in the future allow the transfer of the capacity to fix dinitrogen to plant species other than legumes. There are a large number of structural and functional similarities between AM and nodule symbiosis. For example, both AM development and nodule organogenesis take place in the cortical parenchyma cells. In both interactions, a plant-derived membrane delineates the physiologically active interface between the

Molecular Root Bioengineering

symbionts in both systems. Several nodulation-defective mutants are also defective in the establishment of the AM symbiosis (Catoira et al., 2000). There is evidence accumulating that genes that are induced during AM formation are also expressed during nodule formation (Gianinazzi-Pearson, 1996; Albrecht et al., 1998). Thus, there probably exist common regulatory mechanisms that are involved in both the establishment of AM and nodule symbiosis. D.

Differential Gene Expression in Nematode-Induced Feeding Structures

Root knot nematodes and sedentary cyst nematodes are obligate plant parasites, obtaining nutrients only from the cytoplasm of living root cells. During root infection, they induce development of a specialized feeding structure within the vascular cylinder (see Chapter 51 by Koltai et al. in this volume). Root knot nematodes feed from multinucleate giant cells developed by the expansion of cambial cells within the differentiating vascular cylinder, whereas cyst nematodes feed from a syncytium which results from protoplast fusion after partial dissolution of the cell walls of neighboring cells (Jung et al., 1998). Feeding-site formation results from a complex interaction between the pathogen and the host plant in which the nematode alters patterns of plant gene expression within the cells destined to become the feeding site (Sijmons et al., 1994; Williamson and Hussey, 1996). Except for natural resistance genes, plants have probably not evolved genes specifically expressed only in pathogen-induced tissue. This may be the reason why all the nematode-inducible genes detected so far are not expressed in a root-specific manner, but rather are expressed in various tissues, with highest activities in roots. Differential cDNA screening led to the identification of Lemmi9, a tomato gene that is upregulated in giant cells after Meloidogyne incognita infection (Van der Eycken et al., 1996). Lemmi9 is related to cotton Lea14-A, a putative desiccation protectant. In related work, differential display allowed the cloning of 13 Arabidopsis cDNA clones the corresponding transcripts of which were more abundant in infected and 11 transcripts that were more abundant in the uninfected root sections (Hermsmeier et al., 2000). For two of the respective genes, in situ hybridization experiments confirmed the changes in mRNA abundance in Arabidopsis roots predicted by the differential display analyses. The tobacco gene TobRB7 encoding a putative water channel protein is upregulated during feeding site development (Opperman et al., 1994). The

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protein probably facilitates the passage of water molecules to the feeding sites, which are characterized by a high metabolic rate. Expression of a potato gene encoding a catalase, Cat2St, is locally induced upon infection with the cyst nematode Globodera pallida being highest at the adult stage of the parasite. Moreover, Cat2St expression is systemically induced in uninfected organs of infected plants. Localized and systemic induction of Cat2St has also been observed upon root knot nematode and in root bacteria infections (Niebel et al., 1995). Thus, straightforward detection of transcript abundance of the genes described so far did not result in the identification of nematodespecific expression of plant genes and more refined approaches were needed.

IV.

TISSUE-SPECIFIC PROMOTERS

Once a candidate gene has been identified, genomic fragments from the promoter region can be cloned. To visualize promoter activity, the promoter fragment is fused to the -glucuronidase (GUS) or the jellyfish green fluorescent protein (GFP) reporter gene (Jefferson et al., 1987; Haseloff et al., 1997). The chimeric gene is then introduced into plants, and promoter activity is finally assessed by histological analysis of reporter gene expression in the transgenic plant. A combination of RNA expression analysis and promoter-reporter gene studies provides detailed information on the spatial and temporal expression pattern of the respective gene and, in combination with sequence data, allows predictions of its presumed function. Vector molecules used for the efficient transformation of higher plants contain genes to be transferred bordered by the left and right T-DNA border sequences of the vir region of the Agrobacterium tumefaciens Ti (tumor-inducing) plasmid. This synthetic TDNA integrates into the nuclear genome of transformed plants by Agrobacterium-mediated T-DNA transfer. T-DNA carrying the GUS reporter gene was shown to act as an enhancer trap. In this elegant approach, an enhancer trapping cassette is created by fusion of the GUS coding sequence to the TATA region of the 35S minimal promoter from the cauliflower mosaic virus (CaMV). This minimal promoter does not confer detectable GUS expression in transformed Arabidopsis plants. However, its presence allows other upstream elements to direct GUS expression in a developmental and/or cell-specific manner. The use of a minimal promoter rather than a promoter less construct allows GUS expression even if the

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enhancer trap cassette inserts at a distance from the enhancer element in the plant genome. This approach made the characterization of tissue-specific expression of unknown genes and the subsequent cloning of the corresponding promoters possible (Klimyuk et al., 1995; Sundaresan et al., 1995; Campisi et al., 1999). Promoter trapping has now become widely used in the model plant Arabidopsis, but is also applicable to other plant species such as tomato (Meissner et al.,1997, 2000), which is one of several target species for functional genomics. A prerequisite for successful application is a good transformation protocol which enables large-scale insertional mutagenesis. For example, see Trieu et al. (2000) for Medicago truncatula. A.

Enhancers Directing Root TissuePreferred Expression

A cis element of the 35S CaMV promoter, the B2 subdomain, has been shown to direct expression to the root tip of the seedling primary root (Benfey et al., 1990) when fused to a minimal promoter containing the TATA box. Comparison of the expression patterns conferred by the individual subdomains of the 35S promoter or combinations of several subdomains provided evidence for synergistic interactions among cis elements within the 35S enhancer. Promoter regions from genes involved in nutrient transport have been isolated and studied for cell-specific transcriptional activity. In Arabidopsis plants expressing the chimeric gene construct containing the promoter of the sulfate transporter Sultr1;1 gene fused to GFP, GFP was localized in the lateral root cap, root hairs, epidermis, and cortex of roots (Takahashi et al., 2000). As determined by fluorimetric and histochemical tests, the promoter of the Arabidopsis potassium channel AKT1 directs preferential expression in the peripheral cell layers of mature root regions. Expression in root tissue exceeding that in the aerial part of the plant by 50 times was detected. The discrete activity found in leaves relates to leaf primordia and to a small group of cells, the hydathodes (Lagarde et al., 1996). Enhancers of genes encoding structural cell wall proteins, such as proline-rich proteins (PRPs) or hydroxyproline-rich glycoproteins (HRGPs), direct predominant expression to root hairs. These proteins confer special physical properties and structures to cell walls and may serve an important role in hair formation. Histochemical staining of GUS activity in transgenic tobacco carrying a soybean SbPRP1/GUS chimeric gene indicated that it is expressed in the apical

and elongating regions of both primary and lateral roots, most strongly in the rhizodermis. A similar localization pattern was found in transformed hairy roots when this construct was introduced into Vigna aconitifolia (cowpea) using Agrobacterium rhizogenesmediated transformation (Suzuki et al., 1993). Several lines of evidence support a direct relationship between expression of the AtPRP3 gene and root hair development in Arabidopsis. AtPRP3/GUS expression correlated with root hair formation in transgenic roots treated with chemicals known to influence the biosynthesis of the hormones ethylene and auxin, both promoting root hair formation. In addition, AtPRP3/ GUS activity was enhanced in ttg and gl2 mutant backgrounds exhibiting ectopic root hairs, but was reduced in root-hair-less mutant seedlings (Bernhardt and Tierney, 2000). LeExt1 encoding an extensin-like protein was cloned from Lycopersicon esculentum root hairs (Bucher et al., unpublished). In situ hybridization and promoter/GUS expression studies provided evidence for a direct correlation between LeExt1 expression and cellular tip growth in root hairs and pollen tubes. Root hair-specific activity of LeExt1/GUS was comparable in roots of transgenic tomato, potato, and tobacco plants (Fig. 1, see color insert). Gene expression is also directed to the rhizodermis by promoter elements originating from genes controlling cell patterning during root epidermis development in Arabidopsis. Expression of the GUS reporter gene fused to promoter fragments from GL2 and WER was localized to the differentiating hairless cells of the root tip of transformed wild-type plants, during a period in which epidermal cell identity is believed to be established (Galway et al., 1994; DiCristina et al., 1996; Masucci et al., 1996; Lee and Schiefelbein, 1999). GUS expression was also detectable in epidermal cells in the seedling shoot, suggesting a role for the corresponding genes in root and shoot epidermal patterning. The cell layers of the Arabidopsis primary root are arranged in a simple radial pattern. The outermost layer is the lateral root cap and is localized outside the rhizodermis that surrounds the ground tissue. Genes specifically expressed in the root cap or in root cap border cells have been identified. The root cap-specific enhancer in the gene designated RCP1 was initially identified in an enhancer–trap line (Fedoroff and Smith, 1993; Smith et al., 1996). Here, a transposon carries a promoterless GUS reporter gene that can be activated by insertion into or near a promoter or enhancer. GUS gene expression in roots of

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the RCP1 enhancer–trap line is observed only in root cap cells of primary, lateral, and adventitious roots (Tsugeki and Fedoroff, 1999). The Arabidopsis gene designated SCARECROW (SCR) was shown to be expressed in the root cortex/ endodermal initial cells and in the endodermal cell lineage (Di Laurenzio et al., 1996). In mature roots, expression was localized primarily to the endodermis. The deduced amino acid sequence of SCR suggests that it is a member of a novel family of putative transcription factors. In the scr mutant root, a cell layer is missing due to a disruption of the endodermis/cortex cell differentiation. The SHORT-ROOT (SHR) gene was shown to be required for the asymmetric cell division responsible for formation of ground tissue (endodermis and cortex) as well as specification of endodermis in Arabidopsis roots. SHR encodes a putative transcription factor with homology to SCR. Confocal laser scanning microscopy of SHR/GFP, or histochemical staining of SHR/GUS reveal SHR expression in the stelar tissue, but not in the ground tissue lineage, thus supporting a role for SHR in a radial signalling pathway (Helariutta et al., 2000). In summary, several enhancers directing root-preferred gene expression have been identified. Further analysis is required to show whether they can serve as promoters for root bioengineering approaches in suitable

plant species. Enhancers reported to direct gene expression to distinct root tissues are listed in Table 1. B.

Enhancers Directing Gene Expression to Nematode Feeding Sites

Several promoter fragments exhibiting activity in nematode feeding sites have been isolated. Promoter trapping allowed the isolation of RPE, an upregulated nematode-responsive gene from Arabidopsis (Favery et al., 1998). RPE encodes D-ribulose-5-phosphate-3-epimerase (EC 5.1.3.1), a key enzyme in both the reductive Calvin cycle and the oxidative pentose phosphate pathway. Histochemical localization of GUS activity in the RPE T-DNA tagged Arabidopsis line showed early and strong GUS expression in the galls. During plant development, the GUS gene was expressed in the root meristem and in the elongation zone. Expression analysis of several nematode-inducible genes has shown that many genes normally expressed at different developmental times or in different cell types are upregulated in giant cells (Barthels et al., 1997; Moller et al., 1998; Moller and McPherson, 1998). The gene TobRB7 is normally expressed in the meristem and immature central cylinder regions of tobacco roots, and its expression is induced during feeding site development. The cis-acting sequences that mediate induc-

Table 1 Genes Whose Corresponding Enhancers Exhibit Root-Preferred Expression Gene 35S CaMV, mutated domain A 35S CaMV, subdomain B2 TobRB7 AKT1 AtPRP3 SbHRGP3 LeExt1 GL2 WER ARSK1 SbPRP1 RCP1 HRGPnt3 SCR SHR

Preferred site of expression Root, stem Root tip Root meristem, root central cylinder, nematode feeding site Rhizodermis (including root hairs) Rhizodermis (including root hairs) Rhizodermis (including root hairs), hypocotyl Rhizodermis (including root hairs) Differentiating atrichoblasts, shoot epidermis Differentiating atrichoblasts, hypocotyl epidermis Outer root tissues, weak in root central cylinder, inducible by dehydration Root tip, rhizodermis in elongation zone Root cap Pericycle and endodermis Cortex/endodermal intials, endodermis Stele tissue

Plant origin

Reference

(CaM virus) (CaM virus) Tobacco Arabidopsis Arabidopsis Soybean

Lam et al., 1989 Benfey et al., 1990 Yamamoto et al., 1991; Opperman et al., 1994 Lagarde et al., 1996 Fowler et al., 1999 Ahn et al., 1996

Tomato Arabidopsis

Bucher et al., unpublished Masucci et al., 1996

Arabidopsis

Lee and Schiefelbein, 1999

Arabidopsis

Hwang and Goodman, 1995

Soybean Arabidopsis Tobacco Arabidopsis Arabidopsis

Suzuki et al., 1993 Tsugeki and Fedoroff, 1999 Keller and Lamb, 1989 Di Laurenzio et al., 1996 Helariutta et al., 2000

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tion by the nematode are separate from those that control normal root-specific expression. Reporter transgenes driven by the nematode-responsive promoter sequences exhibit expression exclusively in the developing feeding site (Yamamoto et al., 1991; Opperman et al., 1994). Thus, dissecting promoter regions of genes, which are normally upregulated during feeding site development, may yield suitable enhancers which could then be used for interfering with feeding site-specific gene expression.

V. ROOT BIOENGINEERING The root is a largely unexplored frontier for genetic engineering. Root processes influence rhizosphere chemistry, nutrient acquisition, and interactions with beneficial and pathogenic organisms. There is interest in root bioengineering for numerous reasons. Engineered roots may be better adapted to nutrientpoor soils, be better hosts to beneficial organisms, resist soilborne pathogens more effectively, remediate toxic waste, or be a cost-effective alternative for the production of biomolecules. A.

Citrate Secretion and Agricultural Consequences

Secretion is a basic function of all plant cells and organs and is especially developed in plant roots. Roots of white lupine form proteoid (clustered) roots as a response to P deficiency. These roots have the remarkable capacity of secreting large amounts of organic acids (citric acid, malic acid) which accumulate naturally up to 50–90 mmol/g soil in the rhizosphere soil of cluster roots (Marschner, 1995; see also Chapter 55 by Pate and Watt in this volume). Secretion was suggested to be the result of P deficiency-induced changes in carboxylate metabolism and anion channel mediated secretion of the organic acids from cluster roots (Neumann et al., 1999, 2000). Citrate secretion is thought to lead to a ligand exchange with P-adsorbing Fe/Al humic acid complexes and reduction of the abundance of P sorption sites in the soil matrix (Marschner, 1995). Acid soils rich in metal oxides cover 40% of the world’s arable land. Here, not only P deficiency but also Al toxicity is a problem (see also Chapter 33 by Gerendas and Ratcliffe and Chapter 46 by Matsumoto in this volume). In alkaline soils, P tends to precipitate as low soluble calcium and magnesium salts.

Owing to limited availability of P to crop plants, large amounts of fertilizers are normally put on soils each year to ensure crop productivity. The generation and cultivation of crop plants exhibiting a higher P efficiency could minimize fertilizer input and reduce the risk of environmental pollution due to runoff, leakage, or soil erosion. Moreover, it could be of great importance for subsistance farmers who are unable to apply high amounts of fertilizer to their fields owing to high costs or little infrastructure. Overproduction of citrate was achieved in transgenic tobacco plants expressing a bacterial citrate synthase gene. Citrate-secreting transgenic plants exhibit enhanced aluminum tolerance on acid Al-rich media (de la Fuente et al., 1997) and grow better on alkaline calcium-phosphate-rich medium (Lo´pez-Bucio et al., 2000). Thus, overproduction and secretion of citrate in transgenic crop plants is a promising approach to alleviate P deficiency in agricultural soils. B.

Protein Secretion and Molecular Farming

Besides secretion of low-molecular-weight molecules, plant roots have the capacity for protein secretion. Proteins are secreted when targeted to the lumen of the endoplasmic reticulum by signal peptide-mediated translocation, followed by migration through the exocytotic pathway (Denecke et al., 1990). This allows modulating plant protein secretion by transferring chimeric genes to plants, which encode recombinant proteins carrying a secretory signal peptide. However, low yields and difficulties in extraction and purification of the recombinant proteins have so far limited the utilization of plants as bioreactors. An elegant way offering great potential for the production of industrially or pharmacologically important proteins has been termed rhizosecretion and involves continuous secretion of recombinant proteins from roots into a hydroponic medium (Borisjuk et al., 1999). The root cells perform correct posttranslational processing of recombinant proteins required for the latter’s activity such as glycosylation, phosphorylation, and other modifications. In addition, proteins secreted from root cultures are less likely contaminated with pathogenic viruses, which may be present in animal production systems. Using this system, it was calculated that up to several milligrams of protein/g root dry weight could be secreted during the life span of a tobacco plant. The purification of the secreted proteins can be performed using standard procedures. A combination of rhizosecretion of recombinant proteins, or

Molecular Root Bioengineering

of small biomolecules, with root cultures of almost unlimited growth potential, such as the hairy root system (see also Chapter 58 by Vivanco et al. in this volume), could lead to an increased production rate. In addition, the use of strong tissue-specific promoters, such as the constitutive tomato LeExt1 root hair promoter (Fig. 1) or inducible promoters, may be advantageous for root development in case of the production of compounds that could potentially reduce overall root growth. In soils rich in organic matter, 20–50% of the organic P may be bound in phytate (myo-inositol hexakisphosphate). There is a great variation among crop species, and even among different varieties of a given species, in their capacity to secrete phosphohydrolases exhibiting phytase activity, and thus to have access to phytate P (Asmar, 1997; Li et al., 1997b). Adding phytase to the root medium of maize plants had a marked effect on the P availability of added phytate and the growth of the plants (Findenegg and Nelemans, 1993). An Aspergillus niger phytase cDNA has been used to achieve constitutive accumulation and secretion of the phytase in transgenic cell cultures or whole plants (Li et al., 1997a; Verwoerd et al., 1995; Brinch-Pedersen et al., 2000). Very recently it was reported that the growth and P nutrition of Arabidopsis plants grown in phytate containing agar supplied with phytase is improved significantly by the expression of the A. niger phytase gene (Richardson and Hayes, 2000). The development of crop plants with an increased capacity to secrete phytase may enhance acquisition of orthophosphate originating from organic P, and may lead to improvements in fertilizer management in agriculture. C.

Phytoremediation

Global heavy-metal contamination of soils and aquatic systems is increasing. Plants that can process and sequester heavy metals might provide efficient and ecologically sound approaches to remove these metals from soils. In contrast to bacteria used for bioremediation, plants grow autotrophically and are physiologically adapted to extract metal micronutrients from the environment with a dense root system that penetrates into large soil volumes. In addition to the ease of plant cultivation, genetic engineering offers the possibility of transferring the components of detoxification mechanisms from adapted species to transgenic plants, which can thus be optimized for phytoremediation purposes. Recent developments towards an in situ detoxification strategy for mercury-polluted wetlands and aquatic sediments reveal the potential offered by this technol-

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ogy to solve environmental problems. Methylmercury can be produced from the more abundant toxicant, ionic mercury, Hg2+, by bacteria present in aquatic sediments. Both mercurials are absorbed by plankton and higher organisms, with methylmercury exhibiting much higher uptake rates and retention times. Methylmercury then moves far more efficiently than Hg2+ through the food chain and poses a serious health risk. Rugh et al. (1996) and Bizily et al. (2000) introduced bacterial mercuric ion reductase, MerA, which reduces toxic Hg2+ to the less toxic, relatively inert metallic mercury (Hg0), and organomercurial lyase, MerB, which detoxifies methylmercury in Arabidopsis plants. The two enzymes thus catalyze the following reactions: MerB

R-CH2 -Hgþ þ ! R  CH3 þ Hg2þ MerB

Hg2þ þ NADPH ! Hgð0Þ þ NADPþ þ Hþ Several lines of Arabidopsis expressing the two genes either individually or in combination were resistant to toxic levels of Hg2+ or of methylmercury. Similarly, transgenic yellow poplar plantlets expressing the modified MerA gene were resistant to ionic mercury (Rugh et al., 1998). Both mercury-resistant species, Arabidopsis and yellow poplar, release volatile elemental mercury which is far less toxic than its organic forms. These results indicated that plants expressing modified MerA or MerB constructs may provide a means for the phytoremediation of mercury pollution. Other harmful pollutants, chlorinated solvents, especially trichloroethylene (TCE), are the most widespread groundwater contaminants in the United States. Tobacco plants have been engineered with a notable increase in TCE metabolism by virtue of the introduction of the mammalian cytochrome P450 2E1 (Doty et al., 2000). This enzyme oxidizes major soil contaminants, including TCE. The transgenic plants metabolized TCE at up to 640-fold the rates of the control plants. In addition, uptake and debromination of ethylene dibromide, a toxic, volatile human carcinogen, was increased. These data provide promising perspectives for the use of transgenic plants expressing this enzyme for more efficient remediation of many sites contaminated with halogenated hydrocarbons. Arazi et al. (1999) were able to modulate tolerance of tobacco to heavy metals by overexpression of an endogenous putative cation transporter using the 35S constitutive promoter. Transgenic lines exhibited

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increased tolerance to Ni2+, but were hypersensitive to Pb2+, probably owing to altered affinity for, or allocation of, the two metals in transgenic plants. The physiological role and the function of the gene product remain to be resolved. Moreover, cell-specific expression of the transporter reduced Pb2+ hypersensitivity while Ni2+ tolerance was maintained in the transgenic plants. To investigate the contribution of glutathione and phytochelatin (PC) production to heavy metal tolerance, Brassica juncea (Indian mustard) was genetically modified to express either the Escherichia coli gshI gene encoding -glutamylcysteine synthetase (gamma-ECS), targeted to the plastids, or the E. coli gshII gene encoding glutathione synthetase (GS) (Zhu et al., 1999a,b). Both transgenic lines had an enhanced cadmium accumulation and tolerance correlating with increased concentrations of glutathione and PC. If such promising results can be confirmed under natural conditions at a contaminated site, phytoremediation is likely to become a primary field for ‘‘green’’ gene technology. D.

Nematode Resistance

Current control of plant parasitic nematodes often relies on highly toxic and environmentally harmful nematicides. As their use becomes increasingly restricted, there is an urgent need to develop crop varieties resistant to nematodes. The limitations surrounding conventional plant breeding favor the opportunity for transgenic resistance to reduce current dependence on chemical control (Williamson and Hussey, 1996; Jung et al., 1998; Lilley et al., 1999a,b; Vrain, 1999; see also Chapter 51 by Koltai et al. in this volume). Nematode-responsive promoters may be used to express antinematode proteins, such as proteinase inhibitors (Urwin et al., 1995, 1997), or phytotoxic proteins to inhibit the development of feeding structures. The latter approach, however, calls for a highly specific ‘‘nonleaky’’ promoter, which is active only in the feeding cells (Atkinson et al., 1995). Overexpression of resistance genes could be anticipated to transfer the resistance mechanism from the resistant to the susceptible plant species (Van der Vossen et al., 2000). Alternatively, downregulation of essential genes (Favery et al., 1998) by using posttranscriptional gene silencing approaches (Sharp, 1999; Chuang and Meyerowitz, 2000) could be used to interfere with the development of feeding cells. Feeding-site specific induction of genes involved in resistance to nematodes (Leister et al., 1996; Cai et al., 1997;

Milligan et al., 1998; Shirasu et al., 1999; Williamson, 1999) may activate the defense mechanisms in susceptible crops.

VI.

PATENTING IN THE AREA OF ROOT BIOENGINEERING

In the field of root biotechnology, applicationoriented research can lead to results that might be exploited in the private sector or in collaborations between universities and life sciences companies. Patents covering applications in root bioengineering and which have been discussed in this chapter are listed in Table 2.

VII.

CONCLUSIONS

The increasing amount of experimental data improves our understanding of root biology. Every month, new genetic information on root development, physiology, and biochemistry reaches the scientific community. The growing number of highly ranked publications, including patents, on plant roots reflects the interest and the continuing progress made in this fascinating field of research. The knowledge gained by studying model organisms like Arabidopsis is or will have to be transferred to crop plants to benefit agricultural practices, protection of the environment, or improve animal and human health and nutrition. Molecular genetic tools will allow the development of tailor made crop roots exhibiting a specifically desired morphology, optimized tolerance to adverse environmental influence, resistance to pathogens, or allowing sufficient nutrient uptake while reducing the input of fertilizer and agrochemicals. Exploiting root properties for environmental cleanup, or for large-scale production of pharmaceutical compounds, is certainly of great interest to society and to industry. One may predict that root bioengineering is likely to have a bright future.

ACKNOWLEDGMENTS I would like to thank Dr. Nikolaus Amrhein, ETH Zurich, for very helpful comments and for improving the style of the manuscript. I am grateful to Philip Zimmermann for photographs.

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Table 2 Worldwide and U.S. Patents on Molecular Root Bioengineering Collected from Europe’s Network of Patent Databases (http://ep.espacenet.com/) Publication No. WO0031249 WO0029566 US6018099 US5723751

US5633363 WO0007437 WO9963100

WO9938990 WO0006753 WO9960141 US5824876

Subject Root-preferred promoters and their use

Assignee

Pioneer Hi-Bred International, Des Moines, IA Promoters for gene expression in the Federal Institute of Technology, Zurich, roots of plants Switzerland Tissue-preferential promoters Novartis Finance Corp (U.S.) Expression motifs that confer tissue and Rockefeller University, New York development-specific expression in plants Root preferential promoter University of Iowa Research Foundation A method of identifying and recovering Rutgers University, NJ products exuded from a plant Process for obtaining transgenic plants Centro de Investigacion y Estudios Avanzados del Instituto Politecnico which have an improved capacity for Nacional, Guanajuato, Mexico the uptake of nutrients and tolerance to toxic compounds which are present in the soil Rutgers University, NJ Methods for recovering polypeptides from plants and portions thereof Engineering nematode resistance in Landbouuniversiteit Wageningen, Solanaceae Netherlands Genes and methods for control of Pioneer Hi-Bred International, Des nematodes in plants Moines, IA Plant parasitic nematode control Cambridge Advanced Technologies Ltd, United Kingdom

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18 Root Research Methods Janina Polomski and Nino Kuhn Swiss Federal Research Institute, Birmensdorf, Switzerland

I.

INTRODUCTION

types of study for which they are best suited. There are three possible ways to present the root research methods: (1) in relation to their technical features, (2) with respect to the problems to be solved, and (3) with regard to root attributes (e.g., morphology, architecture, productivity, nutrient uptake, allocation of substances etc.). For the present chapter we have chosen the first approach because many of the other approaches are presented in other chapters of this book.

Unlike airborne plant organs, plant roots are ‘‘hidden’’ in the ground and can be reached in their natural environment only by using special procedures or equipment. Unfortunately, environmental conditions vary with respect to space and time. Experimental layouts and treatments in field research are often masked by unforeseen changes in the environmental conditions. Therefore long-term observations and large numbers of replications are required for meaningful root research in the field. The processes and reactions of root growth can be studied under controlled conditions in laboratories, and by that the cost-intensive investments in such fieldwork can be avoided. However, growth conditions in laboratories can seldom represent natural environments. A compromise between field and laboratory research may be achieved by using so-called growth laboratories. These started out as simple underground glass windows and have since developed into high-technology rhizolaboratories. The application of various methods to root research calls for special skills. Many of the analytic methods used were not originally developed for root research but have been taken over from other sciences. For example, endoscopy adopted from human medicine is today used in root research in minirhizotrons. The experimental design and techniques to be used in a particular study depend on its aims. The following sections of this chapter provide a survey of the different methods currently used in root analysis and discuss the

II.

EXCAVATION METHODS

A.

Excavation of Root Systems

Excavation is a useful technique for exploring the morphological characteristics, architecture, or biomass of root systems of individual plants. The quantitative exploration of such qualitative data means measuring the lengths, diameters, weights, volumes, etc. of particular parts or counting of others—e.g., root tips. It is best done by relating the data per unit area or per unit volume of substrate, and relating them to other soil properties. For large-scale surface or space-related investigations soil core sampling is the more appropriate approach. Results of several excavation studies of annual and perennial plants were published by Kutschera (1960), Kutschera and Lichtenegger (l982, 1992), Kutschera and Sobotik (1992), and Kutschera et al. (1997). The method was discussed by Bo¨hm (1979), Upchurch and 295

296

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Taylor (1990), Kapeluck and Van Lear (1995), Thies and Cunningham (1996), Drexhage et al. (1999), and others. For tree species, root system excavation was used for studying biomass, carbon allocation, and damage caused by windthrow. The biomass, distribution, and quality of coarse roots and the stem diameter at breast height are important parameters in modeling root system architecture and assessing windthrow risks. Assessments of root biomass are often underestimated because root losses during excavations may be considerable. This is most important in the case of uprooted trees. Horizontal roots outside the ‘‘Zone of Rapid Tapper’’ (1–2 m from the stem) may grow at almost constant thickness to several meters in length. Careful excavation of the full length of roots is required, especially when the data are used to calibrate growth simulation models. Roots <5 mm in diameter should be sampled by core sampling. B.

Soil Block (Monolith)

By the soil block method a block of the soil, in its original structure, is extracted from its natural position with a specified depth and cross section. A soil monolith may be used for (1) obtaining a detailed view of the bulk root system of herbaceous plants within their surrounding soil; (2) determining the root mass, solid soil substance, and pore volumes in stony or rocky soils where a traditional core technique is impossible; and (3) assessing other root–soil relationships for plants grown under natural conditions. In clay and sandy soils, soil monoliths can be prepared by digging around a column. Special care is required for soils where the particles do not adhere enough. To visualize or mark the original positions or connections of the roots, pins (Schuurman and Goedewaagen, 1971) or a nylon screen (Gooderham, 1969) can be used. In stony or rocky soils, the use of a grinding machine with a diamond-coated disk blade is indispensable. For disk-blade diameters of up to 40 cm a motor driven, hand-held cutting machine can be used, allowing cuts in the soil down to 15 cm. By applying a second cut with a slightly wider surface area, a larger monolith can be prepared. As most of the fine roots in such soils are located in the upper 10–20 cm, such monoliths are sufficiently large for many field research purposes. For a block with a cross section of 20  20 cm two parallel cuts 20 cm apart and
30 cm long are made. A 0.5- to 1-cm-thick wood fiber board can be laid on the ground to protect the soil surface. To pre-

vent the grooves filling up again, folded plastic tapes are inserted immediately after the cut has been made. The procedure is then repeated perpendicular to the first two cuts. A second square 30–40 cm larger than the first can be cut around the first to facilitate the excavation of a ditch. The block is best removed by wrapping it up with cardboard (especially around the corners) and sticking plastic tape around it. This method has been successfully applied for stands with steep slopes with Rhizomull Rendzina soil (Fig. 1). Excavation of deeper monoliths requires cutting machines and the application of a solid mantle (Buchter, 1986). As an alternative to the traditional soil block, soil coring may be used. However, core diameters are usually restricted to a maximum of 10 cm. For larger cores, heavier drilling machines, which are difficult to transport, would be necessary. Another alternative to the common soil block is pinboard sampling (‘‘fakir bed’’). A slice of soil 10– 15 cm thick is detached from a soil profile using a needle board. Stainless-steel needles, regularly arranged in a 5  5 cm grid, are driven through a plywood board. The pin grid keeps the roots approximately in their original position, when the soil is washed out. What remains then represents a root profile (Oliveira et al., 2000). C.

Soil Core Sampling

This method is based on sampling of a cylinder-shaped core of undisturbed soil. Soil core samples are frequently used for estimating the spatial distribution

Figure 1 Soil monolith cut off from a rocky Rhizomull Rendzina soil on a 60% slope by using a Stihl TS 400 separating-grinding machine. Soil properties and root densities have been estimated in the soil monoliths (see Polomski and Kuhn, 2000).

Research Methods

(biomass, necromass, root tips) and the volumetric relation of fine roots, preferably with diameters <5 mm. The resulting data can be used as references in investigating many other aspects of root analysis. Taking a soil core sample involves pushing, pressing, or drilling of a steel tube, preferably with an integrated PVC, plastic sampling casing or plastic liner vertically inserted into the soil. The method is appropriate for peat and other soft soils without stones and can produce cores with various diameters. Nowadays a variety of soil samplers and drilling machines, as well as hammering or vibrating machines, are available. An advisory service can be mediated by Braun and Flu¨ckiger (1998). The larger and heavier the borer, the more restricted is its use. Good results for most purposes can still be obtained with hand-driven steel cylinders with a hammering head. Care should be taken to ensure that the edges of the cylinders are well sharpened because a blunt edge does not cut roots but rather drags them along the wall of the cylinder as it penetrates the soil and causes underestimation of the results. In stony or rocky soils, core samples need to be drilled by a drilling machine. Types of grinding crowns (saw edges) up to 100 mm in diameter are available. The cutting ability of the saw edge needs to be checked repeatedly since fine roots may cause problems as they are often withdrawn. If this happens, they will curl up and destroy the core. Sampling design, procedure, equipment, and the preparation of roots when using the soil core sample method are described in detail by Upchurch and Taylor (1990), Mackie and Atkinson (1991), Vogt and Persson (1991), and Persson (1996). D.

In-Growth Core (Mesh Bag)

An in-growth core consists of a cylindrical gauze bag with a specified volume filled with root-free substrate. It is inserted into a properly drilled hole and left to become colonized by the roots of neighboring plants. An alternative to the cylindrical bag is the alignment of flat-sided cuboid bags along a properly prepared soil miniprofile (Fig. 2). The in-growth technique yields an estimate of the root growth dynamics of a plant (root biomass, production) and for the time of exposure (root growth rate). The method is suitable for (1) comparing root growth activities at different sites or stands, (2) investigating the seasonal variation, or (3) comparing the effects of different experimental treatments. Root production may be determined as dry weight or as total

297

Figure 2 An alternative allocation of the in-growth bags in a prepared soil miniprofile in a Rhizomull Rendzina. The bags are not cylindrical but rather shaped as flat-sided cuboids (see Polomski and Kuhn, 2000).

root length, using a scanning technique and/or as the number of root tips per unit volume and time. The main objects involved and features of the method are shown in Table 1. Particular attention was paid to choosing a sufficiently large mesh size of the bags for the roots to pass through (cf. Table 3), removing the bags from the hole where care must be taken to ensure that roots that have grown into the bag are not pulled out, and interpreting in-growth data for root production: the manipulated soil conditions in the bags may affect root growth (cf. Vogt and Persson, 1991; Steen, 1991; Persson, 1996). A critical review of the method was published by Vogt et al. (1998).

III.

DIRECT MONITORING IN SITU

Direct observation is a reliable technique for investigating interactions between root systems and their environmental conditions. Roots can be directly observed by the trench wall technique. Direct monitoring of soil profiles or root windows allows estimations of the quantity and the development of both fine and coarse roots of the same plant. Interactions between the root growth pattern and soil environment, such as water, temperature, or nutrient status, can be studied using rhizotrons or minirhizotrons. The roots visible on the profile are measured by various procedures: (1) counting root tips or root intersections with a line grid drawn on the window; (2) hand-tracing onto acetate sheets with fine-point colored markers or by photography; (3) observing

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Table 1 Indications for the Use of the In-Growth Core Method Object Core bag

Attribute, property, quality Polythene, nylon gauze Shape and measures Mesh opening

Substrate

Allocation of samples Sampling design

Local soil from corresponding soil horizon Controlled material for experiment Soil horizon Soil profile wall Areal distribution

Special features Nondecaying material Cylindrical for hole, cuboid for wall (soil mini profile) Free root growth, fixing substrate Free of roots Particle size according to mesh opening Tight bottom of the hole Proper contact to wall

Vertical distribution

Sampling

Earliest Sampling

According to growth intensity

Interval Withdrawal

According to growth intensity Care about pulling out roots from bag

with a microscope, scanning and digitizing the images, and calculating the desired root parameters by computer.

A.

Profile Wall Technique (Trench Wall)

The profile wall technique is particularly useful for estimating coarse roots, which can be distributed through the heterogeneous soil profile. The estimation should be carried out quickly because the roots, especially root tips, are very sensitive to desiccation. By monitoring tree roots, which are often widely spaced and connected by root graftings (Polomski and Kuhn, 1998), the effects on neighboring trees should be considered. A trench at a suitable distance from the trunk is excavated and a slice of the soil removed from the trench wall. The roots passing through the wall profile are recorded. This technique was described in detail by Bo¨hm, (1979), Upchurch and Taylor (1990), and Mackie and Atkinson (1991).

B.

Frequently used size Diameter (20)-40-(60) mm, 60  60  30 mm (2)-3-(5) mm Sifted 5 mm or according to mesh opening Quartz, perlite Corresponding diameters of hole and bag Coordinate No. of samples, No. of samplings and No. of replicats Deeper horizons with less frequent roots need more samples! Tropical: 3–6 weeks (see also Table 2) Boreal: 6–12 months after installation Term of withdrawal before or after growth season

Root Window

The root window is a transparent 6- to 8-mm-thick glass or a plexiglass plate pressed onto the soil profile. It is a good method for investigating the morphological development of roots as well as phenological changes and the life span or mortality of individual roots. However, in practice only very few replications of root windows can be installed. The optimal location, orientation, and size of root windows depend on the research aims. For observation of the fine roots and mycorrhizae of forest trees, a depth of 10–30 cm at 1–2 m distance from the trunk is usually recommended. However, the fine-root distribution varies with species, age, and environmental properties (Polomski and Kuhn, 1998). Therefore a preliminary distribution pattern should be estimated. The main advantage of this simple, relatively cheap technique is that it allows investigations in vivo over several vegetation periods and in remote locations (Ha¨sler et al., 1999). One of the problems of this method, which occurr during long-term observations,

Research Methods

is water condensation on the inner side of the glass plate. Careful warming of the plate for some few minutes during observation is a useful remedy (Egli and Ka¨lin, 1991). If absolutely necessary, the plate can be removed carefully from the wall for cleaning. But it is wise to prevent condensation in the first place by ensuring the observatory system is well insulated. Condensed water causes the so-called border effect— i.e., preferential root growth on the glass surface. Good insulation is also advisable to reduce the exposure to light. In some sites, intensively growing grass roots can cover the window within 2–3 years which impedes the observation of the fine roots of trees. In such a case it is worthwhile cleaning up the soil profile and changing the glass plate (Streule, personal communication, 2000). Plastic plates (6–8 mm) are installed with a slight lean. Egli and Ka¨lin (1991) first pressed a steel plate with a whetted cutting edge, then excavated a ditch behind it and replaced the steel plate with a glass plate. In nonstony clay soils the plate can be fixed directly onto the smoothed soil profile. In other soils a thin layer of sieved soil material should be placed between the intact soil profile and the glass plate. In the first version the observation can begin immediately after installation. Intensive mycorrhizal colonization in natural soil cavities can then be observed. For technical details see Bo¨hm (1979), Egli and Ka¨lin (1991), and Ha¨sler et al. (1999). One modification of the window technique which allows observation of roots growing more horizontally than vertically is to use horizontally installed windows on the soil surface of several small glass sheets. Small windows are more suitable for studies requiring more replications. It is even possible to estimate the growth of horizontal fine roots directly in vivo without a window by excavating the root from its tip up to the desired length, marking the measuring points and covering the place with a protective sheet after measurement (cf. Ladefoged, 1939). The procedure developed by Espeleta et al. (1999) for studying the mycorrhizal fungi effects on roots of mature trees can be considered as a modification of classical root window observation. In a citrus orchard in Florida wooden chambers (40  45  50 cm) were buried in the soil at a distance of 1 m from the tree trunks (Fig. 3). Inside every chamber two pairs of vertically arranged, 500-mL plastic split pots were installed. They were filled with mycorrhizae inoculated substrate in one compartment and with sterile substrate in the other. On each pot transparent windows

299

(25 cm2) were fixed to observe and trace regrown roots. The windows as well as the chambers were covered with insulated lids to protect the roots from sunlight, heat, and rainwater. Roots 5 mm in diameter were excavated and passed through an aperture in the chamber wall and through both vertically separated split pots. Careful excavation, avoiding mechanical damage to roots, helped to ensure successful regrowth. Within 10 months new roots were observed and traced. The plastic windows with the root traces were then collected, the images scanned, and the root length calculated using ‘‘Rootlaw’’ software (Pan and Bolton, 1991). C.

Rhizotron

1.

Classical Rhizotron

Rhizotrons are subterranean glass chambers used for root studies. The classical rhizotron is a root observation laboratory in which several glass plates are installed on the soil walls on both sides of a tunnel. The roots are grown in glass bins filled with soil, and the shoots grow on top in the air. The use of rhizotrons combines the controlled conditions of laboratory experiments with the advantages of field-oriented investigations, even when the field environment is artificial. The dynamics of root development under such conditions can be continuously measured over a long period with the same plants undergoing the experimental treatments (Upchurch and Taylor, 1990; Sackville et al., 1991; Box, 1996). 2.

Rhizolab

The rhizolab can be seen as a highly automated modification of a rhizotron. It allows fully controlled above- and underground environmental conditions for testing models of optimal crop conditions. Root and shoot relationships in interactions with the soil and the atmosphere can be studied and diverse parameters measured simultaneously. The results obtained from such artificial experimental conditions are useful for specific investigations in high-tech agriculture or for developing theories of plant growth. However, they do not provide a realistic picture of the relevant ecological variables and require careful verification (Van de Geijn et al., 1994; Smit et al., 1994; Box, 1996). 3.

Soil Biotron

The soil biotron is a kind of rhizotron located in a natural ecosystem. One example of a biotron con-

300

Polomski and Kuhn

Figure 3 Field root chambers consisting of two vertically arranged plastic split pots which can be used for testing effects of various experimental treatments on fine root growth in vivo. (From Espeleta et al., 1999.)

structed in a mixed hardwood forest in Michigan was 2:5  2:2  31 m in size and contained 34 1:2  1:2 m window bays. Each bay contained 16 removable glass windows in a 4  4 array (Fogel and Lussenhop, 1991; Pregitzer et al., 1993). Between the native soil horizon and the windows, sieved soil material was separately excavated from every horizon and then repacked. Before experimental treatments were carried out, the root systems of the surrounding vegetation were allowed to re-colonize the disturbed soil. Biotron is a good alternative for experimental field research of root-soil relationships under artificial laboratory conditions. D.

Minirhizotron (MR)

This technique is based on observing and recording roots in situ through a transparent tube inserted into the substrate through which the roots spread. Since the same root segments can be measured directly and repeatedly, this technique is particularly appropriate in assessing root production, phenological processes,

turnover, or longevity. It is, however, not suitable for determination of the root topology or architecture because of its limited observation space. MR equipment is compact and easily transportable so that it can be used in natural ecosystems, even in remote research areas. However, studies in natural plant communities are still scarce and the results are controversial (Aerts et al., 1989; Hendrick and Pregitzer, 1992a,b; Majdi and Nylund, 1996; Steinke et al., 1996). The MR technique has been used frequently in agricultural monocultures or laboratory containers as the root growth pattern is relatively uniform and the installation of MR tubes relatively simple (Box, 1996; Majdi and Nylund, 1996; Hendrick and Pregitzer, 1996; Smit et al., 2000a,b) MR equipment consists of a transparent tube inserted into the soil, an optical system introduced into the tube, and a video-processing system for storing and analyzing the recorded images. Several procedural steps are described below in order to help in choosing optimal equipment and the most appropriate method of analysis.

Research Methods

1.

MR Tubes

a.

Material and Size

The tubes are made of polycarbonate, acrylates, Teflon, Pyrex glass, or quartz glass. None of these materials have yet been evaluated in comparative studies. Transparent acryl is used extensively since it is cheap and more durable than glass tubes, which are not frost resistant. Moreover, acryl tubes can easily be labeled manually or even automatically with a self-constructed etcher to quickly recognize the root locations at the successive sampling dates (Kloeppel and Gower, 1995). Teflon or quartz glass tubes are recommended, particularly when UV light is used for root photography (Box, 1996). Commonly used tubes vary between 60 and 200 cm in length and between 3.0 and 6.0 cm in diameter, depending on the root distribution in the growing substrate and on the experimental approach. Tubes as small as 6–15 mm are also recommended since the smaller the tube the larger can their number be and the more accurate the estimation of root density (Itoh, 1985). Both ends of the tube are closed with stoppers to prevent water leakage. The part above ground is protected against light and solar heating. b.

Insertion Techniques

To insert a MR tube, an initial hole has to be drilled in the soil. Only into sieved rooting substrates used for laboratory experiments can the MR tubes be inserted manually. The drilling technique depends on the applicability of the equipment, particularly on the soil properties and on the working conditions. Success in insertion of MR tubes is largely a question of manual skill. However, when installing large numbers of tubes, even in arable land, a power-driven hydraulic soil sampler, ship auger, or rotary auger is applied (Liedgens et al., 2000a,b). The diameter of the hole in soft soils should be 2 mm less than that of the MR tube. The tube with a bullet-shaped plug in front is pressed into the hole with the help of a hydraulic press. In forest ecosystems inaccessible to vehicles, a portable system was developed consisting of chain-sawmounted tree planter equipped with carbide-tipped auger for drilling the holes into compact, stony soils (Kloeppel and Gower, 1995). Smit et al. (2000a) suggest the use of flexible or pressurized (inflatable) tubes with a sophisticated inserting technique, using a pilot metal tube. The technique was successfully tested in stony or heavy clay soils (Gijsman et al., 1991; Merrill, 1992).

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Root growth monitoring in rocky soils was avoided for a long time because of technical problems. Nevertheless, Phillips et al. (2000) have managed to install large numbers of MR tubes in desert soil with a high rock content. They used a pneumatic rock drill, mounted on a pneumatic screwdrive guide system. To minimize soil compaction and plant trampling, the drill was installed on a platform. A special modification of the MR technique is the installation of tubes in pits, dug in experimental plots, down to 100- or 150-cm depths. The soil material is then refilled, with an attempt to maintain the original stratification, so as to establish good contact between the tube surface and the soil. c.

Installing Position

MR tubes can be placed horizontally, vertically, or at any other angle to the soil surface (Goins and Russelle, 1996; Smit and Zuin, 1996). Various investigators have obtained much better results with 458 angled MR (Bragg and Cannell, 1983). On the contrary, no significant differences in root lengths were found by Ephrath et al. (1999) when comparing vertical and 458 tilted tubes. Preferential root growth along vertical tubes has been frequently observed (Majdi and Nylund, 1996; Steinke et al., 1996). This depends on soil bulk density, however, and these phenomena can be much more pronounced in heavy silty or clayey soils than in sandy ones. In general, angled insertion between 308 and 458 can be recommended for most soil types as it gives good results, is practical, and reduces attaching growth along the tube to an excessive extent. Table 2 summarizes various parameters which can be investigated with the MR technique in natural ecosystems. It also contains some technical information about using MR. 2.

Optical Systems

Several optical systems are in use in MRs, ranging from a simple tilted mirror lowered into the MR tube and fiber optics to more elaborate approaches involving a camera coupled to an endoscope or batterypowered periscope (Kloeppel and Gower, 1995). The endoscope produces spherical, slightly deformed pictures compared to the rectangular images of high resolution by a telescope system (Poelman et al., 1996; Steinke et al., 1996). Color video minicameras have been extensively used. The camera can be moved within a 100- to 150-cm-long tube, which is usually marked at 10- to 25-mm intervals. The minicamera

Great spatial variation.

a

Ponderosa pine (Pinus ponderosa)

Open-top chamber

Silt loam vulcanic ash Entic Eutrandept (Montana, USA) Sandy loam Haplic podzol (Sweden)

Typic haplorthod (Michigan, USA)

Sandy rocky soil Aridosol (Mojave Desert, USA) Typic Paleuduls (USA)

Salt marsh (Niedersachsen, Germany) Heathlands (Netherlands)

Soil type (location)

Productivity Mortality Seasonal pattern Mycorrhiza New root number Life span

Length density Total production Seasonal pattern Phenology Elongation

Elongation Biomass

N. collection1 Day

Root number N.month1

mm.day1

mm.cm2 mm.cm2yr2

mm/tube kg.ha1

45

90

45

45

45

46

85

110

60–90

100

30–60

45

cm/image gm2 yr1 30

20

90

Root number N.cm2

Densitya Vertical distribution Seasonal pattern Length Biomass Turnover rate Phenology Technical parameters

3/chamber

3–7/pit

80

4 per 900 m2

30

294

5/community

600

Number

about 16

20

4

10

1

3–5

about 2

Start after installation (month)

3

2

1

1

2

1

2

3

Total (year)

Observation period

Methodological parameters Tubes

Depth (cm)

Unit

Angle (8)

Root parameters

Applications of the Minirhizotron Technique in Natural Ecosystems

Salt marsh (Astero tripolii– Agropyretum repentis) Wet heathlands (Erica tetralix Molinia caerulea) Xerophytic shrubs (Larrea tridentata, Ambrosia dumosa) Hardwood forest (Quercus prinus, Q. alba, Acer sacch.) Hardwood forest (Acer saccharum, A. rubrum, Quercus r.) Mixed larch-pine stand (Larix occidentalis, Pinus contorta) Norway spruce (Picea abies)

Stand

Table 2

Johnson et al. (2000)

Majdi and Nylund (1996)

Kloeppel and Gower (1995)

Hendrick and Pregitzer, (1992a, 1993)

Joslin and Wolfe (1999)

Phillips et al. (2000)

Aerts et al. (1989)

Steinke et al. (1996)

References

302 Polomski and Kuhn

Research Methods

system provides images of high quality and allows the images to be recorded for further processing.

3.

Analysis of Root Images

Images from optical systems are commonly stored on film or video tapes, which have a large storage capacity. They can even be viewed in the field (Smucker et al., 1987; Box 1996), as a means of checking, on the monitor. Processing the images is in many cases still performed manually by tracing the individual roots on transparencies or by counting the observed roots in a frame (Hendrick and Pregitzer, 1992a,b; Majdi and Nylund, 1996; Ephrath et al., 1999). Digitized or scanned drawings of roots are computed, e.g., for root lengths, using specific PC-based software. Tedious and time-consuming manual labor can be avoided by employing automatic image-analyzing procedures. The main problem with the image analysis remains the discrimination of the roots from extraneous objects and soil background. The accuracy of this procedure also depends (1) on the quality of the optical equipment and (2) on the characteristics of the studied objects, such as (a) the homogeneity and color of the background and (b) the color, diameter, and branching appearance of the roots. When identifying the roots and interpreting the results, all of these features should be carefully considered. a.

Distinguishing Between Roots and Soil Background

How much the appearance and characteristics of the background affect root recognition varies according to soil properties. For the discrimination procedure, the morphological characteristics of the roots should be investigated with respect to the image resolution (Box, 1996). The best images have been obtained for roots in clay soils due to the relative homogeneity. Organic soils also yield good results for automatic measurements if the roots are bright and the contrast between roots and background is strong (Vamerali et al., 1999). However, if the roots are dark, a stronger contrast is achieved in sandy soils. According to Richner et al. (2000), the resolution of most of cameras is 500  700 pixels per picture. With this resolution roots with diameters 70–100
m can be discriminated. As shown in Table 3, most of the roots of monocot and dicot plant species can be discriminated as long as the soil background is uniform. But soil background may be a complex mixture of mineral and organic particles, animals, water droplets, etc. Moreover, it is hetero-

303 Table 3 Diameter Ranges of Roots, Root Hairs, and Fungal Hyphae in Soils

Object

Diameter min (mm)

Diameter max (mm)

110

4200

100 35

4000 1500

2000 50 1

4000 2000 25

3 2 1

12 20 2

Herbaceous plant Monocotyl Dicotyl Laterals Ist order Laterals IInd order Trees: nonwoody roots Long roots Fine roots Root hairs Fungal hyphae Ectomycorrhiza VA mycorrhiza Other hyphae in soil Source: Polomski and Kuhn (1998).

geneous in color and texture, and it can change on a microscale level and during the observation period. The luminosity of the soil itself is stable but it changes with soil moisture and with the intensity and the quality of the light source. The luminance of the roots decreases with time as they senesce, making it difficult to distinguish them from the soil background. b.

Distinguishing Between Roots and Extraneous Objects

To distinguish the roots from extraneous objects in the background, such as bright organic debris, droplets of water, or insects, a threshold procedure is commonly used. It provides a binary image (black and white) on which roots and other bright particles are interpreted as white and the soil background as black. Some investigators propose using color images or cameras equipped with blue or UV light, thus allowing more precise analysis (Heerman et al., 1993). When the luminances of the roots and the background are very similar, root distinction is better in the blue band of color images. The accuracy of distinguishing roots from nonroot objects can also be increased by applying a specific minimum root length (MRL) as a background reference (Vamerali et al., 1999). c.

Distinguishing Between Living and Dead Roots

If root turnover is to be estimated accurately, living roots must be distinguished from dead ones. Usually roots are classified manually on the basis of color, or by the degree of degradation of the cortex. However,

304

the procedure is time-consuming, and the parameters change according to root age and environmental conditions (Vogt and Persson, 1991; Bloomfield et al., 1996). Using a UV illumination system helps to distinguish between dead and live roots better than relying on visible light alone. Under UV light the fluorescence of dead roots is not so intensive as that of living roots. However, the contrast between dead root fluorescence and that of the background soil is rather low. The UV illumination method has other weaknesses; e.g., it is more expensive to use than visible light, and the intensity of fluorescence and the degree of contrast may be affected by root properties or by the type of tube used. Moreover, the fluorescence intensity is primarily determined by the genetic properties of the plant (Smit and Zuin, 1996). Both light methods tend to overestimate the number of dead roots in herbaceous species and to underestimate them in woody plant species. The reason is that the color of the woody species roots is dark due to suberization or the mycorrhiza mantel. Under both UV and visible light, such roots may be misidentified as dead. The UV light method is, however, useful for identifying living roots in plants, which develop transparent roots (Wang et al., 1995; Smit and Zuin, 1996). Vital staining of roots with various dyes such as TTC, Congo Red, or Trypan Blue is an alternative or supplementary procedure to the morphological criteria for distinction. d. Algorithms for Calculating Root Parameters The digitized images can be analyzed using different algorithms. The most common method is to count the number of roots with the help of a grid, and convert the numbers to root lengths (Newman 1966; Tennant, 1975) or determine the numbers by other algorithms (Kimura et al., 1999; Vamerali et al., 1999). These methods require samples with well-distributed roots and little overlap. When thick roots overlap, the number counted is lower than when thin ones overlap (Box, 1996). Several methods have been proposed to minimize such a problem. 4. What Should Be Considered When Using the MR Technique? a. Underestimation of the Root Density in Top Soil MR is an established method for investigating production, turnover, or the phenology of roots in relatively loose, nearly stone-free soils in areas well accessible to

Polomski and Kuhn

machines. Figures for root density that were obtained by MR and by core samples show a good correlation for soil layers deeper than 20–30 cm. Root density estimates near the surface were lower when using MR than those that were determined by soil coring (Samson and Sinclair, 1994; Franco and Abrisqueta, 1997; Ephrath et al., 1999). However, with respect to those findings, when such a method is applied in nature, one has to notice the following: In most natural plant communities of the nemoral and the boreal climatic zones, the bulk of fine roots are located in the uppermost soil horizons. This has been shown statistically at a high level of significance. The soil color, its tendency to dry and shrink, disruption of soil capillary continuity, detachment of the contact between the soil and the tube, and the formation of light leaks—all of these phenomena may lead to measurement errors. The accuracy of the estimations can be improved by installing angled tubes, by using many tubes with small diameters (6–15 mm) and by providing protection against excessive light and desiccation. b.

Soil–Tube Contact

The soil along the installed tube should be tight. Soil from the same hole should be packed after the tube has been installed and then pressed down slightly. c.

Disturbance of Soil

Once the soil structure along the tube has been disrupted, sufficient time is required for the soil environment to reach equilibrium and for the roots to recolonize it (Majdi and Nylund, 1996; Steinke et al., 1996). A time lag of between 1 month and 2 years is required before the situation is back to the original conditions. One year seems to be sufficient for most applications. However, the correlation between the observed root growth dynamic and the roots under nondisturbed conditions is not clear. d.

Intensive Root Proliferation

Cutting the roots during tube installation causes excessive root proliferation since carbohydrates and hormones are translocated to the cut roots or mobilized there. e.

Heterogeneity of Root Distribution

Root distributions in natural ecosystems vary in time and space, causing high variability within sample replications or plots (Hendrick and Pregitzer, 1992a). Therefore, a large number of MR tubes are required for reliable results, which increases the affects of installation and the costs of equipment. Small-scale varia-

Research Methods

tions in the color and structure of the soil as well as mycorrhiza diversity, even within small root segments, makes the automatic discrimination of roots and other soil particles unreliable. f.

Effects of Light

Root reactions to light during investigations has rarely been evaluated. Therefore, preventive measures are required to minimize the light leaks, especially through gaps in the soil–tube interface or near the soil surface. The aboveground part of the tube should be painted black or laminated to prevent light penetration and covered with a white coating or aluminum foil to reduce heat by solar radiation. For this purpose installation of protective collars is recommended (Box, 1996; Majdi and Nyluan, 1996; Ephrath et al., 1999).

E.

Scanning

Scanning combined with computerized image analysis is a fairly rapid method for assessing morphological root patterns, such as root length and diameter, topology, or branching. Computerized scanning complements manual (Newman, 1966) or camera-based estimations (Pan and Bolton, 1991; Farrell et al., 1993; Kirchhof and Pendar, 1993; Murphy and Smucker, 1995). Scanning can be done using photos (Ostonen et al., 1999), traces of roots, as well as of intact root systems from hydroponic cultures or of root segments isolated from in-growth bags or soil core sampling (Kaspar and Ewing, 1997; Bauhus and Messier, 1999; Nielsen et al., 1999; Berkelaar and Hale, 2000; Escamilla and Comerford, 2000). The digital output of root images is stored in computer as a TIFF file. Images are then analyzed using appropriate software. However, the accuracy of scanning and measuring depends not only on the software but even more on (1) the sample preparation and (2) the scanning protocol. 1.

Sample Preparation

The roots are cleaned to remove soil particles and then spread out over a transparent, water-filled tray (2–4 mm water depth). The tray is then placed on the scanner bed and the roots are scanned. The maximal root length density in the sample should not exceed 0.5 mmmm2, the optimum being between 0.1 and 0.3 mmmm2. Large samples should be divided into subsamples and the root bunches cut into small segments. This procedure decreases the number of root overlaps,

305

which are considered to be the major error source in root length estimation (Bouma et al., 2000). To improve the contrast between the roots and the background, staining can be useful, particularly when the roots are pale. Dark-colored roots should not be stained. Various dyes, such as Neutral Red (Nielsen et al., 1999; Bouma et al., 2000), Methyl Violet (Kimura et al., 1999), Congo Red (Yoder, 2000), or heated (508C) Crystal Violet solution (Kaspar and Ewing, 1997), are recommended. A fixed staining time and optimal concentrations of solutions are important for obtaining reproducible results. A staining period of 24 h is recommended to ease the selection of an appropriate threshold. If the roots cannot be stained because of further analysis, an underestimation of root length is expected. This error can be corrected, although unfortunately not entirely, by selecting a correction routine (Adoptive-Lagarde’s) for very pale roots, which is usually included in commercial software such as WinRHIZO. A fluorescent lamp can also be helpful in distinguishing the darker roots against the brightly illuminated background (Kimura et al., 1999). 2.

Scanning Protocol

The scanning resolution and transformation thresholds are very sensitive parameters and should be listed in the methodological part of publications to allow results to be compared. For commercial software such as WinRHIZO or Delta T-Scan (Bouma et al., 2000), a resolution of 400 dpi is recommended. The effects of the threshold selection on the scanning results are not yet known. The original gray-scale images obtained by most digitizing procedures is transformed by thresholding to binary (black-and-white) images. Pixels of gray-scale value greater or the same as that of the threshold are considered to be part of the object and the value of the pixel is set to black. All pixels of gray-scale value less than the threshold represent the background and are set to white (zero). The next step is the skeletonization process, whereby the skeleton axis of roots is obtained by repeatedly removing edge pixels from the object until only one single chain of pixels represents the center line of the sample. WinRHIZO software allows a flexible selection of the threshold range and offers some options for automatic threshold selection. Estimating root diameters in combination with staining and setting the threshold is a sensitive procedure. Validations of the root length and root diameter estimations should be repeated for every new species measured, using a large

306

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number of observations. Bouma et al. (2000) recommended >400.

principle of this technique is similar to that of the rhizotron.

3. Measuring Root Length and Diameter

A.

Root lengths are usually calculated using the line intercept principle. This principle is based on the relationship between the length of roots and the number of intersections of randomly placed lines or a grid system, over which the roots were spread (Newman, 1966; Tennant, 1975). The measurement can be carried out manually or by using various methods of computerized image analysis (Box, 1996; Kimura et al., 1999). Such analysis assumes a random arrangement of the roots and few overlaps, which requires a time-consuming preparation of the roots. In a new algorithm suggested by Kimura et al. (1999) direct counting of the overlap points reduces the underestimation error and makes the measurements largely independent of random placement of the roots and of the sample size. However, this algorithm was developed for rice hydroculture and has not been verified for the topologically more complex roots of dicots under natural soil conditions. Commercial software usually includes a corrector routine to reduce the overlapping effect. However, the careful preparation of root samples, as well as the choice of a suitable scanning resolution and threshold value, is decisive for ensuring accurate results. When estimating the root diameters, a high transformation threshold can cause an overestimation of the diameter. Therefore, a compromise between the lowest root length value that is still sufficient for the threshold value and the value that is optimal for estimating diameters should be found. An inappropriate gray-scale threshold can cause shadowing, resulting in overestimation of the root diameter, which seems to be the main source of errors. The effect of root hairs should also be taken into account, particularly when root hairs are long and dense or when mycorrhizae with dense hyphae occur. No effect of root hairs on diameter estimation was found with three hydroponical cultivated grass species (Bouma et al., 2000). However, the appearance of root hairs is not constant and changes depend on environmental conditions.

IV.

EXPERIMENTAL APPROACHES USING VARIOUS ROOT CONTAINERS

A variety of containers for direct observation of root growth under laboratory conditions are in use. The

Hydroponic Approach

Hydroponics involve a series of containers in which plants are cultivated under soilless conditions in controlled nutrient solutions. Subdividing the interior of the cylinder into radial partitions allow the distribution of root segments to be studied (Crick and Grime, 1987). PVC plates can be welded or sealed with silicone rubber to form watertight compartments. A plant is placed in the center of the container and the individual roots are distributed around it in the compartments containing nutrient solutions according to the experimental layout. B.

Root Tubes or Root Boxes

Plastic or wooden containers, of various depths and diameters, are filled with an appropriate growth substrate. A front panel, made of glass or Plexiglas (3–6 mm), is installed. The roots can be observed through such windows. The front wall can be removed for replacement of the soil–root–plant system. The front panel can be covered with transparent sheaths on which the positions of each of the roots can be marked. If the boxes are filled with soil as a growth substrate, a few weeks’ incubation is required before starting the experiments. During the experiments the boxes are kept in a rack at 30–458 angles, with the transparent window facing downward, to enhance root growth along the panel. The container should be protected from light and temperature fluctuations by insulation. The boxes can be equipped with measuring instruments, such as microelectrodes, to estimate pH or redox potential, or with microtensiometers for measuring variations in soil water potential (Wilcox, 1968; Marschner et al., 1982; George et al., 1992; Ma¨der et al., 2000). The gas composition and nutrient status can be controlled by ensuring there is a constant flow circulating through the box (Fig. 4). During experiments the boxes are placed under controlled conditions in a greenhouse or growth chamber. For special experimental treatments, split boxes can be used, in which different parts of the root system are led into separated zones. Such zones can be separated by watertight PVC, nylon mesh, or membranes of appropriate pore size. The aim of such separation is to impede the growth of roots from one compartment into other ones, but also to allow free access of fungal hyphae etc. The mesh or pore size combinations of

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Figure 4 Schematic diagram of Plexiglas boxes (in literature also cited as rhizotron) used for observations on roots growing under controlled conditions. The front wall of the box is fixed with screws, allowing removal of the the soil and plant after the experiments. For using a liquid substrate the box can be made air- and watertight by silicone sealant. The boxes can be equipped with microelectrodes or microtensiometers, and gas composition and the nutrient status can be controlled by circulating through the box. (From Bartsch, 1985; Stepniewski et al., 1992.)

separating membranes have to be selected according to the experimental aim and the given diameters of roots, root hairs, fungal hyphae, etc. (see Table 3). Staining the root system of the plants before transplanting them into the boxes allows the new roots to be more easily distinguished from previously grown roots. The roots can be dyed in a 1% Methyl Violet solution for 15 s, dried 15 min between paper sheets, and finally washed with water. This staining procedure has no effect on the subsequent root growth (Bartsch, 1985). Graphical representation of root growth, established through one of the direct observation boxes, is presented in the form of a root density map (Fig. 5). The patterns of root production of gray birch (Betula populifolia) in space and time are calculated as relative root density changes. The roots visible on the windows of the containers were traced weekly by hand onto acetate sheets. The images were then digitized and skeletonized to a single pixel width. Total root length was calculated as the sum of pixels in 1 cm2 cells of a grid laid over the image. The relative change in root density (RRDC) is calculated as: RRDC ¼ ðlogðD2 Þ  logðD1 ÞÞ=t where Di is the length of roots within each cell at time i, and t is time interval between sequential traces (Berntson et al., 1995).

V.

LABELING METHODS

Such techniques involve an injection of labeled substances into the plant tissue or the growth medium to assess their translocation paths or changes in colorinferring root properties. This approach can replace, compensate, or complete the time-consuming direct estimations of root distribution and extension. It is indispensable for investigating processes such as nutrient acquisition and flux, carbon allocation and dynamics, and competition or connections between underground organs. The following markers are commonly used: (1) isotopes; (2) plant toxicants (De Byle, 1964; Bedeneau and Pages, 1984; Robertson et al., 1985; Aymard and Fredon, 1986); (3) dyes (Joslin and Henderson, 1984; Sattelmacher et al., 1983); or (4) fluorescence dyes (Donaldson and Robinson, 1971; Dyer and Brown, 1983; McGowan et al., 1983).

A.

Radioisotopes

Radioisotopes, such as 3H, 14C, 35S or 32P, are widely applied in short-term controlled experiments in the laboratory, greenhouse, or field studies. Particularly, such methods were used for investigation of herbaceous plants. The distribution patterns of the markers within the plant or their binding to particular compounds can be measured rapidly and accurately.

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pared 14C translocation in the rhizomes of two forest herbs (Aster acuminatus and Clintonia borealis). The roots of woody plants have seldom been studied with this method. 2.

Phosphorus-32

32

Figure 5 Distribution of the relative root densities of gray birch seedlings elevated by direct observation method (root boxes). The figure illustrates the periodical development pattern of the root system within the soil volume. The weekly traced root images were digitized by scanner, skeletonized to a single pixel width. The relative change in the root density was calculated based on the sum of pixels within each cell. (From Berntson et al., 1995.)

Isotopes can be either injected into the plant tissues and the radioactivity measured in the different compartments of the growth system, or applied to the growth medium allowing the subsequent detection of the radioactivity in the plant tissues. Measurement is done by scintillation counting or by autoradiography. An autoradiograph is particularly useful for mapping the tracer in the plant tissues (Rogers, 1979; Ashmun et al., 1982; Marschner, 1995; Chiariello et al., 1989; Isebrands and Dickson, 1991). 1. Carbon-14 Usually carbohydrates are labeled by exposure of the entire plant to 14CO2 . The 14C flux to the root system and the C distribution within the roots and between roots and rhizosphere are measured. Another method to study C translocation involves labeling the various organic compounds such as sugars, organic acids, or herbicides applied directly to leaves or to roots in a hydroponic solution. Relationships between sink strength of the underground organs of herbaceous plants were assessed by Pitelka and Ashmun (1985). Ashmun et al. (1982) com-

P has been used for estimation of the activity of roots, particularly of herbaceous species. The isotope is applied to the soil at different depths and finally determined in the plant tissues. The root activities of clover and ryegrass within the soil profile were monitored in mixed and monocultures by labeling the soil at different depths with 32P (Goodman and Collison, 1982). The 32P and 33P isotopes can be used simultaneously to assess the contribution of different sources through time. Christians et al. (1981) observed P acquisition using this double-label technique. This was later modified and used by Caldwell et al. (1985) in root competition experiments on P acquisition by Artemisia tridentata, Agropyron desertorum, and Agropyron spicatum. The transport pathway of 32P between rhizomes and shoots was detected by autoradiography (Noble and Marshall, 1983). When 32P is injected into an intact soil, its mobility characteristics should be taken into account. P is not uniformly distributed within the soil profile because it frequently fluctuates between the solid and liquid phases of the soil and the organic and mineral phases. It can be particularly affected by soil microorganisms. A fairly uniform distribution of P within the soil profile can be accomplished by applying the tracer at different soil depths instead of exclusively at the top or the bottom of the root zone. A preferential absorbtion and uptake within the root system can also change the distribution of P. 3.

Hydrogen-3

Tritium has been used in studies of root activity and transport processes. Lewis and Burgy (1964) injected tritiated water into wells in an oak forest in California. By measuring the radioactivity of the leaves, a root activity was traced down to 38 m. Tritium is a particularly suitable tracer for the microautoradiography, provided that the location of labeled organic molecules does not change (Bingham et al., 2000). B.

Stable Isotopes

Stable isotopes are nonradioactive forms of elements— e.g., carbon (13C), nitrogen (15N), oxygen (18O), and sulfur (34S). Most of the elements have two or more

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stable isotopes, with one of them occurring in much higher concentration than the others. For example, the mean terrestrial abundance for 12C is 98.89%, whereas for 13C it is 1.11%. For 14N it is 99.63% and for 15N it is 0.37%. The isotope ratio (R) is defined as the ratio of heavy to light isotopes and is expressed with the symbol R (e.g., the R 13C/12C is 1.11/98.89 = 0.01123). To assess the natural variation in isotopic abundance of stable isotopes (natural abundance), the relative difference between the isotopic ratio in the sample and the standard is calculated and expressed in @ notation as part per thousand (&) according to the formula: @sample = [(Rsample  Rstandard)/ Rstandard]1000

(1)

where @sample = natural abundance in sample, Rsample = isotope ratio of sample, and Rstandard = isotope ratio of standard. For example, for C: @13Csample ¼ [(0.01137-0.0112372)/0.0112372]  1000 ¼ 11:8& A negative value means the heavy isotope is less abundant than the standard, and vice versa. The currently accepted values of isotope ratios in the international standard materials for deuterium, 13C, 15N, 18O, and 34 S are listed by Ehleringer and Osmond (1989) and Bingham et al. (2000). Using this approach, a very small difference in the isotopic composition of two samples can be determined much more accurately than if just the absolute abundance is measured. In order to calculate the isotope contribution of one of two different sources (A and B), the notation of mixing ratios is used. It is assumed that the isotopic composition of the sources is both constant and different, and that the isotopic fractionation during the transfer from sources to sink is low. The mixing ratio is calculated as follows: fA ¼ ½ð@sample  @B Þ=ð@A  @B Þ  100

ð2Þ

where fA = percentage of isotope derived from A, @sample = @ of sink, and @A and @B = @ of sources A and B. Since stable isotopes are safe to use, this technique can be safely applied in ecological scale field studies and quite as well in laboratory experiments. Stable isotopes can be measured by various types of mass spectrometers: continuous-flow MS, gas chromatograph MS, HPLC-MS, or secondary ion MS (Bingham et al., 2000). The stable isotope technique

is more precise than the conventional analytical methods, but less precise than the radioisotope approach. Nevertheless, the accuracy of measurements in ecological studies is less affected by analytical errors than by variations in plants and in the environment. For example, the analytical error when estimating 15N was calculated to be
0.001 atom%, but was 10 times higher due to plant variation (Bledsoe and Atkinson, 1991). 1. 15

Nitrogen-15

N has successfully been applied as a label in agricultural research of the nitrogen metabolism of crops in assessing fertilizer efficiency and environmental affects in studies of the benefits of N-fixing legumes in crop rotation, of organic matter turnover, etc. (Stevenson et al., 1998). 15N-enriched inorganic and organic substances, 15N-enriched gas, and 15N-depleted material are commercially available. Applying 15N-labeled nitrogenous compounds for defining root depth or the spatial distribution of roots, for uptake kinetics, or for 15N-translocation can be used by measuring the incorporated nitrogen. 15 N has been used less frequently for studies of natural ecosystems, and there are hardly any studies using 15 N in forests because 15N-enriched material is expensive and the analysis usually has to be carried out in commercial laboratories. Nevertheless, 15N uptake has been measured in the roots of tree seedlings (Nambiar and Bowen, 1986; Rygiewicz et al., 1984) and in young trees (Heilman et al., 1986). The contribution of mycorrhizal hyphae to 15N translocation from the soil to plant roots, vice versa, has been studied in split-root boxes. 15N-NH4NO3 was first applied to the fungal hyphae, which were separated from the roots by a nylon or polytetrafluoroethylene membrane. 15N transport by the hyphae contributed 24–40% of the N supplied to the shoots (Ma¨der et al., 2000). The 15N tracer technique has some disadvantages which should be considered when planning root experiments and interpreting the results. 15N occurs in all plants and organic matter at concentrations slightly above the atmospheric abundance of 0.365 atom%. This natural background concentration should be accurately analyzed, particularly if low 15N levels are planned. Transport experiments require a high N concentration in the solution because 15N uptake by roots is a relatively slow process (Bledsoe and Atkinson, 1991). Since the highly mobile NO3 is leached rapidly from soil and is, at the same time, immobilized by microbial processes, its distribution in the soil is not

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uniform. Such dynamic properties mean that the results need careful interpretation. 2. @15N Approach The 14N/15N ratio in the atmosphere is relatively stable and therefore used as a standard for @15N analysis. All plants show a natural abundance of 15N, slightly higher than that of the atmosphere. Usually, 15N is enriched in the soil organic matter more than in the aboveground plant tissues (Ehleringer and Osmond, 1989). Based on these significant deviations between natural 15N abundance in the atmosphere and the soil (@15N value between 5& and 15&), the symbiotically fixed N2 can be detected. How far the root depth can be inferred by comparing the @15N of the N2fixing with the non-N2-fixing species from the same site, would be a challenge for future research. The resulting method would particularly be useful for monitoring deep-rooting N2-fixing plants. Shoot/root relationships of the deep-rooting woody legume, Prosopis glandulosa, and of a shallow-rooting woody non-N2-fixing reference plant, Atriplex polycarpa, have been studied in the Californian Sonoran Desert (Shearer and Kohl, 1986; Virginia et al., 1989). The @15N value of leaves provided information about the root activity and root depth. Moreover, the recolonization strategy of underground organs after damage to the shoots, e.g., by fire, has also been studied using this technique.

The authors recommend the @15N procedure as an important approach in monitoring the vertical distribution of deep-rooting, N2-fixing plant species. However, Bingham et al. (2000) urge caution when interpreting the results because of the natural variation in @15N in plants. 3.

@13C Approach

@13C is a widely used tool in ecosystem research. It is used to discern the root biomass of plants of different assimilation groups, especially between C3 and C4 plants. Plant enzymes discriminate against the heavier and less reactive 13CO2 (13C depletion) during photosynthesis such as (1) the Calvin cycle typical of C3 plants, (2) the Hatch-Slack-cycle dominant in C4 plants, or (3) the Crassulaceae acid metabolism characteristic of CAM plants. This process contributes to natural variation in the isotopic composition of plants (Table 4). C4 plants have less negative @13C values (between 9& and 14&) than C3 plants (between 20& and 35&), whereas CAM plants show a wide range of intermediate values. Isotope ratios vary not only among the plant species but also among the plant organs or tissue types depending on tissue composition. The reason is that some compounds, such as pectine, hemicellulose, starch or sugars, contain more 13C (are ‘‘heavier’’) than cellulose, lignin, and, in particular, lipids. Such

Table 4 Ranges of @13C Values in Various Plant Groups Plant groups Plant material Aquatic plants Seagrass species Terrestial C3 plants (dominant at mesic sites) C3 desert perennial C3 Poaceae grassland (East Africa) C3 Poaceae in forest (East Africa) C3 woody at grassland C3 woody at forest Terrestial C4 plants (oft high WUE) C4 marsh grasses Peat deposit Plant on saline soils (C4 dominante) CAM: facultative or obligate (dry location)

@13C/12C

References

27.0 8.0 to 30.0 15.0 to 23.0 3.0 to 23.8 23.0 to 30.0

Craig (1954) Ehleringer and Rundel (1988) Keeley (1988) Fry and Sherr (1988) Fry and Sherr (1988)

20.6 to 30.7 12.3 14.0 27.8 28.8 10.0 to 14.0 12.0 to 14.0 12.0 to 28.0

Ehleringer (1988) Tieszen and Boutton (1988) Tieszen and Boutton (1988) Tieszen and Boutton (1988) Tieszen and Boutton (1988) Ehleringer and Rundel (1988) Fry and Sherr (1988) Fry and Sherr (1988) Fry and Sherr (1988)

10.0 to 22.0 12.0 to 3.00

Ehleringer (1988) Keeley (1988)

Research Methods

variations can be used for distinguishing between various root compartments or for studying root/shoot relationships. The correlations between environmental factors and stable isotope compositions, especially for C3 and CAM plants, are used widely in ecological studies (Tieszen and Boutton, 1988; Ehleringer and Osmond, 1989). The @13C technique is not applied so widely in root research as it is in other types of ecological research, such as tree ring analysis (Leavitt and Long, 1988; Tognetti et al., 2000), water use efficiency, or photosynthetic pathways (Ehleringer, 1988). However, in recent years several interesting applications for root research have been published, particularly (1) in relation to C allocation, biomass, and the competitive ability of roots; (2) in order to distinguish the photosynthetic pathways of C3 and C4 plants (Svejcar and Boutton, 1985; Mordacq et al., 1986; Wong and Osmond, 1991); and (3) with respect to climatic change (Robinson and Scrimgeour, 1995; Cheng, 1996; Rochette and Flanagan, 1997; Andrews et al., 1999). Root respiration, which contributes to a large part of the soil respiration, is thus an important parameter affecting the global carbon metabolism. Root respiration was separated from the total soil respiration in a 15-yr-old loblolly pine plantation in a FreeAir Carbon-dioxide Enrichment (FACE) experiment (Andrews et al., 1999). The CO2 used for enrichment was strongly depleted in 13C. By measuring the depletion of 13CO2 in the soil system, they found that roots contribute 55% of the total soil respiration and that this reflected the fine-root distribution within the soil profile. The C derived from the microbial respiration of added C4-sucrose and CO2 from the roots was also distinguished using the @13C technique (Ekblad and Ho¨gberg, 2000). Another interesting recent variation of this approach was its use in studies of the metabolic interaction between pathogeneous fungi and roots (M. Hess, personal communication, 2000). 4.

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the groundwater was near that of @D in precipitation (about 20&). Deep-rooted plants have @D values between groundwater and rainfall water. In a different approach, isotopes such as 25Mg, 26Mg, 41K, 42Ca and 44 Ca were used in single-root and split-root labeling experiments to study the effects of Al and pH on nutrient acquisition by spruce trees (Kuhn et al., 1995). C.

Dye Methods

The classical labeling techniques are no longer frequently used in the present era of digital technology. Nevertheless, such methods are useful because they are simple, inexpensive, and suitable for ecosystem research in remote locations. Some applications of various markers in root research are described below. Various dyes, such as safranin, acid, and basic fuchsin, have been used to study water translocation pathways (Waisel et al., 1972), connections between underground organs, growth rate (Carman, 1982), and root vitality. Dyes can be applied in situ to the plant organ or to isolated plant segments in the laboratory (cf. Sauter, 1984; Zimmermann, 1983). 1.

Vitality Test—TTC

TTC (2,3,5-triphenyltetrazolium chloride) is an indicator used for testing the respiratory activity of tissues. TTC, which is colorless in the oxidized form, is easily taken up by living cells and reduced by dehydrogenase to red formazan (Fig. 6). The intensity of formazan production, measured as optical density, corresponds well with tissue vitality. The TTC method was successfully applied for many years in various biological tests and has also been adopted by root researchers. Root activity of herbaceous plants was tested by Knievel (1973), who calculated the ratio of living to dead roots of grass (Bouteloua gracilis) and maize. The viability of fine roots (<1 mm) of various tree species, such as Quercus alba (Joslin and Henderson, 1984), Picea

Other Stable Isotope Approaches

The natural abundance of 18O and D (deuterium) in rainwater differs from that in the soil groundwater. The contribution of groundwater and surface water to plants can be determined by measuring @D in the transpiration stream of plants. This provides information about root activity and location. White et al. (1985) compared the @D of xylem water of white pine (Pinus strobus) trees with that of the groundwater. The @D value of trees whose root systems did not contact

Figure 6 Formulae of the reaction from triphenyltetrazoliumchloride to triphenylformazan.

312

abies (Raspe, 1992), or Fagus sylvatica (Polomski and Kuhn, 2000), was estimated by this procedure. A possible use of TTC for studies of freezing damage to tree seedling roots yielded controversial results and need further investigation (Sattin and Lindstro¨m, 1999). The following procedure is used: Water-washed roots are cut into 2- to 3-mm small pieces. Root samples (100–200 mg fresh weight) are mixed with a solution of 0.6% TTC, 0.06M phosphate buffer, and wetting agent (Tween 20) and infiltrated under vacuum. The root samples are finally incubated at 308 C for 20 h. The produced formazan is extracted from the samples with 95% ethanol in 808C water bath, and the optical density measured spectrophotometrically at 520 nm. Root samples are used after extraction for determination of oven dry weight (1058 for 24 h). The quantity of formazan produced is correlated with the dry weight of the root tissue of both dead and live roots. The slope of the regression curve is, however, distinctly lower for dead roots. This difference is still visible even in considerably darker extracts from tree roots. TTC reduction is a sensitive process varying according to plant species, root age, and root diameter. The slope of the curve is also affected by other factors, such as temperature. For oak roots (Quercus alba) a maximum slope of 9.5 at a soil temperatuire of 228C in August and a minimum slope of 7.1 at 11:58C in April was registered (Joslin and Henderson, 1984). Therefore, care should be taken when comparing root samples. Samples should be homogenous with respect to species, the diameter of roots, and sampling time. The TTC method is simple and cheap. Moreover, it is practical when testing large numbers of samples to compare the root activities and the percentage of living roots in mixed root samples at different ecological sites or under different experimental treatments. However, standard curves describing the relationships between the root dry weight and the optical density of the formazan produced need to be first determined and the regression calculated. Separate curves should be determined for living and dead roots. Dead root tissues can be obtained by air-drying or by boiling. To obtain living tissue, the white tips of growing roots are sampled, but only the samples with the highest absorbance/weight ratios (the top 25% of the samples) should be considered for the standard curves. In this way the potential error arising from the visual identification of living roots can be minimized. This verifying procedure is sensitive and time-consuming, but important, since 75% of the variance can be explained by

Polomski and Kuhn

differences between living and dead roots (Joslin and Henderson, 1984). 2.

Vitality and Vigor Tests—Starch Content

The technique based on iodine staining of starch granulates has been used for evaluation of root vitality. This procedure is easy to perform, accurate, easily replicable, and suitable even for field applications. The visual method is, of course, less precise than chemical analysis, but it is still precise enough to evaluate the effects of stress on plants or to be used to supplement other vigor-tests. The following procedure has been used by Wargo et al. (1972, 1975): Small root pieces are frozen at 208C until cutting. Then they are thawed rapidly and 100- to 150-mm-thick cross sections are cut. The sections should be kept moist all the time. For staining 15 g KI and 3 g crystalline I2 are dissolved in 1000 mL distilled water. Two cross sections are placed on a glass slide and rinsed with the solution for 5 min. After the stain is blotted, the sections are rinsed twice with distilled water and the excess water is removed. Glycerin is added and the slide is covered with a cover glass. The samples can then be photographed within 48 h. Roots can easily be categorized, either visually or digitally, according to their starch content into four groups: high, medium, low, and null. The variability in starch content among species, locations, phenological status, and root diameter should be considered when evaluating and comparing results. Starch content and distribution patterns in the underground organs of herbaceous and woody plants are useful indicators of plant vigor because they reflect the plant’s photosynthetic capacity. Stressed roots have lower starch contents than unstressed ones (Wargo et al., 1972, 1975). The parenchymatic cells of the xylem are specialized tissues for accumulating starch in plant roots. In some cases, e.g., Tamarix roots, starch is accumulated also in fibers (Fahn, 1990). The distribution patterns and concentrations of starch in seeder and in resprouter species differ. However, the latter have greater reserves of starch in their underground organs. This potential source of energy allows efficient regeneration after damage to shoots. Bell and Ojeda (1999) found a strong relationship between starch accumulation and distribution in roots and regeneration after fire in Erica species. The starch content of roots is not only a useful index of plant vitality, but has also been used as an important variable for modeling fine root production and turnover (Marshall and Waring, 1985; Vogt et al., 1985).

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Changes in starch content depend on the phenological status of the plants (Krueger and Trappe, 1967; Dubroca and Saugier, 1988) and on environmental stress, such as defoliation, drought, increased shade, or air pollution (Crookshanks et al., 1998). 3.

Staining of Root Profiles

a.

Quantifying the Root Distribution and Biomass

Root distribution and production can be directly quantified using a nondestructive dye technique. The dye should preferentially be absorbed by the roots but not by soil particles. Rhodamin water tracing (RWT) has been used for staining roots of Populus tremuloides (Ruark and Bockheim, 1988). The profile faces of soil monoliths were sprayed with dye solution (1%), and the roots became darkly colored against a more lightly stained soil matrix. The profiles were then photographed with infrared color film, the photos scanned, and the total cross section areas, root length, root biomass, branching, etc. calculated using computer software. The RWT dye method combined with infrared photography seems to be a useful technique, with best results obtained with (1) larger roots (300 and 3000 mm in diameter), (2) relatively light roots compared to the background soil matrix, and (3) relatively homogeneous stands with low understory cover. b.

Staining Technique for Quantification of Root Growth Rates

The growth rate of roots can be visualized and measured by using rinsed with various colored dye solutions at different time intervals. To obtain the best staining results, the experimental variables such as

the dye, buffer system, staining time, rooting media, and type of leachate should be selected carefully. The color of the dye should remain stable during the observation period, and the dye and leaching solution should not be toxic to the plants. The staining exposure depends not only on the dye type and concentration, but also on the root properties and the state of the rooting medium. The most favorable growth media for staining are porous substrates, such as sand, perlit, or vermiculte, because they allow a high flow rate, which reduces the staining time and allows deep roots to get into contact with the dye solution rapidly. To minimize injuring the roots in repeated staining, the dye should be leached from the rooting medium immediately after the staining procedure is finished. Carman (1982) recommended using the red, blue, and yellow chlorotriazinyl (CI) dyes in a nondestructive staining technique for estimating the root growth rate. Table 5 summarizes the optimal staining conditions tested for Sorghum bicolor. Under the proposed staining conditions the roots remain colored for more than 2 months, and no growth anomalies on the roots have been observed. After 21 days, the roots were collected separately in the soil increments, washed, and floated in shallow Plexiglas dishes over a 5  5 cm grid. Total root growths per week for each color were estimated (Tennant, 1975). 4.

The Agar Technique with pH Indicators

The agar technique is a nondestructive, simple laboratory method for measuring pH changes and redox processes (Mn, Fe) in the rhizosphere. This procedure was developed by Weisenseel et al. (1979) for visualizing

Table 5 Parameters for Chlorotriazinyl (C.I.) Dye Used as a Soil Drench Procedure Tested on Sorghum bicolor Cultivated in Cylinders (10 cm diameter, 80 cm long) Filled with 13.4 kg Air-Dried Sand Solution composition Buffer NaCl NaHCO3 Stains C.I. Reactive Red 2 C.I. Reactive Blue 163 C.I. Reactive Yellow 86 Leachate HNO3-KOH

Concentration (glitre1H2O) 1.500 1.500 (glitre1 buffer) 0.100 0.175 0.250

Treatment time Minutes

Solution quantity Litrecontainer1

50

4

40

4

The cylinders were leached in three 7-day intervals with red, blue, and yellow dye solutions, successively. Source: Carman (1982).

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the participation of H+ in the ionic composition of plants. It is frequently used in rhizosphere research of Fe deficiency, P mobilization, or N sources. The pH value in the soil can vary by >2 units, not only between bulk soil and the rhizosphere, or between primary and lateral roots, but also according to distance along the roots (Marschner et al., 1982; Marschner and Ro¨hmheld, 1983). Moreover, local acidification of the rhizosphere caused by Fe or P deficiency has often been observed. For example, in the root zone of rape (Brassica napus), the soil pH decreased in the absence of phosphate from 6.5 to 4.1 within 2 weeks (Hoffland et al., 1989). a. pH Indicators The first step involves choosing a suitable indicator for the planned experiment. To measure pH changes Bromocresol Purple (0.006%) is used. Its color changes to purple at pH 7.0, red at pH 6.0, and yellow at pH 4.5. Bromocresol has, at the concentrations needed, no effect on root growth, unlike other indicators like Methyl Red or Bromothymol (Weisenseel et al., 1979). To measure iron reduction at the root surface, the nutrient solution is mixed with FeIII EDTA at a concentration of 0.1 mM and with 0.3 mM BPDS (chelator for FeII). Under Fe deficiency, a red-colored FeII complex (Fe2[BPDS]3) is formed. The reactions of seedling roots to Fe deficiency can be observed by comparing the pH changes along roots growing in Fe-enriched solution to roots growing in Fe-free solution. The solution should be adjusted to pH 6.0 with NaOH (0.1 M). Within 20 min, the acidified root zones begin to change colors from purple to yellow (Ro¨hmheld et al., 1984). For measuring manganese reduction in a root system growing in Fe-deficient solution, 1 mM KMnO4 is used as indicator. The solution is adjusted to pH 6.0, mixed with agar, and kept warm at 508C for 2 h. During this time MnO2 is formed and becomes dispersed within the agar, changing its color to brown. However, it takes up to 3 days for the MnO2 to be reduced to MnII. The sensitivity of this test depends also on the plant species (Marschner et al., 1982). b. Preparing an Agar Medium A suitable indicator is mixed with distilled water or test nutrient solution and the pH value adjusted. Purified agar is then added at 508C to a final agar concentration between 0.5% and 1.0%. The solution is kept fluid at 408C in a water bath until used for the experiments. The precultivated seedlings or small plants are inserted into small Plexiglas boxes or dishes filled either with

Polomski and Kuhn

prepared soil material or nutrient solution. Finally, the boxes are placed for a few days in the growth chambers, with the soil-filled boxes fixed at angles between 308 and 458, to the vertical. To minimize the light effects upon roots and loss of water, the boxes are covered with glass plates and with black plastic sheets. The boxes are turned upside-down to inject the agar solution into the solid growth substrate, the screwed lid at the bottom is removed, and the fluid agar (temperature between 358C and 388C) is enriched with an indicator’s injected ‘‘drop by drop’’ into the substrate (Marschner and Ro¨hmheld, 1983). The typical change in color along the roots appears within 2 h. The hydroponic cultivated plants are removed from the solution, the intact roots are spread out carefully in the flat, and transparent boxes or petri dishes are filled with fluid agar media. The boxes are then rapidly cooled to 258C. Color changes along the roots can be observed by microscope or can be recorded on color film or by scanner. H+ ion influx can also be calculated by measuring the volume of color change in time by using the formula JH ¼ VC=At, where JH is the H+ ion influx (mol cm2s1), V the volume of color change near the growth zone (cm3), C the specific capacity of the dye (mol cm3), and t the time needed (s). Control trials, using known quantities of applied acid, should be tested in advance. The use of bromocresol purple (pH 5.0) was demonstrated by Weisenseel et al. (1979). This indicator turns yellow at pH 5.2 and becomes purple at pH 6.8. The pH bands along the roots were recorded on color film. The qualitative agar technique can be combined with measurements of pH by microelectrodes inserted into the agar medium or with a chemical analysis of the roots and substrate after experiments. For measuring the root exudates, small plastic rings (diameter 1.2 cm) can be placed on the roots spread over a glass plate to collect the nutrient solution during the experiments. One part of the rings is placed just behind the root tips and the other ones are placed close to the root base. Finally, the roots are covered with liquid agar. Nutrient solution 0.25 mL is pipetted into every ring, which is then incubated, and sampled to estimate the concentrations of organic acids or nutrient elements (Hoffland et al., 1989). c.

Advantages and Disadvantages of the Agar Technique

This simple, nondestructive method allows root study in vivo. This is an important advantage compared to studies of isolated plant tissues or of root

Research Methods

segments. The visual test with indicators can be combined with quantitative measurements such as measuring pH with a microelectrode or collecting roots or growth substrate for chemical analysis. The agar technique is sensitive to pH measures, even in such a small volume of solution as 150 mL per plant. It is simple, reliable, and cheap (Marschner, 1995). However, this method is only appropriate for seedlings or small plants growing under laboratory conditions, not for larger, naturally growing plants. Furthermore, the proton distribution pattern observed along the roots growing in solution is uniform compared to that of those grown in an undestroyed soil where the pattern is usually extremely heterogeneous. These aspects should be borne in mind when interpreting results.

5.

Phytocides (Toxicants)

Injecting a toxic solution, which causes visible stress in plants, is a useful way of qualitatively investigating some processes in the field, such as root grafting, shoot/root development of tree coppices, or regeneration strategy after fire. However, some phytocides are very poisonous and therefore require careful handling. Moreover, phytocides do not cause changes in color, so their appearance in tissue is not so well defined as that of dyes. Aymard and Fredon (1986) applied the phytocide armitrol directly to one root of several stools belonging to a coppiced chestnut stand (Castanea sativa) to observe the relationship between the roots and the growth of new shoots with respect to the root system of the stump. The intensity and distribution of leaf damage showed the translocation pathways of stored resources. De Byle (1964) monitored the translocation of sodium arsenite, a dye (Eosin Bluish), and 86Ru within roots of aspen (Populus grandidentata) coppiced to detect the underground connections. They found that both the phytocide and the dye were effective tracers, but they recommended using the dye because it is safer to use. The tracers were transported up to 14 m from the trunk to 10 neighboring stems and indicated a large number of interconnected stem groups as well as transport pathways between stems. Bormann and Graham (1959) used acid fuchsin dye, 86RuCl, Na131I (both 1% solutions), and ammonium sulfamate poison as tracers in Pinus strobus stands. They found that of 84 donor trees injected, 41 were found to be naturally grafted through roots to 54 receptor trees.

315

6.

Fluorescence

Fluorescence dyes (e.g., eosin, erothrysin, trypaflavin, acridin, fuorescein, rose bengal) have been used as tracers for studying the water transport from roots to shoots. The dye solution is applied into the root zone and fluorescence intensity is measured in the foliage tissue by a Turner fluorometer. A very sensitive step in the analytical procedure is a complete removal of the dye from the plant tissues. Rhodamine WT and Pontacyl Brilliant Pink, in dosage between 0.03 and 0.15 g kg soil, have been recommended for such studies (Donaldson and Robinson, 1971). Four to 24 h after the dye solution was injected into the soil, the dyes were detected in the leaves. No negative effects were observed in the plants. An interesting approach to studies of root growth is to observe the natural fluorescence phenomena of plants. Dyer and Brown (1983) studied soybean plants cultivated in porous plastic membranes and observed a natural fluorescence of the root parts near the apex. Occult chlorophyll or contamination by algae can be excluded (cf. also Smit and Zuin, 1996) The authors were then able to measure the rate of new root production by documenting this phenomenon. Unfortunately, the fluorescence method has so far proved to be suitable only for plants with chlorophyll-containing tissue. The fluorescence tracer technique is a simple, rapid, and inexpensive method for studying of transport and the root/shoot relationship.

ACKNOWLEDGMENTS We thank Mrs. Verena Fataar for drawing the figures, and Mrs. Silvia Dingwall and Yoav Waisel for their help in improving the English language of the manuscript.

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319 Pregitzer KS, Hendrick RL, Fogel R. 1993. The demography of fine roots in response to patches of water and nitrogen. Plant Soil 125:575–580. Raspe S. 1992. Biomasse und Mineralstoffgehalte der Wurzeln von Fichtenbesta¨nden (Picea abies Karst.) des Schwarzwaldes und Vera¨nderungen nach Du¨ngung. Freiburger Bodenkundliche Abhandlungen. Freiburg im Breisgau 29. Richards JH. 1984. Root growth response to defoliation in two Agropyron bunchgrasses: field observations with an improved root periscope. Oecologia 73:486–489. Richner W, Liedgens M, Bu¨rgi H, Soldati A, Stamp P. 2000. Root image analysis and interpretation. In: Smit AL, Bengough AG, Engels C, Van Noordwijk M, Pellerin S, Van de Geijn SC, eds. Root Methods. A Handbook. Berlin; Springer-Verlag, pp 305–341. Robertson BM, Hall AE, Foster KW. 1985. A field technique for screening of genotypic differences in root growth. Crop Sci 25:1084–1090. Robinson D, Scrimgeour CM. 1995. The contribution of plant C to soil CO2 measured using @13C. Soil Biol Biochem 27:1653–1656. Rochette P, Flanagan LB. 1997. Quantifying rhizosphere respiration in a corn crop under field conditions. Soil Sci Soc Am J 61:466–474. Rogers AW. 1979. Techniques of Autoradiography. 3rd ed. Amsterdam; Elsevier. Ro¨hmheld V, Mu¨ller C, Marschner H. 1984. Localization and capacity of proton pumps in roots of intact sunflower plants. Plant Physiol 76:603–606. Ruark GA, Bockheim JG. 1988. Digital image analysis applied to soil profiles for estimating tree root biomass. Soil Sci 146:119–123. Rygiewicz PT, Bledsoe CS, Zasoski RJ. 1984. Effects of ectomycorhizae and solution pH on 15N nitrate uptake by coniferous seedlings. Can J For Res 14:885–892. Sackville Hamilton CAG, Cherrett JM, Ford JB, Sagar GR, Whitbread R. 1991. A modular rhizotron for studying soil organisms: construction and establishment. In: Atkinson D, ed. Plant Root Growth. An Ecological Perspective. Oxford, UK: Blackwell Scientific, pp 49– 59. Samson BK, Sinclair TR. 1994. Soil core and minirhizotron comparison for the determination of root length density. Plant Soil 161:225–232. Sattelmacher B, Klotz F, Marschner H. 1983. Vergleich von zwei nicht destruktiven Methoden zur Bestimmung der Wurzeloberfla¨chen. Z Pflanzenerna¨hr Bodenkd 146:449–459. Sattin E, Lindstro¨m A.1999. Influence of soil temperature on root freezing tolerance of Scots pine (Pinus sylvestris L.) seedlings. Plant Soil 217:173–181. Sauter JJ. 1984. Detection of embolization of vessels by double staining technique. J Plant Physiol 116:331– 342.

320 Schuurman JJ, Goedewaagen MAJ. 1971. Methods for the Examination of Root Systems and Roots. 2nd ed. Wageningen, Netherlands: PUDOC. Shearer GB, Kohl DH. 1986. N2-fixation in field settings: Estimations based on natural 15N abundance. Aust J Plant Physiol 13:699–756. Smit AL, Zuin A. 1996. Root growth dynamics of Brussels sprouts (Brassica olearacea var. gemmifera) and leeks (Allium porrum L.) as reflected by root length, root color and UV fluorescence. Plant Soil 185:271–280. Smit AL, Groenwold J, Vos J. 1994. The Wageningen rhizolab—a facility to study soil–root–shoot–atmosphere interactions in crops. II. Methods for observations. Plant Soil 161:289–298. Smit AL, George E, Groenwald J. 2000a. Root observations and measurements at (transparent) interfaces with soil. In: Smit AL, Bengough AG, Engels C, Van Noordwijk M, Pellerin S, Van de Geijn SC, eds. Root Methods. A Handbook. Berlin; Springer-Verlag, pp 235–271. Smit AL, Bengough AG, Engels C, Van Noordwijk M, Pellerin S, Van de Geijn SC. 2000b. Root Methods. A Handbook. Berlin; Springer-Verlag. Smucker AJM, Ferguson JC, De Bruyn WP, Belford RK., Ritchie JT. 1987. Image analysis of video-recorded plant root systems. In: Taylor HM, ed. Minirhizotron Observation Tubes: Methods and Applications for Measuring Rhizosphere Dynamics. Spec Publ 50. Madison, WI: Am Soc Agron, pp 67–80. Steen E. 1991. Usefulness of the mesh bag method in quantitative root studies. In: Atkinson D, ed. Plant Root Growth. An Ecological Perspective. Oxford, UK: Blackwell Scientific, pp 75–86. Steinke W, Von Willert DJ, Austenfeld FA. 1996. Root dynamics in salt marsh over three consecutive years. Plant Soil 185:265–269. Stepniewski W, Pezeshki SR, De Laune RD, Patrick WH Jr. 1992. Root studies under variable redox potential in soil using laboratory rhizotrons. In: Bo¨hm W, Kutschera L, Lichtenegger E, eds. Wurzelo¨kologie und ihre Nutzanwendung. Irdning, Germany: Bundesanstalt fu¨r alpenla¨nische Landwirtschaft, pp 353–356. Stevenson FC, Walley FL, Van Kessel C. 1998. Direct vs. indirect nitrogen-15 approaches to estimate nitrogen contributions from crop residues. Soil Sci Soc Am J 62:1327–1334. Svejcar TJ, Boutton TW. 1985. The use of stable carbon isotope analysis in rooting studies. Oecologia 67:205– 208. Tennant D. 1975. A test of a modified line intersect method of estimating root length. J Ecol 63:996–1001. Thies WG, Cunningham PG. 1996. Estimating large-root biomass from stump and breast-height diameters for Douglas-fir in western Oregon. Can J For Res 26:237– 243.

Polomski and Kuhn Tieszen LL, Boutton TW. 1988. Stable carbon isotopes in terrestrial ecosystem research. In: Rundel PW, Ehleringer JR, Nagy KA. eds. Stable Isotopes in Ecological Research. New York; Springer-Verlag, pp 167–195. Tognetti R, Cherubini P, Innes JL. 2000. Comparative stemgrowth rates of Mediterranean trees under background and naturally enhanced ambient CO2 concentrations. New Phytol 146:59–74. Upchurch DR, Taylor HM. 1990. Tools for studying rhizosphere dynamics. In: Box JE Jr, Hammond LC, eds. Rhizosphere Dynamics. Washington, DC: AAAS Selected Symposium, pp 83–115. Vamerali T, Ganis A, Bona S, Mosca G. 1999. An approach to minirhizotron root image analysis. Plant Soil 217:183–193. Van de Geijn SC, Vos J, Groenwold J, Goudriaan J, Leffelaar PA. 1994. The Wageningen rhizolab—a facility to study soil–root–shoot–atmosphere interactions in crops. I. Description of main functions. Plant Soil 161:275–287. Virginia RA, Jarrell WM, Rundel PW, Shearer G, Kohl DH. 1989. The use of variation in natural abundance of 15N to assess symbiotic N2-fixation by woody plants. In: Rundel PW, Ehleringer JR, Nagy KA. eds. Stable Isotopes in Ecological Research. New York; Springer-Verlag, pp 375–394. Vogt KA, Persson H. 1991. Measuring growth and development of roots. In: Lassoie JP, Hinckley TM, eds. Techniques and Approaches in Forest Tree Ecophysiology. Boston; CRC Press, pp 477–501. Vogt KA, Vogt DJ, Bloomfield J. 1998. Analysis of some direct and indirect methods for estimating root biomass and production of forests at an ecosystem level. Plant Soil 200:71–89. Vogt KA, Vogt DJ, Moore EE, Littke W, Grier CC, Leney L. 1985. Estimating Douglas-fir fine root biomass and production from living bark and starch. Can J For Res 15:177–179. Waisel Y, Liphschitz N, Kuller Z. 1972. Patterns of water movement in trees and shrubs. Ecology 53:520–523. Wang Z, Burch WH, Mou P, Jones RH, Mitchell RJ. 1995. Accuracy of visible and ultraviolet light for estimating live root proportions with minirhizotrons. Ecology 76:2330–2334. Wargo PM. 1975. Estimating starch content in roots of deciduous trees—a visual technique. USDA Forest Service Research NE-313, p 8. Wargo PM, Parker J, Houston DR. 1972. Starch content in roots of defoliated sugar maple. For Sci 18:203–204. Weisenseel MH, Dorn A, Jaffe LF. 1979. Natural H+ currents traverse growing roots and root hairs of barley (Hordeum vulgare L.). Plant Physiol 64:512–518. White SWC, Cook ER, Lawrence JR, Broecker WS. 1985. The D/H ratios of sap in trees: implications for water

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321 different CO2, N nutrition irradiance, with emphasis on below-ground responses using @13C value of root biomass. Aust J Plant Physiol 18:137–152. Yoder CK, Vivin P, Defalco LA, Seemann JR. 2000. Root growth and function of three Mojave Desert grasses in response to elevated atmospheric CO2 concentration. New Phytol 145:245–256. Zimmermann MH. 1983. Xylem Structure and the Ascent of Sap. Berlin; Springer-Verlag.

19 Aeroponics: A Tool For Root Research Under Minimal Environmental Restrictions Yoav Waisel Tel Aviv University, Tel Aviv, Israel

I.

INTRODUCTION

this volume. However, neither of those facilities renders the possibility for nondestructive and uninterrupted investigations of root development for prolonged periods. Some of the setups, e.g., root boxes or minirhizotrons, suffer from inherited methodological disadvantages: The observed roots represent only a small fraction of the total root population, without disclosing the class of roots to which they belong, their age, or their hierarchy. The only method that enables comprehensive studies of root systems is aeroponics. Aeroponics is not just a Disneyland feature. It is one of the more elegant research systems that had been developed for continuous studies of root development and function, and nowadays constitutes an important tool for root research. Aeroponics is not a new idea. Plants were grown with their roots in moist air, already >120 years ago (Sachs, 1874). However, the serious use of aeroponics for root research started some 80 years ago (Barker, 1922) and was further developed later on (Carter, 1942; Klotz, 1944). Since then, several types of aeroponic facilities were developed and used for a variety of topics. Aeroponics was used for investigations of root growth, mineral nutrition, root excretions, toxicity of NaCl or heavy metals, and comparisons of root systems of different varieties of economic plants (cf. Vyvyan and Travel, 1953; Clayton and Lamberton, 1964; Zobel et al., 1976; Vincenzoni, 1979;

Root research has been hampered for a long time by methodological difficulties. Most roots are hidden in the soil, are out of sight, and therefore must be excavated before any observation, specific treatment, or analysis can be done. However, it is practically impossible to excavate the whole system of plant roots without destroying the delicate root hairs, damaging and losing the fine roots, and destroying some of the larger ones. Thus, research that is based on such a methodology (see Chapter 18 by Polomski and Kuhn and Chapter 11 by Persson in this volume) yields some general information regarding the location of various roots, but is under a handicap and has limited meaning when the developmental or functional aspects of roots are studied. Root research has suffered from the inability to investigate whole root systems in a setup where the roots can be continuously observed for prolonged periods. As a result, most root system studies have been limited to roots of seedlings or small plants. To avoid such hindrance and to enable meaningful studies of growth, development, and function of roots, various research devices had been developed. These included glass front observation boxes, minirhizotrons (transparent observation tubes), lysimeters, hydroponics, etc. Details of such methods were discussed by Box (1996) and in Chapter 18 by Polomski and Kuhn in 323

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Engenhart, 1984; Waisel and Breckle, 1987; Waisel, 1988; Eshel and Waisel, 1992). Most of the aeroponic chambers that were used in the past were rather small and therefore could be employed only for short-term experiments (Hendrix and Lloyd, 1968; Shimura, 1970). The improvement of the mechanics and reliability of the mist chambers, and especially the introduction of the large ones, has enabled the study of small annual plants but also of large perennials and of trees (Waisel, 1996). To date, scores of plant species were grown in aeroponics, including evergreen and deciduous trees, crop plants, xerophytes, hygrophytes, lithophytes, ornamental plants, etc. Most of the plants exhibited outstanding growth under such conditions and develop substantial root systems. However, whether the observed architecture that develops under such conditions is the genuine expression of the plant’s capability to develop roots or it is only the outcome of the growth potential under the specific combination of environmental restrictions remains unanswered and should be addressed in the future.

II.

TECHNICAL DETAILS

Basically, an aeroponic system is composed of a root chamber, in which mist is intermittently produced and sprayed. Mist can be produced by mechanical foggers, by sprayers of the Ventury type, by ultrasonic foggers, or by pressurizing the solution through very fine nozzles. The duration of the spray pulses has an important effect on the development of the exposed plants (Peterson and Krueger, 1988; Chung et al., 1993) and should be adjusted for each species. Apparently, too long an interval between sprays affects the water availability to the plants and constitutes one of the limitations of the system. Infrequent spraying might cause water stress whereas, too frequent spraying might cause leaching of various essential elements out of the roots. However, infrequent spraying pulses may also cause precipitation of salt layers on the root surfaces. Indeed, the use of diluted solutions is recommended for many of the aeroponically grown plants. The nutrient solution is usually collected at the base of the chamber and recirculated. In such cases it is recommended to incorporate devices for the solution filtration and online UV sterilization in order to reduce bacterial buildup and assure fault-free operation of the system. Control of the temperature of the solution and of its composition, concentration, and pH can be done

automatically by computer-controlled devices or manually, at cerain time intervals. A special nutrient–mist bioreactor was designed that separates the nutrient medium of the root chamber from the electronic components of the system by an acoustic window, thus allowing sterile growth conditions (Buer et al., 1996; Correll and Weathers, 1998). Aeroponic devices for home use are now commercially available (see ‘‘Future Garden’’ at http://www.howtohydroponics.com/aero.html or ‘‘Aeroponic Systems’’ at http://www.thegrowroom.com/aero.html).

III.

SPECIFIC USES

Aeroponics was used for a variety of research projects, a few examples of which are presented in the following. A.

Growth Studies

The capability of plants to grow in aeroponic systems, and of stem cuttings to root, was studied with a variety of species and under various growth conditions (Nir, 1982; Waisel, 1988). Growth of most plants was highly improved under such conditions, and only very few species (e.g., Pinus halepensis) have shown negative responses to aeroponics. Aeroponics enables direct access to each of the roots, thus allowing direct determination of their elongation rate and lifespan—i.e., the potential of roots to remain alive without interference of diseases or of grazing animals. B.

Mineral Nutrition

Aeroponics was used in studies of nutrient consumption by seedlings (Ingestad and Lund, 1979). The advantage of such a system for the study of plant nutrition is augmented by the capability to reach individual roots and to compare the behavior of different types of roots, under a variety of conditions (Waisel and Breckle, 1987; Eshel et al., 1992). Aeroponics was used in studies of plant responses to phosphorous deficiency (Biddinger et al., 1998), calcium nutrition (Quintana, 1999), and for studies of the capacity of roots to exchange ions, of K fluxes, and of pH changes (Garrido et al., 1998a,b). For long-term studies, aeroponics has also weak aspects. When the mist supply of the plants is not precisely controlled, accumulation of salts on the root surfaces may result in poisoning or in an osmotic stress (Engenhart, 1987).

Aeroponics

C.

Studies of Water Use

Aeroponics was also utilized for demonstrating that roots are heterogeneous organs and that water uptake along and among them differs greatly. Rates of water uptake by lateral roots was found to be eight times faster than those of uptake by the taproot. The hydraulic conductivity of excised roots of Lotus japonicus was found to vary over fivefold during a day/ night cycle. A marked diurnal variation was also seen in root pressure (Henzler et al., 1999). Aeroponics was also used for studies of plant–water relations and for the determination of xylem ABA and nitrate concentrations (Dodd et al., 2000). Such differential features can be demonstrated in an aeroponic system. For example, the volume of absorbed water can be estimated from the increase in concentration of an applied dye, Sulphorhodamine G, on the root surface (Varney and Canny, 1993). Water penetrates the roots much faster than the dye. Therefore, the dye accumulates on the surface of those roots that are active in water uptake but not on roots of nontranspiring plants. The same method can be used for studies of the water uptake efficiency of various root segments. D.

Effects of the Root Atmosphere

Aeroponics was used for the study of the effects of pO2 and of pCO2 on root performance (Strausberg and Rakitima, 1970; Shimura, 1970). Roots were found to develop in different angles under the various concentrations of O2 and CO2. At very low CO2 concentrations, roots tend to grow downward, whereas at 30% CO2 they grow almost horizontally. This might have an important ecological implication, i.e., control of the development of vertical and of horizontal components of root systems (Ycas and Zobel, 1983). Roots that grow horizontally in air (high pO2) tend to bend downward in water (low pO2). However, the high pO2 that characterizes the atmosphere of aeroponic systems may also have negative effects. High pO2 reduced the uptake rates of K by wheat plants, as compared to the uptake by hydroponically grown plants. Similarly, Ca stimulated the uptake of K in hydroponics but inhibited it in aeroponics (Zsoldos et al., 1987). Aeroponics was used for studies of the effects of inhibition of ethylene production on nodulation of alfalfa roots (Caba et al., 1998) and in the study of the effects of pCO2 on roots and on ethylene production. Roots of sunflower were investigated showing

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that 2% CO2 treatment reduces ethylene evolution and that this effect operates by lowering the availability of 1- aminocyclopropane-1-carboxylic acid (ACC) (Finlayson and Reid, 1996). Special aeroponics root chambers were designed to evaluate the influence of low pO2 on disease development in various clones of Eucalyptus marginata susceptible or resistant to infection by Phytophthora cinnamomi (Burgess et al., 1998, 1999). E.

Nitrification

The effects of nitrifying bacteria on the use of ammonium by plants has been demonstrated in aeroponics (Padgett and Leonard, 1993). Plants of maize and peas provided only with ammonium chloride have produced some nitrate after a few days, following the inoculation of the roots by nitrifying bacteria. Thus, in nonaseptic aeroponic systems, conversion of ammonium to nitrate by airborne bacteria is a fast process that must be taken into account (Padgett and Leonard, 1993). F.

Disease Research

Aeroponics was used for the estimation of rates of spreading of root diseases and for the evaluation of applied countermeasures (Wagner and Wilkinson, 1992; Wagner et al., 1993; Rao et al., 1995). The control of the soybean root rot (Phytophthora sojae) depends on the development of disease resistant cultivars. Classically, those were divided into hypersensitive varieties and into varieties with rate-reducing resistance. Nevertheless, the efficiency of reduction of the fungal dispersal and of lesion development can be accurately estimated only from continuous observations and by using nondestructive methods. Moreover, identification of the specific roots that are infected and of the time course of infection are highly important. Attempts to inoculate roots with Phytophthora sojae in the soil did not yield reliable results. Inoculation can be controlled only under the accurate conditions of aeroponic systems. Aeroponics was used for studies of the effects of hypoxia on the growth of Eucalyptus marginata, on its root morphology, and on its responses to infection by Phytophthora cinnamomi (Burgess et al., 1999). Aeroponics enabled the study of the effects of foliate application of fungicides on Eucalyptus root inoculation by Phytophthora, and of the inoculation mechanism (Jackson et al., 2000).

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Aeroponics was used for studies of the effects of barley yellow dwarf virus on wheat root and shoot growth, and in comparisons of root and shoot growth among cultivars (Hoffman and Kolb, 1997). Acacia mangium seedlings were grown and nodulated with selected elite strains of Bradyrhizobium sp. under aeroponic conditions. The aeroponics system induced rapid growth and enhanced performance of the Acacia seedlings under greenhouse conditions (Martin-Laurent et al., 1997, 2000). G.

Mycorrhizae

Aeroponic cultures were used for studies of inoculation of mycorrhizal fungi. Aeroponically grown plants of Paspalum notatum and Ipomoea batatas were colonized by various species of Glomus and developed normal arbuscules and vesicles. The aeroponic treatment did not alter the normal behavior or the characteristics of the infectious fungus (Sylvia and Hubbell, 1986; Hung and Sylvia, 1988). Differences were found in mycorrhizal development in response to sprays of the root system or exposure to a nebulizer system. Roots from the nebulized system had more infective propagules than the sprayed ones (Mohammad et al., 2000). Aeroponics was used to produce Acacia mangium saplings associated with arbuscular mycorrhiza (AM) fungi. Aeroponics was found to be a better system than soil for such studies, allowing the production of tree saplings twice as high as those grown in soil. Moreover, aeroponically grown saplings inoculated with AM fungi exhibited significantly different rates of mycorrhization, resulting in an increase in phosphorus and chlorophyll in the plant shoot tissues (Martin-Laurent et al., 1999). Study of the nutrient requirement for the production of mycorrhizal associations was also done with aeroponics (Jarstfer et al., 1998). H.

Root Exudates

Aeroponic facilities can be kept aseptic, thus providing a reliable system for the investigation of root metabolism, including the release of volatile substances (Smucker and Erickson, 1976). Aeroponic systems (fog boxes) were used for investigation of root exudates by Tagetes and Albizia plants. The advantage of an aeroponic system for such studies lies in the fact that the contaminated solution is washed away from the roots’ surface as soon as the organic material

is excreted (Clayton and Lamberton, 1964). Such a system was also used for the study of bacterial inhabitation of various root types of faba beans (for details see Chapter 9 by Waisel and Eshel in this volume).

I.

Mechanical Impedance

Mechanical impedance of root growth is one of the major obstacles that roots encounter in nature (see Chapter 45 by Masle in this volume). To elucidate the effects of soil impedance on root growth and function, it is essential to compare the growth of roots impeded and unimpeded conditions. Aeroponics is the only system that enables such a comparison. Exposure of aeroponically grown tomato roots to mechanical impedance created by a column of glass beads caused a dramatic change in their size and architecture (Fig. 1). Such a change in root system architecture was expressed by other plant species as well. In the aeroponic laboratory, roots of Tamarix nilotica grew down to 6 m with practically no branching. Only when the roots confronted the mechanical impedance of the floor did they start to branch. Moreover, a study of gene expression in roots of tomato plants, grown in the aeroponic facility, has shown that touch-related genes (XET and CAL) are upregulated in the tips of roots that have confronted a solid surface or by roots that were exposed to mechanical pressure for 1 h, as compared with other roots of the same plants that were hanging freely in the aeroponic chamber (Eshel, Katz, and Ohad, unpublished results). The physiological effects of mechanical impedance can thus be revealed only when compared with a nonrestrictive environment.

J.

Effects of Temperature

Aeroponics was used for studies of the effects of root temperature on the growth, photosynthesis, and development of lettuce plants (Lee and Cheong, 1996; Jie and Kong, 1998a,b). A high root zone temperature prevented plants from forming dense heads. Aeroponics enables a fast switch between constant and fluctuating ambient temperatures (25–408C) without the buffering effects of the soil. It was used for studies of the growth of Capsicum annuum, for its stomatal conductance, water relations, and for determination of xylem ABA and nitrate concentrations (Dodd et al., 2000).

Aeroponics

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Figure 1 Differences in development of two similar roots; one free-hanging in the aeroponic chamber, and the other one confronting the mechanical impedance produced by a column of glass beads.

K.

Screening for Root Mutants

The search for plant mutants with a preferred root system was hampered in the past by the lack of proper facilities. Those require easy access, observation, and screening of large numbers of plants but still with a restricted budget and in a limited laboratory space. This was now designed for selection of banana cultivars by Dr. Draye of the KUL in Belgium (see details below). Aeroponics was used for screening of maize genotypes for resistance to Fusarium (du Toit et al., 1997). L.

Tissue and Organ Culture

Aeroponics can be used for sterile growth of plant tissues, provided the mist is formed beyond a dividing window (Buer et al., 1996). M.

Gravireactions

Aeroponics was used also for exceptional purposes, e.g., for the supply of nutrients to plants grown in a microgravity facility (Hessel et al., 1993). Aeroponics was used for growing cucumber plants during space flight (Takahashi et al., 1999) and verified that the lateral positioning of a peg in the germinating cucumber seedling and the gravitropism of lateral roots are

modified by gravity (see also Chapter 29 by Porterfield in this volume). N.

Structural Modifications

Aeroponic culture has affected maize root structure by induction of exodermal layers, i.e., development of thick walls and Casparian strips in the hypodermal layer of such roots (Freundl et al., 2000; Zimmermann et al., 2000). Such a layer may reduce the plants’ hydraulic conductivity. Aeroponics was also used for the application of ABA and of ABA analogs (Fuchs et al., 1999) and for the radial flow of ABA from the soil (Freundl et al., 2000).

IV.

LARGE AEROPONIC FACILITIES

The understanding of the development and function of mature root systems requires the proper experimental facilities where roots can be investigated in a nondestructive setup, and where they can be repeatedly reached for observations and measurement for prolonged periods of time (several months or years). Large-scale aeroponic rhizotrones overcome many of these obstacles by enabling continuous observation of each root of fully grown crop plants and of trees, the measurement of its development, and the study of its

328

performance under a variety of environments (Waisel and Eshel, 1998). To the best of our knowledge, the largest aeroponic root research laboratory to date is in the Sara Racine Root Laboratory at Tel Aviv University (Fig. 2). The Racine laboratory was planned for long-term experiments with arboreal species, where ‘‘long-term’’ means at least several years. It includes four 6-m-deep root chambers and four upper greenhouses. Mist is produced by pressurizing (4 atm) the required nutrient solution through fine atomizers. Usually mist is sprayed for 20s every minute, an interval that enables a sufficient water and nutrient supply to all the investigated plants. The sprayed solution is drained and collected below the root chamber, filtered, sterilized, and reused. The temperature of the root chamber is controlled by the temperature of the nutrient solution.

Waisel

Each of the four chambers provides a uniform root environment and enables uninterrupted observations of roots, even of large arboreal plants. Apparently, time is not limiting; trees have been grown in this laboratory for >10 years. We are aware of two large aeroponic research facilities that were constructed recently and are now in operation: 1. A relatively large aeroponic facility was constructed at the University of Bielefeld, Germany, by Prof. S.W. Breckle. It constitutes 20 individual planting sites, with a depth of the root chamber of 2.5 m. The plants are watered and nourished through four fogging systems, with the nutrient solution of each pumped and recycled through a pressurized container. A pH-stat controls the pH of the recycled solution, and the temperature of the root chamber is controlled by a water-heating system. 2. A compact aeroponic facility, designed for large-scale mutant screening experiments, was constructed at the Universite´ Katholique de Louvain in Belgium by Dr. Xavier Draye. Plants are placed on top of a 1-m-long, large-diameter plastic tube, with their roots allowed to grow into the tube. A nutrient solution is sprayed through a fine atomizer by a pulse of compressed air. The fogger is located near the top of the tube, and the runoff solution is collected at the base the tube. A small chamber under the collector, installed with two opposite one-way valves, serves as a volumetric pump. A single magnetic valve and a single pressure source are used for simultaneous driving of hundreds of such experimental units. The measurements and analysis of the collected solution allows the calculation of the water and nutrient uptake by the tested plants. An aeroponic facility for commercial growing vegetables was recently established in the Philippines (see http://members.tripod.com/aerogreen/).

V.

Figure 2 Cross section of the Sarah Racine Root Research Laboratory at Tel Aviv University. From top to bottom, 12 m.

SYNOPSIS

The development of each of the roots within a root system is determined by its individual genetic potential as well as by several environmental determinants. As a rule, development can be restricted by the availability of water and nutrients, by the presence of a hostile ionic composition, by the pH, by the gas composition (pO2 and CO2), by the environmental temperature, by the neighboring biota, and by the pressure against soil particles and the friction with them. All those determinants can be controlled in an aeroponic system, thus

Aeroponics

enabling the study of the potential development of root systems in an environment with minimal restrictions. Aeroponics overcomes many of the difficulties that have hampered root research in other growth systems. It is used for observation of root growth and spatial organization without the mechanical, physical, and electrochemical limitations which are imposed by the soil or by an aqueous environment. Aeroponic rhizotrons have several unique advantages: 1.

2.

3. 4.

5.

6.

7.

8.

9.

They enable continuous observation of each of the roots and the measurement of their development and function under a variety of environments. They enable manipulation, observation, and measurement of the growing roots of perennial plants through several annual cycles. Aeroponics enables the distinction, in real time, between various growth patterns of various roots. It enables comparison between the behavior of different roots of a similar/different status, and/or location of one plant. Aeroponic enables sampling of selected roots without causing damage to any of the others. Aeroponics provides information regarding the growth potential of roots, rather than mere measurements of growth under the limitations imposed by the mechanical impedance of the soil or by the aeration stresses of the environment. Aeroponics is the only research system where roots are subjected to a uniform environment without a limiting supply of minerals and without the formation of depletion zones. Roots are grown under what seem to be optimal conditions (high pO2, lack of mechanical impedance, optimal water conditions, good and constant mineral nutrition, etc.). Roots of aeroponically grown plants are free of slimes of fungi and bacteria, so common for roots grown in hydroponics. Root measurement in an aeroponic system is nondestructive; it enables frequent observation of each of the roots of the whole root system. The use of aeroponics enables repetition of measurements of the same roots, thus reducing the sampling errors. Aeroponics give a uniform environment to all the roots of the system, with the possibility to alter the nutrient milieu, and the gas composition of the atmosphere around the roots, very rapidly. It subjects all the roots to a uniform

329

10.

environment but concomitantly enables the induction of spatial changes in the nutrient milieu or the gas composition of the ambient atmosphere. Aeroponic chambers can be constructed in various sizes and can fit the specific requirement and budget limitations of any experimental project.

REFERENCES Barker BTP. 1922. Studies of root development. Long Ashton Res Sta Rep 1921:9–20. Biddinger EJ, Liu CM, Joly RJ, Raghothama KG. 1998. Physiological and molecular responses of aeroponically grown tomato plants to phosphorus deficiency. J Am Soc Hort Sci 123:330–333. Bohm W. 1979. Methods of Studying Root Systems. Berlin; Springer-Verlag. Buer CS, Correll MJ, Smith TC, Towler MJ, Weathers PJ, Nadler M, Seaman J, Walcerz D. 1996. Development of a nontoxic acoustic window nutrient-mist bioreactor and relevant growth data. In Vitro Cell Dev Biol Plant 32:299–304. Burgess T, McComb J, Hardy G, Colquhoun I. 1998. Influence of low oxygen levels in aeroponics chambers on eucalypt roots infected with Phytophthora cinnamomi. J Plant Dis 82:368–373. Burgess T, Hardy GES, McComb JA, Colquhoun I. 1999. Effects of hypoxia on root morphology and lesion development in Eucalyptus marginata infected with Phytophthora cinnamomi. J Plant Pathol 48:786–796. Caba JM, Recalde L, Ligero F. 1998. Nitrate-induced ethylene biosynthesis and the control of nodulation in alfalfa. Plant Cell Environ 21:87–93. Carter WA. 1942. A method of growing plants in water vapor to facilitate examination of roots. Phytopathology 32:623–625. Chung SJ, Chi SH, Shinohara Y, Ikeda H, Suzuki Y. 1933. Effect of misting intervals of nutrient solution on the growth and fruit yield of tomato. J Korean Soc Hort Sci 34:91–98. Clayton MF, Lamberton JA. 1964. A study of root exudates by the fog-box technique. Aust J Biol Sci 17:855–866. Correll MJ, Weathers PJ. 1998. Studies on hyperhydration of Dianthus caryophyllus in an acoustic window mist reactor. In Vitro Cell Dev Biol Anim 34:3. Dodd IC, He J, Turnbull CGN, Lee SK, Critchley C. 2000. The influence of supra-optimal root-zone temperatures on growth and stomatal conductance in Capsicum annuum L. J Exp Bot 51:239–248. du Toit LJ, Kirby HW, Pedersen WL. 1997. Evaluation of an aeroponics system to screen maize genotypes for resistance to Fusarium graminearum seedling blight. Plant Dis 81:175–179.

330 Engenhart M. 1987. Der Einfluss von Bleiionen die Produktivitat und den Mineralstoffhaushalt von Phaseolus vulgaris L. in Hydroponik und Aeroponik. Flora 175:273–282. Eshel A, Zilberstaine M, Waisel Y. 1992. Characterization of various root types of avocado. In: Kutschera L, Huebel E, Lichtenegger E, Persson H, Sobotik M, eds. Root Ecology and its Practical Application. Proc 3rd ISRR Symposium, Vienna, pp 691–694. Finlayson SA, Reid DM. 1996. The effect of CO2 On ethylene evolution and elongation rate in roots of sunflower (Helianthus annuus) seedlings. Physiol Plantarum 98:875–881. Freundl E, Steudle E, Hartung W. 2000. Apoplastic transport of abscisic acid through roots of maize: effect of the exodermis. Planta 210:222–231. Fuchs EE, Livingston NJ, Rose PA. 1999. Structure–activity relationships of ABA analogs based on their effects on the gas exchange of clonal white spruce (Picea glauca) emblings. Physiol Plantarum 105:246–256. Garrido I, Espinosa F, Paredes MA, Alvarez-Tinaut MC. 1998a. Effect of some electron donors and acceptors on redox capacity and simultaneous net H+/K+ fluxes by aeroponic sunflower seedling roots: evidence for a CN-resistant redox chain accessible to nonpermeative redox compounds. Protoplasma 206:141–155. Garrido I, Espinosa F, Paredes MA, Alvarez-Tinaut MC. 1998b. Net simultaneous hydrogen and potassium ion flux kinetics in sterile aeroponic sunflower seedling roots: effects of potassium ion supply, valinomycin, and dicyclohexylcarbodiimide. J Plant Nutr 21:115– 137. Henzler T, Waterhouse RN, Smyth AJ, Carvajal M, Cooke DT, Schaffner AR, Steudle E, Clarkson DT. 1999. Diurnal variations in hydraulic conductivity and root pressure can be correlated with the expression of putative aquaporins in the roots of Lotus japonicus. Planta 210:50–60. Hessel MI Jr, Reichert GE Jr, Nevill GE Jr. 1933. Airflowcontained aeroponic nutrient delivery for a microgravity plant growth unit. Biotronics 21:33–38. Hoffman TK, Kolb FL. 1997. Effects of barley yellow dwarf virus on root and shoot growth of winter wheat seedlings grown in aeroponic culture. Plant Dis 81:497– 500. Hung LLL, Sylvia DM. 1988. Production of vesicular-arbuscular mycorrhizal fungus innoculum in aeroponic culture. App Environ Micribiol 54:353–357. Ingestad T, Lund A-B. 1979. Nitrogen stress in birch seedlings. I. Growth technique and growth. Physiol Plantarum 45:137–148. Jackson TJ, Burgess T, Colquhoun I, Hardy GES, Jackson TJ. 2000. Action of the fungicide phosphite on Eucalyptus marginata inoculated with Phytophthora cinnamomi. Plant Pathol 49:147–154.

Waisel Jarstfer AG, Farmer-Koppenol P, Sylvia DM. 1998. Tissue magnesium and calcium affect arbuscular mycorrhiza development and fungal reproduction. Mycorrhiza 7:237–242. Jie H, Kong LS. 1998a. Growth and photosynthetic characteristics of lettuce (Lactuca sativa L.) under fluctuating hot ambient temperatures with the manipulation of cool root-zone temperature. J Plant Physiol 152:387– 391. Jie H, Kong LS. 1998b. Growth and photosynthetic responses of three aeroponically grown lettuce cultivars (Lactuca sativa L.) to different rootzone temperatures and growth irradiances under tropical aerial conditions. J Hort Sci Biotechnol 73:173–180. Klotz LGA. 1944. A simplified method of growing plants with roots in nutrient vapors. Phytopathology 34:507–508. Lee SK, Cheong SC. 1996. Inducing head formation of iceberg lettuce (Lactuca sativa L.) in the tropics through root-zone temperature control. Trop Agric 73:34–42. Martin NE, Hendrix JW. 1967. Comparison of root systems produced by healthy and stripe rust-inoculated wheat in mist-, water-, and sand-culture. Plant Dis Rep 51:1074–1076. Martin-Laurent F, Lee SK, Tham FY, He J, Diem HG, Durand P. 1997. A new approach to enhance growth and nodulation of Acacia mangium through aeroponic culture. Biol Fertile Soil 25:7–12. Martin-Laurent F, Lee SK, Tham FY, Jie H, Diem HG. 1999. Aeroponic production of Acacia mangium saplings inoculated with AM fungi for reforestation in the tropics. For Ecol Manage 122:199–207. Martin-Laurent F, Tham FY, Lee SK, He J, Diem HG. 2000. Field assessment of aeroponically grown and nodulated Acacia mangium. Aust J Bot 48:109–114. Mohammad A, Khan AG, Kuek C. 2000. Improved aeroponic culture of inocula of arbuscular mycorrhizal fungi. Mycorrhiza 9:337–339. Nir I. 1982. Growing plants in aeroponics growth system. Acta Hort 126:435–445. Padgett PE, Leonard RT. 1993. Contamination of ammonium-based nutrient solutions by nitrifying organisms and the conversion of ammonium to nitrate. Plant Physiol 101:141–146. Peterson LA, Krueger AR. 1988. An intermittent aeroponics system. Crop Sci 28:712–713. Quintana JM, Harrison HC, Palta JP, Nienhuis J, Kmiecik K, Miglioranza E. 1999. Xylem flow rate differences are associated with genetic variation in snap bean pod calcium concentration. Studies of Ca nutrition. J Am Soc Hort Sci 124:488–491. Rao A, Gritton ET, Grau CR, Peterson LA. 1995. Aeroponic chambers for evaluating resistance to Aphanomyces root rot of peas (Pisum sativum). Plant Dis 79:128–132.

Aeroponics Sachs J. 1874. Ueber das Wachstum der Haupt- und Nebenwurzlen. Arbeiten Bot Inst Wurzburg 4:586– 589. Shimura K. 1970. Root research phytotron. Jpn Agric Res Q 5:54–57. Smucker AJM, Erickson AE. 1976. An aseptic mist chamber system: a method for measuring root processes of peas. Agron J 68:59–62. Sylvia DM, Hubbell DH. 1986. Growth and sporulation of vesicular–arbuscular mycorrhizal fungi in aeroponic and membrane systems. Symbiosis 1:259–267. Shtrausberg DV, Rakitima EG. 1970. On the aeration and gas regime of roots in aeroponics and water culture. Agrokhimiya 4:101–110. Takahashi H, Mizuno H, Kamada M, Fujii N, Higashitani A, Kamigaichi S, Aizawa S, Mukai C, Shimazu T, Fukui K, Yamashita M. 1999. A spaceflight experiment for the study of gravimorphogenesis and hydrotropism in cucumber seedlings. J Plant Res 112:497– 505. Varney GT, Canny MJ. 1993. Rates of water uptake into the mature root system of maize plants. New Phytol 123:775–786. Vincenzoni A. 1979. Aeroponics: method of soilless culture (hydroponics). Coltura Protette 8:51–59. Vyvyan MC, Travell GF. 1953. A method of growing trees with their roots in a nutrient mist. Annu Rep East Malling Res Station, pp 95–98. Wagner RE, Wilkinson HT. 1992. An aeroponics system for investigating disease development on soybean taproots

331 infected with Phytophthora sojae. Plant Dis 76:610– 614. Wagner RE, Carmer SG, Wilkinson HT. 1993. Evaluation and modelling of rate-reducing resistance of soybean seedlings to Phytophthora sojae. Phytopathology 83:187–192. Waisel Y. 1988. Aeroponic observatories: useful tools for teaching and for long term studies of root behavior. Proc 4th ISSR Meeting, Uppsala, Sweden, 4:27. Waisel Y, Breckle SW. 1987. Differences in responses of various radish roots to salinity. Plant Soil 104:191–194. Ycas JW, Zobel RW. 1983. The response of maize radicle orientation to soil solution and soil atmosphere. Plant Soil 70:27–35. Zimmermann HM, Hartmann K, Schreiber L, Steudle E. 2000. Chemical composition of apoplastic transport barriers in relation to radial hydraulic conductivity of corn roots (Zea mays L.). Planta 210:302–311. Zobel RW. 1989. Steady state control and investigation of root system morphology. In: Torrey JG, Winship LJ, eds. Applications of Continuous and Steady State Methods to Root Biology. Dordrecht, Netherlands: Kluwer, pp 165–182. Zobel RW, Del Tredici P, Torrey JG. 1976. Methods for growing plants aeroponically. Plant Physiol 57:344– 346. Zsoldos F, Vashegyi A, Erdei L. 1987. Lack of active K+ uptake in aeroponically grown wheat seedlings. Physiol Plantarum 71:359–364.

20 Use of Microsensors for Studying the Physiological Activity of Plant Roots D. Marshall Porterfield University of Missouri-Rolla, Rolla, Missouri

I.

INTRODUCTION

To fully understand the physiological activity of plant roots, one must understand the physical and chemical properties of the rhizosphere immediately near the root surface. For the root this is the direct environment for physiological interaction with the bulk soil. The attributes of the rhizosphere are determined not only by the soil, but also by the biochemical and physiological activity of the root. The properties of the rhizosphere can vary in size, shape, and composition, even from root to root on the same plant. Root biologists have sought to explore the rhizosphere and understand plant-mediated rhizosphere activities by utilizing microsensors to probe this environment. These sensors have included various types of ion-selective microelectrodes, as well as polarographic electrochemical oxygen sensors to determine the concentration of important molecules within the rhizosphere. Advanced microsensor techniques now make it possible to measure dynamic flux of these molecules in real time with a relatively high degree of spatial and temporal resolution. This technique has been referred to as the microelectrode ion flux estimation (MIFE) technique, the vibrating probe (VP), and more recently the self-referencing microsensor (SRM) technique. The use of such microsensors and microsensor techniques to study the plant root and rhizosphere processes is discussed in this chapter.

II.

THE ION-SELECTIVE MICROELECTRODE (ISM)

A.

ISM Basics

For this general class of sensors the term ‘‘selective’’ is preferred because the sensors tend to favor one ion species over others. In some cases the sensors are specific, but that is rare and care must always be taken to consider the sensor’s capabilities and limitations. Ionselective electrodes are constructed based on the ion selective properties of various inorganic soluble salts, organic ion exchangers (ionophores), and glass materials. These ion-selective components are used to create an effective barrier or membrane to separate the electrode’s internal electrolyte solution from the solution being measured. This ion-selective membrane is electrically capacitive, and the voltage potential that develops across the membrane is based on the ion concentration differential across the membrane. This voltage potential is measured using a pair of Ag/ AgCl electrodes on each side of the electrode membrane, where one of the electrodes is contained within a defined solution separated by the membrane from an undefined medium into which the second electrode (reference electrode) is placed. Ion-selective microelectrodes are constructed utilizing ionophore-based membranes. The ionophores are typically incorporated into lipophylic ion exchange (LIX) cocktails that are in most cases commercially 333

334

Porterfield

available. The details of how to build these types of microelectrodes have been described (Amman, 1986; Miller, 1995; Smith et al., 1999), and a brief description of the protocols is reviewed in Fig. 1. For intracellular probes the liquid membranes of the electrodes can be plasticized using polyvinyl chloride (PVC), but this tends to slow the response time of the sensor. For non-invasive measurements of ion activities around plant roots this is not necessary. Since the selectivity of the sensor is mediated by the properties of the LIX, the basic electrode construction (Fig. 1) can be utilized to build any number of ion-selective microelectrodes simple by changing the electrode electrolyte backfill solution and the LIX (Table 1). The response of these electrodes is commonly termed to be ‘‘Nernstian’’ as the relationship between ion concentration and the resulting electrode potential conforms to the Nernst equation (28 mV or 56 mV change in electrode potential per 10-fold change in divalent or monovalent ion concentration, respectively). B.

Use of ISM Techniques to Study Roots and Root Cells

There has been fairly broad application of ISMs in measurements of ion activities in, or at the surface

of, plant cells and tissues. This is in contrast to the use of ISMs as noninvasive self-referencing flux sensors as discussed in subsequent sections. Discussion here will be limited to the use of ISMs as invasive sensors to probe the activities of ions in individual root cells and in root tissues. That is not to say that the measurement of ion activities at the root surface using a static probe does not provide valuable information. In fact a recent study has shown a strong relationship between surface pH and profiles of elongation in maize roots (Peters and Felle, 1999). This study does, however, illustrate some limitations in the use of static sensors to characterize the surface of a root. Static measurements do not provide any direct signal relating to the polarity of ion movements. Thus, pH scale changes may not accurately describe the magnitude of physiological ion flux activity from the root and significant differences in ion transport activity may be overlooked (Table 2). Despite the limitations of the use of static ISMs, they definitely are valuable tools. While surface concentration and transport activity can be best measured using self-referencing ion-selective (SRIS) microelectrodes, static ISMs are effectively used for determination of intracellular ion concentrations, and in some cases offer a good alternative to the use of ratiometric

Figure 1 Diagram depicting the construction and calibration of a calcium-selective microelectrode. The specificity of this microelectrode is based on the characteristics of the commercially available lipophyllic ion exchange cocktail (Ca2+ Ionophore I Cocktail A) that forms an organic phase liquid membrane barrier in the tip of a silanized glass microelectrode. This membrane separates the internal electrolyte (100 mM CaCl2) from the solution being measured. The selectivity of an ionophore is defined by the Nikolsky-Eisenman equation (Ammann, 1986). Other types of ISMs are constructed filling the glass micropipette with the appropriate backfill solution and tipped with a membrane made from a particular ion-selective cocktail (Table 1). All of these types of microelectrodes have Nernstian calibration responses; a 10-fold change in concentration generates a constant voltage difference.

Microsensors

335

Table 1 Ionophore Cocktails and Appropriate Backfill Solutions for Several Ion-Selective Membranes Used to Construct Ion-Selective Microelectrodes for Use as a Self-Referencing Sensor Ion-selective Membrane

Column length (mm)

Backfill solution

200 30 150 30 80 200 200 50 100

500mM NH4C1 100 mM CaCl2 100 mM NaCl 100 mM KCl 500 mM MgCl2 500 mM KNO3 500 mM KNO2 100 mM KCl 100 mM NaCl

Ammonium Fluka cat. #09879 Calcium Fluka cat. #21048 Chloride Fluka cat. #24902 Hydrogen Fluka cat. #95293 Magnesium Fluka cat. #63048 Nitrate Fluka cat. #72549 Nitrite Fluka cat. #72549 Potassium Fluka cat. #60398 Sodium Fluka cat. #I7397

dyes. This would be the case when microinjection is required to get the dye into the cell or when there is not a dye available for the particular ion of interest. Developmentally important gradients in the concentration of calcium in root hairs of Sinapis alba were measured using a combination of intracellular calcium ISM and ratio imaging. Generally, the two techniques showed good agreement. Using ISMs’ intracellular calcium levels have also been measured in individual root hairs and in the stele of maize roots (Felle, 1998) and in root hairs of alfalfa and soybean during the transduction of the Nod factor signal (Felle et al., 1999a,b). The use of ISMs really has an advantage over ratio metric dyes is in the ability to measure more than one ion at a time using separate and multi barreled microelectrodes. Both H+ and Ca2+ and H+ and Cl concentrations were measured in a study of tip growth in root hairs of Sinapis alba (Felle, 1994; Herrmann and Felle, 1995) using multibarreled microelectrodes that combine measures of the separate ions with measures of membrane potential. Triple-barreled electrodes have

also been used to combine measurements of multiple ions and membrane potential into a single unit (Walker et al., 1995). In such a unit H+, membrane potential, and either K+ or NO3 were measured simultaneously in barley root epidermal cells. III.

POLAROGRAPHIC ELECTROCHEMICAL SENSORS

A.

Theory and Operation of Electroanalytical Sensors

Electrochemical sensors mediate analyte detection by reducing or oxidizing an analyte. The redox changes in the analyte are driven by a polarized potential on the electrode’s sensing surface that facilitates electron transfers between the surface and the molecule being detected. The flow of electrons is therefore dependent on the concentration of the analyte, and these sensors typically show a linear relationship between analyte concentration and the resulting current (Bard and

Table 2 Relationship Between pH and H+ in the Bath and at the Root Tip as Measured Using a H+ Ion-Selective Microelectrode Bath pH 7.8 6.5 4.2

Root tip surface pH

Bath H+ (mM)

Root tip surface H+ (mM)


pH (root tip–bath)


(H+) (root tip–bath) (mM)

6.7 5.8 4.45

0.015848932 0.316227766 63.09573445

0.199526231 1.584893192 35.48133892

1.1 0.7 0.25

0.18368 1.26867 27.6144

Notice that the largest changes in H+ are associated with relatively small pH changes in the range of 4.2 and 4.45 pH. It is also important to note that the decrease in pH occurring in the bath at pH 7.8 and 6.5 relates to an increase in the H+ ion concentration, presumably due to H+ efflux and H+/ATPase activity. The pH increases (decrease in H+) associated with the pH 4.2 bath is conversely presumed to be associated with a complete reversal of the direction of H+ ion flux back into the root. Source: Peters and Felle (1999).

336

Faulkner, 1980). Since the primary electronic signal that relates to the analyte concentration is a current, these sensors are sometimes referred to as amperometric sensors. This is in contrast to ion-selective electrodes where the primary electronic signal is a voltage potential and the term voltametric sensor is often used. Specificity of analyte detection by electroanalytical sensors is mediated by the redox properties of the analyte, the polarization potential of the electrode, and membrane coatings on the electrode surface that may facilitate phase or size exclusion of potential interferants. Electroanalytical sensors have been widely applied in animal and biomedical research. These techniques have been used to measure compounds ranging from simple gases (oxygen and nitric oxide [NO]) to complex hormones like insulin (Huang et al., 1995). In

Porterfield

plant sciences, electroanalytical techniques have been applied to measure oxygen in relation to photosynthesis, respiration, and soil oxygenation (Armstrong, 1994). There are basically only two types of electroanalytical oxygen electrodes—the Whalen- and the Clark-style microelectrodes (Fig. 2). It is important to note that all polarographic oxygen sensors are variations of these two basic designs. The Whalen-style electrode is a platinum or gold cathode that is directly membrane coated and that utilizes a separate reference electrode, whereas the Clark electrode has the noble metal cathode (Au or Pt) and the reference electrode contained within a KCl solution behind a common membrane. The membrane is important in both of these designs as it acts not only to protect the noble metal surface from fowling by compounds in the measuring media (most commonly proteins and

Figure 2 Construction and calibration of Clark (A) and Whalen (B) style oxygen-selective microelectrodes. The Clark oxygen electrode is constructed by building a noble metal (Au or Pt) cathode and sealing this inside of a membrane-tipped electrolyte containing micropipette body along with an Ag/AgCl reference electrode. The Whalen style electrode is simply a noble metal cathode with a recessed tip that contains an oxygen-permeable membrane. For either style electrodes, the cathode can be constructed by filling a micropipette with a low melting point alloy such as Wood’s metal. This metal-filled electrode is then etched and plated with gold in order to create an electrode with the desired tip geometry. In the case of the Whalen electrode, the recessed tip is filled with Pt or gold but gold is reported to be less susceptible to redox changes itself when reducing oxygen to water. Finally, the electrode tip recess is filled with a gas-permeable membrane. The electrode is calibrated (C) against solutions of known oxygen concentrations made by bubbling with gases of known oxygen partial pressures.

Microsensors

sulfides), but it alters the oxygen consumption (resistance) of the electrode in such a way as to render the electrode stir insensitive. This quality of stir insensitivity is very important because the electrode, in the process of reducing oxygen to water, is consuming oxygen. If the consumption of oxygen by the electrode were not modified in this way, the electrode could potentially alter the concentration of oxygen in the local area where it is actually measuring. The membrane effectively solves this problem by increasing the resistance to oxygen transport through the membrane. A properly functioning electrode should have 99% of the electrode oxygen depletion gradient contained entirely within the electrode’s membrane (Sneiderman and Goldstick, 1978). Some Whalenstyle electrodes utilize a deep recess in the electrode tip that is not membrane coated to contain the depletion gradient. While this does effectively deal with the problem of stir sensitivity, it does not adequately protect the electrode surface from fowling.

B.

Use of O2-Selective Microelectrodes in Root Research

Roots are invariably composed of heterogeneous cell types characterized by different levels of metabolic activity. Understanding cellular and tissue metabolism in roots turns out to be the key to understanding normal physiology and various types of stress responses. This can be accomplished using spatial oxygen measurements that will provide a general indicator of many aspects of cellular metabolism and physiological status. Davies and Brink (1942) first described the use of oxygen microelectrodes for direct measurement of oxygenation biological tissues. Later the electrodes were miniaturized with the goal of measuring intracellular O2 concentrations (Whalen et al., 1967). Electrode designs have been subject to further development (Forstner and Gnaiger, 1983) and widespread use, including the study of metabolism and aeration in plant roots (Armstrong, 1994) and in plant aerial tissues (Porterfield et al., 1999). The first notable use of an oxygen electrode to study root tissue oxygenation was by Bowling (1973), who studied the oxygen concentration profiles across excised sunflower roots. He used a commercial electrode (tip diameter 1 mm) and revealed a shallow radial oxygen gradient across the root cortex. Tjepkema and Yocum (1974) studied oxygenation in soybean root nodules using glass-insulated Pt microelectrodes and documented that the major resistance to oxygen trans-

337

port in root nodules lies in the cortex. This was followed by studies of oxygen distribution in the root nodules of pea and French bean (Witty et al., 1987) and of oxygen profiles in the barley rhizosphere (Hojberg and Sorensen, 1993). Much of what we know about root oxygenation and oxygen transport during hypoxia and anoxia come from the use of in vivo oxygen microelectrodes. Studies of radial oxygen distribution in maize roots have showed that during low oxygen stress, oxygen was supplied from the shoot (Armstrong et al., 1993). Steep radial diffusion gradients were characteristic of the nonporous epidermal/hypodermal shell and stele, while shallow profiles were generally found in the cortex (Armstrong et al., 1990). Anoxia in the stele was inducible by manipulating the oxygen concentrations around the shoot (Armstrong et al., 1990). Measurement of oxygen profiles obtained by slowly advancing a microelectrode through the meristemic zone and the root cap of roots grown in stagnant anaerobic medium, suggests that the bulk of the root tip tissue is likely to be anoxic. (See also Chapter 42 by Armstrong and Drew in this volume). Root oxygenation in solution and vermiculite grown maize roots were studied using a Clark-style microelectrode (Ober and Sharpe, 1996). Treatments used to alter water potential in solution grown maize roots by application of polyethylene glycol reduced the availability of oxygen in the medium by altering the solubility and diffusion of oxygen in the system. This reduction in oxygen availability produced substantial decreases in root tissue oxygenation (Versules et al., 1998). This suggests that experimental artifact associated with simulated water stress could occur, and indeed it was noted that the hypoxic enzyme alcohol dehydrogenase (EC 1.1.1.1) is induced under simulated water stress situation.

IV.

THE SELF-REFERENCING MICROELECTRODE (SRM) TECHNIQUE

A.

Background and History

A very important consideration in the use of any microsensor for studying biological transport activity is the mode of operation of the sensor. The use of static measurements to characterize a component of the roots dynamic physiology are thus subject to limitations and sources of artifacts as previously discussed. In many cases the signal-to-noise characteristics of the recording instrumentation and electrodes hinder the

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ability to detect the intricate biological patterns that are present. The use of invasive microelectrodes can introduce experimental error (Silver, 1967; Whalen, 1974; Schneiderman and Goldstick, 1978), the most significant of which in plants is the induction of an oxidative burst in the tissue being probed (Lamb and Dixon, 1997). Such limitations in the use of microelectrodes are in many ways overcome by the self-referencing microelectrode (SRM) technique. This technique allows noninvasive monitoring of physiological transport activity (flux) from single cells or whole tissues in real time. Apparently this approach has been reinvented several times, and with each new rediscovery a new name has been coined. The basic approach has been referred to as the vibrating probe, the self-referencing microelectrode, the microelectrode ion flux estimation (MIFE), and the microelectrode flux estimation (MFET) techniques. What these sometimes subtle variations all have in common is that a single microelectrode is used to sample two distinct positions within a biologically derived gradient. The self-referencing approach (Zisman, 1932) used a metal electrode as a probe to measure low-magnitude currents by determining voltage differences between two distinct points in an electric field using a single electrode. This is what is specifically referred to as the vibrating voltage probe and is used to measure bulk ion fluxes. Despite the nonspecificity of the technique this approach has provided a practical method to show total electrical currents in a number of biological systems. The first biological use of the technique was for plant research (Bluh and Scott, 1950). Later it was used to study gravitropic responses of corn coleoptiles (Hertz, 1960; Grahm, and Hertz 1962, 1964; Grahm, 1964). It was even used to measure net ion fluxes on skeletal muscle fibers (Davies, 1966). A notable rediscovery of the vibrating probe (Jaffe and Nucitelli, 1974) resulted in significant improvements of the technique based on the use of a commercial lock-in amplifier. The vibrating voltage probe was used to investigate the presence of ionic currents during barley and clover root development (Weisenseel et al., 1979; Miller et al., 1986), gravitropism in cress roots (Weisenseel et al., 1992), and wounding responses in Nicotiana (Miller et al., 1988) and pea roots (Hush et al., 1992). The limitation of the self-referencing vibrating voltage probe is the inability of the sensor to determine the actual ionic makeup of the measured biological currents. The ability to measure the active flux of specific ions came with the development of the self-refer-

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encing ion-selective (SRIS) microelectrode. This approach differs from the vibrating probe in that it is based on the use of ion-selective microelectrodes instead of the original metal electrodes, and the frequency of probe movement is substantially slower (0.1–0.3 Hz vs 300–1000 Hz). Initially, investigators drove the probe movements manually to sample the biological gradients (Newman et al., 1987). Later, Khu¨trieber and Jaffe (1990) used computer-driven electrode movement and data acquisition to automate the measurement of calcium ion flux. The automated SRIS approach was subsequently diversified to measure other ions like H+ and K+ (Kochian et al., 1992) and even heavy metals like Cd2+ (Pineros et al., 1998). The self-referencing technique has also recently been used to measure the biological flux of nonionic compounds through the development of the self-referencing electrochemical microelectrode (SREM) technique (Porterfield et al., 1998; Land et al., 1999; Porterfield and Smith, 2000).

B.

SRM Theory and Operation

As previously stated, the goal of the use of SRM is to actively measure compounds fluxing near the surface of single cells or whole tissues based on the diffusionary movement of molecules within the gradient (Newman et al., 1987; Khu¨trieber and Jaffe, 1990; Shabala et al., 1997; Porterfield et al., 1998; Land et al., 1999; Porterfield and Smith, 2000). This is accomplished by controlling the translational movement of a selective microelectrode in a gradient. A recording system is constructed using a microscope and a head-stage electrode amplifier driven by a translational motion control system. Such an assembly is mounted on an antivibration table and housed within a Faraday cage. The amplifier and the electrical components of the motion control system are commercially available, whereas in the past automated systems were custom built and required custom software development. The motion control system allows the electrode tip to be moved through the gradient at a known frequency and between known points (commonly 10–50 mm apart). The automated electrode movement induces a phasedependent waveform on the electrode output signal, where the amplitude of said waveform is proportional to the differential analyte concentration within the gradient (Fig. 3). This effectively turns a static concentration sensor into a dynamic flux sensor, all while minimizing the impact of random noise and drift on the differential electrode output (Fig. 4).

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Figure 3 Diagrams showing the basic procedure for conducting a self-referencing microelectrode flux measurement. In panel (A) a schematic of an experiment is depicted where a generated diffusional gradient is established outside of a micropipette. The relationship between probe positions (A) and electrode output (B) during the probe movement cycle is plotted to illustrate how a waveform is induced on top of the electrode output signal. For automated systems a PC typically controls the translational frequency allowing signal analysis software to extract voltage changes in phase with the probe moving through the gradient. The relationship between the concentration differential (C1–C2) is directly proportional to the amplitude of the waveform of the differential electrode output value (
fA for amperometric electrochemical sensors and
mV for potentiometric ion selective sensors).

There are two main approaches to acquiring and analyzing amplitude of the waveform or the electrode differential signal. Some of the first automated systems utilized an approach sometimes referred to as AC coupling. Here the electrode signal coming from the preamplifier (mV) is passed through a capacitor

that nondiscretely subtracts off the baseline output signal so that the differential waveform signal (mV) can be amplified enough to digitally analyze these minute signals. The problem with this approach is that the capacitor discharges exactly when the differential signal is being analyzed, invariably resulting in

Figure 4 Data showing how the self-referencing technique effectively filters out noise based on the basic principles of phasesensitive detection. For this experiment a SRIS-H+ sensor was operated at a frequency of 0.3 Hz and over an excursion distance of 10 mm. The artificial gradient was created outside of a micropipettes with a 10-mm tip diameter that was filled with a pH 4 buffer immobilized in an agar gel matrix (1.0%). Data were collected only during the time when the probe was stopped at the two positions and the data that were collected are plotted here as a scatter plot. This clearly shows that the data segregate into two distinct groups representing the two measuring positions. Note how electrode drift and noise patterns are common to both groups of data while the differential (
V) between the two populations stays relatively constant.

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degradation of the differential signal (Khu¨trieber and Jaffe, 1990), and in an underestimation of the true flux values (Smith et al., 1999). Despite the fact that the signal degradation problem was described and quantified a decade ago (Khu¨trieber and Jaffe, 1990), the approach is still used today (Smith et al., 1999). The problem can be overcome by subtraction of the baseline signals before amplification. This is referred to as DC coupling, and it allows for baseline signal subtraction without the associated problems of differential signal degradation due to capacitive discharge (Shipley and Fiejo, 1999). Once the measurements of the differential electrode signals within the gradient have been acquired, they must be converted into a measure of differential concentration before flux can be calculated. The formulas for doing the conversion from differential electrode output to differential concentration are another area where the AC-coupling and DC coupling approaches diverge. Because of the signal degradation and the way that the AC-coupled method logs data, it is impossible to directly calculate these values (Smith et al., 1999). Because the DC-coupling discretely subtracts off the baseline signal, this allows for logging of the actual electrode output values at both measuring positions. It thereby allows calculation of these concentration differentials without having to make any types of assumptions regarding background electrode signals and measurement efficiency. Flux calculations are all based on the Fick equation J ¼ Dð
C=
XÞ where J ¼ flux, D is the diffusion coefficient,
C is the differential concentration, and
X is the distance between the two electrode measuring positions. This version of the formula assumes flat planar geometry of the surface being analyzed. Some researchers, using a variation of the Fick equation, have gone to great lengths to calculate flux using cylindrical diffusion geometry for the plant roots. Obviously a geometric cylinder is an oversimplification of a plant root, especially when considering a high-resolution scan of the heterogeneous root tip. This approach was compared to the planar flux model using actual root recording data and quantitatively shown to be unnecessary (Kochian et al., 1992). The planar model yielded almost identical results to the cylindrical model, as would be expected given the small electrode tip size and excursion distance, and the fact that any surface can be described mathematically as a series of flat planar surfaces. When possible, the less complex planar approach is preferred as estimations of some of

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the values required for the cylindrical formula may actually introduce analytical error.

C.

Self-Referencing Ion-Selective (SRIS) Microelectrode Root Studies

The SRIS technique has been used in investigations of various plant systems, including studies of ion transport mechanisms, in plant roots. All plant cells have sophisticated ion transport mechanisms, but the structural characteristics of the cell wall and the presence of a large osmotically active central vacuole limit the application of standard intracellular electrodes and electrophysiological studies of these processes. Given the noninvasive nature of the SRM technique, membrane ion transport properties can be measured in real time without disrupting the cell or tissue. Roots and root hairs have been the subject of many important studies. Calcium and hydrogen ion fluxes have been measured in the root hairs of Sinapis alba and shown to correlate with intracellular events and other properties of growth (Hermann and Felle, 1995; Felle and Hepler, 1997). Root hair calcium fluxes have been investigated in relation to normal development (Schiefelbein et al., 1992) in Arabidopsis, and under the influence of the Nod factor in leguminous roots (Allen et al., 1994). Calcium fluxes in the root apex and in root hairs of wheat and in Limnobium stonoloniferum were studied under the influence of aluminum in toxic concentrations (Huang et al., 1992; Jones et al., 1995). Proton and potassium flux in maize roots and maize suspension cells (Kochian et al., 1992) has been characterized. Ammonium and nitrate ionophores have been used to measure and map the uptake of these ions along the axis of a barley root (Henricksen et al., 1992), and ammonium, nitrate, and proton fluxes have also been measured along maize roots (Taylor and Bloom, 1998). Ionophores have recently been developed for the measure of heavy-metal ions, and a cadmium-selective SRIS was used for plant toxicity studies (Pineros et al., 1998) by measuring the flux of cadmium into the roots of Thlaspi and wheat. Using advanced hardware and software that includes digital electrode position tracking with video feedback, high-resolution scans of ion flux activities are now possible (Fig. 5). A root tip of Typhya latifolia was scanned using an H+ ion-selective microelectrode. The electrode positioning data was used to reconstruct the

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Figure 5 H+ flux around an actively growing root tip of Typha latifolia. Flux measurements were made using a SRIS-H+ sensor (10 mm excursion/0.3 Hz). At closest approach the probe was within 1 mm of the root surface and the excursion distance of 10 mm was entirely within the rhizosphere gradient as shown in a step-back experiment (data not shown). The experiment was done using an IPA-2 DC-coupled amplifier (Applicable Electronics, Forestdale, MA) and a PC running ASET software (ScienceWares, Falmouth, MA). The 3D profile of the root tip was reconstructed using the microelectrode positioning data that were collected along with the electrode output by the computer data acquisition system.

3D root surface and to plot a contour map of the flux from the surface. D.

Self-Referencing Electrochemical Microelectrode (SREM) Techniques

The SREM sensors are a recent development (Porterfield et al., 1998; Land et al., 1999; Porterfield and Smith, 2000) that was originally based on the use of a Whalen-type oxygen microelectrode used as a selfreferencing sensor (SREM-O2). Given that this is a recent development, it has seen only limited application in plant science research but already has demon-

strated very high sensitivity and special resolution. Oxygen and proton fluxes have been studied in single algal cells of Spirogyra and Acetabularia using a combination of probes (Porterfield and Smith, 2000; Serikawa et al., 2000, 2001) and demonstrated the subcellular resolution of the technique. The SREM-O2 sensor has been used in a study of metabolic oxygen consumption patterns along the root of Elodea (Mancuso et al., 2000). However, care must be taken in interpreting these results because a stir-sensitive bare cathode was used as an oxygen sensor. The SREM-O2 oxygen microsensor has great promise as an important tool for root physiology research.

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Like other SRM sensors it is noninvasive and allows for direct measurement of rhizosphere oxygen transport into and even out of roots. Perhaps the most significant applications of the sensor will come from

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simultaneous SREM-O2/SRIS recordings. An example of such application can be seen in Fig. 6. High-resolution scans of the surface of a maize root were done using a combination of oxygen- and proton-

Figure 6 Metabolic oxygen flux patterns and H+ flux around the roots of Zea mays (cv Kandy Korn) as measured using a dual sensor arrangement. Here the plants were exposed to different forms of nitrogen (A=200 mm (NH4)2SO4, 100 mM Ca2SO4; B=200 mm Ca(NO3)2, 100 mM Ca2SO4), and the resulting differences in flux patterns were scanned using two separate sensors mounted together (tips within 2 mm of one another) on a single computer-driven motion control system. The two separate preamplifiers were operated using a dual-channel DC-coupled amplifier (model IPA-2, Applicable Electronics, Forestdale, MA) and a PC running ASET software (ScienceWares, Falmouth, MA). When the roots are exposed to nitrogen in the form of NH4, the H+ efflux values are typically higher, along the entire root, than the root exposed to NO3. Although the flux patterns were virtually identical in the two treatments, the H+ efflux values ranged between 10 to 20 pmoles cm2 sec1 along the NH4 roots whereas the NO3-exposed roots had flux values that ranged between –4 and 14 pmoles cm2 sec1. Note that the negative values associated with the positions in the range of 600–2400 mm from the root tip indicate that this was a zone of net H+ influx. Oxygen consumption patterns were again very similar in the two treatments; however, the flux values tended to be almost three times higher in the NO3-exposed roots. The differences in root metabolic oxygen influx and H+ flux are thought to be related to the additional metabolic energy and H+ required to reduce NO3 to NH4.

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selective microelectrodes. The sensors were mounted together and driven using a single motion control system. The flux patterns were mapped around roots when exposed to either ammonium or nitrate. Results reveal significant differences in hydrogen ion flux and metabolic oxygen consumption resulting from the differences in transport and assimilation of these different nitrogen forms. While the SREM-O2 sensor will be an important tool, perhaps the most significant use of the SREM sensor technology will result from application of other types of electroanalytical sensors. Already the SREM technique has been expanded to include sensors for NO, ascorbic acid, and hydrogen peroxide (Heck et al., 1999; Kumar et al., 1999; Pepperell et al., 1999a,b; Billack et al., 2001; Kumar et al., 2001; Porterfield et al., in press). While these sensors have not been used in plant research, they do have great potential as tools for root research, possibly advancing our understanding in many fundamental areas. NO has been suggested to be involved in plant signaling (Pfeiffer et al., 1994), stress physiology (Leshem et al., 1997), root nodule biochemistry in Lupinus albus (Cueto et al., 1996), soil biology (Vermoesen et al., 1996), and pathological responses in potato tubers (Noritake et al., 1996). There are also studies that suggest NO might regulate growth and cell elongation in the root of pea (Sen and Cheema, 1995) and maize (Gouvea et al., 1997). In the root system, ascorbate plays a significant role in protecting the root system from free radicals produced as a result of exposure to metals like aluminum (Lukaszewski and Blevins, 1996), copper (Gupta et al., 1999), lead (Renata et al., 1999; Mishra and Choudhuri, 1998), and mercury (Mishra and Choudhuri, 1998). Ascorbate-mediated antioxidant protection has also been implicated in root tolerance and recovery from salinity (Meneguzzo et al., 1999), chilling (Queiroz et al., 1998), and hypoxic stresses (Biemelt et al., 1998). Ascorbate also has been shown to be involved in mineral nutrition as iron deficiency induces changes in both ascorbate concentrations and enzyme activities related to ascorbate metabolism (Zaharieva et al., 1999). There is now evidence that inhibition of root growth associated with boron deficiency is a result of impaired ascorbate metabolism (Lukaszewski and Blevins, 1996). Depending on the concentration H2O2 can induce cell protective responses, programmed cell death (apoptosis), or necrosis in plant systems. Low concentrations of H2O2 can induce antioxidant responses, whereas a higher level of H2O2 triggers apoptosis (Levine et al.,

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1994). One of the best-studied and best-understood roles of H2O2 in plants is the hypersensitive response (HR), which is induced in plant tissues during pathogen infection (Lamb and Dixon, 1997). The SREM probes that are now being developed will be invaluable tools in these areas. E.

Developing Self-Referencing Biosensor Technologies

Development of enzyme-coupled electrochemical sensors (biosensors) for use as self-referencing microelectrodes has a great potential for application in plant science. The basic concept is to utilize an enzyme to convert the presence of an undetectable substance into a detectable signal via the production of a measurable reporter. For the sake of brevity this discussion will focus on electrochemical detection of the reporter species and will not deal with other types of reporter detection such as the use of ion electrode, colorimetric, photometric, luminometric, or enthalmetric coupled techniques. There are two classes of electrochemical enzyme electrodes and these are based on the uses of either oxidase or dehydrogenase enzymes. The most common type of these biosensors is the oxidase-based biosensor. Oxidase activity can be coupled to the electrochemical detection of hydrogen peroxide. During the enzymatic step the analyte reacts with oxygen producing the enzymatic product, which itself is electrochemically inactive, and H2O2. The hydrogen peroxide, however, is detected electrochemically by oxidation at 0.65 V with a platinum electrode. This approach has been used to develop biosensors capable of detecting compounds such as bilirubin, creatinine, dextrin, glucose, glucose-6-phosphate, glutamate, maltose, and sucrose (Danielson and Mosbach, 1988). Oxidase base biosensors have also been built upon oxygen electrodes, as was done in the development of the first enzyme electrode (Updike and Hicks, 1967) for the measurement of glucose. Here, increases in analyte concentration are correlated with decreases in measured oxygen availability. Sensors capable of detecting biotin, creatinine, d-lactate, l-lactate, lactose, l-lysine, phenol, and sucrose have been built using this approach (Danielson and Mosbach, 1988). The second type of enzyme-based biosensors is those that utilize NAD+- or NADP+-dependent dehydrogenase enzymes. In the enzymatic step, the analyte is converted to the product along with the requisite reduction of NAD+ to NADH. NADH is subsequently oxidized to produce an electrochemical signal that relates to the concentration of the analyte. While

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direct electrochemical measurement of NADH oxidation is possible, this requires such high overpotentials that the electrode surface will become fouled. Using a mediator to facilitate the transfer of electrons from NADH to the electrode or the direct transfer of electrons from the enzyme to the electrode can solve this problem. Numerous mediator-based strategies for modifying electrodes for the detection of NADH have been reported (Albery et al., 1987; Hin and Lowe, 1987; Nowall and Kuhr, 1995; Nakamura et al., 1996; Silber et al., 1996; Alvarez-Crespo et al., 1997; Curulli et al., 1997; Pandey et al., 1998; Lorenzo et al., 1998). Many of these strategies involve the use of both conducting and nonconducting polymers that are electrodeposited on the active surface of the electrode. The potential of biosensors is in allowing direct measurement, and it will be of value for identification of compounds that have not previously been measurable because they are nonionic and electrochemically inactive. Biosensors should allow for direct measurement of any number of low-molecular-weight organic compounds in and around plant roots. These include glucose, sucrose, and ethanol, as well as low-molecular-weight organic acid exudates, like malate and citrate, that are believed to mediate root tolerance to soil metals like aluminum.

V. SUMMARY Microelectrode sensor technologies have proven to be important tools in root biology. The importance of the application of microelectrodes to root biology will increase as application grows from the use of ISM sensors to include electroanalytical and biosensorbased microelectrodes. For root surface transport studies, the SRM technique is an extremely powerful technique as it allows for noninvasive mapping of dynamic flux from individual plant roots in real time. Furthermore, these fluxes can be scanned and mapped along the entire surface of an active tissue, such as a root tip or root hair, and correlated spatially with subsequent analysis of the morphology and anatomy. The utility of the SRM technique will also be enhanced by the application of various types of analytical sensor techniques that include new types of ISMs, electroanalytical sensors, and biosensors. In addition to the application of existing analytical technologies, there is much to be gained in actually exploring the development of new classes of electroanalytical and biosensors that are specific for com-

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pounds that are unique to and important in plant systems. These new techniques could then be used to develop new classes of SRMs that could provide detailed information for the flux of a diverse array of compound that may even include plant hormones like auxin and ethylene. REFERENCES Albery WJ, Bartlett PN, Cass AE. 1987. Amperometric enzyme electrodes. Phil Trans R Soc Lond B Biol Sci 316:107–119. Allen NS, Bennet MN, Cox DN, Shipley A, Ehrhardt DW, Long SR. 1994. Effects of nod factors on alfalfa root hair Ca++ and H+ currents and on cytoskeletal behavior. In: Daniels MJ, Donnie JA, Osbourn AE, eds. Advances in Molecular Genetics of Plant–Microbe Interactions, Vol. 3. Dordrecht, Netherlands: Kluwer, pp 107–113. Alvarez-Crespo SL, Lobo-Castanon MJ, Miranda-Ordieres AJ, Tunon-Blanco P. 1997. Amperometric glutamate biosensor based on poly(o-phenylenediamine) film electrogenerated onto modified carbon paste electrodes. Biosens Bioelectron 12:739–747. Ammann D. 1986. Ion Selective Micro-electrodes. New York; Springer-Verlag. Armstrong W. 1994. Polarographic oxygen electrodes and their use in plant aeration studies. Proc R Soc Edinb 102B:511–527. Armstrong W, Beckett PM, Justin SHFW, Lythe S. 1990. Modelling and other aspects of root aeration. In: Jackson MB, Davies DD, Lambers H, eds. Plant Life Under Oxygen Stress. The Hague; SPB Academic Publishing, pp 267–282. Armstrong W, Cringle S, Brown M, Greenway H. 1993. A microelectrode study of oxygen distribution in the roots of intact maize seedlings. In: Jackson MB, Black CR, eds. Interacting Stresses on Plants in a Changing Climate. NATO ASI Series I; Global Change, Vol 16. Berlin; Springer-Verlag, pp 287– 304. Bard AJ, Faulkner LR. 1980. Electrochemical methods: fundamentals and applications. New York; John Wiley and Sons, pp 142–145. Billack B, Heck DE, Porterfield DM, Malchow RP, Smith PJS, Gardner CR, Laskin DL, Laskin JD. 2001. Minimal amidine structure for inhibition of nitric oxide biosynthesis. Biochem Pharmacol 61:1581–1586. Biemelt S, Keetman U, Albrecht G. 1998. Re-aeration following hypoxia or anoxia leads to activation of the antioxidative defense system in roots of wheat seedlings. Plant Physiol 116:651–658. Bluh O, Scott BIH. 1950. Vibrating probe electrometer for the measurement of bioelectric potentials. Rev Sci Inst 10:867–868.

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346 H+, and Ca2+ fluxes in maize roots and maize suspension cells. Planta 188:601–610. Khu¨trieber WM, Jaffe LF. 1990. Detection of extracellular calcium gradients with a calcium-specific vibrating electrode. J Cell Biol 110:1565–1573. Lamb C, Dixon RA. 1997. The oxidative burst in plant disease resistance. Annu Rev Plant Physiol Plant Mol Biol 48:251–257. Land SC, Porterfield DM, Sanger RH, Smith PJS. 1999. The self-referencing oxygen-selective microelectrode: detection of trans-membrane oxygen flux from single cells. J Exp Biol 202:211–218. Leshem YY, Haramaty E, Iluz D, Malik Z, Sofer Y, Roitman L, Leshem Y. 1997. Effect of stress nitric oxide (NO): interaction between chlorophyll fluorescence, galactolipid fluidity and lipoxygenase activity. Plant Physiol Biochem 35:573–579. Levine A, Tenhaken R, Dixon R, Lamb C. 1994. H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 79:583–593. Lorenzo E, Pariente F, Hernandez L, Tobalina F, Darder M, Wu Q, Maskus M, Abruna HD. 1998. Analytical strategies for amperometric biosensors based on chemically modified electrodes. Biosens Bioelectron 13:319– 332. Lukaszewski KM, Blevins DG. 1996. Root growth inhibition in boron-deficient or aluminum stressed squash may be a result of impaired ascorbate metabolism. Plant Physiol 112:1135–1140. Mancuso S, Papeschi G, Marras AM. 2000. A polarographic, oxygen-selective, vibrating-microelectrode system for the spatial and temporal characterisation of transmembrane oxygen flux in plants. Planta 211:384–389. Meneguzzo S, Navari-Izzo F, Izzo R. 1999. Antioxidative responses of shoots and roots of wheat to increasing NaCl concentrations. J Plant Physiol 155:274–280. Miller AJ. 1995. Ion-selective microelectrodes for measurement of intracellular ion concentration. Methods Cell Biol 49:275–291. Miller AL, Raven JA, Sprent JI, Weisenseel MH. 1986. Endogenous ion currents traverse growing roots and root hairs of Trifolium repens. Plant Cell Environ 9:79– 83. Miller AL, Shand E, Gow NAR. 1988. Ion currents associated with root tips, emerging laterals and induced wound sites in Nicotiana tabacum: spatial relationship proposed between resulting electrical fields and phytophtoran zoospore infection. Plant Cell Environ 11:21–25. Mishra A, Choudhuri MA. 1998. Amelioration of lead and mercury effects on germination and rice seedling growth by antioxidants. Biol Plant 41:469–473. Nakamura Y, Suye S, Kira J, Tera H, Tabata I, Senda M. 1996. Electron-transfer function of NAD+-immobilized alginic acid. Biochim Biophys Acta 1289:221–225.

Porterfield Newman IA, Kochian LV, Grusak MA, Lucas WJ. 1987. Fluxes of H+ and K+ in corn roots. Plant Physiol 84:1177–1184. Noritake T, Kawakita K, Doke N. 1996. Nitric oxide induces phytoalexin accumulation in potato tuber tissues. Plant Cell Physiol 37:113–116. Nowall W, Kuhr B. 1995. Electrocatalytic surface for the oxidation of NADH and other anionic molecules of biological significance. Anal Chem 67:3583–3588. Ober ES, Sharpe RE. 1996. A microelectrode for direct measurement of O2 partial pressure within plant tissues. J Exp Bot 47:447–454. Pandey PC, Upadhyay S, Upadhyay BC, Pathak HC. 1998. Ethanol biosensors and electrochemical oxidation of NADH. Anal Biochem 260:195–203. Pepperell J, Porterfield DM, Liu L, Smith PJS, Keefe DL. 1999a. Ascorbic-acid regulation in eggs and zygotes of the hamster. Biol Reprod 60:418. Pepperell J, Porterfield DM, Liu L, Smith PJS, Keefe DL. 1999b. Noninvasive detection of ascorbate fluxes in individual, oxidatively-stressed early embryos. Human Reprod 14:157. Peters W, Felle HH. 1999. The correlation of profiles of surface pH and elongation growth in maize roots. Plant Physiol 121:905–912. Pfeiffer S, Janistyn B, Jessner G, Pichorner H, Ebermann R. 1994. Gaseous nitric oxide stimulates guanosine-3 0 ,5 0 cyclic monophosphate (cGMP) formation in spruce needles. Phytochemistry 36:259–262. Pineros MA, Shaff JE, Kochian LV. 1998. Development, characterization and application of a cadmium-selective microelectrode for the measurement of cadmium fluxes in roots of Thlaspi species and wheat. Plant Physiol 116:1393–1401. Porterfield DM, Smith PJS. 2000. Characterization of transcellular oxygen and proton fluxes from Spirogyra grevilleana using self-referencing microelectrodes. Protoplasma 212:80–88. Porterfield DM, Trimarchi J, Keefe DL, Smith PJS. 1998. Metabolism and calcium homeostasis during development of the mouse embryo to the blastocyst stage in M2 culture medium. Biol Bull 195:208–209. Porterfield DM, Kuang A, Smith PJS, Crispi ML, Musgrave ME. 1999. Oxygen-depleted zones inside reproductive structures of Brassicaceae: implications for oxygen control of seed development. Can J Bot 77:1439–1446. Porterfield DM, Laskin J, Smith PJS, Malchow RP, Billack B, Heck D. Direct measurement of nitric oxide fluxes from single cells using a novel self-referencing microsensor. Am J Physiol (in press). Queiroz CGS, Alonso A, Mares-Guia M, Magalhaes AC. 1998. Chilling-induced changes in membrane fluidity and antioxidant enzyme activities in Coffea arabica L. roots. Biol Plant 41:403–413.

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21 Rooting of Micropropagules Geert-Jan de Klerk Centre for Plant Tissue Culture Research, Lisse, The Netherlands

I.

INTRODUCTION

Micropropagation, i.e., vegetative propagation of plants in tissue culture, has been broadly applied starting since the early 1980s. This chapter deals first briefly with general principles of micropropagation, and then focuses on adventitious root formation from cuttings produced in tissue culture (microcuttings).

The term tissue culture is used to describe culture of plant tissues under sterile conditions on top of or submerged in an artificial medium. Such media are composed of an aqueous solution of organic nutrients (usually sucrose), inorganic nutrients (often based on the formulation of macro- and micronutrients published by Murashige and Skoog [1962]), plant growth regulators (mostly a cytokinin and/or an auxin), and vitamins. The medium may be either liquid, or by addition of agar semisolid. In tissue culture, excised plant organs (shoots, roots, embryos, meristems), callus, cells and even protoplasts can be cultured and the direction of their development can be controlled relatively easily. Tissue culture has become an important tool for studies of physiological and developmental processes because of two major advantages: the conditions during experiments can be strictly controlled, and research can be carried out in simplified systems. As an example of the latter, for research of adventitious root formation a system was developed consisting of 1-mm slices excised from stems of apple microcuttings (Van der Krieken et al., 1993). In this system, the interferences by other organs of the plants, in particular by leaves and by apical and axillary buds, are avoided. Because the explant is very small, the factors under examination have an almost direct access to the founder cells from which the adventitious roots develop.

II.

METHODS IN MICROPROPAGATION

First practical applications of tissue culture concerned freeing plants from diseases. The potential of propagating plants (orchids) through tissue culture was recognized by Morel in 1960. Major breakthroughs were made when a generally applicable nutrient medium was devised by Murashige and Skoog in 1962, and when the use of benzylaminopurine (BAP) was introduced in order to force outgrowth of axillary buds (Sachs and Thimann, 1964). Micropropagation occurs in three steps: establishment (in which plant tissue is transferred to the in vitro environment and growth is initiated), multiplication, and reestablishment ex vitro. In the establishment phase, tissues from plants growing ex vitro in a glasshouse or in the field are surface-sterilized and transferred to a tissue culture tube with nutrient medium and plant growth regulators. Either buds or tissues without preexisting meristems may be taken as starting material. Buds are allowed to grow out to shoots, and tissues without preexisting meristems are induced to produce callus or to generate adventitious shoots. Two major prob349

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lems are met. First, the chlorine that is used to kill microorganisms on the surface of explants cannot reach endophytic bacteria and fungi. This problem of endogenous contamination can be met by keeping the mother plants under special conditions—for example, by growing them in a greenhouse and by avoiding wetting the shoots. Endogenous contamination is also reduced when meristems are excised and cultured instead of complete buds. In addition, antibiotics can be added to the initiation medium. In tissues that are capable of resisting environmental stresses, i.e., bulbs, tubers, corms, and dormant buds of perennial plants, a warm-water treatment may kill endogenous contaminants (Langens-Gerrits et al., 1998). The second major problem is that the explants may not resume growth. This can be solved by mother plant pretreatments, by starting the cultures in the proper season or by medium adaptations. Multiplication may be carried out in three ways: 1. Via axillary branching from shoots. Apical dominance is broken in blocked axillary buds by addition of cytokinin to the medium or by removal of the apical bud. The released buds develop into side shoots. For the next cycle, the newly formed side shoots are excised and apical dominance is broken again, resulting in the formation of secondary side shoots. The secondary side shoots are excised and the process is repeated until sufficient shoots have been produced. These shoots are reestablished ex vitro. Axillary branching can be applied in many crops and is, because of its ease and reliability, the most frequently applied method in tissue culture companies. A major disadvantage, though, is that it is a very labor-intensive method. Problems that are met include a low rate of propagation, hyperhydricity, decline in vigor, necrosis, loss of the chimeric structure, and, after reestablishment, bushiness. 2. Via adventitious shoot formation. Shoot meristems are regenerated from tissues without preexisting meristems (e.g., from stem, leaf, or scale fragments). The shoots may develop directly from cells of the explants or after an intermediate phase of callus. This method is applied commercially for a few crops, among others, for lilies and African violets. In many crops, however, regeneration of shoots at large numbers is difficult to achieve. It should be noted that plants originating from adventitious meristems may suffer from a high incidence of mutations, in particular after an intermediate callus phase. This phenomenon is denoted as somaclonal variation (reviewed in De Klerk, 1990).

De Klerk

3. Via somatic embryogenesis in liquid medium. By this method, new meristems are also being formed from tissues without preexisting meristems. A major difference from the previous method is a practical one: the embryos are singulated in liquid medium, allowing automation. Since major problems are met, this method is being used on a commercial scale for a few crops only. The three types of propagules that are produced in tissue culture—viz., microcuttings, microstorage organs (bulblets, tubers, corms), or somatic embryos—have to be reestablished in soil. This is problematic for somatic embryos because they are often tiny and vulnerable, and for microcuttings. Microcuttings have a phenotype different from that of normal cuttings: They have a diminished stature, a cuticle with reduced wax, poorly functioning stomata, poorly developed palisade tissue, ample spongy parenchyma tissue with large intercellular air spaces, and hypolignified stems (Ziv, 1995). These features are related to the in vitro environment that is very different from the normal environment. In the headspace of tissue culture containers, the atmosphere has a very high humidity, high diurnal fluctuations of CO2 and O2, and high levels of organic gases, among others of ethylene. Other notable characteristics of the in vitro environment are the medium that contains high concentrations of sucrose and plant growth regulators, low light intensity, and the incompleteness of the cultured plants. With respect to the latter, in shoot cultures the absence of roots may have serious consequences: roots are uptake organs for water and nutrients, and they may produce compounds that are necessary for the shoots. After transfer from tissue culture, microcuttings require a transitional environment for acclimatization to the ex vitro conditions. In the transitional environment in which the plantlets are cultured for a few days to several weeks, relative humidity is at first close to saturation and then gradually brought down to ambient values. The light intensity is kept low at first but is raised later. The transitional period allows the microcuttings to develop an adequate root system, to improve the water retention capacity of the persistent leaves, to form new leaves, and to become autotrophic. For example, microcuttings of a Juglans hybrid remain heterotrophic for the first 7 days after transfer to soil (Chenevard et al., 1997). Just after transfer from the in vitro environment, microcuttings are often very vulnerable to mechanical damage and to attacks by pathogens.

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Like normal cuttings, microcuttings require a rooting treatment. They may be either rooted like normal cuttings, e.g., by giving them a dip in rooting powder (an auxin, usually IBA, with talc as carrier), or they may be rooted in vitro after which microcuttings with roots are transferred to soil. Microstorage organs usually do not receive a rooting treatment, but such treatment may be crucial, e.g., in Narcissus bulblets (Langens-Gerrits and Nashimoto, 1997). III.

THE ROOTING PROCESS

A.

Steps in the Rooting Process

Differentiated somatic cells may reinitiate the developmental program and give rise adventitious shoots, adventitious roots, or somatic embryos. Just like the formation of new organs from somatic cells of animals, this phenomenon is referred to as regeneration. The formation of shoots (caulogenesis) or roots (rhizogenesis) are also specified as ‘‘adventitious organogenesis’’ whereas ‘‘somatic embryogenesis’’ describes the formation of adventitious (somatic) embryos. Regeneration occurs frequently during a normal plant life, but its occurrence is strongly enhanced in tissue culture, among others because plant hormones may be easily applied via the sterile tissue culture medium. Addition of cytokinin, often together with a low concentration of auxin, promotes adventitious shoot formation. Addition of auxin promotes adventitious root formation. Addition of certain auxins, in particular 2,4-D, increases the incidence of somatic embryogenesis. The most important achievement in the study of adventitious root regeneration during the last decade is a conceptual one (Kevers et al., 1997): rooting is now envisaged as a process composed of well-defined, successive phases each with its own requirements. The timing of these phases has been examined in histological and biochemical studies. However, physiological experiments using differential sensitivities to pulses with specific plant growth regulators are more instructive (De Klerk, 1996). We have proposed a time schedule for three successive phases of the rooting process in apple microcuttings (Fig. 1) based on experiments in which 24-h pulses with auxin (indolebutyric acid; IBA), cytokinin (BAP), or a genuine antiauxin (p-chlorophenoxyisobutyric acid; PCIB) were given (De Klerk, 1995; De Klerk et al., 1995): 1. In an initial dedifferentiation phase that lasts up to 24 h after excision, certain cells in the base of the stem develop competence to respond to the rhizogenic signal.

Figure 1 Dissection of the rooting process in apple Jork 9 microcuttings. For the timing of these phases, their differential sensitivities to 24-h pulses with auxin, cytokinin, and antiauxin were used.

2. Subsequently during an induction phase (between 24 and 96 h), these founder cells become determined to form roots by the rhizogenic action of auxin. Histological studies have shown that during this period the root meristems are being formed. 3. Thereafter, a phase of morphological differentiation occurs during which the roots develop. During this phase the rhizogenic signal is no longer required, and is—at the concentration used to induce root meristems—even strongly inhibitory. B.

Auxin

Auxin is known to enhance adventitious root formation from cuttings (Thimann and Went, 1934). In the practice of vegetative propagation, application of auxin is still the only method used to achieve rooting. When auxin is supplied to normal cuttings, uptake occurs almost exclusively via the cut surface (Kenney et al., 1969). The cuticle of microcuttings is often in a very poor condition (Ziv, 1995). Nevertheless, auxin uptake by microcuttings also occurs predominantly via the cut surface (Guan and De Klerk, 2000). Uptake of auxin into the cells occurs very rapidly either by diffusive permeation of the membrane of the relatively lipophilic undissociated molecule or by mediated uptake of the anion (Lomax et al., 1995). To my knowledge, the rate of auxin uptake from rooting powder has never been examined. In tissue culture, IAA uptake from solidified medium is very rapid,

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depleting the medium close to the cut surface (Guan et al., 1997). Therefore it is likely that auxin is taken up very rapidly from the rooting powder, probably within a few hours. It should be remembered that auxin is rapidly deactivated after uptake, leaving 2% or less in the active, free form (De Klerk et al., 1999). There are two major pathways of auxin deactivation: by conjugation to sugars or amino acids, or by oxidation. Conjugation is reversible but oxidation is not (Smulders et al., 1990). As noted above, auxin exerts its rhizogenic action from
24 h to 72 h after excision of apple microcutting cuttings (Fig. 1). It is important to stress again that after the induction phase, once the cells have been determined to root formation, a high auxin level is no longer required. Actually, the high concentration of auxin becomes deleterious since it blocks the outgrowth of root primordia, the growth of roots, and the development of the shoots (De Klerk et al., 1990, 1997; see also Chapter 23 by Gaspar et al. in this volume). By enhancing ethylene production, auxin also promotes senescence of shoots. C.

Ethylene

All other hormones, viz., cytokinins, gibberellins, ethylene, and abscisic acid, and the ‘‘new’’ hormones (e.g., brassinosteroids, jasmonate), influence adventitious root formation, sometimes having a vast effect. However, with the exception of ethylene their mechanism of action is not well understood and research is still too fragmentary. Before discussing the effect of ethylene, two essential features of this hormone should be stressed. First, ethylene is a gas. This has various implications. Whereas plants deactivate other hormones by enzymatic conversion (oxidation or conjugation), such metabolic deactivation systems do not exist for ethylene, except for a low rate of oxidation. Nevertheless, plants do not accumulate ethylene since it simply diffuses away from the tissue into the atmosphere. However, since the rate of ethylene diffusion in water is approximately 10,000 times less than the rate in air (Jackson, 1985), ethylene may accumulate in submerged tissues. This has an important consequence for in vitro rooting: Microcuttings are submerged with the basal portion of the stem in solidified medium, and therefore ethylene may accumulate in the tissue where the adventitious roots are mostly formed. How much ethylene accumulates depends on how far the stem is submerged, and on the rate of ethylene transport in the plant. Secondly, synthesis of ethylene is enhanced by auxin, by wounding, the orientation of

the cuttings as well as by many other factors (De Wit et al., 1990). The type of effect of ethylene depends on the phase of the rooting process (compare Fig. 1). 1. During the dedifferentiation phase, just after the cuttings have been excised from the mother plant, ethylene is stimulatory. This became apparent from 24h pulse treatments with the ethylene antagonist siverthiosulfate (STS) or with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). It has been found that elicitors enhance rooting (De Klerk et al., 1999). Thus, it is tempting to speculate that ethylene acts as an elicitor making cells capable of responding to the rhizogenic signal of auxin. 2. During the induction period, when the meristems are formed and auxin has its rhizogenic effect, ethylene becomes inhibitory. This was also shown by pulse treatments with ACC and STS. The reason why ethylene is inhibitory at this stage is unclear. Possibly, ethylene disrupts the establishment of polarity during meristem formation (cf. Kalev and Aloni, 1999). 3. During the differentiation period, ethylene is required for the formation of root hairs (Tanimoto et al., 1995). Thus, in the presence of STS, no root hairs are formed (Fig. 2). Furthermore, ethylene is required for the formation of aerenchyma in the roots of waterlogged plants (see Chapter 42 by Armstrong and Drew in this volume). The same may apply for aerenchyma of normal roots. Ethylene may also inhibit xylem formation (Zobel and Roberts, 1978). D.

Improvement of Rooting

In spite of much research, the standard rooting treatment for normal cuttings is still the one that was developed in 1936, using the auxin IBA with talc as carrier. Apparently auxin is still the only growth regulator that has a consistent, enhancing effect. Recently developed methods, viz., application of slow-release type of auxins or elicitors, may improve rooting of many crop plants (De Klerk et al., 1999). Rooting may also be promoted by enhancement of the rooting capability of (micro)cuttings. A major factor is the use of juvenile cuttings. In normal plants, rejuvenation is obtained by repeated pruning of the apex so that successively primary, secondary, tertiary, etc., branches are formed. As noted above, micropropagation is mostly based on repeated outgrowth of axillary buds. This may be the reason why during micropropagation rejuvenation occurs (e.g., Webster and Owen, 1989). For normal cuttings it is known that rooting capability of etiolated stems is improved

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Figure 2 Microcuttings of apple Jork 9 were rooted with 1
M IBA without (A) or with (B) addition of 10
M STS. Note that the roots formed in the presence of STS have hardly any root hairs and that they are much thinner. The bar is 0.5 mm.

(Kawase, 1965). In microcuttings of apple, long internodes root better than short ones (Fig. 3B) and elongation obtained by a dark treatment at the end of propagation or by using double layer, i.e., a layer of liquid medium on top of the semisolid medium, strongly enhances rooting (Fig. 3A). IV.

ROOTING AND PERFORMANCE

A.

Ex Vitro or In Vitro Rooting

As noted above, leaves of microcuttings have a very poor water retention capacity because of poorly functional stomata (Santamaria and Kerstiens, 1994). Thus, it is essential that the new root system of microcuttings functions as soon as possible after transfer to ex vitro conditions so that the extensive water loss from the leaves can be compensated for by water taken up by the roots. Often, microcuttings do not require application of auxin to achieve rooting, but treatment with auxin increases the rooting percentage, the number of roots, and the speed and synchronicity of rooting (Marks and Simpson, 2000). Microcuttings of some plants can be rooted like normal cuttings by dipping them in rooting powder and planting them in a suitable ex vitro medium, for example in a peat–perlite mixture. Microcuttings may also be rooted in vitro on a semisolid nutrient medium containing auxin and, after roots have been formed, transferred to soil. It is surprising that no critical comparisons have been made between in vitro and ex vitro rooting. Many published reports

involve major pitfalls because they monitor only survival and not growth performance, and/or because the in vitro rooting treatment was not optimized. In particular, investigators did not search for the appropriate auxin treatment and did not consider the detrimental effect of accumulation of ethylene in the headspace during in vitro rooting. In vitro rooting is a controversial issue. It was stated that in vitro roots die after planting in soil (Debergh and Maene, 1981; McClelland et al., 1990; Ziv, 1995) or that they do not function properly (Grout and Ashton, 1977). On the other hand, it was shown that the compensation for water loss by Picea microcuttings correlates with the number of in vitro formed roots (Mohammed and Vidaver, 1991). Van Telgen et al. (1993) describe that performance after transfer to soil is correlated with the number of roots formed during in vitro rooting. Almost all in vitro formed roots of the apple rootstock Jork 9 resumed growth after transfer to soil, and root growth resumed almost immediately after the transfer (Fig. 4). Performance ex vitro was related to the number of roots (albeit only weakly; De Klerk, 2000). Ex vitro rooting of apple microcuttings ensured survival but did not result in rapid resumption of growth. Microcuttings rooted in vitro showed much better growth, in particular when ethylene had been removed from the headspace (Fig. 5). The situation was different for roses where ex vitro rooting gave good results. It should be noted here that the differences between rooting treatments diminish when the conditions in the greenhouse have been optimized (Anthonis, 2001).

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Figure 4 Length of the longest root of apple Jork 9 microcuttings after transfer to soil. The microcuttings had been rooted for 3 weeks in vitro.

Figure 3 Effect of shoot elongation on rooting of apple. (A) Effect of stem elongation in a ‘‘double-layer’’ system. Microcuttings of the apple rootstock M26 were examined for their rooting capability after a large number of subculture cycles (data are from C. Denissen, Lisse). (B) Effect of stem internode length on rooting of Jork 9 microcuttings. Onemillimeter stem slices were cut from these internodes and rooted with 3 mM IBA.

96 h in apple microcuttings. Thus, when auxin is supplied as a brief, early pulse, it is important to supply the cutting with a stable auxin that remains present for a sufficiently long period of time. Indeed, when auxin was supplied as a brief 1-h pulse, the concentration of applied IAA (unstable) to achieve sufficient rooting was so high (Fig. 6) that the shoots suffered. In such a case, IBA (stable) was the preferable auxin. During in vitro rooting, though, uptake of auxin occurs during an extended period of time of at least 3 days (Guan et

In vitro rooting is also advantageous because microcuttings are often tiny and vulnerable at the end of the propagation cycle, and considerably increase in size during the rooting treatment. Dry weight of the shoots of apple microcuttings increases about fourfold during the 3-week rooting treatment (De Klerk, 2000). The obvious disadvantages of in vitro rooting are that, because of the additional labor, the price of rooted microcuttings is approximately twice as high and the rooted microcuttings are more difficult to plant in soil. B.

Choice of the Type of Auxin

As noted above, rooting requires presence of a high level of auxin for a protracted period, e.g., for 72–

Figure 5 Weight of Jork 9 shoots that had been rooted either ex vitro (1 h exposure to 1 mM IBA) or 3 weeks in vitro with 10 mM IAA. When the shoots were rooted in vitro, grains coated with KMnO4 were also added. Weight was measured after 4–7 weeks in soil.

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Figure 6 Concentrations of auxin that gave optimal rooting after exposure of Jork 9 cuttings to auxin for 1 h or 1 d, or after continuous exposure. Note that for a 1-h exposure a very high concentration of IAA had to be supplied.

al., 1997). As auxin is taken up over such a long period, in in vitro rooting an unstable auxin may result in high rooting. Moreover, because the high concentration of auxin, necessary for the induction of rooting, inhibits the outgrowth of root primordia and is detrimental for both root and shoot growth, the use of an unstable auxin for in vitro rooting may be even preferable. Indeed, IAA is the preferential choice for in vitro rooting of apple microcuttings in comparison with the stable auxins IBA and NAA: The maximum number of roots is the same as with IBA but it is reached over a much wider range of concentrations, callus formation was little, the roots are longer, and the shoots hardly show signs of aging (De Klerk et al., 1997). C.

Figure 7 Microcuttings of Jork 9 that had been rooted with 3 mM NAA. To reverse the effects of ethylene, either 10 mM STS had been added or ethylene had been captured from the headspace by grains coated with KMnO4.

Removal of Ethylene

Shoots exposed to a high concentration of auxin show leaf senescence, indicating an effect of ethylene (De Klerk et al., 1997). When the ethylene inhibitor STS was applied together with auxin, the shoots recovered almost completely (Fig. 7). However, upon planting out, many of the shoots died, possibly because of a carryover of STS that inhibited root hair formation (Fig. 2). Thus, STS does not seem to be appropriate to counteract ethylene. Significant improvement of performance was observed when ethylene was removed from the headspace of the Petri dish by KMnO4. STS has also a tremendous positive effect on shoot quality of roses but at the same time blocks rooting (De Klerk, 2000). KMnO4 treatment strongly increases both survival and growth of rose microcuttings (Fig. 8).

Figure 8 Survival and growth of surviving shoots of rose microcuttings (Madelon) rooted in vitro with 10 mM IAA. Ethylene was allowed to accumulate in the headspace or was removed by grains coated with KMnO4.

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V. PROSPECTS The central theme of this chapter is that there are two main differences between rooting in vitro and ex vitro. First, for ex vitro rooting a brief pulse with a high concentration of a stable auxin is supplied. Rooting of microcuttings in vitro involves a protracted uptake of auxin. Therefore, unstable auxins may perform well in in vitro rooting. Actually, they may be preferable because stable auxins remain present for a too long time. Thereby, they inhibit outgrowth of root primordia and affect the quality of shoots. Secondly, because of the wounding effect and of the auxin medium, ethylene may accumulate in the tissue culture containers during the rooting treatment and affect the microcuttings. This can be overcome by addition of KMnO4. The interface between in vitro and ex vitro has hardly been explored. In addition to adapting the rooting conditions, there are many other possibilities that may greatly improve performance after establishment. Acclimatization may be carried out to some extent in vitro, the shoots may be bacterized before transfer to soil, additional nutrients may be added before transfer, and hormonal or dormancy breaking treatments can be used. Such research can greatly enhance growth performance of microcuttings and is therefore of high importance for horticulture and the micropropagation industry. The treatment used for rooting of normal cuttings was developed some 70 years ago. New molecular techniques, e.g., DNA microarrays and studies on mutants, and the rapid developments in molecular research on lateral root formation and auxin action, will have significant impact on the understanding of rooting. This should result in new rooting treatments. Tissue culture systems, e.g., root formation in vitro from excised stem segments, constitute an excellent and indispensable tool for such research.

REFERENCES Anthonis B. 2001. The commercial practice of rooting in a nursery. In: De Klerk GJ, Van der Krieken W, eds. Root Formation. Proceedings of the Third International Congress on Adventitious Root Formation. Dordrecht, Netherlands: Kluwer (in press). Chenevard D, Frossard JS, Jay-Allemand C. 1977. Carbohydrate reserves and CO2 balance of hybrid walnut (Juglans nigra No. 23  Juglans regia) plantlets during acclimatisation. Sci Hort 68:207–217.

De Klerk Debergh PC, Maene LJ. 1981. A scheme for commercial propagation of ornamental plants by tissue culture. Sci Hort 14:335–345. De Klerk GJ. 1990. How to measure somaclonal variation: a review. Acta Bot Neerl 39:129–144. De Klerk GJ. 1995. Hormone requirements during the successive phases of rooting of Malus microcuttings. In: Terzi M, Cella R, Falavigna A. eds. Current Issues in Plant Cellular and Molecular Biology. Dordrecht, Netherlands: Kluwer, pp 111–116. De Klerk GJ. 1996. Markers of adventitious root formation. Agronomie 16:563–571. De Klerk GJ. 2000. Rooting treatment and the ex vitro performance of micropropagated plants. Acta Hort 530:277–288. De Klerk GJ, Ter Brugge J, Smulders R, Benschop M. 1990. Basic peroxidases and rooting in microcuttings of Malus. Acta Hort 280:29–36. De Klerk GJ, Keppel M, Ter Brugge J, Meekes H. 1995. Timing of the phases in adventitious root formation in apple microcuttings. J Exp Bot 46:965–972. De Klerk GJ, Ter Brugge J, Marinova S. 1997. Effectiveness of indoleacetic acid, indolebutyric acid and naphthaleneacetic acid during adventitious root formation in vitro in Malus ‘Jork 9’. Plant Cell Tissue Org Cult 49:39–44. De Klerk GJ, Van der Krieken W, De Jong J. 1999. The formation of adventitious roots: new concepts, new possibilities. In Vitro Cell Dev Biol 35:189–199. De Wit L, Liu JH, Reid DM. 1990. Production of ethylene by gravistimulation; a potential problem with the interpretation of data from some experimental techniques. Plant Cell Environ 13:237–242. Grout BWW, Aston H. 1977. Transplanting of cauliflower plants regenerated from meristem culture. 1. Water loss and transfer related to changes in leaf wax and to xylem regeneration. Hort Res 17:1–7. Guan H, Huisman P, De Klerk GJ. 1997. Rooting of apple stem slices in vitro is affected by rapid decline of indoleacetic acid in the medium. J Appl Bot 71:80–85. Guan H, De Klerk GJ. 2000. Stem segments of apple microcuttings take up auxin predominantly via the cut surface and not via the epidermis. Sci Hort 86:23–32. Jackson MB. 1985. Ethylene and responses of plants to soil waterlogging and submergence. Annu Rev Plant Physiol 36:146–174. Kalev N, Aloni R. 1999. Role of ethylene and auxin in regenerative differentiation and orientation of tracheids in Pinus pinea seedlings. New Phytol 142:307–313. Kawase M. 1965. Etiolation and rooting in cuttings. Physiol Plant 32:170–173. Kenney G, Sudi J, Blackman GE. 1969. The uptake of growth substances. XIII. Differential uptake of indole-3yl-acetic acid through the epidermal and cut surfaces of etiolated stem segments. J Exp Bot 20:820–840.

Micropropagules Kevers C, Hausman JF, Faivre-Rampant O, Evers D, Gaspar T. 1997. Hormonal control of adventitious rooting: progress and questions. J Appl Bot 71:71–79. Langens-Gerrits M, Nashimoto S. 1997. Improved protocol for the propagation of Narcissus in vitro. Acta Hort 430:311–313. Langens-Gerrits M, Albers M, De Klerk GJ. 1998. Hotwater treatment before tissue culture reduces initial contamination in Lilium and Acer. Plant Cell Tissue Org Cult 52:75–77. Lomax TL, Muday GK, Rubery PH. 1995. Auxin transport. In: Davies PJ, ed., Plant Hormones: Physiology, Biochemistry and Molecular Biology. Dordrecht, Netherlands: Kluwer, pp 509–530. Marks TR, Simpson SE. 2000. Manipulation of rooting competence in vitro in a range of difficult and easy-to-root woody plants. Plant Cell Tissue Org Cult 62:65–74. McClelland MT, Smith MAL, Carothers ZB. 1990. The effects of in vitro and ex vitro root initiation on subsequent microcutting root quality in three woody plants. Plant Cell Tissue Org Cult 23:115–123. Mohammed GH, Vidaver WE. 1991. Plantlet morphology and the regulation of water loss in tissue-cultured Douglas-fir. Physiol Plant 83:117–121. Morel G. 1960. Producing virus-free Cymbidium. Am Orchid Soc Bull 29:495–497. Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 95:814–821. Smulders MJM, Van de Ven ETWM, Croes AF, Wullems GJ. 1990. Metabolism of 1-naphthaleneacetic acid in

357 explants of tobacco: evidence for release of free hormone from conjugates. J Plant Growth Regul 9:27–34. Santamaria JM, Kerstiens G. 1994. The lack of control of water loss in micropropagated plants is not related to poor cuticle development. Physiol Plant 91:191–195. Sachs T, Thimann KV. 1964. Release of apical buds from apical dominance. Nature 201:939–940. Tanimoto M, Roberts K, Dolan L. 1995. Ethylene is a positive regulator of root hair development in Arabidopsis thaliana. Plant J 8:943–948. Thimann KV, Went FW. 1934. On the chemical nature of the root forming hormone. Proc Kon Akad Wetensch 37:456–459. Van der Krieken WM, Breteler H, Visser MHM, Mavridou D. 1993. The role of the conversion of IBA into IAA on root regeneration in apple: introduction of a test system. Plant Cell Rep 12:203–206. Van Telgen HJ, Van Mil A, Kunneman B. 1992. Effect of propagation and rooting conditions on acclimatization of micropropagated plants. Acta Bot Neerl 41:453– 460. Webster CA, Owen OP. 1989. Micropropagation of the apple rootstock M. 9: effect of sustained subculture on apparent rejuvenation in vitro. J Hort Sci 64:421–428. Ziv M. 1995. In vitro acclimatization. In: Aitken-Christie J, Kozai T, Lila Smith M, eds. Automation and Environmental Control in Plant Tissue Culture. Dordrecht, Netherlands: Kluwer, pp 493–516. Zobel RW, Roberts LW. 1978. Effects of low concentrations of ethylene on cell division and cytodifferentiation in lettuce pith explants. Can J Bot 56:987–990.

22 Modeling Root System Architecture Loı¨c Page`s INRA, Centre d’Avignon, Avignon, France

I.

INTRODUCTION

Many root system models were developed as part of larger models which aimed at describing either the crop functioning in relation to its environment, including the soil (Ritchie et al., 1985; Whisler et al., 1986; Jones and Kiniry, 1986; Klepper and Rickman, 1990; Chapman et al., 1993; Adiku et al., 1995; Asseng et al., 1997) or some aspects of the soil–plant–atmosphere system (Hansen, 1975; Skiles et al., 1982). In such models, the root system was considered as an uptake system (water and minerals) and/or as a sink for photoassimilates (Reynolds and Thornley, 1982; Huck and Hillel, 1983; Cannell, 1985; Hoogenboom and Huck, 1986; Ho, 1988). Representing the uptake function of the root system requires a minimal representation of its morphology. For this purpose, the concept of root density profile has been used extensively (Huck and Hillel, 1983). Root density profiles describe the amount of roots in terms of biomass, length, surface area, etc., in horizontal layers of the soil. In relation to this very simple representation of the root system, the functional assumptions of the uptake models are also very simplified. They generally assume the spatial distribution of the roots to be homogeneous in the soil layer, and the uptake to be similar between roots. They formulate the uptake function as related to root length and to soil layer water potential (for instance) and do not take into consideration transport and resistance in pathways. (See Chapter 37 by Silberbush and Chapter 35 by Jungk in this volume for details.) The role of the root system as a sink for photoassimilates is sometimes

The main purpose of this chapter is to discuss the interest of using mathematical simulation models to study the development and the architecture of root systems. Methodological difficulties and the development of new techniques and methods for studying root systems have long been, and still are, a dominant subject in the scientific literature on roots (see review by Bo¨hm, 1979, and Chapter 18 by Polomski and Kuhn in this volume). These efforts have led to significant advances in the way of conducting experiments on roots, and obtaining more reliable data at various scales and in various conditions. Another major issue, often overshadowed until now because data acquisition appeared as the true bottleneck, is the parallel development of concepts and models to acquire, interpret, and use these new data. The set of roots, organized into a root system, can either be considered per se or as part of the larger soil–plant system (soil–plant system), as a complex system requiring specific tools and methods to be studied (Legay, 1997). The behavior of each component is the result of numerous interactions, many of which cannot be discarded without taking the risk of distorting the image of the system under study. Therefore, I would like to emphasize the methodological purpose of this chapter. The models will be mainly reviewed as tools corresponding to research steps, associated with various viewpoints on the investigated system. 359

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considered in these global models, using very simple rules. The root system either receives a given proportion of the total amount of assimilates produced by the plant, is used as a spillway for excess of assimilates after consumption by the shoots, or is provided according to its overall calculated demand (e.g., as a function of its biomass and soil conditions). These oversimplifications in describing the root system are justified for these models which have predictive objectives, or aim at describing the functioning of a larger system, including roots, while taking several additional aspects of its functioning into account. Whenever the objective of a model is to investigate the development and function of the root system, the framework imposed by root depth or root density models is unsuitable. The major simplifications in these models are generally designed for calculation simplification and for matching data availability (root density profiles as outcome of a long tradition of soil coring) rather than for the achievement of a clear hierarchy between interacting mechanisms. Therefore, it is necessary to develop models that include details of the architecture and dynamics of root systems in order to gain new insight. In this respect, the word ‘‘architecture’’ needs to be clarified by specifying its two complementary meanings: shape and structure. Shape refers to the root system geometry, or to the spatial distribution of the roots. Shape, as well as its time-dependent variations, is most important during the first phase of soil resource acquisition because at that stage, uptake is limited by the soil transport an interception of the resources by the growing roots. The shape has sometimes been characterized by a functional criterion: distribution of distances between sample points in the soil and their nearest neighboring root (Tardieu, 1988). Root distribution often presents non uniform but clumped patterns (Tardieu, 1988; Logsdon and Allmaras, 1991; Pellerin and Page`s, 1996) which may have significant effect on the uptake processes (Van Noordwijk and Brouwer, 1991; Bruckler et al., 1991; Tardieu et al., 1992). The significance of the kinetics of root spatial distribution depends on the mobility of the resources considered (Baldwin et al., 1973). The less mobile the resources, the more crucial is this distribution pattern. The root system geometry is also a major component of the anchoring function (Coutts, 1983; Ennos and Fitter, 1992; Guingo and He´bert, 1997; see also Chapter 11 by Persson and Chapter 10 by Stokes in this volume). Root orientations, as well as the spatial distribution of their diameters and mechanical properties, are in this case the most important geometric characteristics.

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The term structure refers to the differentiation of components within the root system, and to their mutual relationships: differences in their ontogenetic characteristics which are organized along the axes (gradients) and differences between the connected roots (e.g., daughter roots are necessarily younger and generally finer than the mother roots). Connection relationships define the topology of the branching system (Fitter, 1982; Fitter, 1986; see also Chapter 2 by Fitter in this volume). The morphogenetic and functional differences between and along the roots significantly influence the root-foraging potential and resource acquisition (see Chapter 9 by Waisel and Eshel in this volume). The issues concerning transport pathways within the root system, organization, and effective conductance are still points of debate and new analyses (Fitter, 1987; Berntson, 1994a; Fitter, 1996). They cannot be dismissed just because of the calculation problems they raise. It is also evident that the structure of the root system plays a prime role in other functions of the root system, such as sink and storage functions, deposition and excretion of various biochemical compounds, sending of signals toward the plant or the soil, or association with symbiotic organisms. Thus, we must consider strong relationships between architecture and function and, from a dynamic point of view, among the time-dependent geometric, developmental, and functional properties. It shows the need for integration of the various aspects of the dynamics of the root system architecture into a consistent framework, such as a simulation model based on developmental processes. Such a model can be used to simulate these close links between the generation of the root system components, their architectural position, and their functions. In this chapter we present dynamic architectural models, i.e., models simulating both the shape and the structure of the root system and its progressive development. Some static models have been developed (Henderson et al., 1983a,b), which will not be discussed, because they rely on very different objectives and design. Fractals (Mandelbrot, 1983) can be used to simulate the dynamics of the root system architecture, but they will not be specifically considered here. Moreover, in respect to the purpose of this chapter, they can be considered as a particular case among the models that we are presenting, having more restrictive hypotheses regarding developmental processes in order to meet the autosimilarity assumption (Tatsumi et al., 1989; Fitter and Stickland, 1992; Berntson, 1994b; Shibusawa, 1994; Van Noordwijk et al., 1994; Eshel, 1998; Ozier-Lafontaine et al., 1999).

Modeling Root System Architecture

The chapter will obviously focus on the root system, with only few references to the shoot system. Still, it is worth noting that the approach and tools are not fundamentally different from those applied to shoots, and some concepts have only been translated. However, the main basic processes, as well as the constraints considered, are quite different and justify specific considerations. This review will complement and update other interesting reviews on the subject (e.g., Klepper and Rickman, 1990; Jones et al., 1991; Lynch, 1995; Lynch and Nielsen, 1996). We shall successively analyze the basic developmental hypotheses of these models and discuss how the interaction with various environmental characteristics has been considered. Then, we shall illustrate various aspects of architecture modeling using models which have been developed according to different viewpoints. This will lead us to look at the present and future links between simulation of the root system architecture and various aspects of the research conducted on root system functioning. II.

MODEL DEVELOPMENT

A.

Basic Principles

The basic principle of most root system architecture models from the first (Hackett and Rose 1972a,b; Lungley, 1973) to the most recent ones (Fitter et al., 1991; Clausnitzer and Hopmans, 1994; Somma et al., 1998) has been to mimic developmental phenomena of individual roots using very simple rules. This idea resulted from the observation that part of the global complexity is actually the result of a rather limited number of axis types and dynamic processes that are repeated many times in space and time, with some quantitative variations. Thus, the plasticity of the whole root system contrasts with the high organization of the basic processes and the relative invariance of structural properties. Therefore, the modeling approach has been (1) to identify and categorize within the root system a reduced number of components having a homogeneous developmental behavior, and (2) to characterize and formalize the dynamics of appearance, time variation, and sometimes disappearance of these components. This approach is somehow close to what Halle´ and Oldeman (1970) and Halle´ et al. (1978) have called l’analyse architecturale (architectural analysis), which they applied to the shoots of a large number of tropical tree species. The various combinations of axis types and developmental processes led them to define about 20 qualitatively different mode`les architec-

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turaux (architectural models), which described all the observed species. It is noteworthy that the relevance of such an approach relies on the relative invariance of the components and basic processes during development. 1.

Components of the Root System

In many models the components that have been defined are the roots, or the root apices, belonging to different developmental branching orders (order 1 being directly connected to the shoot system, and order i giving rise to order i+1 by branching). The first papers, which have mainly worked on young plants (Hackett and Rose, 1972a,b; Lungley, 1973; Diggle, 1988; Page`s and Arie`s, 1988), have grouped roots according to their branching order, considering only the three or four first branching orders representing in this case almost the whole root system. This root categorization is an essential assumption, contrasting with what had been assumed before in root modeling. It resulted from the observation that roots of different branching orders have very different developmental characteristics (e.g., appearance, growth, and branching). For example, Diggle (1988) considered growth rates decreasing by a factor of 10 from order 1 to 3. Later on, several authors (Page`s et al., 1989; Le Roux and Page`s, 1994; Jourdan et al., 1995) extended the approach, defining general root types which are not strictly associated to branching orders. Apparently, root growth rates, as well as other morphogenetic properties, may be highly variable according to the origin of the root, and not only because of variations in the soil or substrate (see Chapter 9 by Waisel and Eshel in this volume). The classification of root types is based on several criteria that describe the morphogenetic properties of the roots: growth rate, growth duration, branching ability and density, and tropisms (cf. Wilcox, 1962, 1968; Charlton, 1967; Hackett, 1969; Varney et al., 1991; Atger and Edelin, 1992; Le Roux, 1994; Jourdan and Rey, 1997; Vercambre and Page`s, 1998). Coutts (1987), and then Page`s (1995) and Vercambre and Page`s (1998), showed that the apical diameter was a reliable synthetic criterion to distinguish roots of different types and to locate them on a common scale. Schematically, the big roots (those having a large apical diameter) have a high morphogenetic potential (axial and radial growth, branching, gravitropism), whereas fine roots have low developmental potential. In conclusion, it is possible to generalize the concept of root components by considering not only roots

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grouped into types, by also types of apices and segments (which are pieces of axes). Thus, we define root system modules by analogy with the modules which make up the shoot system, as defined in the LSystem models (Prusinkiewicz and Lindenmayer, 1990). 2. Morphogenetic Rules and Their Formalization Identifying root system components and their connection relationships (topology) is not sufficient in itself to make dynamic simulations of its development. It is also necessary to investigate the processes by which the components change of state from their initiation onward. Three basic processes, involved in the development of root systems have first been identified and modeled: emission, resulting in the appearance of firstorder roots, at the base of the plant that is directly connected to the shoot; branching, by which new order i+1 roots are produced laterally on order i mother roots; and axial growth, leading to the elongation of existing roots. The emission process has been studied in cereals (Klepper et al., 1984; Picard et al., 1985; Nemoto and Yamazaki, 1986) and then included as submodels (modules) in the simulation of wheat (Diggle, 1988) and maize (Page`s et al., 1989). Cereals continuously produce nodal roots according to a highly organized sequence in space and time. As the phyllochron, which is generally tightly correlated to the cumulated sum of temperatures since sowing, nodal roots are emitted similarly from the phytomers at a regular rate, from the base upward. In these models, devoted to cereals, a simple linear function was used to link the rank of the phytomer where emergence occurs to cumulated thermal time. The branching process that has been studied the most is the acropetal branching, which results in the emergence of lateral roots in a limited zone along the bearing axis that shifts acropetally, following at a given distance the apex of the bearing axis (see Chapter 8 by Lloret and Casero in this volume). This acropetal branching process is of prime importance in the development of most root systems because it results generally in a large number of roots and it arranges lateral roots according to their age. Therefore, such branching leads to a certain spatial organization of root age. In order to formalize the spatial and temporal aspects of such a branching process, the simplest models (Hackett and Rose, 1972a,b; Lungley, 1973) were based on two rules: (1) successive lateral roots appear

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at fixed distance from one another (parameter interbranch distance), and (2) they cannot appear closer to the apex than a given threshold distance (parameter length of the apical nonbranching zone). In later models (Diggle, 1988; Page`s et al., 1992; Clausnitzer et Hopmans, 1994), the apical nonbranching zone was simulated using a temporal parameter, which is the time duration before emergence of the lateral primordia that are initiated close to the apex of the bearing axis. Using this formalization, which defines duration by a parameter, the length of the apical nonbranching zone varies according to the growth rate of the bearing axis, in agreement with observations made on several species (Page`s and Serra, 1994; Pellerin and Tabourel, 1995; Aguirrezabal and Tardieu, 1996). In the model of Jourdan and Rey (1997), as well as that of Vercambre and Page`s (1998), the type of lateral axis is not strictly determined by the type of bearing axes, but is simulated using a stochastic process. These models are more flexible regarding the topological structure of the branching system. The general idea is that the type of an axis is only partly determined by its bearing axis (e.g., a fine root cannot give rise to big lateral roots), but it also depends on random factors (e.g., among the branches of a big root, fine roots can also be observed). The initial branching direction is calculated in threedimensional models by considering an insertion angle and a radial angle. The radial orientation of laterals was sometimes simulated in relation to the internal structure of the roots (Berntson, 1994a), forcing lateral roots to emerge only in front of xylem poles along orthostichies (Esau, 1989). Axial growth, or axis elongation, was assumed to be primarily dependent on the type of axis, with strong differences reaching a factor of 10–20, showing the significance of an architectural factor on elongation. In the simplest versions (Hackett and Rose, 1972a; Lungley, 1973; Fitter, 1987; Diggle, 1988; Fitter et al., 1991), growth rate was constant for each root type and calculated from a single parameter per root type. In a slightly different way, Page`s et al. (1989), simulating maize root systems, considered that root types did not differ only in their growth rates, but also in their growth patterns (determinate or undeterminate depending on the root type, according to Cahn et al., 1989; Varney et al., 1991; Varney and McCully, 1991). For this purpose, they used a common growth function allowing the simulation of both growth patterns. In their model, Clausnitzer and Hopmans (1994) and then Somma et al. (1998) also used predetermined growth functions, and calculated a growth potential on

Modeling Root System Architecture

this basis. Environmental factors are used to calculate reduction factors (values between 0 and 1), which are then multiplied by the potential growth rate to obtain the actual growth rate. Other authors, such as Page`s et al. (1992) or Jourdan and Rey (1997), have used stochastic elongation models to account for growth variations, even for a given root type, which cannot be modeled on the basis of environmental variations. Lastly, the model of Thaler and Page`s (1998) did not use a predetermined growth function for each root type, but calculated a potential growth rate from the root apical diameter using a single function. In this model, the apical diameter, indicating the meristem volume of the root, varies with time according to carbohydrate availability. This growth model predicts large variations in growth patterns from one root to another. Growth directions were also specifically considered in several three-dimensional models, since the trajectory of the root is generally not straight and is considered as a major feature of the root system architecture. Growth direction, for the faster-growing roots especially, contributes along with growth rate to define the actual volume of soil explored. Growth direction is affected by soil constraints, which will be discussed in the next section, but also depends on the ability of the different root types to direct themselves according to specific mechanisms, the tropisms. For example, different gravitropic behaviors of roots have been described and classified by Riedacker et al. (1982). In addition to this well-known gravitropism, root orientation by various mechanisms is a very common and major feature in plants (see Chapter 29 by Porterfield in this volume). From an architectural point of view, it gives the main roots a globally organized trajectory and, simultaneously, an important local tortuosity (see Kutschera, 1960; Fig. 1). The gravitropic trends of roots were first modeled by Lungley (1973) and then formalized with more detail in several three-dimensional models (Page`s and Aries, 1988; Berntson, 1994a; Clausnitzer and Hopmans, 1994). The general principle is to give, at each elongation step, an additional vertical component to growth direction. Tardieu and Pellerin (1990) have shown the ability of this type of model to account for the general trajectory of the maize nodal roots under field conditions. Le Roux (1994) has also proposed a representation of the plagiogravitropism of some axes, i.e., their tendency to come back to a horizontal direction after deflection, by gravitropism (Riedacker et al., 1982; Le Roux and Page`s, 1996). In his model, a horizontal directional component is calculated and added to the growth

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direction. Other submodels of tropism based on the same method could be included, in order to help in analyzing and quantifying other mechanisms which may concur to the growth direction of roots (chemiotropism, thermotropism, hydrotropism, etc.) (Coutts, 1989; see also Chapter 29 by Porterfield in this volume). In addition to these main developmental processes, which were the bases of root system architectural models, some authors (Clausnitzer and Hopmans, 1994; Page`s et al., 1995; Vercambre and Page`s, 1998) have suggested application of the same approach to other processes that affect root system dynamics: reiteration, decay and abscission, and radial growth. The reiteration concept was suggested by Oldeman (1974) to describe the dynamics of shoot architecture in tropical trees. He considered the architectural development as a sequence (the ‘‘architectural unit,’’ according to Barthe´le´my et al., 1989), in which axes of defined types appear in ordered steps at predictable locations both in time and space. Reiteration is the development of a new sequence within the first one. The new sequence may start from the beginning (in case of complete reiteration) making the initial (youngest) axes, or at a later stage (partial reiteration). This phenomenon occurs spontaneously, with aging of the tree crown, giving rise to a set of small treelike structures (complete reiterated structures) within the old tree (Halle´ et al., 1978). It may also be an adaptive response to various pruning events and changing conditions. The same phenomenon is observed in root systems. It has been described by Lyford and Wilson (1964) and by Kahn (1978), who interpreted for example the ‘‘sinkers’’ as new ‘‘architectural units’’ developing within the overall root system, starting from a typical taproot, which is the initial axis of the sequence (complete reiteration). Atger and Edelin (1992) have also used this concept to describe the dynamics of the root systems of several tree species. Vercambre and Page`s (1998) have formalized and quantified this phenomenon in a model applied to the peach tree (Fig. 2). They observed that roots not only branched by acropetal branching, but also by reiteration, creating forks in which the bearing axes were duplicated. The reiterations were partial, since the new sequences were not started from the beginning. These authors showed that the process occurred periodically, as a result of drastic variations in the growing conditions (e.g., winter or dry season). In their model, a set of new axes of the same type were generated reiteratively on the bearing axis, substituting it in the process. The number of

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Figure 1 Drawing of a root system, drawn from a field excavation of a Polygonum lapathifolium plant. The general organization of the trajectories of the main roots contrasts with their local tortuosity. (From Kutschera, 1960.)

reiterated axes was calculated using a predetermined distribution law. Decay and the subsequent abscission are common phenomena of the architecture genesis in long-term development of perennial plants. Clausnitzer and Hopmans (1994), Le Roux (1994), Page`s et al. (1995), and Vercambre and Page`s (1998) have developed modules to account for these decay phenomena. In their model, Vercambre and Page`s (1998) assumed that the root tip has a given life expectancy (parameter defined for each type of axis) after which it dies. Later, the axis generated by this apex disappears, either completely if it bears no living branch, or partly—i.e., its distal part—if it bears living branches. This decay module, which is very simple mainly owing to the lack of understanding of this complex phenomenon, allowed a first realistic representation of the spatial

and temporal organization of decay and abscission. This phenomenon is not randomly distributed within the root system, and its complex distribution can be described, at least roughly, by this algorithm in an efficient way. This point illustrates once more the relevance of linking local and basic developmental processes to the whole root system architecture using simulation models. Radial growth, and the production of secondary xylem and phloem, are also of major significance in the aged root system of dicotyledon species. Their functional consequences are obvious on several whole plant processes: sink for carbohydrates, axial water conductance, storage, and anchorage (see also Chapter 6 by Chaffey in this volume). In their architectural model, Vercambre and Page`s (1998) developed a radial growth submodel based on the strong allo-

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Figure 2 Simulations of the peach tree root system in orchard conditions (after the model of Vercambre and Page`s, 1998). A. Main roots (macrorhizae) 2 (left), 3 (center), and 4 (right) years after plantation. B. Top view of these main roots, 4 years after plantation. Forks are the consequence of the reiteration process (see text), which is typical of several tree species. Radial growth, represented by the line width, was simulated using the principles of the pipe model (Shinozaki et al., 1964). Root decay and abscission concerns the fine roots (enlargement) and results in a decrease of the density near the base.

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metric relationships of root section areas observed at the branching points of lignified roots. The cross-sectional area of the mother root was proportional to the sum of the cross-sectional areas of the daughter roots, with a proportion coefficient close to unity. Moreover, this type of relationship is quite common to various branching systems, and is the basis of the pipe model proposed by Shinozaki et al. (1964), as well as of the fractal model of Van Noordwijk et al. (1994). More specifically, Vercambre and Page`s (1998) used this close relationship concerning root cross-sectional areas and simulated radial growth on any root segment as a function of the emergence of new roots at distal position relative to this segment. These new roots are assumed to require new pipes to connect to the shoot system (Eshel et al., 2000). Thus, radial growth is linked during the simulation to the branching processes (acropetal branching and reiteration in this case). Such a simulation renders the empirical allometric relationship at each branching point and contributes to realistic representations (Fig. 2). 3. Driving the Simulation and Coding the System Most models developed until now rely on almost the same principles and approximately the same coding system. Time is divided into time steps; at each time step, the model calculates the structure alterations induced by the application of the development modules, and the possible interactions with the environment and its variations during the considered time step (Porter et al., 1986; Clausnitzer et Hopmans, 1994; Bengough, 1997). The simulation operates typically on individual plants, clearly identified, and not at the crop scale. However, several authors produced outputs by subsequent calculations at the crop scale (Lungley, 1973; Porter et al., 1986; Bengough, 1997), considering that the simulated plant is an average plant. The characteristics of such plant could be converted to surface or volume per ground area units, knowing the plant density of the crop. Some models (Page`s et al., 1989) allow the simulation of a small crop, via the simulation of several plants located as they are in the field, in order to produce two- or three-dimensional outputs, which can be compared to plane maps (Pellerin and Page`s, 1996) or three-dimensional density maps (Grabarnik et al., 1998). The time step of the simulation varies from one model to another, from 12 h (Bengough, 1997) to several days (Vercambre and Page`s, 1998), depending on

the simulation duration, the considered processes and the expected accuracy. It can sometimes be adjusted by the user (Somma et al., 1998). In several models, some of the modules run using a shorter time step to increase numerical accuracy within these specific modules, without increasing by too much the duration of the program execution, or enlarging unnecessarily the size of the simulated structures. The root system is generally coded in the computer memory as a set of components, which represents the segments in most models, and sometimes also the generating apices. The segments code the parts of axes generated at each time step. They contain more or less detailed information on their location and development (spatial coordinates, age, diameter, type of the bearing axis) as well as pointers keeping the connections with the rest of the structure (originating segment, set of segments originating from it). Lynch and Nielsen (1996) and Lynch et al. (1997) called this structure the ‘‘extensible tree structure’’ because it actually represents a branching structure (a tree) during its development. Some authors (Vercambre and Page`s, 1998; Bidel et al., 2000a) have proposed a more elaborated structure in which they also defined an axis structure containing several segments and identified apices with specific characteristics (e.g., type, state, and diameter). When these simulated structures are stored in permanent files, they can later be represented by several different graphical or numerical outputs, using other software. When explicitly included in the system, the soil is also divided and coded into elements, either by horizontal soil layers (e.g., Porter et al., 1986; Bengough, 1997) or by cubic volume elements (Clausnitzer and Hopmans, 1994; Somma et al., 1998). It is then possible to use numerical methods to solve differential equations concerning water and mineral transport in the soil (Somma et al., 1998; see also Chapter 37 by Silberbush, Chapter 34 by Glass, and Chapter 35 by Jungk in this volume). The shoot system, when considered, was modeled as a global compartment (Clausnitzer and Hopmans, 1994) or as a simplified structure with several organs (Thaler and Page`s, 1998) providing carbohydrates and taking up water and nutrients. B.

Interactions with the Soil Environment

The first part of this chapter describes the means of simulating various aspects of the developmental program of the plant in architectural models. The common feature of these models is that they focus on the

Modeling Root System Architecture

plant root system, with rules attached to its components. It contrasts with other models predicting root density profiles, which use as basic elements the soil elements organized in space. This structuring approach is the result of a viewpoint which naturally accounts for the numerous features of root system architecture. However, the root system is also known for its plasticity, or even its opportunism, in relation to the soil spatial and temporal heterogeneity. Thus, the extension of the root is subject to numerous conditions and constraints which are non uniformly distributed, in space or time, and have several interacting effects on the whole architecture. Several authors have underlined this difficulty and have shown the relevance of architectural models to help integrating, at the root system level, the response of its components in relation to the diversity of situations they may experience. The general idea formulated in the models is that the root component that appears at a given time and in a given location is faced to specific environmental conditions which it responds to or interacts with. The environment can be described as a fixed map, or can be modeled itself as a dynamic system, with some of its properties changing with time, particularly under the root system influence. A large number of physical, chemical, or biotic characteristics may have a more or less significant impact on root development. Therefore, the first steps in modeling are to sort and select factors among all the possible influences, to give priority to the major ones (which are not always those for which the effects are best known), and to identify the most affected processes. Thus, architectural models are integrating frames for modules that relate soil variables to developmental variables, at the root scale. Up to now, the process of root growth has received the main attention. 1.

Temperature

Soil temperature is an extremely variable parameter, to which root systems are known to respond (Porter et al., 1986; Diggle, 1988; Clausnitzer and Hopmans, 1994). Given the time step of these models, generally about 1 day, seasonal variations only were considered. Diurnal variations, whose amplitude may be large in surface horizons, with maximal values typically higher than the optimal temperatures, were usually left out. In the Rootmap model, for example (Diggle, 1988), time is expressed in cumulated temperature for each apex, and growth rates are expressed in unit length per unit of thermal time (degreeday). Thus, tempera-

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ture variations were taken into account according to a linear model, between a threshold temperature and an optimal temperature, provided that a dynamic map of temperatures is supplied. The model of Porter et al. (1986) relies on the same assumption of a linear influence of temperature and includes a module to calculate the variations of temperature with depth. In the model of Clausnitzer and Hopmans (1994), a maximal growth rate is defined at an optimal temperature. The actual growth rate is calculated by reducing this value with a multplicative factor (between 0 and 1) calculated from a sine function of the temperature, with a minimum and maximum located at the threshold and optimal temperatures. In these models, temperature also determines the progress of acropetal branching, since the time lag between initiation and emergence of a lateral root is expressed in thermal time. The emergence kinetics for the nodal roots of cereals was also linked to thermal time (e.g., Page`s et al., 1989). Even though temperature may have many other influences on root development, including growth directions and branching densities (Coutts, 1989; Chapter 41 by McMichael and Burke in this volume), such relationships have not yet been integrated into the models. 2.

Soil Strength

The mechanical impedence of the soil to root penetration is a major factor in many soils, which has to be considered to understand the root system architecture and especially the rooting depth (Bengough, 1997; Chapter 45 by Masle in this volume). Unfortunately, this mechanical resistance is not easy to characterize by a relevant variable. It depends on several other soil characteristics, among which some are complex or exhibit short-term variations. They include soil texture, structure, bulk density, and water content. However, several authors (e.g., Bengough and Mullins, 1990) have obtained clear experimental relationships between root growth rate and penetration resistance, as measured with a penetrometer. These empirical relationships, as well as relationships between soil resistance and other soil characteristics (e.g., bulk density and water content), were used as basic functions to integrate this aspect of root growth in architectural models (Clausnitzer and Hopmans, 1994; Bengough, 1997). Regarding elongation, the general trend was to reduce the potential growth rate by a multiplicative coefficient (beween 0 and 1), calculated by a predictive function of the soil penetration resistance (Clausnitzer and Hopmans, 1994; Bengough, 1997).

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Mechanical constraints also influence the direction of root growth, leading to tortuous trajectories and sometimes global directional trends (Fig. 3). Such effects have been modeled by combining several influences in the growth direction, each influence being represented by a vector contributing through a vectorial sum to the resulting growth direction (Diggle, 1988; Page`s et al., 1989; Clausnitzer and Hopmans, 1994). The general anisotropic mechanical constraint is represented by a random vector, generating noise in the tra-

jectory, whereas local resistance gradients are represented by a directed vector, giving an organized trend to the trajectory (Clausnitzer and Hopmans, 1994; Fig. 3). 3.

Water and Mineral Availability

Combining root system architecture modeling and water uptake modeling, Bengough (1997) and Clausnitzer and Hopmans (1994) studied the influence

Figure 3 Observed (top) and simulated (bottom) root systems of Juncus squarrosus in a heterogenous soil environment. The model includes functions of root growth rate and growth direction according to mechanical impedance. (From Clausnitzer and Hopmans, 1994.)

Modeling Root System Architecture

of soil water content via the mechanical impedance associated with soil drying. Thus, the simulated effect of water content variations is indirect. The assimilate supply to the root system is also indirectly dependent on water availability in the model of Clausnitzer and Hopmans (1994). Concerning mineral distribution in the soil, Somma et al. (1998) suggested a module simulating a direct influence of mineral concentration on root elongation. According to this module, growth is optimal in a given range of concentration. Below and beyond this opimal range, growth is linearly reduced, until two other limits, below and beyond which there is no more growth. It is worth mentioning that the influence of nitrate availability on root branching density, although shown by several authors (e.g., Drew, 1975; Drew and Saker, 1975; Granato and Raper, 1989), has not yet been included in such models. However, its role is probably important for simulating adaptation of the root system to heterogeneous distribution of the nitrogen resources (Robinson, 1994). C.

Interactions Within the Plant (Photoassimilate Availability)

In addition to interactions with the external environment, the root system components have numerous relationships, between each other and with the rest of the plant. These endogenous interactions have often been neglected or have not been specified in models, for obvious reasons of simplification, although their role in architecture genesis is fundamental. Considering independently local interactions between the development of the root system components and their surrounding environment may lead to lack of realism. Developmental correlations within the plant were described long ago (e.g., Dyanat-Nejad and Neville, 1972; Champagnat, 1974; Champagnat et al., 1974; Wightman and Thiman, 1980; Favre, 1985). They lead to various phenomena (growth compensation, synchronization, competition, inhibition, promotion), the determining factors of which are complex. Within the root system particularly, growth reallocation occurs when axis growth has stopped (Torrey, 1976; Lamond et al., 1983; Amin et al., 1987; Atzmon et al., 1994a,b) or when different parts of the root system are subjected to variable conditions of water and mineral availability (Drew and Saker, 1975; Coutts and Philipson, 1976; Granato and Raper, 1989). Interactions with the shoot system have also been observed. For example, the alternating growth of shoots and roots, in the rythmically growing

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species Hevea brasiliensis, was described by Thaler and Page`s (1996a). It revealed tight interactions between the shoot and the root system, showing a clear control of the root system development and architecture by interactions within the plant (see also Chapter 12 by Reich in this volume). In order to describe such interactions, phenomenological models were designed to synchronize the development of the nodal roots through the development (Porter et al., 1986). Close relationships between the rate of leaf emergence and the rate of nodal root emergence have been shown in several cereals, such as wheat (Klepper et al., 1984), maize (Picard et al., 1985; Pellerin, 1993), and rice (Nemoto and Yamazaki, 1986). In such plants the phyllochron can be considered as a plant internal clock that determines nodal root initiation. Among the numerous factors likely to lead to interactions within the whole plant, photoassimilate availability has been considered by root system modelers to be the most significant (Clausnitzer and Hopmans, 1994; Nielsen et al., 1994; Thaler and Page`s, 1998; Bidel et al., 2000a). The dependence of the root system on carbohydrate supply from the shoot and the distance of the sinks from the carbohydrate sources have a profound effect on root system architecture (Aguirrezabal et al., 1993). In contrast with some crop models which formalized this interaction via a global sink function of the root system, architectural models have contributed to specify a more realistic sink function (distributed in time and space) and to assign it to certain structures within the root system. Using this approach, Nielsen et al. (1994) evaluated and located, using a modeling tool, the carbon cost of the root system contruction. The model was used to estimate the overall construction cost as it can be measured locally by respiration (Bidel et al., 2000b), biomass deposition, and exudation. This approach showed the spatial variations in carbon allocation, and the effect of architectural diversity on carbohydrate cost. In the model of Clausnitzer and Hopmans (1994), the interaction between the root and the shoot systems is considered in terms of water uptake (by the roots) and photoassimilate supply (by the shoots). Assimilate production is related to the amount of water absorbed and transpired, through a water use efficiency coefficient. The part of biomass allocated to the roots is shared between axes, proportionately to their demand. This is calculated from their individual potential growth rate after taking into account the possible reductions related to the soil conditions surrounding

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the root tips. This model makes it possible to represent time variations in terms of supply and demand, and growth compensations. Roots located in the most favorable environment will also have the largest demand, and subsequently the largest supply, at the expense of less favorably located roots. In the model of Thaler and Page`s (1998), carbohydrate allocation is also proportionate to the demand, but demand is not calculated from typical growth functions associated to each root type. It uses the apical diameter of each root at each time step. The basic assumption is that the larger the meristem volume (tightly related to apical diameter), the stronger the meristemic sink and the higher the potential elongation of the root. The meristem volume is not definite, but subjected to buffered variations at each time step: it may increase when carbohydrate supply is high and decrease when it is low. Moreover, the initial apical diameter of the root is the result of both its architectural location (size of the mother root) and carbohydrate availability during the primordium development. This model also represents growth compensation, because the assimilate resources are shared and may limit growth under specific conditions. It may also account for the large plasticity in root thickness and in their carbon demand according to the availability within the plant. In contrast to the previous two models, which consider an overall amount of carbon resource shared between the demanding sinks and give a common availability, the model of Bidel et al. (2000a) specifies the transport of carbohydrate resources, thus calculating a local availability for each sink. This approach accounts for local compensation phenomena, which operate for example between a mother root and its daughter roots because they are proximal sinks. D.

Tools for Translating and Simulating

As in other fields using modeling and simulation, advances in simulating root system architecture are clearly consistent with those made in computer science concerning hardware and software. The first model published by Hackett and Rose (1972a,b) was an algebraic model devoted to the analytical calculation of a number of state variables, such as root length, root number, and root surface or volume, classified by branching order. Though this approach did not include the spatial features of the architecture, the general idea of the model was based on an architectural vision of the root system. It is highly probable that the analytical formulation of the model, with strong simplifica-

tions such as the uniformity of growth rates, was imposed by the lack of computer technology available. The model proposed by Lungley (1973) took advantage of advances in this field, allowing numerical formulation, coding in a programming language (Fortran), and graphical representation of outputs. This model was in two dimensions and was used to calculate one-dimensional root density profiles. It is worth mentioning that Lungley’s work was consistent with this traditional representation of the root system, and therefore ensured the link between an architectural representation and a density profile representation. The first three-dimensional models of root system architecture were published independently by Diggle (1988) and by Page`s and Aries (1988). Both these models were written in programming language Pascal, which made this sort of programming easier, by using structured programming and user-adapted data structures. Since then, various significant advances have been made concerning modeling tools, which make developing and using elaborated models much easier. Regarding programming languages first, recent models make extended use of object oriented modeling methods (such as OMT, or UML—Rumbaugh et al., 1991; Muller, 1997) and associated languages, also object oriented (Smalltalk, C++, Java). These tools are particularly adapted to complex applications (Muller, 1997; Bidel et al., 2000a). The biological objects that we call the components (such as the apices, segments, roots) have a computer representation closer to the real (biological) world, being modeled as entities containing both attributes (object characteritics) and computer functions (permitting to account for the behavior). These autonomous objects (computer scientists say ‘‘encapsulated’’) can also interact with each other by sending messages. The recent development of computer graphics associated with an increased computing power allows a direct and realistic representation of the simulated structures. These software programs carry out image synthesis, by modeling the visual effects produced by the simulated volumes and surfaces, and therefore significantly contribute to the development of biological models. Such programs are now available in the public domain (e.g., Geomview—Phillips, 1996). In addition to these advances, a specific language for simulating biological structures was developed by Lindenmayer (1968, 1971), called the L-System. This language uses an axiom (i.e., an initial structure as starting point), modules (i.e., elementary components of the structure), and production rules operating on

Modeling Root System Architecture

the modules (i.e., formal translation of morphogenetic rules). It allows translation quite directly of the various concepts presented previously, even though it has not been applied that much to root systems (the hidden half!). Thanks to subsequent improvements, now included in the syntax, this language also permits to capture a number of interaction models (context-sensitive L-System), either between the system modules or with the environment. Moreover, this formal language, which was rather theoretical at the beginning, has been complemented with specific software (Pruzinkiewicz and Lindenmayer, 1990; Kurth, 1994). It enables the direct interpretation of the models and their simulation, and the production of graphical outputs (tool Grogra, by Kurth, 1994; tools L-Studio and VLab, by Pruzinkiewicz, 1998).

III.

EXAMPLES OF MODELING APPROACHES

After this analytical presentation of the knowledge and hypotheses which were put together in architectural models, we will now illustrate the application of the approach, by concrete examples of models or pieces of modeling approach, showing more specifically some research topics in which models may be helpful. A.

Estimation of Parameters (Calibration)

One of the most recurrent criticisms against architectural models is the large number of parameters included (Passioura, 1996). This number is actually highly variable, depending on the level of detail and the number of interactions taken into account. More specifically, it first depends on the number of different types of axes which have been identified, because each type is generally associated with a set of parameters. It also depends on the number of developmental processes described: the simplest models considered only two or three basic processes (emission, elongation, acropetal branching), whereas more complete models integrated other processes, described previously (e.g. reiteration, decay-abscission, radial growth). Each new process is formalized using a module, which requires new parameters to be included. Lastly, each process runs in relation to the environment sensu largo (i.e., endogenous and exogenous), using specific functions which can be rather complex. Thus, the total number of parameters may vary greatly, from 20 to 200. However, the total number of parameters only gives a limited vision on this question, the actual objec-

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tives of the modeling approach being more conclusive. Moreover, it seems essential to distinguish on one hand the parameters that are globally fitted, matching overall outputs with data, and on the other hand those parameters that are measured directly, or fitted locally on particular pieces of data, independently from one another. This last type is mostly used in architectural models, and therefore the large number of parameters is a less acute disadvantage. Moreover, these parameters, which have a concrete biological meaning, are easier to use and compare in relation to various situations. Among the concrete parameters, we find for example branching densities, or time lag before branching. Parallel to this calibration step, it is generally appropriate to use sensitivity analyses, which help to establish a hierarchy among these parameters, and to yield a better idea of the expected accuracy according to the objectives determined for the main variables of interest. Measuring empirical parameters, i.e., those directly derived from actual measurements, refers to the methodological problems that are associated to root system studies (see also Chapter 18 by Polomski and Kuhn in this volume). Some of these parameters are typically geometrical (e.g., branching density, trajectory parameters, branching angles) and are therefore directly measurable on single observations (destructively if necessary), whereas others include a temporal dimension (e.g., time lag, growth rate or duration, life expectancy) therefore requiring continuous or repeated observations using temporal markers. Tardieu and Pellerin (1990), Pellerin and Page`s (1994), and Page`s and Pellerin (1994) illustrated these procedures, applied to calibration of a model of maize root system architecture under field conditions. The parameters defining the emergence rate of nodal roots according to cumulated temperatures were estimated from counting nodal roots at the base of plants which were periodically excavated during the season. Periodic excavation of primary roots (seminal and nodal) made it possible to estimate the parameters concerning their trajectories and branching density, as well as growth rates using both length (measured directly on each excavated root) and age (estimated from the emission module). Similarly, the geometrical parameters of lateral roots (angles, trajectories, branching densities) were estimated from photographs of individually excavated roots, whereas growth parameters were calibrated from the length and position relative to the apex of the mother root. The distance to the apex made it possible to estimate the date of emergence, and therefore their age at excavation time. To

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complement these data obtained in the field, other data from the literature or independent observations in rhizotrons were used. Tsegaye et al. (1995a,b) have used a very similar approach to estimate the parameters of the Rootmap model (Diggle, 1988) for potted plants. B.

Evaluation of Outputs (Validation)

The validation of these models, although it is a vital part of the modeling approach, has not been much dealt with (Page`s et al., 1992; Tsegaye et al., 1995b; Page`s and Pellerin, 1996; Pellerin and Page`s, 1996). There are probably several reasons for that which are worth analyzing. Irrespective of the model, validation cannot be made in the absolute, but has to be related to a predetermined objective, the model being considered valid as long as it satisfies this initial objective. In many cases, however, the objectives assigned to the model were numerous, and not always well specified, because architectural models are potentially rich tools whose abilities are to be further investigated. Consequently, it is impossible to define clear validation criteria. Moreover, even when the objectives are predetermined, the formalization of validation criteria is not always a simple and straightforward step per se. A further difficulty arises from the comparison with data. The data sets used to test such models are rather scarce because they are difficult and costly to obtain. The diversity of the conditions required to refine validation makes it an acute problem, and limited data sets do not permit a powerful (discriminative) evaluation, given the intrinsic variations of the root system and the rhizosphere. Moreover, the data may also be subjected to numerous errors or uncertainties. Although subject to difficulties, validation is made, at least partially, by evaluating separately parts of the model. Some of these aspects can be illustrated by the work of Pellerin and Page`s (1996) and Page`s and Pellerin (1996), whose specific objective was to evaluate the capacity of a root system architectural model to predict the dynamics of the spatial distribution of maize roots. Previous studies showed the root distribution heterogeneity in various crops (e.g., Van Noordwijk and Brouwer, 1991) and more specifically in maize (Tardieu, 1988; Logsdon and Allmaras, 1991). Root distribution exhibits a spatial structure: at a large scale (dm to m), gradual variations can be observed, versus depth and distance from the row, and at a small scale (mm to cm), strong local variations are related to the aggregated pattern of root distribution. The question was to determine whether a simple model of the

root system architecture (Page`s et al., 1989) could simulate these main features of spatial distribution, as observed in the field. The data compared were horizontal root maps at different depth levels and at different dates (Pellerin and Page`s, 1996) as well as vertical maps at the flowering stage (Page`s and Pellerin, 1996). These works have shown the general ability of the model to simultaneously represent the various aspects of the data set, containing both spatial and temporal variations (Fig. 4). They have also shown that the main discrepancies between observed and simulated patterns were related to local compaction phenomena (especially in the plough pan). This pointed out that a better agreement could be expected by integrating the mechanical effect in the model. Using the model, it is also possible to evaluate the effect of the method used to draw vertical root maps. For example, some millimeters of soil are generally excavated around the roots to make them more visible, which increases the risk of overestimation of the root density, in comparison to what would be obtained on a strictly plane cross section. C.

Addressing Methodological Problems

Methodological problems encountered in the study of the root system come from its complexity, its fineness and fragility, and the opacity and cohesion of the medium in which it develops. In order to obtain data on its spatial distribution or development, many methods have been suggested, in which sampling procedures (and subsequent extrapolations) and indirect evaluation of target variables (via easier methods of measurement) take an important place. Such procedures rely more or less explicitly on theories and a priori assumptions on root system architecture, some of which have not been validated. The dynamic model of root system which can be produced by simulation models can help to improve the data acquisition procedures. Bengough et al. (1992) and Grabarnik et al. (1998) have studied some major geometrical characteristics of the root systems using model root systems and have evaluated the consequences of these characteristics on sampling schemes and indirect evaluation procedures. Page`s and Bengough (1997) tried to model root depth observations, as they are obtained from minirhizotron, using model root systems combined with cylinders of variable configuration. Their combined model made it possible to test theoretically several sampling designs considering variations in diameter, position, and inclination of the tubes. The simulation approach allowed

Figure 4 Distributions of the number of primary root intersections with horizontal trench walls in a maize stand versus the orthogonal distance to the plant row, at three dates after sowing, and three depths. These distributions were obtained from observed maps (solid lines) after excavation of horizontal trench walls, and from simulated maps (dashed lines) calculated from three-dimensional simulated root systems. (From Pellerin and Page`s, 1996.)

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comparison of a large number of different theoretical situations. In addition to their contribution to the design of sampling schemes, architectural models can help to fill the gaps in observations that are often scattered, partial, or at least discontinuous, either in time or space. Spek and Van Noordwijk (1994), for example, showed the relevance of their fractal model to simulate whole root systems using local measurements, achieved on very limited parts of the real root systems. This is particularly relevant for long-living and large root systems, such as tree root systems. D.

Consequences of Soil Constraints

The highly dynamic and interactive character of the soil–root system justifies developing integrated models among which are the characteristics of the root system architecture, its functioning, and its responses to soil constraints. The questions raised by modern agriculture, in a context of environmental protection, require new models with increased performance. The strong mechanical constraint imposed by soils on the root systems is an example of interaction between the root and the soil dynamics, with water acting as a mediator. In Bengough’s model (1997), some of the major dynamic features are combined in order to study the contribution of the soil mechanical and hydrodynamic properties, climatic demand, and root uptake and response within the overall system functioning. The model could account for great differences between crops regarding their rooting depth. The model of Somma et al. (1998), an extension of that of Clausnitzer and Hopmans (1994), generalized this approach in three-dimensional space and included more details about the effects of the mechanical constraint on root growth. It revealed additional aspects of the architectural plasticity, which emerged from the integrated system functioning. A complementary approach was suggested by Doussan et al. (1998a,b) for studying the consequences of the hydraulic organization of the root system architecture, in relation to the heterogeneity of water availability in the soil. This model permits evaluation of the spatial heterogeneity of water uptake and water potential within the root system, as a function of the variations in the soil water potential. E. Episodic Growth of the Root System Unlike episodic development of the shoot, which is well known and has been evidenced in many plants,

the temporal variations (phenology) of the root development are much less documented. Growth rate variations, not explainable by the heterogeneity of the rhizosphere, have been noticed on several species—on trees particularly (Head, 1967; Lyr and Hoffman, 1967; Riedacker, 1976; Atkinson, 1983). In young rubber trees, having a typical rhythmic shoot development (Halle´ and Martin, 1968), timedependent growth variations were also marked in some roots which exhibited alternations between elongation and rest periods, whereas others roots had a nearly continuous growth (Thaler and Page`s, 1996a). These variations also had consequences on the shape of the whole architecture, which also exhibited some episodic features. Along the main axes, zones where roots were dense and long alternated with zones where roots were rather short and scarce (Thaler and Page`s, 1996a). These complex phenomena with major architectural consequences emphasize the need to include within the models of the root system architecture some way of representing interactions between the root and the shoot system. Following this objective, Thaler and Page`s (1998) developed a model based on competition for carbon assimilates within the plant. This model combines a dynamic architectural description of the whole plant, with rules that govern the allocation of carbon assimilates among sink organs, in the shoot and root systems. It enables quantification of the organ requirements and consumption, and the kinetics of carbohydrate availability. It therefore helps to validate the hypothesis of carbohydrate competition in the determinism of an episodic root system development. F.

Simulating Infection and Disease Transmission

The root system is also the support and vector of many plant diseases. Several authors have shown the interest of combining models of the root system architecture with models of pathogen distribution and growth to investigate this complex and dynamic system (Bloomberg, 1979; Reynolds et al., 1986; Brown and Kulasiri, 1994, Gilligan et al., 1994; Page`s et al., 1995). In Hevea brasiliensis, for example, the Fomes fungus, causing root rot, is transmitted from one root system to another, through interroot contacts or close proximity. Then, it can develop along the roots and reach very quickly other parts of the root system (Tran Van Canh, 1982). In young plantations, the symptoms of the disease can generally be observed on some trees, from which the disease then spreads

Modeling Root System Architecture

as soon as root systems are large enough to be in contact with their neighbors. Such a phenomenon has been simulated by Page`s et al. (1995) using a root system architecture model which made it possible to calculate outbreak zones where roots from neighboring trees were very close to each other, and therefore predict the theoretical spread of the disease from the first infected parts. Such a model helps to evaluate the risks and measures that should be taken in order to control the disease.

IV.

FUTURE TRENDS AND NEW ISSUES

Modeling of root system architecture is aimed at understanding either root system development, function, or the methodological aspects required for root research. These models have now reached a level of maturity which makes their development and utilization credible in relation to several scientific issues. It is also a rather new approach, with a strong potential of development in several complementary directions that we shall now discuss briefly. A.

Integration of the Root System into the Soil–Plant System

When referring to former models developed in the 1970s and 80s to describe the development of root systems, we have mentioned the strong duality between ‘‘root density’’ models, starting from the soil and oversimplifying the root system, and root system architecture models, based on morphogenetic processes described from the plant, hardly tackling the interactions between the roots and their environment. This duality was justified, because morphology of soils with high constraints and heterogeneity clearly affects the root systems that show plasticity, whereas the internal processes of architectural genesis provide the structure of the root system, which is evident mostly in favorable or homogeneous soils. Since then, the two points of view (soil and plant) have got closer, especially because of the advances achieved in simulating root system architecture based on a morphogenetic approach. Gradually, these models have become capable of integrating the interactions between root components and their surrounding environment, with the later being either exogenous (soil, including biota) or endogenous (plant). However, there is still much to do in this area of soil–plant interactions.

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1.

Merging with Models of the Soil and Biotic Environment

Regarding the soil properties to be first considered, we have seen that some variables have been emphasized because they play a major role in many situations, but not universally of course. Simulation in other specific conditions, for which the limiting factors are different, e.g., salinity, nitrogen, or oxygen availability, should involve the formalization and integration of other interactive mechanisms. Moreover, the soil variables considered up to now exhibit almost continuous variations in the soil. Therefore, in addition to a simple substitution, the type of variable considered can also differ in its distribution and effect, leading to a possibly new approach (Greene, 1991). For example, the integration of biotic interactions requires taking into account the architectural diversity within the root system, as well as the complex and time-dependent distribution of soil organisms. The interaction with soil structure elements, such as different pore types (cracks, textural pores, biopores) which have dimensions close to that of root tips, may result in exciting and promising modules, in which architectural diversity in root diameters is a major factor. Another great challenge regards the description of the spatial rhizosphere environment (Hinsinger, 1998), instead of an average environment (bulk soil), as it can be measured at the decimeter scale. Local measurements, when possible, or local models reveal large gradients between overall water and mineral availability and availability at the soil–root contact area, experienced by the root. From both the experimental and modeling points of view, it raises many methodological problems. The finite element method, as suggested by Lafolie et al. (1991) and Clausnitzer and Hopmans (1994), calls for division of the soil space into very fine elements around the roots, i.e., according to a highly complex geometry, which is a complicated numerical problem. Therefore, alternative approaches are needed in association with several levels of details during simulation. 2.

Merging with Models of ‘‘Endogenous Environment’’ Within the Plant

The concept of environment can easily be extended to internal conditions, which are found and perceived by the meristems (Page`s, 2000). Thus, the ‘‘endogenous environment’’ includes resource availability for growth (mainly carbohydrates, water, and nitrogen compounds) and possibly the presence of signaling compounds.

Page`s

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Current models have started to link the fate of root components to carbon resource availability, and therefore contribute to specify a carbohydrate availability environment (Clausnitzer and Hopmans, 1994; Thaler and Page`s, 1998). Other resources needed for the development of new structures should be included in the future, especially water and nitrogen. The approach of Doussan et al. (1998a,b) shows some means of including the endogenous water environment by integrating the combined effects of climatic demand, availability in the soil, and transport within the root system. The endogenous environment has been considered as an overall environment by Clausnitzer and Hopmans (1994) and by Thaler and Page`s (1998). In these models, all the root components experience identical assimilate availability at the same time. To account for local competition phenomena, or apparent priority rules between meristems, it seems necessary to consider the spatial variation of the availability variable. Bidel et al. (2000a) proposed an approach in this direction for carbohydrates by modeling carbohydrate transport and consumption throughout the root system architecture.

about carbohydrates (Muller et al., 1998; Bidel et al., 2000b), water (Fraser et al., 1990; Durand et al., 1997), and nitrogen (Gastal and Nelson, 1994). Regarding mechanical constraints, architectural models have mainly integrated empirical relationships as submodels. New insight could probably be gained using architectural models to provide an organized local information on root tips, such as diameter and turgor pressure (from their endogenous environment). The influence of the apical movement, as well as exudation, is probably a major key for a better understanding of elongation and growth direction of the roots. Architectural models have highlighted the need for modules describing root growth direction. A number of studies have dealt with the positive gravitropism in radicles just emerging from the seed, whereas other roots, which seem to exhibit numerous ways of orienting themselves (Coutts, 1989; see Chapter 29 by Porterfield in this volume), have not been studied so extensively.

B.

A large majority of existing architectural models classify emerging roots according to predetermined types, which tightly control their subsequent development. The plasticity of root systems tends to be underestimated by this simulation method, particularly in very heterogeneous and constraining soils, where lateral roots are much affected by the fate of their mother root. It is very important to make significant progress in modelling the genesis of the type of axis, or even to re-evaluate these constraining categories. This point requires the study of early developmental stages of the root meristems, from initiation to emergence. Most of the variations in meristematic volume are acquired during these early stages, with significant consequences on subsequent growth of axes (Coutts, 1987; Page`s, 1995; Thaler et Page`s, 1998). The availability of carbon and nitrogen resources is probably critical during these stages, contributing both to the number and growth characteristics of the promeristems (Zhang and Forde, 1998).

Modeling the Response of Root Meristems to Their Local Environment

Modeling the architecture also raises new relevant questions for studies at the lower organization level—that of the root system components, and particularly meristems. Architectural models should be considered as tools for integrating knowledge on individual meristems, as well as for specifying variations in the situations experienced by meristems in a root system. This variability is built at the root system level, during its development and functioning (see also Chapter 9 by Waisel and Eshel in this volume). It regards both the types of roots emerging during development, and the actual environmental conditions (exogenous and endogenous) in which they develop. 1. Root Growth In terms of main resources, architectural models need to specify the root component requirements according to developmental stages and the effects of availability on these stages. Therefore, studies are required at the root segment level (Nielsen et al., 1994; Bidel et al., 2000b) or even at finer scales, using the theory and techniques of spatial analysis (Silk and Erickson, 1979; Silk, 1994). Such studies have been carried out recently, and have provided many interesting results

2.

3.

Root Initiation and Primordium Development

Interactions with Microorganisms, Mycorrhizae

There is much to gain by considering the root with its surrounding microorganisms, and particularly mycorrhizal fungi, which have a considerable functional significance in a large number of plant families (see

Modeling Root System Architecture

Chapter 50 by Kottke in this volume). No doubt that in this field also, the valuable dialog between specialists and the description of interactions between roots and symbionts require a detailed description of the root system architecture. Such a description should make it possible to specify favorable sites on the root system and the specialized effects of the microorganisms on root development (Schellenbaum et al., 1991; Wullscheleger et al., 1994). C.

New Prospects of Development and Applications

In parallel to these possible improvements, modeling the root system architecture makes it possible to investigate new fields of application. For example, modeling the anchorage ability of the root system should benefit from combining the architecture of the main root axes, the variation of their mechanical properties, and their response to the mechanical constraints transmitted from the shoot to the root system (cambium functions especially). Such models should also renew the study of various fluxes, either within the plant, from soil to plant, or from plant to soil, thus permitting a spatial representation of phenomena occurring at various scales in the plant and the soil. In addition to quantitative overall aspects, regarding water and carbon budget especially, this refined integration of fluxes and signals from the root system should be helpful to better understand some behaviors of the shoot organs, with various possible applications to morphogenesis, stomatal regulation, fruit quality, etc. In the general context of a modern agriculture, stressing environmental protection, such models should also contribute to genetic studies and to selecting adapted genotypes to many adverse situations. To develop models able to fulfill this crucial issue, it is necessary to contribute to the identification of most genetically controlled mechanisms and to integrate them into models. Provided such an integration is successful, architectural models will become major tools to make the necessary link between fundamental mechanisms and the architectures generated under sets of environmental constraints. Further advances will be made by increasing the power of computing tools. These tools should make biological (or biophysical) models easier to formalize, and particularly the consistent association between plant structures and their specific functioning traits (including genetic regulation) and the simulation of various interactions with the environment. Ergonomics and graphical visualization are obviously

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23 Auxins in the Biology of Roots Thomas Gaspar, Odile Faivre-Rampant, Claire Kevers, and Jacques Dommes University of Lie`ge, Lie`ge, Belgium

Jean-Franc¸ois Hausman CRP-Gabriel Lippman, Luxembourg

I.

INTRODUCTION

cycles (cf. John et al., 1993; Ormrod and Francis, 1993). Moreover, the level of any one hormone affects the levels of the others by affecting their biosynthesis, degradation, conjugation, or transport (Itai and Birnbaum, 1991). This is called the hormonal crosstalkings (Rodrigues-Pousada et al., 1999). It seems that determining the level and effect of one hormone may yield information with a limited value. No single hormone has an overriding role. Hormone metabolism and action cannot be dissociated from the primary metabolic pathways with reciprocal influences (Gaspar et al., 2000a,b). This means that the effects of an externally applied hormone, or of an analog, cannot be interpreted simply through an increase of its endogenous bulk (Pilet, 1996) but that changes in metabolism and the role as an exterior signaling molecule have to be considered. Auxins and cytokinins were originally thought to produce growth responses at distances from their sites of synthesis, and thus to fit the definition of transported chemical messengers. It is now clear that none of the recognized five main classes of phytohormones (auxins, cytokinins, gibberellins, abscisic acid, ethylene) fulfill the requirements of a hormone in the mammalian sense, i.e., chemical messengers at low concentration, involving a localized site of synthesis, transport to a target tissue and control of a precise physiological response in a target tissue via the concentration. The synthesis of all plant hormones, as a rule, occurs or can occur in any type of living cells even if

The concepts in plant hormonology have changed dramatically with the progressive discovery of new phytohormones. It can now hardly be claimed that a single hormone is responsible for one growth or development process. Growth and development processes have been dissected into successive interdependent physiological phases with different requirements. Moreover, in many cases, it was shown that the control of these events is due to the simultaneous interaction of different plant hormones, acting synergistically or antagonistically, rather than to the effect of a single hormone. Distinct cell types respond differentially to various signals. It is clearer now that the hormonal controls act in a developmental and in a tissue-dependent manner. Thus, the former claimed specificity of one hormone may simply be the result of its preponderance in a balance with another one; for example, the ratio of auxins to cytokinins is determining growth and development. In neoplastic tissues, the sensitivity to this couple of hormones is shifted to the tandem polyamines/ethylene (Kevers et al., 1999c; Gaspar et al., 2000a). The possibility that different hormonal receptors control growth and development (De Klerk et al., 1997) or that different hormones compete for a common receptor or operate in separate signalling pathways (Timpte et al., 1995) is being investigated. Apparently, different hormones play in very tightened sequential events in the control of cell division

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certain tissues are privileged sites of synthesis and export for some hormones (e.g., aerial meristemic tissues for auxins or growing root parts for cytokinins). Thus, phytohormones may be transported and participate in some precise physiological processes at a distance. However, it is far from being the general case; they also act in the tissue or even within the cell in which they are synthesized. Furthermore, experimental results strongly argue that phytohormone control is not only by concentration but also by changes in sensitivity of the cells to the compounds (Trewavas and Cleland, 1983). Thus, the responses evoked by plant hormones are rarely proportional to their concentration. Furthermore, the countercurrents of hormones between aerial and underground plant parts create varying gradients and continuously change organ cross-talks. II.

THE DIVERSITY OF AUXINS AND THEIR METABOLISM IN ROOTS

A.

Naturally Occurring Auxins

No root-specific auxin has been discovered, and the most common natural auxin is indole-3-acetic acid (IAA). However, depending on the species, age of the plant, season, and the conditions under which it has been growing, other natural auxins have been identified, such as 4-chloroindole-3-acetic acid, indole-3acrylic acid, and indole-3-butyric acid (Marumo, 1986; Gaspar et al., 1996). In addition to these indolic auxins, various phenolic acids (such as phenylacetic acid) that appear also in roots have low auxin activity. However, a physiological role for such nonindolic compounds in auxin regulation has not been established (Bandurski et al., 1995). Auxin precursors may also have auxinlike properties and can sometimes replace IAA. Sometimes they may be more effective than auxin itself in stimulating growth or inducing organised development. Auxins are found in plants both as the free acid (which is thought to be the primary ‘‘active form’’) and as conjugated forms. Conjugation appears to be a mechanism for storing auxin in cells and stabilizing the level of free auxin by metabolizing its excess. Auxin in conjugated molecules is protected from oxidative breakdown and may provide a readily accessible and easily source of free IAA without de novo synthesis. One type of conjugated form is linked through carbon–oxygen–carbon bridges and these compounds are referred to generically as ‘‘esters,’’ although some 1-O sugar conjugates such as indole-3-acetyl-1-O--D-

glucose (1-O-IAGluc) are actually linked by acyl alkyl acetal bonds. True esters include compounds such as 6O-1AGluc and indole-3-acetyl-myo-inositol (IAInos). The other type of conjugates are linked through carbon–nitrogen–carbon amide bonds, as in the IAA– amino acid and peptide conjugates. All native auxins are found in both free forms and conjugated forms. However, in most tissues the conjugated forms predominate. Various conjugates of IAA, both ester and amide, have been used as ‘‘slow-release’’ forms of IAA for tissue cultures and for rooting of cuttings. IAA conjugates, each differing in ease of hydrolysis by the plant’s enzymes and having conjugating moieties of varying degrees of lipophilicity, could be used to ‘‘target’’ the IAA to a particular tissue or particular cell organelle with delivery of the hormone at the required rate. The conjugating moiety might thus be used as a ‘‘zip code,’’ to bring the IAA to the desired location, with simultaneous protection against peroxidative attack (Bandurski et al., 1995).

B.

Auxinlike Growth Regulators

Several indole derivatives, both naturally occurring and synthetic, are active in plant cultures. For instance, indole-3-acetaldehyde, indole-3-acetamide, indole-3acetonitrile, indole-3-lactic acid, indole-3-propionic acid, indole-3-pyruvic acid, indole-3-glycolic acid, 5OH-tryptamine (serotonin), and tryptophan were shown to support root, callus, or shoot formation and growth (Gaspar and Hofinger, 1969; Maeda and Thorpe, 1979; Gatineau et al., 1997). Several other substances can mimic auxin activity, in roots notably: this is the case of acetylcholine (Penel et al., 1976), which has been found in many plant roots (Tretyn et al., 1992). The most commonly used synthetic auxins are 2,4dichlorophenoxyacetic (2,4-D) and 1-naphthaleneacetic acid (NAA), but others like dicamba (3,6-dichloroo-anisic acid) and pichloram (4-amino-3,5,6-trichloropyridine-2-carboxylic acid) have applications in tissue cultures or as selective herbicides. BSAA [benzo(b)selenienyl-3 acetic acid] and its chloro and methoxy forms are new synthetic auxins with powerful effects on root growth and adventitious root formation (Hofinger et al., 1980; Lamproye et al., 1990; Gaspar, 1995; Kevers et al., 1997). Such a powerful auxin as BSAA is able to induce and sustain the growth of hairy roots in the absence of Agrobacterium rhizogenes (Kevers et al., 1999a).

Auxins

Auxins affect root formation and/or growth either directly or indirectly by affecting the level of endogenous auxin. The greater efficiency of IBA versus IAA in root formation is probably due to its progressive conversion (-oxidation) into IAA, thus working as slowrelease source for IAA. This has led Van der Krieken et al. (1997) to design synthetic slow-release auxin sources (IAA bound to bovine serum albumine, indolehexanoic acid, IBA-anhydride, IBA-aminoacids, IBA-polyamine-IBA, or IAA-polyamine-IAA). Most of these slow-release compounds are stable and can be autoclaved. They are more effective than the standard auxins in the induction of adventitious roots. C.

Auxin Protectors and Elicitors of Auxin Action

Monophenolic substances enhance the so-called IAAoxidase system but polyphenolic substances inhibit the same system (Gaspar, 1965; Pilet and Gaspar, 1968). Substances such as the soil humic acids can influence growth of the roots indirectly by modifying their auxin level. Other substances, e.g., breakdown products of the cell membrane (nonanoic acid and jasmonate), of the cell wall (lignosulfonates), or of fungal origin (Pythium extract), have the capacity to increase the sensitivity to auxins and thus the rooting of ‘‘recalcitrant’’ cutting types (Van der Krieken et al., 1997; Kevers et al., 1999b). D.

Auxin Metabolism and Distribution in the Roots

Bandurski et al. (1995) have addressed the different aspects of auxin metabolism. Apparently the pathways of auxin anabolism and catabolism of roots are the same as in other plant organs. However, it can be expected that the rates of turnover, the pool sizes, and their inputs and outputs are different, namely through the contribution of bacteria and mycorrhizae. The auxin–oxidase system, through its adaptation capacity, plays an important role in the regulation of the auxin levels. This seems particularly true along the roots where an inverse relationship between the auxin activity and the auxin level has been shown (Pilet and Gaspar, 1968). Some peroxidases, at least, are involved in auxin catabolism and hence in growth (Gaspar et al., 1982, 1991). It is therefore not astonishing that peroxidase activity and secretion also vary along roots (Bouchet et al., 1980). Plants synthesize, inactivate, and catabolize IAA by multiple pathways, and multiple genes can encode a

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particular enzyme within each of the pathways. A number of these genes are now cloned, which greatly facilitates the future deciphering of IAA metabolism (Normanly and Bartel, 1999).

III.

CONTRIBUTION OF RHIZOBACTERIA AND MYCORRHIZAE TO ROOT AUXIN METABOLISM AND GROWTH

Saprophytic free-living bacteria and fungi, as well as symbiotic endophytic or nonendophytic rhizobacteria and mycorrhizae, exert a beneficial effect on plant growth. They increase the tolerance to abiotic constraints and to phytopathogens. This is generally an indirect result of an improved development of the root system (Mosse, 1957; Nelsen and Safir, 1982; Gianinazzi-Pearson, 1996). Root-colonizing bacteria and fungi produce plant growth substances, including auxins, gibberellins, and cytokinins (Brown, 1974). IBA concentrations in mycorrhizal and in nonmycorrhizal maize roots differed (Ludwig-Muller et al., 1997). Young roots of maize colonized with Glomus intraradices had a higher content of IBA while the total IAA content of such roots was lower. In older roots, total IBA content was slightly lower in arbuscular mycorrhizae-infected roots than in controls. However, the activity of IBA synthetase was threefold higher in infected roots than in control ones (Ludwig-Muller et al., 1997). Since it is unknown whether fungi possess similar enzymes as higher plants, it was assumed that IBA is synthesized by the host plants, though the observed increase was probably induced by the arbuscular mycorrhizal fungus (Kaldorf and Ludwig-Muller, 2000). However, in ectomycorrhizal symbiosis, IAA is produced by the fungal partner, resulting in an increased initiation of lateral roots (Karabaghli-Degron et al., 1998). Such production may also contribute to the enhancement of plant growth (Frankenberger and Arshad, 1995). Esch et al. (1994) showed that hyphae of Glomulus intraradices produce abscisic acid (ABA). This plant growth regulator increases the IBA synthesis (Ludwig-Muller et al., 1995). Thus it was hypothesized that auxin production may be increased via fungal ABA. On the other hand, the higher endogenous content of ABA could lead to an increase in the number of receptor sites, as indicated by the induction of auxinbinding protein 1 by IBA (Kaldorf and LudwigMuller, 2000). Another interesting example is the free-living, plant growth–promoting rhizobacterium Pseudomonas.

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These bacteria enhance plant growth by one or several mechanisms including production of plant growth regulators. Following the binding of P. putida to the root, the bacterial deaminase degrades ACC that is released from the plant’s root. This would lower the level of rhizogenic ACC as well as that of ethylene, and promote plant growth (Glick et al., 1994). Moreover, it was shown that some strains of P. putida overproduce IAA that is able to enter the plant’s root cells. This fungal IAA is then able to promote cell elongation and proliferation as well as to stop ethylene synthesis, by a combined decrease of ethylene and increase of endogenous auxin content enhance rooting (Xie et al., 1995; Glick et al., 1994). Nitrogen-fixing bacteria inducing the formation of root nodules produce auxins, but the involvement of the excreted auxins in nodulation remains debated. Nodulation may result either from production of phytohormones by the microsymbiont or the promotion of hormone synthesis in the cells of the host roots (Crozier et al., 1988).

IV.

AUXIN-RELATED GENES IN ROOTS

Questions regarding which genes are involved in the biosynthesis of auxin, how the level of auxin is regulated in the plant, and what is the basis for the differential sensitivity of different tissues to auxin remain to be answered. The isolation of mutants with altered responses to auxin and the cloning of the corresponding genes are a valuable strategy with which to address such questions. The following genes are involved in IAA metabolism. 1. PIN2 (protease inhibitor II). The protein PIN2 was found to be similar to members of the major facilitator family of transport proteins (Mu¨ller et al., 1998). It was localized in membranes of cortical and epidermal cells of the meristemic and of the elongation zones. The polar localization of such proteins and its function was specific in roots. The loss of PIN2 function impairs basipetal auxin transport. The authors have suggested that PIN2 plays an important role in control of gravitropism regulating the redistribution of auxin from the stele toward the elongation zone of roots. PIN2 was shown to encode a root-specific member of a novel membrane protein family, supporting the idea about its role in the transport of auxin (Chen et al., 1998; Utsuno et al., 1998; Luschnig et al., 1998). 2. AXR1 and AXR2 (altered-auxin response 1 and 2). AXR1 encodes a protein related to ubiquitin-activating enzyme E1, which catalyzes the first step in the

biosynthesis of ubiquitin–protein conjugates. The existence of relationships between AXR1 and E1 suggests that through the action of AXR1, auxin may stimulate ubiquitin-mediated degradation of a putative repressor of auxin-regulated genes (Abel et al., 1994). AXR1 gene is required very early in an auxin response pathway in mature roots. Sabatini et al. (1999) have shown that a reduced AXR1 activity is correlated with an incomplete cell division program in the distal root cap. AXR2 protein is likely to play a role very early in signal transduction (Wilson et al., 1990; Timpte et al., 1992, 1994). Karlsson et al. (1996) have shown that the AXR2 gene product has a different function in roots than in hypocotyls but may be more important for IAA responses in the hypocotyls than in roots. Since responses to IAA differ in roots and hypocotyls of wild-type plants, it seems likely that they should be regulated differently and the AXR2 gene product may be subject to or involved in this differential regulation. 3. AUX1 (auxin-resistant 1). The AUX1 polypeptide exhibits sequence similarities to a family of plant and fungal amino acid permeases (proteins facilitating transport of amino acids), suggesting that AUX1 mediates the transport of an amino acid–like signaling molecule. IAA, which is structurally similar to the amino acid tryptophan, is thus a likely substrate (Bennett et al., 1996). It was proposed that AUX1 may mediate proton-driven IAA uptake, as plant amino acid permeases mechanically function as proton-driven symporters (Bush, 1993). Bennett et al. (1998) showed that AUX1 is expressed in root apical tissues that control the root gravitropic response and also in root epidermal cells. According to Marchant et al. (1999), the AUX1 gene is expressed predominantly in the cap of lateral roots and in the epidermis of the root meristem of Arabidopsis thaliana. 4. RSI-1 (root system inducible-1). This gene is inducible by auxin and is expressed very early in lateral root development (Taylor and Scheuring, 1994). A specific role for RS1-1 is still unknown. 5. TIR1 (auxin transport inhibitor resistant 1). Expression of TIR1 is strongest in the primary root— and in lateral root—meristems, which is consistent with a key role for the protein in root development (Del Pozo and Estelle, 1999). TIR1 is required for pericycle cells to respond to the inductive signal, presumably auxin. 6. RML (root meristemless). The RM1 genes are involved in signalling cell proliferation at the root tip (Cheng et al., 1995). Their products are involved specifically in activating the cell division cycle in the root apical cells. They are required for cell proliferation

Auxins

during postembryonic root growth. RML genes regulate cell proliferation not only of primary roots but also of laterals and adventitious roots. Functional differentiation along the root axis may be accomplished by the regulation of the cell division cycle via the RNL gene products. Several other mutants overproducing IAA have been isolated, but the corresponding genes have not been cloned yet. Boerjan et al. (1995) have described the isolation of Arabidopsis mutants overproducing free and conjugated IAA designated superroot (sur). Seven allelic Arabidopsis mutants sur1-1 to sur1-7 developed excess adventitious and lateral roots. The authors hypothesized that the SUR1 gene encodes a regulator of auxin biosynthesis in Arabidopsis. Another Arabidopsis mutant exhibiting a phenotype similar to superroot mutant has been selected by King et al. (1995) and called rooty (rty). The authors suggested that the regulation of auxin levels is done by the wild-type RTY gene product. Another promising candidate for analysis of auxinregulated growth and development is the tomato mutant diageotropica (dgt). dgt plants failed to form lateral roots and do not respond to gravity (Zobel, 1974). According to Kressin Muday et al. (1995), the defect in dgt is in the ability to respond to auxin, rather than in auxin uptake or altered endogenous auxin concentrations. The analysis of Arabidopsis mutants has been instrumental in linking root hair formation to the action of plant hormones, particularly ethylene and auxin. Mutations affecting the CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) locus, which encodes a Raf-like protein kinase negatively regulate the ethylene signal transduction pathway (Kieber et al., 1993), cause root hairs to form on epidermal cells that normally are hairless (Dolan et al., 1994). The hairless root phenotype of the dwarf (dwf; auxin resistant) and auxin resistant2 (axr2; auxin, ethylene, and abscisic acid resistant) mutants implicate that auxin is another possible regulator of root hair formation (Mizra et al., 1984; Wilson et al., 1990). In addition, the hairless phenotype of the root hair defective6 (rhd6) mutant can be suppressed by the inclusion of 1-aminocyclopropane-1-carboxylic acid (ACC; an ethylene precursor) or IAA in the growth media. This is another implication of ethylene and auxin action in root hair initiation (Fig. 1; Masucci and Schiefelbein, 1994). Given the involvement of hormones in epidermal cell differentiation, one attractive possibility is that ethylene and/or auxin may act as a diffusible signal responsible for epidermal cell–type patterning. According to

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Figure 1 Tentative model for the control of cell-type differentiation in Arabidopsis root epidermis. T-bars indicate negative regulation, question marks indicate unclear relationships. (From Schiefelbein et al., 1997.)

this notion, TTG and GL2 may represent downstream transcription factor genes that are regulated by the hormones in a cell position–dependent manner. Masucci and Schiefelbein (1996) have shown that the ethylene/auxin pathway does not regulate the TTG/ GL2 pathway. It acts upstream of or independently from the ethylene/auxin pathway to define the pattern of cell types in the root epidermis (Fig. 1). All newly formed epidermal cells in the Arabidopsis root may initially have the capacity to differentiate into root hair cells, and the action of the TTG/GL2 pathway may be required to induce the hairless cell fate (i.e., a root hair cell may represent the default fate). Other root auxin-related genes are those introduced by plasmids, notably the ones of Agrobacterium rhizogenes. The best studied plasmid is the agropine pRiA4 plasmid that carries and can mediate the transfer of two T-DNAs, denoted TL and TR, to the plant cell (White et al., 1985). Hairy roots induced by agropine strains frequently contain only the TL-DNA (Jouanin et al., 1987). White et al. (1985) showed that insertions in only four of the 18 potential loci on the TL-DNA noticeably affected the morphology of the hairy roots that were produced. These loci were denoted root locus A-D (rolA-D). The gene rolB (ORF 11) contains an ORF of 777 bp encoding a 259-amino acid protein with a molecular mass of 30 kDa. It has been observed that the biological effects of the rolB expression, like root initiation, are reminiscent of auxin-mediated effects (Schmu¨lling et al., 1988; Estruch et al., 1991). Two main theories

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concerning the mechanism of RolB action were proposed. The first theory hypothesized that RolB acts by increasing the pool of free, active auxin in transgenic plants (Estruch et al., 1991). This was shown by using a RolB-mediated hydrolysis of inactive IAA conjugates. The second theory for RolB action suggests that RolB is regulating the cell sensitivity to IAA—i.e., making normally unresponsive cells responsive to IAA (Maurel et al., 1994). Further support for a role of RolB in an IAA signal perception/transduction pathway is the recent finding that RolB has a tyrosine phosphatase activity (Filippini et al., 1996). The rolB gene showed a tissue-specific expression pattern being mainly confined to root meristems. In hybrid aspen, the rolB promoter exhibited highly specific expression in groups of pericycle cells prior to and during lateral root initiation (Nilsson et al., 1997) and strong expression during the initiation and growth of tobacco adventitious roots (Altamura et al., 1991). This exhibits a high correlation between the expression of this gene and the initiation of root growth. V. AUXIN CONTROL OF ROOT GROWTH A.

Auxin Dose–Growth Response Curve

Root growth is stimulated by ‘‘low concentrations’’ of auxins, whatever their nature is, and is inhibited by ‘‘high concentrations.’’ This classical hormonal dose response curve differs from what is known for buds and stems (cf. Fig. 2). This has led to the consideration

Figure 2 Schematic representation of the growth responses of roots, buds, and stems to a range of auxin concentrations, each organ having a promotive and an inhibitory range. (From Thimann, 1969.)

of roots as more sensitive to auxins than the other organs, but still left some questions unanswered: 1. Do root and stem growth responses really correspond to different levels of endogenous auxins? The natural levels of auxin in these two organs have not really been investigated; when treated by the same exogenous auxin concentrations, do these organs absorb auxin in the same proportion and at the same rate? Does their adaptative auxin-oxidase system react in the same manner? 2. Are the different responses caused by changes in the number of receptors, changes in receptor‘s affinity, or changes in the subsequent chain of events, including possible changes in the level of other endogenous hormones that affect the response? For example, growth and ethylene production by lentil root tips, treated with varying concentrations of indolylacetic and indolylacrylic acids were measured. Stimulation of growth by low concentrations of auxin was accompanied by a reduced ethylene production. Growth inhibition by high levels of auxin induced increased ethylene evolution (Fig. 3A). If the ethylene synthesis is prevented by ethylene synthesis inhibitors, if ethylene is removed by hypobaric conditions, or if the action of ethylene is opposed by silver ions, then auxin is no longer inhibitory. This suggests that root growth stimulation by auxins may be mediated by ethylene (Davies, 1995). But simultaneous application of auxin and rhizobitoxine analog on the lentil root reduced ethylene evolution even though growth was inhibited (Fig. 3B). On the contrary, this might prove that auxin-controlled root growth is independent of ethylene production (Dubucq et al., 1978). In any case, this means that growth of roots as well as growth of aerial organs or calluses results in sequential events where auxins and ethylene play some roles (Kevers et al., 1984; Gaspar et al., 2000a). Thus, root growth inhibition can no longer be explained simply by a decrease in auxin content. Kinetin, light, and dinitrophenol, three factors inhibiting root growth, cause a decrease in auxin content together with a rise in the activity of the so-called inhibitor (Gaspar, 1973). Methyleneoxindole, the main degradation product of IAA, chromatographically runs next to ABA, and was shown to inhibit root growth. Thus, physical or chemical treatments which enhance auxin biodestruction may cause root growth inhibition through the regulation of an auxin ‘‘inhibitor’’ balance. Auxin oxidase must therefore be considered not only as a regulatory destructive system (Gaspar, 1965; Pilet and Gaspar, 1968) but also as a system that generates inhibitory substances. In this context, the role of peroxidases pre-

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B.

Figure 3 (A) Effect of varying concentrations of indolylacrylic acid (IAcrA) on growth (*—*) and ethylene production ~  ~) by lentil root tips. Results expressed in percent of the control. (B) Compared effect of IAA and rhizobitoxine (AAR), alone or combined, on root growth and ethylene production. Open column, growth; hatched column, ethylene. (From Dubucq et al., 1978.)

sent in the roots and not in the aerial parts (Gaspar et al., 1974) should be further considered. Moreover, it is still unclear whether the concentrations measured in extracts of whole plant organs or tissues reflect active hormone pools. There is an urgent need for a reliable method for differentiation between active hormone pools and pools that are inactive either because of compartmentation or because of conjugation.

Auxin–Cytokinin Interactions

Skoog and Miller (1957) discovered that both auxins and cytokinins can act synergistically in the induction of cell division and growth in plant tissue cultures but can also antagonistically control lateral bud and root outgrowth (Fig. 4). Molecular work on the links between hormone action and cell division led to the cloning of several genes that are responsive to auxins, to cytokinins, or to both hormones. However, the function of the majority of these genes either is not yet known or has no obvious connection with cell cycle control. A more direct link between plant hormones and cell cycle control is now being uncovered by analyzing the expression patterns and activity of proteins that are homologous to those that control the cell cycle in yeast (Coenen and Lomax, 1997). Lateral root primordia are generally initiated through the commencement of cell divisions in the pericycle opposite the xylem arches of the root vascular system. The antagonistic relationships of auxins and cytokinins in this process is similar to their interaction in regulation of the expression of a cdc2-like protein (Fig. 5; John et al., 1993). Although auxins increase immunologically detectable cdc2-like protein in extracts, cytokinins reduce the levels of the cdc2kinase. Recent papers on apical dominance (Bangerth, 1994; Li et al., 1995) have demonstrated that decapitation, and thus removal of the endogenous auxin source, leads to a large (up to 40-fold) increase in the cytokinin content of xylem exudate. Such an increase can be eliminated by application of the synthetic auxin -NAA to the apex of the decapitated plants. This effect of auxin on cytokinin concentrations in the xylem suggests that auxin can influence apical dominance via inhibition of cytokinin synthesis or export from the roots. However, bud outgrowth can also be inhibited by apically applied auxin in isolated stem segments, which indicates that this is not the only mechanism available. Coenen and Lomax (1997) have proposed the scheme of Fig. 6 with the potential points of control of active cytokinin pools by auxin, namely through a control of cytokinin oxidase by auxin. The auxin and cytokinin interactions in root growth were interpreted through a cytokinin control of isoperoxidases with the latter supposed to function as auxin oxidases (Darimont et al., 1971). However, increases in free IAA are observed both in roots of cytokinin-overproducing lines of Nicotiana glutinosa transformed with the ipt gene (Binns et al., 1987), after exogenous

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Figure 4

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Control of different organogenic programs by the balance between auxins and cytokinins. (From George, 1993.)

Figure 5 Speculative model for control of the cell cycle through auxins and cytokinins. Both cytokinin and auxin have been reported to regulate the expression of cdc2 kinases and the cyclins that are required for their activation. The interaction between auxin and cytokinin in regulating the cell cycle is synergistic in undifferentiated cells, such as callus or protoplasts, with both auxin and cytokinin stimulating expression of the cdc2 kinase and cytokinin treatment increasing expression of a cyclin. In lateral root primordia the interaction is antagonistic: auxins stimulate and cytokinins reduce levels of the cdc2 kinase, and the expression of at least one cyclin is increased by auxin. (From Coenen and Lomax, 1997.)

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Figure 6 Potential points of control of active cytokinin pools by auxin. Open arrows indicate the steps where auxin or auxin conjugates are thought to regulate enzyme activity, resulting in changes in conjugate or metabolite levels. (From Coenen and Lomax, 1997.)

application of cytokinins to maize (Bourquin and Pilet, 1990) or in pea roots (Bertell and Eliasson, 1992). The different possibilities of a mutual control of auxin and cytokinin abundance can be explained even before the enzymes involved in their metabolism have been isolated and cloned and while a debate persists on the anabolic and catabolic pathways of both hormone types (cf. Schmu¨lling et al., 1997).

VI.

AUXIN TRANSPORT WITHIN AND FROM THE ROOTS

A phytohormone does not act like an animal hormone—i.e., as a signal that carries information from source cells to specific target cells or tissues. The observation that auxin replaces all the correlative effects of a shoot apex led to the conclusion that the activity profile of auxin may be better interpreted as the integrative signal by which the growing shoot tissues influence the development of the rest of the plant, including adventitious and lateral root formation, growth, and vascular patterning (Davies, 1995; Aloni, 1995; Palme and Ga¨lweiler, 1999; Jouve et al., 1999). Auxin acts in plants over long distances and throughout the plant’s life. Auxin triggers all developmental steps, beginning with the differentiation of the embryonic axis, and later with the transport to growing shoot and root (Bandurski et al., 1995). Auxin is transported acropetally from the shoot through the stele up to the meristemic region of the root tip. There it is redistributed into the various root tissues

and is transported basipetally toward the elongation and differentiation zone (Palme and Ga¨lweiler, 1999). Specific growth processes can be manipulated using naturally occurring or synthetic auxin transport inhibitors (Lomax et al., 1995). (For additional information see Chapters 31 by Poovaiah et al., 29 by Porterfield, and 30 by Pilet, in this volume). The idea that transport was an essential part of the role of plant hormones originated from experiments on the control of tropisms. Such growth movements, including the root gravitropic response, are based on an altered lateral auxin transport within plant tissues. Current models indicate that the gravity signal is perceived at the root cap and transmitted via the meristem to the zone of elongation. There, at the lower flank of the tissue (oriented toward the gravity stimulus), an increase in the auxin concentration can be found, leading to an overall increase of growth rate of the upper side and a decrease in the lower side (see also Chapter 30 by Pilet in this volume). The basipetal directionality of auxin transport from shoot apices and young leaves to roots is thought to result from the polar distribution of specialised carrier molecules in the plasma membranes. The discovery of mutants affecting polar transport or root gravitropism allows a molecular approach of the auxin efflux and influx carriers (Palme and Ga¨lweiler, 1999). This can answer the question whether such auxin efflux carriers represent the elusive auxin receptors. Furthermore, the new possibilities to modulate auxin distribution and response using genetic tools will allow direct tests of the role of auxin in patterning through the threshold-

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dependent activation of secondary responses (Sabatini et al., 1999; Tsukegi and Fedoroff, 1999). It must finally be recalled that auxin transport and metabolism are interdependent. This is illustrated by the relationship shown in Fig. 7 between auxin transport and degradation, in stems and roots. VII.

ROOT AUXINS AND THE GROWTH AND DEVELOPMENT OF AERIAL PLANT PARTS

Evidently, the development of seminal and adventitious roots sustains and enhances growth and vigour of the aerial plant parts. The importance of root-toshoot communication has been specifically emphasized in studies of root responses to environmental stresses (Davies and Jeffcoat, 1990). The contribution of root auxins, in addition to water and nutrients provision, has not been much addressed in the literature, probably because the problem is complex. Each hormone interacts with the others, and the development of the root system is also dependent on the actively growing aerial parts. Textbooks still teach that the ratio of shoot-derived auxin to root-derived cytokinin controls apical dominance and branching. Reexamination of this problem, using mutants, reveals that hormonelike signals other than auxin and cytokinin are also involved (Beveridge et al., 1997; see also Chapter 26 by Hose et al. in this volume).

A.

Contribution of Root Auxins in Stem Vascular Differentiation

Roots do not induce vascular differentiation nor must they be present in order to obtain vascular tissues in stems. However, the roots have two major functions in vascular differentiation, namely: (1) roots orient the pattern of vascular differentiation towards their tip by acting as a sink for the flow of auxin derived from young leaves; and (2) root apices are sources of inductive stimuli that promote vascular development (Aloni, 1995). The major developmental signals of roots are cytokinins and ABA. It should be emphasized however, that cytokinin alone, or root apices in the absence of an auxin source, do not induce vascular differentiation in stem tissues. B.

Auxin-Induced Adventitious Root Formation and Wood Formation

The lignin content of walnut shoots during their in vitro multiplication did not practically change but started to increase as soon as they were transferred to a rooting medium supplied with auxin (Fig. 8) (Kevers et al., 2000). Exogenous auxin provoked a temporary elevation of the endogenous free IAA level which allowed the completion of the rooting inductive phase before any visible histological event. This means that either exogenous or endogenous

Figure 7 IAA transport and IAA degradation compared in the stem and in the root. (From Pilet and Gaspar, 1968.)

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Figure 8 Variation of the lignin content of a whole population (*) of micropropagated walnut shoots, compared with the 20% shoots (*) which will not form roots, in the course of the multiplication (M) phase followed by the successive rooting inductive (Id), initiative (In) and expressive (Ex). (From Kevers et al., 2000.)

IAA, besides its rooting-inducing role, serves as a signal for an increased lignification. Indeed, auxin was shown to be a determining factor in cell wall lignification and differentiation (Bolwell, 1997). Sustained lignification in the shoot depends on the initiation and expression of rooting, since lignification was positively correlated with root emergence (Fig. 8). Continued increase of stem lignification with accentuated xylem proliferation and diameter increase were further shown to depend on the development of the root system (Fig. 9). This means that roots not only bring about essential substances for wood formation but that they also serve as coordinating organs. The cytokinins exported from growing roots are also known to be involved in cell wall growth and differentiation (Montague, 2000; see also Chapter 25 by Emery and Atkins in this volume), but this is probably part of a more complex interacting network between roots and aerial organs.

VIII.

AUXIN INVOLVEMENT IN ADVENTITIOUS ROOT FORMATION

It happened that the first discovered and identified phytohormone, IAA, was shown to promote adventitious rooting. Later identified natural auxins and synthetic compounds of this category had the same effects (Jackson, 1986; Altman and Waisel, 1997). Rooting property of auxins appeared to be specific to this

Figure 9 Evolution of peroxidase activity (A), and lignin level (B), and stem diameter (C) of micropropagated walnut shoots growing with a different number of roots. (From Kevers et al., 2000.)

class of growth regulators since no clear-cut effect was obtained by exogenous application of other phytohormones. Some hormones, such as cytokinins and gibberellins, were even classified as rooting inhibitors (Jackson, 1986; Davis et al., 1988; Davis and Haissig, 1994). This matter needs further investigation because the necessity of cytokinins and gibberellins for rooting, under certain circumstances, was also reported (Letham, 1978; Gaspar et al., 1977). Moreover, the application of exogenous auxins resulted in a series of wrong concepts: that auxin is the major triggering agent in rooting, that the application of exogenous auxin is needed to augment the endogenous bulk of

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auxin, that rooting necessitates the maintenance of a ‘‘high’’ level of endogenous auxin for a certain time, etc. Because there are inductive/adaptative enzymes that regulate the exogenously fed hormones and because application of a hormone may induce modifications in the metabolism of other ones, no simplistic conclusions can be drawn. Another associated error was to consider rooting as a single developmental process. One of the main achievements in the studies of adventitious root formation has been the recognition of successive interdependent physiological phases (Mitsuhashi-Kato et al., 1978; Jarvis et al., 1983; Moncousin et al., 1988; Blakesley, 1994). These were generally called induction, initiation, and expression (Fig. 10) (Gaspar et al., 1992, 1994). Although some other terminologies can be used (De Klerk et al., 1995), a consensus tacitly emerged to define the rooting inductive phase as the time necessary for the biochemical events to precede the initiation of cell divisions and which lead to the formation of root primordia (Jarvis, 1986; Moncousin, 1991). Another definition, and a practical way to estimate the duration of the inductive phase, is the minimum time required for the presence of the external signal for rooting to proceed from competent cells (Hand, 1994). Competence itself is defined as a cell reactivity state allowing response to a stimulus that finally leads to a specific developmental pathway. Induced cuttings which do no longer require the rooting signals are said to be determined, even if other environmental factors are required for the completion of the successive developmental phases (Mohnen, 1994). The inductive rooting period sometimes appears to be very short, being achieved in <24 h from the time of application of the external auxin (Moncousin et al., 1988; Hausman et al., 1994). Taking into account these considerations, a role for each of the phytohormone

Figure 10 Successive interdependent phases of adventitious root formation. (From Hausman et al., 1997.)

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types in the various phases of the rooting process is to be anticipated. The indissociable involvement of endogenous auxins, polyamines, and peroxidases in the rooting inductive phase will be discussed. A.

IAA Level and Peroxidase Changes in the Inductive Phase of Adventitious Rooting

The different physiological phases in rooting experiments were not determined precisely. The variations in IAA levels measured in cuttings before and in the course of the rooting process were quite inconsistent (Gaspar and Hofinger, 1988; Blakesley et al., 1991). It appeared in recent years that, whatever the cutting types were, the endogenous level of free IAA always rose during the inductive phase of rooting (Fig. 11). This early IAA peaking during the inductive phase has been causally related to rooting because (1) it is not observed in nonrooting cuttings (Hausman et al., 1995a); (2) it is measurable only in the rooting zone (Hausman et al., 1995a); (3) treatments, such as with riboflavine, which inhibit rooting, hinder IAA peaking (Fig. 12); and (4) PCIB, a competitor of IAA for auxin receptors or auxin-binding proteins (Marumo, 1986), when applied at inductive phase, inhibits rooting without hindering IAA peaking (Fig. 12). The involvement of free IAA in the rooting inductive phase does not exclude the participation of other auxins, such as IAA aspartate (Blakesley, 1994) and serotonin (Gatineau et al., 1997). The study of peroxidase activity and isoperoxidases, firstly as possible enzymes mediating IAA catabolism, and later as markers of the successive rooting phases, allowed the

Figure 11 Generally occurring changes of peroxidase activity and of endogenous free IAA level along successive, initiative, and expressive rooting phases. (From Gaspar et al., 1997.)

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Figure 12 Changes in free IAA level in poplar shoots during their inductive phase in the presence of NAA (0.3 mg l1, *) alone, and supplemented either by riboflavine (0.3 mM, &) or by PCIB (0.1 mM, ~). The respective rooting percentages are indicated. (From Gaspar et al., 1997.)

establishment of a general time course of activity. It included a typical minimum at rooting inductive phase, and a typical maximum at initiative phase (Moncousin, 1991; Gaspar et al., 1992, 1994). The levels of soluble peroxidase activity and of endogenous free IAA in the course of the successive inductive, initiative, and expressive phases of rooting are presented in Fig. 11. These two curves appear to be approximately the reverse of one another (cf. Gaspar et al., 1992). B.

Polyamine Changes at the Rooting Inductive Phase

It was often suggested that polyamines play a role in the rooting process (Friedman et al., 1982, 1985; Jarvis et al., 1983; Tiburcio et al., 1989; Torrigiani et al., 1989, 1993; Biondi et al., 1990; Altamura, 1994; Hausman et al., 1995b). However, only a few investigations dealt with precise rooting phases where polyamines were really involved. A series of works on rooting of poplar shoots showed an early typical elevation of putrescine, close to the IAA peak, and to termination of the inductive phase (Fig. 13). The content of spermidine and spermine did not change significantly. The role of putrescine in the rooting inductive phase of poplar was implied because of the following arguments (Hausmann et al, 1994; 1995a): 1. 2.

The transient elevation of putrescine did not occur in nonrooting cuttings. Putrescine was measurable only in the basal rooting zone.

Figure 13 Changes in free putrescine (*), spermidine (&), and spermidine (~) during the rooting inductive phase of poplar shoots, in the presence of NAA, as rooting auxin. In the absence of NAA, no such transient increase of putrescine is measured. (From Gaspar et al., 1997.)

3. Inhibitors of putrescine biosynthesis, such as DFMO and DFMA (-difluoromethylornithine and -difluoromethylarginine, respectively), applied prior to or at the beginning of inductive phase, inhibited rooting. 4. An inhibitor of putrescine conversion into spermidine and spermine (cyclohexylamine, an inhibitor of spermidine synthase), which promoted the accumulation of endogenous putrescine, favored rooting, in the absence of exogenously supplied auxin. 5. Exogenously applied putrescine, prior to or at the beginning of inductive phase, had a positive effect on rooting. Additional results point to the role played by putrescine catabolism through its
1 -pyrrolineGABA (-aminobutyric acid) pathway (Hausman et al., 1994, 1995a, 1997b). Indeed, treatment of poplar cuttings with AG (aminoguanidine, an inhibitor of diamine oxidase, DAO, which converts putrescine to GABA) inhibited rooting (Hausman et al., 1994, 1995a). Similar arguments (Kevers et al., 1997) suggest an involvement of putrescine and its catabolic pathway to GABA, in the rooting inductive phase of walnut shoot cuttings, where endogenous IAA and peroxidase

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had also been implicated (Ripetti et al., 1994; Heloir et al., 1996). C.

Possible Interrelationships Among IAA, Peroxidases, and Putrescine

Because the activity of the IAA oxidase system parallels the activity of peroxidase, at least during the rooting initiation phase (Gaspar et al., 1992, 1994), it was tempting to relate the changes in auxin level to the peroxidase-mediated auxin catabolism. All peroxidases can catalyze auxin destruction in vitro (Gaspar et al., 1982). Alternatively, it can be hypothesized that prior changes in IAA levels have modulated peroxidase activity. IAA is known to control the activity of peroxidases involved in lignification of xylem cells (Ros Barcelo and Munoz, 1992), an obligatory process involved in root formation. Other factors involved at different steps of root formation, such as phenolics (Curir et al., 1990; Berthon et al., 1993), cytokinins (Vidal et al., 1994), and ethylene (Moncousin et al.,

1989), are also known to influence peroxidase activity. Investigations of the changes of the spectrum of isoperoxidase during rooting did not solve the problem of the relationships between IAA and peroxidase. The main difficulty stems from the polyfunctionality of the isoperoxidases (Pedreno et al., 1995). Figures 12 and 13 show that the transient increases of free IAA and free putrescine at the rooting inductive phase of poplar cuttings occur at about the same time. Concomitantly a peroxidase increase precedes the typical minimum of the rooting inductive phase (Fig. 14A). Exogenous application of putrescine or of CHA, in the absence of auxin, promotes rooting (see above) and induces a peroxidase peak (Fig.14B and C). It is tempting to suggest that putrescine controls the IAA level, mediated by some peroxidases. The reverse is also true. Rooting inhibition by riboflavine is correlated with the absence of the characteristic IAA peak at the inductive phase, but also of the temporary putrescine increase. The transient rise and decline of IAA and putrescine thus seem closely related. It is hypothesized that IAA

Figure 14 Changes in peroxidase activity of poplar shoots (A) in the presence of NAA as rooting auxin (RM, rooting medium) or in the absence of NAA (NRM, nonrooting medium); (B) in the absence of auxin but in the presence of putrescine 104 M (NRM + Put); (C) in the absence of auxin but in the presence of CHA 104 M (NRM + CHA). (From Gaspar et al., 1997.)

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and putrescine, which are known to control cell division cycles (Del Duca and Serafini-Fracassini, 1993), are required to initiate cell divisions at the end of the rooting inductive phase. However, the considerable peroxidase variation that occurs in the same time period may indicate a possible role of peroxidase in the relationship between IAA and putrescine. Putrescine degradation through its
1 -pyrroline pathway is accompanied by the formation of H2O2. H2O2 can be utilized by some (iso)peroxidases. Therefore, the following hypotheses regarding the roles of peroxidases in the interplay between IAA and polyamines can be formulated: 1.

2.

Putrescine can inactivate some peroxidases involved in auxin catabolism by providing H2O2 through its catabolic effects, thus favoring an increase in the level of endogenous IAA. An increase in putrescine level might enhance peroxidase activity and hence bring about a decrease in IAA level; a possible putrescine control of peroxidase activity via the GABA shunt and the Shemin pathway must not be excluded.

Indeed, a GABA shunt of tricarboxylic acid cycle, from glutamate (Shelp et al., 1995) or from polyamines (Bisbis et al., 1997) to succinate, has been shown to operate under certain stress conditions. Succinate is a precursor for the biosynthesis of tetrapyrrole-containing compounds such as peroxidase via the Shemin pathway (Castelfranco and Beale, 1983). Rooting might in some way be considered as a response to stress—e.g., wounding due to cutting plus imposed hormonal imbalance in favor of auxin. GABA alone, in the absence of auxin, is indeed able to promote rooting to some extent in poplar shoots (Hausman et al., 1997b). Finally, it becomes evident that many transduction pathways may not be initiated without the participation of the membrane wall-associated peroxidases (and NAD(P)H oxidase) and the generation of active oxygen species. Among the latter, H2O2 has been proposed to be involved directly in the regulation of gene expression (Mehdy, 1994). Results of the presented investigations have shown the indissociability of auxins and polyamines just like the interrelationships between auxins and cytokinins, and between auxin and ethylene (Gaspar et al., 2000b). As for auxins and cytokinins, the catabolism and the catabolic products of polyamines seem to play important roles and cannot be dissociated from primary biochemical pathways such as the Krebs cycle.

D.

Hormonal Characterization of a Nonrooting Mutant of Tobacco

Auxin-resistant mutant shoots of tobacco were derived from the protoplasts and called rac mutants owing to their inability to root, even in response to an auxin or polyamine treatment (Faivre-Rampant, 2000b). The auxin resistance of the mutant cells and tissues was not correlated with an increased rate of conjugation or breakdown of auxins or with a perturbation of auxin transport (Caboche et al., 1987). Using the auxin-induced hyperpolarization response, the mutant protoplasts were shown to be 10 times less sensitive to auxin than the wild-type protoplasts (Ephritikhine et al., 1987). Histological analyses indicated that 3–4 d after seed germination, the root meristem degenerated and was transformed into a callus (Pele`se et al., 1989). In stem cuttings treated with IBA, perivascular cells divided, but never formed adventitious root meristems (Lund et al., 1996). According to these authors, the rac mutation did not disrupt the auxin concentration, but implied changes in the receptor affinity for auxin and/ or in the efficiency of the transduction pathways. However, a reappraisal of the auxin concentrations did not lead to the same conclusions: the tobacco rac mutant showed hyperauxiny as compared to the wildtype (Faivre-Rampant et al., 2000c). This would result from an overaccumulation of phenolic compounds inhibiting the auxin catabolism in the rac mutant (Faivre-Rampant et al., 2000c). In addition, the nonrooting shoots grow at a lower rate in vitro (Faivre-Rampant et al., 1998); we have compared on a hormonal basis the rac and wild-type shoots (these latter root spontaneously at the end of the culture cycle without auxinic treatment; FaivreRampant et al., 1998, 2000a–c). Variations of endogenous hormone levels during the culture cycle have shown differences between the two genotypes. But using peroxidase, auxins, and polyamines as markers the rooting phases, the results have presumed the rooting induction phase, the process of root formation being blocked after this phase in the mutant. This once more emphasizes the interdependence of the hormonal controls in the successive phases of a developmental process such as rooting.

IX.

CONCLUSIONS

Auxins are involved in the biology of roots, in root growth, with and without symbionts, in lateral and adventitious root formation, and in whole plant devel-

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opment through the root partnership with shoots. However, to the best of our knowledge, phytohormones act in an integrated manner. They can act either cooperatively or antagonistically, in a sequential manner, or by influencing the metabolism of each other. Thus, it is wrong to claim that, in the biology of roots, auxins play a specific or a more preponderant role than that of other phytohormones.

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Auxins Schmu¨lling T, Scha¨fer S, Romanov G. 1997. Cytokinins as regulators of gene expression. Physiol Plant 100:505– 519. Schmu¨lling T, Schell J, Spena A. 1988. Single genes from Agrobacterium rhizogenes influence plant development. EMBO J 7:2621–2629. Shelp BJ, Walton CS, Snedden WA, Tuin LG, Oresnik IJ, Layzell, DB. 1995. GABA-shunt in developing soybean is associated with hypoxia. Physiol Plant 94:219–228. Skoog F, Miller CO. 1957. Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Soc Exp Biol Symp 11:118–131. Taylor BH, Scheuring CF. 1994. A molecular marker for lateral root initiation: the RSI-1 gene of tomato (Lycopersicon esculentum Mill) is activated in early lateral root primordia. Mol Gen Genet 243:148–157. Thimann KV. 1969. The auxins. In: Wilkins MB, ed. Physiology of Plant Growth and Development. London; McGraw-Hill, pp 1–45. Tiburcio AF, Gendy CA, Tran Tanh Van K. 1989. Morphogenesis in tobacco subepidermal cells: putrescine as a marker of root differentiation. Plant Cell Tiss Org Cult 19:43–54. Timpte C, Wilson AK, Estelle M. 1992. Effects of the axr2-1 mutation of Arabidopsis on cell shape in hypocotyls and inflorescence. Planta 188:271–278. Timpte C, Wilson AK, Estelle M. 1994. The axr2-1 mutation of Arabidopsis thaliana is a gain-of-function mutation that disrupts an early step in auxin response. Genetics 138:1239–1249. Timpte C, Lincoln C, Pickett FB, Turner J, Estelle M. 1995. The AXR1 and AUX1 genes of Arabidopsis function in separate auxin-response pathways. Plant J 8:561–569. Torrigiani P, Altamura MM, Capitani F, Serafini-Fracassini D, Bagni N. 1989. De novo root formation in thin cell layers of tobacco: changes in free and bound polyamines. Physiol Plant 77:294–301. Torrigiani P, Altamura MM, Scaramagli S, Capitani F, Falasca G, Bagni N. 1993. Regulation of rhizogenesis by polyamines in tobacco thin layers. J Plant Physiol 142:81–87.

403 Tretyn A, Bossen ME, Kendrick RE. 1992. Evidence for different types of acetylcholine receptors in plants. In: Karssen CM, Van Loon LC, Vreugdenhil D, eds. Progress in Plant Growth Regulation. Dordrecht: Kluwer, pp 306–311. Trewavas AJ, Cleland RE. 1983. Is plant development regulated by changes in the concentration of growth substances or by changes in the sensitivity to growth substances? Trends Biochem Sci 8:354–357. Tsukegi R, Fedoroff NV. 1999. Genetic ablation of root cap cells in Arabidopsis. Proc Natl Acad Sci USA 96:12941–12946. Utsuno K, Shikanai T, Yamada Y, Hashimoto T. 1998. AGR, an agravitropic locus of Arabidopsis thaliana, encodes a novel membrane-protein family member. Plant Cell Physiol 39:1111–1118. Van der Krieken WM, Kodde J, Visser MHM, Tsardakas D, Blaakmeer A, de Groot K, Leegstraa L. 1997. Increased induction of adventitious rooting by slow release auxins and elicitors. In: Altman A, Waisel Y, eds. Biology of Root Formation and Development. New York: Plenum Press, pp 95–104. Vidal N, Ballester A, Vieitez AM, Kevers C, Gaspar T. 1994. Biochemical characteristics of chestnut shoots related to in vitro multiplication and rooting capacities. Adv Hort Sci 8:19–24. White FF, Taylor BH, Huffman GA, Gordon MP, Nester EW. 1985. Molecular and genetic analysis of the transferred DNA regions of the root-inducing plasmid of Agrobacterium rhizogenes. J Bacteriol 164:33–44. Wilson A, Pickett FB, Turner JC, Estelle M. 1990. A dominant mutation in Arabidopsis confers resistance to auxin, ethylene and abscisic acid. Mol Gen Genet 222:377–383. Xie H, Pasternak JJ, Glick BR. 1995. Low temperature growth, freezing survival and production of antifreeze protein by plant growth promoting rhizobacterium Pseudomonas putida GR 12-2. Can J Microbiol 41:776–782. Zobel RW. 1974. Control of morphogenesis in ethylenerequiring tomato mutant, diageotripica. Can J Bot 52:735–741.

24 Gibberellins Eiichi Tanimoto Nagoya City University, Nagoya, Japan

I.

INTRODUCTION

Gibberellin (GA) is one group of plant hormones, which has a chemical structure of gibberellane skeleton (Fig. 1). GA is involved in almost all phases of plant growth and development. GA promotes germination, elongation growth of roots and stems, flowering, and fruit development. Several books and reviews have been published on the physiological role of GA. However, relatively limited references are available for roots, since GA does not strongly promote root elongation of many plants.

1955, three research groups—the Northern Regional Research Laboratory (NRRL) in the United States, the Imperial Chemical Industry Ltd. (ICI) in United Kingdom, and the University of Tokyo in Japan— reported the isolation of active principle from the fungus. The ICI group named their substance gibberellic acid (Curtis and Cross, 1954), The NRRL group found gibberellin A and X (Stodola et al., 1955), and the University of Tokyo Group separated three gibberellins, A1, A2, and A3 (Takahashi et al., 1955, 1986). Takahashi et al. (1957) also isolated a new gibberellin named GA4 from the culture filtrate of the fungus.

A.

B.

Discovery of GA

GA in Higher Plants

Soon after the discovery of GA as a fungal toxin, it was also isolated from higher plants. GA-like substances were found in immature seeds of bean (Mitchell et al., 1951; Radley, 1956; West and Phinney, 1956). GA1 was isolated from immature seeds of runner bean (Phaseolus multiflorus) (MacMillan and Suter, 1958) and from water sprouts of mandarin orange (Citrus unshiu) (Kawarada and Sumiki, 1959).

Gibberellin was named after the Latin name of the fungus Gibberella fujikuroi (Saw.) Wr. that causes the abnormal elongation of rice plants (Hori, 1898). The fungus was named by H. W. Wollenweber, after the name of two Japanese scientists, Yosaburo Fujikuro and Kenkichi Sawada, who studied this pathogenic fungus in 1910s and 1920s. Research on GA started by the discovery that this fungus produces chemicals that induce abnormal elongation in young seedlings of rice. This disease was called bakanae (a foolish seedling in Japanese) disease, since the infected seedlings have abnormally elongated and die without fruit set. The growth-promoting factor was partially isolated from the liquid culture of the fungus as heat-stable substance by Kurosawa (1926). Yabuta and Sumiki (1938) succeeded in crystallizing the GA. In 1954–

II.

BIOSYNTHESIS AND METABOLISM

Owing to diverse structural flexibility of the gibberellane skeleton, 121 kinds of GAs have been identified to date (2000) from higher plants and from microorgan405

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Figure 1 Structure of ent-gibberellane, backbone for all gibberellins.

isms. The biosynthetic pathway has been extensively studied in Gibberella fujikuroi, and the interconversion pathways of these GAs have been investigated (Davies, 1987, 1995; Frankenberger and Arshad, 1995). The biosynthetic pathway and its genetic control have recently been reviewed (Hedden and Kamiya, 1997; Hedden, 1997, 1999; Hedden and Proebsting, 1999). Of the 121 GAs identified to date, relatively few are thought to be physiologically active. These were GA1, GA3, and GA4 for most of the higher plants (Fig. 2). The whole GA biosynthesis pathway is separated into three steps, as shown in Fig. 3. The first step is the formation of ent-kaurene. The ent-kaurene is made from isopentenyldiphosphate (IPP). It has long been believed that IPP is synthesized from mevalonic acid. Recently it was found that IPP is formed from glyceraldehyde 3-phosphate and pyruvate via 1-deoxyxylulose in plastids (Lichtenthaler et al., 1997). Hedden (1999) has recently reviewed the nonmevalonate pathway to IPP. Copalyl diphosphate synthase (CPS) is thought to be the rate-limiting step of ent-kaurene synthesis (West et al., 1982), and the expression of the (GA1) gene was highest in shoot apices, root tips, and developing flowers in Arabidopsis (Silverstone et al., 1997). These facts indicate that not only the shoot apices but also the root tips are the source of the GA precursor. The second step in the GA pathway is oxidation of ent-kaurene to GA12-aldehyde. The ent-kaurene is oxidized by membrane-bound monooxygenases. Several GA-deficient dwarf mutants of Arabidopsis, pea, tomato and rice are defective in ent-kaurene oxidase activity (Fig. 3). The third and final step is the formation

of the bioactive hormones GA1 and GA4 from GA12aldehyde. These reactions involve oxidative removal of C-20 to give a lactone between C-19 and C-10, and hydroxylation at C-3 position. The former step is catalyzed by soluble oxidases that use 2-oxoglutarate as a cosubstrate. The latter reaction is the last and critical reaction to produce active GA1 and GA4. Further hydroxylation at C-2 position results in inactive GA8 and GA34. Not only GA biosynthesis but also the conversion to inactive form determines the active GA level. The genetic deletion of this inactivation step results in the accumulation of active GA that makes the mutant slender and tall like sln mutant of pea (Fig. 3).

A.

Site of GA Synthesis and Transport

GAs are known to move relatively freely from shoots to roots (Prochazka, 1981; Matthysse and Scott, 1984; Kaldewey, 1984). Although at least some GAs were transported from the roots to the shoots (Carr et al., 1964; Crozier and Reid, 1971), the majority of GAs are thought to be transported from young developing tissue to older tissues. Since the enzymes of early GA production such as CPS are expressed in root tips, root is, at least in part, a source site of GA precursors. The gene for 3-hydroxylase is also expressed in meristmatic cells including root tips (Itoh et al., 1999). However, little information is available regarding what percentage of these plant plastid enzymes is operating in roots. Growth-promoting activity of GA3 moves from the shoot or from the cotyledons to the roots and from roots to shoots, since cotyledon-, root-, or shootapplied GA3 exerted growth promotion of roots and of shoots (Tanimoto, 1994). [H3]GA1 is readily taken up and distributed throughout the whole plant (Davies and Rappaport, 1975). Soil-applied GA promotes shoot and root growth of dwarf maize (Frankenberger and Arshad, 1995), revealing that GAs in the soil affect plant growth. GA-producing pathogenic fungi such as Gibberella fujikuroi strongly affect plant growth. Other soil micro-

Figure 2 Structure of GA1, GA3, and GA4, physiologically active GAs for most plants.

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Figure 3 GA-biosynthetic pathway, enzymes, corresponding mutant genes, and site of inhibitor action.

organisms can also synthesize GAs and/or GA-like substances that may influence plants via the rhizosphere. But the stability of soil GA and its availability to plants are obscure. There is considerable evidence for the microbial production of GAs in soil (Frankenberger and Arshad, 1995). B.

Chemical Inhibitors of GA Biosynthesis

Three types of inhibitors are known; onium type (CCC, AMO1618), nitrogen-containing cycle type (ancymidol, paclobutrazole, uniconazole), and cyclohexantrion type (LAB198999) (Fig. 4) (see Rademacher, 1991). The action sites of these inhibitors are shown in Fig. 3. These chemicals are being used for growth retardation of plants, for cultivation of tulips and chrysanthemums with short flower stalks, and for production of small plants. These chemicals also contributed to the investigation of the biosynthetic pathways of GA and of its regulation (Baldev et al., 1965;

Skene and Mullins, 1967; Wylie et al., 1970; Kuo and Pharis, 1975; Coolbaugh and Hamilton, 1976; Coolbaugh et al., 1978, 1982; Izumi et al., 1984). Dwarf cultivars of pea and maize were used for the research of growth-promoting activity of GA, since these plants are deficient in endogenous GA and show high response to externally applied GA. Many of these cultivars were found to be mutants of GA biosynthesis, and these mutants and cDNA clones for GA-biosynthetic enzymes have been reviewed by Hedden and Kamiya (1997). Some of these mutant genes are also indicated in Fig. 3. III.

GA FUNCTIONS IN ROOTS

A.

GA-Mediated Growth Regulation of Roots

GA was discovered as a growth-promoting factor of the shoot, and much more information regarding the

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Figure 4 Synthetic plant growth retardants inhibiting GA biosynthesis. Sites of action of these compounds are shown in Fig. 3.

physiological functions has accumulated for shoots than for roots (Scott, 1984; Davies 1987, 1995; Takahashi et al., 1991). There are conflicting reports of GA effect on root growth (Table 1) (Torrey, 1976; Feldman, 1984; Phinney, 1984). However, basic information regarding GA functions on root growth has been established at least for some plant species such as peas and lettuce (Tanimoto, 1987, 1988, 1991). Although GA showed little effect on root elongation, both in normal and in dwarf peas (Fig. 5), in contrast to the shoot elongation, the requirement of GA for root growth was suggested by the use of inhibitors. The inhibitors of GA biosynthesis, such as CCC, AMO-1618, and ancymidol, suppressed root growth. The requirement of GA for root growth has been argued by the fact that some of the above-mentioned inhibitions were not overcome by the addition of GA (Sachs and Kofranek, 1963; Dunberg and Eliasson, 1972; Crozier et al., 1973). However, a complete recovery from inhibition was shown when the concentration-dependent inhibition in pea and lettuce roots were tested (Tanimoto, 1987, 1988). Concentration dependence for growth promotion by GA was also investigated. Higher concentrations of an inhibitor were required for inhibition of root

growth than for the shoots (Fig. 6). In addition, recovery of growth from inhibition by GA addition was observed at a lower concentration range than those for shoot growth. The elongation rate of roots was traced during the inhibition and recovery process (Fig. 7). The complete recovery of the elongation rate was also recorded by a rhizometer (Tanimoto and Watanabe, 1986; Tanimoto, 1988). When ancymidolpretreated seedlings of lettuce were treated with two different concentration of GA3, 1 nmolar GA3 promoted only root elongation, whereas 1 mmolar GA3 enhanced both root and shoot growth (Fig. 8). The conflicting results of externally applied GA on root growth are thought to be ascribed to the following specific reasons: Other hormones, auxin, ethylene, and cytokinins inhibit root elongation. Thus, GA effect does not appear as long as these hormones limit root elongation. GA is thought to be a saturating-type hormone that is not inhibitory at higher concentration, in contrast to auxin. Since endogenous levels of auxin are thought to be superoptimum for many roots, GA may inhibit root elongation by increasing the endogenous level of auxin (Kuraishi and Muir, 1962) and/or increasing the sensitivity to auxin, as suggested for stems (Ockerse and Galston, 1967; Tanimoto et al., 1967).

Table 1 Example of Conflicting Effect of Externally Applied GA on Root Growth Plant Lactuca sativa Lycopersicon esculentum Oryza sativa Pisum sativum Triticum aestivum Zea mays

Promotion Aspinal et al., 1967 Butcher and Street, 1960 Suge, 1985; Radi and Maeda, 1988 Pecket, 1960 Burstro¨m, 1960 Whaley and Kephart, 1957; Mertz, 1966

Inhibition Krekule and Ullman, 1959 Tognoni et al., 1967

Burstro¨m, 1960 Konings and Wolf, 1984; Svensson, 1972

No effect Sawhney and Srivastava, 1974 Finnie and Staden, 1985 Ogawa et al., 1976 Katayama and Akita, 1989 Manos, 1961 Svensson, 1972; Hirota, 1980

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Figure 5 Effect of externally applied GA3 on the elongation growth of tall and dwarf cultivars of Pisum sativum. (Adapted from Tanimoto, 1988, 1994.)

The mode of root elongation in GA-deficient dwarf mutants has also been an important reason why the role of GA in root elongation has been underestimated. It is well known that root elongation of dwarf peas (le, na) and dwarf maize (d1, d5) were not stunted and remained comparable to those of wild-type counterparts (Fig. 9). The shortage of GA in these plants drastically repressed the shoot elongation, but root elongation was not significantly affected. As indicated in Figs. 5–7, the GA level required for root elongation

is quite low so that roots can elongate while shoot elongation is suppressed at low GA level. This idea is also supported by the fact that genetic mutations of the GA biosynthesis pathways, such as le in pea (see note in proof in Fig. 9), are leaky mutant; i.e., they produce some GA (
10% of WT). However, when the level of endogenous GA became extremely declined by an additional mutation (le^d; personal communication, J.D. Reid) or by the inhibitor ancymidol (Tanimoto, 1994), root elongation became stunted.

Figure 6 Inhibition of root and hypocotyl elongation by ancymidol in lettuce (Lactuca sativa). (Adapted from Tanimoto, 1987.)

Figure 7 Promotion of root and shoot elongation of dwarf pea (cv. Little Marvel) by GA3 in the presence of 3*10-6 M ancymidol. (Adapted from Tanimoto, 1994.)

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Figure 8 Differential promotion of root and hypocotyl growth by two concentrations of GA3 in the presence of 4*106 M ancymidol in lettuce seedlings. Time course of root elongation was recorded every 30 min for 2 d in a rhizometer (Tanimoto and Watanabe, 1986), and the final length of roots and hypocotyls were measured after 4 d (GA3 109 M) and after 2 d (GA3 106 M). Whole seedlings were repeatedly dipped for 2 min in hydroponic solution containing growth regulators every 30 min.

B.

GA-Mediated Control of Root Thickness

When treated by the inhibitors of GA biosynthesis, the elongation zone of the roots becomes thick (Skene and Mullins, 1967; Dunberg and Eliasson, 1972). The inhibitor-induced thickening was reversed by GA (Tanimoto, 1987, 1988). The thickening of roots was mainly due to the expansion of cortex cells (Fig. 10) (Tanimoto, 1987, 1988). Shibaoka (1994) reviewed the control mechanism of plant cell thickening by GA in relation to the orientation of cortical microtubules (CMT). A shortage of GA disorders the orientation of CMT and results in multidirectional orientation of cellulose microfibrils in the cell walls. The latter leads to the thickening or expansion of plant cells rather than elongation. It was also recognized that a GA-deficient dwarf mutant of pea such as Little Marvel has thicker roots than taller varieties and that exogenous GA makes these roots more slender. Thus GA-mediated control of thickness was ascribed to the control of the orientation of cellulose microfibrils in the cell walls. The orientation of CMT and cellulose microfibrils is known to be transverse to the axis of roots (Hogetsu, 1986; Hogetsu and Ohshima, 1986). That orientation causes root cells to elongate, resulting in the slender roots. However, once this GA function was disturbed, CMT orient themselves obliquely or longitudinally to the cell axis as observed in onion leaf sheath cells (Mita and Shibaoka, 1984a,b).

The same function of GA was discovered in roots (Balusˇ ka et al., 1993). They observed a GA-mediated transverse orientation of CMT in the distal elongation zone of the dwarf maize d5. Recently, a comparable effect of GA was observed also in a duckweed, Lemna minor roots (Inada et al., 2000; Inada and Shimmen, 2000). GA promoted root elongation only in the presence of an inhibitor of GA synthesis, uniconazole-P. The transverse orientation of CMT was disrupted by uniconazole-P and resumed by GA (Fig. 11a–c). However, in Lemna, expansion of root by uniconazole-P was not observed. Instead, cell division and cortical cell expansion were suppressed and root became slender when treated by the inhibitor. Cell division and/or cell expansion system of Lamna roots may be more sensitive to an inhibitor of GA synthesis than pea and lettuce roots. GA also plays a role in the regulation of cell division and cell elongation in Lamna roots. C.

GA-Mediated Growth and Cell Wall Extension

Extension growth of plant cell is controlled by the rigidity of cell walls. The mechanical extensibility of cell walls is thought to participate in GA-mediated root elongation. GA increased the cell wall extensibility of pea root cells (Tanimoto, 1994; Tanimoto and Yamamoto, 1997). GA also modified sugar composition and molecular mass of cell wall polysaccharides of pea root cells (Tanimoto, 1988, 1992, 1995; Tanimoto and Huber, 1997).

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ments excised from GA-treated roots showed a stronger acid–growth response than those excised from a GA inhibitor-treated roots (Tanimoto, 1994, 1995). This suggests that GAs enhance acid-induced elongation, probably by increasing the cell wall extensibility that is enhanced by low pH.

D.

Figure 9 Ten-day-old seedlings of dwarf le mutant of pea and its wild counter parts. Stems are stunted but not the roots. Note in proof: Recently, Yaxley et al. (2001) reported that le-l mutant does not reduce root GA1 levels and 3-hydroxylase genes other than le may participate in GA1 production in pea roots.

When excised segments of stems and roots were incubated in a solution of pH 4, the segments quickly elongated. This is caused by the softening of cell walls (increase in cell wall extensibility) (Tanimoto et al., 2000), part of which is catalyzed by acid-activated cell wall proteins such as expansin (Cosgrove, 1998, 1999) and/or yieldin (Okamoto et al., 2000a,b). Root segments also exhibit strong acid growth response (Edwards and Scott, 1974, 1976; Tanimoto and Watanabe, 1986; Tanimoto et al., 1989). Root seg-

GA-Mediated Gene Expression in Roots

A number of genes responsive to GAs have been identified since the -amylase gene (Jacobsen and Beach, 1985). Several genes were found to be transcriptionally upregulated by GAs. Many of the genes are still functionally unknown. GASA gene family in Arabidopsis and GAST1,3,4 in tomato are expressed in seedlings and roots (Aubert et al., 1998). GASA4/GUS expression in transgenic Arabidopsis was found primarily in all meristemic regions, including vegetative, inflorescence, and floral meristems as well as in primary and lateral root tips. The expression pattern suggests that the GASA4 protein plays a role in dividing cells rather than in elongating cells. All those genes are in conserved 60 amino acid C-terminal domain. It is also found in the tomato RSI-1 peptide and the genes for which is upregulated by auxin in young lateral roots (Taylor and Scheuring, 1994). The physiological functions of these genes are under investigation. As far as tissue-specific expression of these genes is concerned, GA-upregulated genes are related to the cell division but less related to elongation growth, since these genes are repressed in elogating roots (Aubert et al., 1998). Further studies on the products of these GA-regulated genes will unravel the GA-regulated growth and development of roots.

E.

Effect of GA on Rooting

Auxin strongly promotes rooting of cuttings and is thought to be indispensable for the differentiation of primodia. In contrast, GA is rather inhibitory for rooting (Goldfarb et al., 1997; Rugini et al., 1997). In fact, GA (Goldfarb et al., 1997) did not enhance the expression of Loblolly Pine Early Auxin-induced (LPEA3) gene above basal level, which may be involved in auxin-induced rooting. The growth retardants such as pacrobutrazol promote rooting of cuttings and while GA reversed it (Porlingis and Koukourikou-Petridou, 1996). These findings suggest that GA is less requisite for rootings and rather inhibitory for auxin-promoted rooting.

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Figure 10 Ancymidol-induced thickening of lettuce root and its prevention by GA3. (Adapted from Tanimoto, 1987.)

IV.

CONCLUDING REMARKS

Auxin and/or Ca2+ movements in root apex conduct signal transduction and gravitropic and hydrotropic differential growth (see Chapters 23 by Casper et al., 29 by Porterfield, 30 by Pilet, and 31 by Poovaiah et al. in this volume). No clear relationship of GA to such signal transduction process has been reported (Mertens and Weiler, 1983; Moore and Dickey, 1985). Gravitropic response is shown by pea and lettuce roots (Tanimoto, unpublished) even when the root growth was severely stunted by treatment with ancymidol—i.e., where endogenous GA level is thought to be low enough to cause abnormal expansion of root cells (Fig. 10). This phenomenon suggests that the depletion of GA down to the level necessary for microtubule orientation does not affect the gravitropic curvature of roots. The signal transfer of IAA and/or Ca2+ from root cap to elongation zone is not affected by the depletion of GA and the curvature takes place at the expanding zone that is normally elongating zone of the root. The curvature may take place by a small difference in growth rate between the two sides of the root, even though the elongation is severely stunted by the depletion of GA. However, the extension growth of roots seems to be regulated by a higher level of GA, at least in pea and lettuce plants. The idea that normal root elongation requires a GA level lower than in shoots is con-

sistent with the genetic retardation of GA biosynthesis pathways, such as le and le^d, in which the endogenous level of GA are 10 times and 100 times lower than in the WT counterparts (see note in proof in Fig. 9). Since GA shows no toxic effect at high concentrations, in contrast to auxin, it promotes elongation growth in a wide concentration range. In case of rosette plants, bolting is induced by massive production of GA in spring. Root growth is thought to take place under very low GA levels before such a massive GA production occurs. Such concentration-dependent regulation of roots and shoots can be applied to the control of the root/ shoot ratio. The ratio of roots to shoots is a very important factor for the productivity of crop plants, in their resistance to harmful environmental conditions, wind, drought, low temperature, etc. Brassinosteroid was recently found to regulate the growth of roots and shoots in studies of mutants and chemical inhibitors of brassinosteroid biosythesis (Nomura et al., 1997; Yokota, 1997; Nomura et al., 1999). Apparently, we have now a possible strategy to manipulate plant shape by controlling hormone levels, particularly of GAs and brassinosteroid by genetic and/or chemical control of their biosynthesis and breakdown. The ratio of the aboveground part of a plant to the belowground counterpart could be manipulated by changing the endogenous level of GA and brasibosteroids.

Gibberellins

Figure 11 Effect of GA and uniconazole-P on the arrangement of cortical microtubules (CMT). Pictures are immunofluorescence micrographs of CMT in root epidermal cells at elongation zone of a duckweed, Lemna minor (a) control, (b) uniconazole-P treatment, (c) GA + uniconazole-P treatment. Long horizontal arrows show the direction of root elongation. Short arrows indicate the direction of CMT oriented perpendicularly to root axis in (a) and (c). CMT was disrupted in (b) and disrupted CMT arranged obliquely (arrowhead). Bar, 10 mm. (Adapted from Inada et al., 2000.)

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Gibberellins Mitchell JW, Skaggs DP, Anderson WP. 1951. Plant growth stimulating hormones in immature seeds. Science 114:159. Moore R, Dickey K. 1985. Growth and graviresponsiveness of primary roots of Zea mays seedlings deficient in abscisic acid and gibberellic acid. J Exp Bot 36:1793–1798. Nomura T, Nakayama M, Reid JB, Takeuchi Y, Yokota T. 1997. Blockage of barassinosteroid biosynthesis and sensitivity causes dwarfism in garden pea. Plant Physiol 113:31–37. Nomura T, Kitasaka Y, Takatsuto S, Reid JB, Fukami M, Yokota T. 1999. Brassinosteroid/sterol synthesis and plant growth as affected by lka and lkb mutations of pea. Plant Physiol 119:1517–1526. Ockerse R, Galston AW. 1967. Gibberellin-auxin interaction in pea stem elongation. Plant Physiol 42:47–54. Ogawa M, Matsunaga E, Yamazaki Y, Pyamada K, Matsui T, Tobitsuka J. 1976. A synergistic effect of 2-ethyl-1isopropoxycarbonyl-3-(4-tolycarbamoyl)isourea with gibberellic acid on growth of rice seedlings. Plant Cell Physiol 17:743–749. Okamoto-Nakazato A, Nakamura T, Okamoto H. 2000a. The isolation of wall-bound proteins regulating yield threshold tension in glycerinated hollow cylinders of cowpea hypocotyl. Plant Cell Environ 23:145–154. Okamoto-Nakazato A, Takahashi K, Kido N, Owaribe K, Katou K. 2000b. Molecular cloning of yieldins regulating the yield threshold of cowpea cell walls: cDNA cloning and characterization of recombinant yieldin. Plant Cell Environ 23:155–164. Pecket RC. 1960. Effects of gibberellic acid on excised pea roots. Nature 185:114–115. Phinney BO. 1984. Gibberellin A1, dwarfism and the control of shoot elongation in higher plants. In: Crozier A, Hillman JR, eds. The Biosynthesis and Metabolism of Plant Hormones. London; Cambridge University Press, pp 17–41. Porlingis IC, Koukourikou-Petridou MA. 1996. Promotion of adventitious root formation in mung bean cuttings by four triazole growth retardants. J Hort Sci 71:573– 579. Prochazka S. 1981. Translocation of growth regulators from roots in relation to the stem apical dominance in pea (Pisum sativum L.) seedlings. In: Brouwer R et al., eds. Structure and Function of Plant Roots. The Hague; Martinus Nijhoff/Dr W. Junk, pp 407–409. Radi SH, Maeda E. 1988. Effect of brassinolide on the cultured rice root growth as modified by Figaron and gibberellic acid. Jpn J Crop Sci 57:191–198. Rademacher W. 1991. Inhibitors of gibberellin biosynthesis: applications in agriculture and horticulture. In: Takahashi N et al., eds. Gibberellins. New York; Springer-Verlag, pp 296–310. Radley M. 1956. Occurrence of substances similar to gibberellic acid in higher plants. Nature 178:1070.

415 Rugini E, Di Francesco G, Muganu M, Astolfi S, Caricato G. 1997. The effects of polyamines and hydrogenperoxide on root formation in olive and the role of polyamines as an early marker for rooting ability. In: Altman A, Waisel Y, eds. Biology of Root Formation and Development. New York; Plenum, pp 65–73. Sachs RM, Kofranek AM. 1963. Comparative cytohistological studies on inhibition and promotion of stem growth in Chrysanthemum morifolium. Am J Bot 50:772–779. Sawhney VK, Srivastava LM. 1974. Cytochalasin-B-induced inhibition of root-hair growth in lettuce seedlings and its reversal by benzyladenine. Planta 119:165–168. Scott TK, ed. 1984. Encyclopedia of Plant Physiology New Series. Hormonal Regulation of Development. Berlin; Springer-Verlag, Vol 10. Shibaoka, H 1994. Plant hormone-induced changes in the orientation of cortical microtubultes: alterations in the cross-linking between microtubules and the plasma membrane. Annu Rev Plant Physiol Plant Mol Biol 45:527–544. Silverstone A, Chang CW, Krol E, Sum TP. 1997. Developmental regulation of the gibberellin biosynthetic gene GA1 in Arabidopsis thaliana. Plant J 12:9– 19. Skene KGM, Mullins MG. 1967. Effect of CCC on the growth of roots of Vitis vinifera L. Planta 77:157–163. Stodola FH, Raper KB, Fennell DI, Conway HF, Sohns VE Langford CT, Jackson RW. 1955. The microbiological production of gibberellin A and X. Arch Biochem Biophys 54:240. Suge H. 1985. Ethylene and gibberellin: regulation of internode elongation and nodal root development in floating rice. Plant Cell Physiol 26:607–614. Svensson SB. 1972. A comparative study of the changes in root growth induced by coumarin, auxin, ethylene, kinetin and gibberellic acid. Physiol Plant 26:115–135. Takahashi N, Kitamura H, Kawarada A, Seta Y, Takai M, Tamura S, Sumiki Y. 1955. Biochemical studies on ‘‘Bakanae’’ fungus. XXXIV. Isolation of gibberellins and their properties. Bull Agric Chem Soc Jpn 19:267. Takahashi N, Seta Y, Kitamura H, Sumiki Y. 1957. Biochemical studies on ‘‘Bakanae’’ fungus. XLII. Bull Agric Chem Soc Jpn 21:396. Takahashi N, Yamaguchi I, Yamane H. 1986. Gibberellins. In: Takahashi N, ed. Chemistry of Plant Hormones. Boca Raton, FL: CRC Press, pp 57–151. Takahashi N, Phinney BO, MacMillan J. 1991. Gibberellins. New York; Springer-Verlag. Tanimoto E. 1987. Gibberellin-dependent root elongation in Lactuca sativa: recovery from growth retardant-suppressed elongation with thickening by low concentration of GA3. Plant Cell Physiol 28:963–973.

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25 Roots and Cytokinins R. J. Neil Emery Trent University, Peterborough, Ontario, Canada

Craig A. Atkins University of Western Australia, Perth, Western Australia, Australia

I.

OVERVIEW

Another reason CK are usually thought of as being associated with roots is that they appear to be synthesized there. This became almost a textbook dogma— i.e., that roots are the predominant, or even sole, organs of CK synthesis. Although aerial organs, especially developing seeds, are thought also to contribute to CK biosynthesis, roots are still presumed to have the leading role (Letham, 1994). There are two consequences of this assumption. The first is that roots supply CK to organs of the shoot. The second is that CK in xylem reflects their synthesis in the root system. As a result, many studies have interpreted changes in the level or form of xylemborne CK in relation to shifts in nutritional status, particularly that of N, changes in soil temperature and stress situations such as water logging, salinity, drought, etc. In this way CK have been perceived to function as ‘‘signal’’ molecules, communicating to the shoot changes sensed in the root environment. However, despite the many apparent correlations uncovered, no direct connection has been made between xylem-mobile CK and a response in the shoot. In fact, Beveridge et al. (1996, 1997) showed that CK in the xylem sap did not control branching of pea. They used a mutant (rms4) in which CK content in xylem is reduced more than 40-fold and concluded that a shoot-derived signal was translocated to roots and regulated CK synthesis in situ. Apart from doubts about their site of synthesis, one of the serious

Historically, research into the biology of cytokinins (CK) has been closely associated with roots and root growth. In one of the earliest examples of hormone interactions in regulating plant differentiation and morphogenesis, Skoog (1994) reported a quantitative relationship between adenine and auxin in the formation of buds and roots. Later he found that CK were much more effective than adenine as potent inhibitors of root formation. Moreover, their ‘‘push-pull’’ relationships with auxin, which determine the degree to which shoots or roots differentiate from dividing callus, have been exploited as well-established procedures for routine manipulation of in vitro tissue culture systems (Krikorkian, 1995).

Abbreviations: CK, cytokinin(s); DHZ, dihydro-zeatin; (OG)DHZ, O-glucoside of dihydro-zeatin; [9R-MP]DHZ, dihydro-zeatin nucleotide; [9R]DHZ, dihydro-zeatin riboside; (OG)[9R]DHZ, O-glucoside of dihydro-zeatin riboside; iP, isopentenyl-adenine; [9R-MP]iP, isopentenyladenine nucleotide; [9R]iP isopentenyl-adenosine; Z, trans-zeatin; (OG)Z, Oglucoside of trans-zeatin; [9R-MP]Z, trans-zeatin nucleotide; cis[9RMP]Z, cis-zeatin nucleotide; [9R]Z, trans-zeatin riboside, cis-[9R]Z, cis-zeatin riboside; (OG)[9R]Z, O-glucoside of trans-zeatin riboside; cis-(OG)[9R]Z, O-glucoside of cis-zeatin riboside.

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uncertainties in assessing a signaling role for CK relates to their movement in long distance translocation channels, especially in the phloem. Although bioassays have shown CK activity in both xylem and phloem, reservations about possible contamination of exudates as a result of the sampling techniques used have arisen (Atkins, 1999). Only recently have definitive GC-MS methods been applied to analysis of transport fluids (Atkins, 1999; Emery et al., 2000). Even though there is an impressive information base of CK ‘‘phenomenology,’’ the precise nature of CK action in regulating processes of development and differentiation, of assimilation or the partitioning of resources in plants, remains largely speculative (Hare and Van Staden, 1997). Much of the data supporting their regulatory role(s) relies on the response of whole plants, explants, tissue cultures, or isolated cells to exogenous application of natural cytokinins or of their more stable analogs, such as benzyl-aminopurine (BAP). In very few cases have endogenous levels of CK been shown to mediate equivalent responses in vivo. Our understanding of the molecular basis for regulation of CK biosynthesis or their role in signaling has lagged well behind those of other hormone systems. Studies of auxin, ethylene, and gibberellin biology have benefited greatly from transgenic technology and the screening of targeted phenotypic mutants. However, this has not been matched in the case of CK (Binns, 1994). On one hand, even mild CK overproduction leads to inhibition of root development, while on the other, severely CK deficient mutants are likely to be lethal. Some Arabidopsis mutants have recently been identified as having altered CK responses, and the initial steps in identifying genes implicated in CK signal transduction pathways have been taken (Deikman and Ulrich, 1995; Cary et al., 1995; Kakimoto, 1996; Vogel et al., 1998a,b). There are also auxin–CK cross-resistance mutants (Coenen and Lomax, 1998). Further breakthroughs may be close at hand, as indicated by the newly characterized shooty mutant lines of Arabidopsis that may be linked to either CK signaling or to a loss in CK oxidase activity (Frank et al., 2000). However, as demonstrated by Auer (1996), better information about CK biochemistry and activity could lead to more effective selection screens for CK mutants. Accordingly, in tests of common methods of seedling screening procedures, results of exogenous application of CK in Arabidopsis root growth bioassays were closely dependent upon the structure of the CK, its concentration, duration of exposure and genotype.

Emery and Atkins

Ever since their initial description as factors that cause or stimulate the proliferation of plant cells in culture, CK have been regarded as factors that function in cell division in planta. More recently their role has been defined in terms of the cell cycle and DNA replication (Jacqmard et al., 1994), and this has been interpreted in relation to developmental processes that rely on cell division or on a change in the cell cycle state of a tissue. Expression of the cell cycle marker cdc2 coincides with a requirement for CK in cultured mesophyll protoplasts of tobacco in which cell division is reactivated (Carle et al., 1998). Both auxin and CK induce transcription of the B1-type mitotic cyclin genes, Cyc1 and Cyc4, in nodule and root meristems of yellow lupin (Jelenska et al., 2000). These studies suggested that while there are separate signal induction pathways for auxin and CK they actually converge before completion of the cell cycle. Furthermore, it has become accepted that because CK stimulate cell division they define in some way the ‘‘sink strength’’ of active meristems and establish ‘‘source-sink’’ relations in the plant. Clearly, the veracity of the root/ shoot signal concept for this group of plant growth regulators is central to establishing their significance in such relationships. CK appear to be involved in two well-characterized signaling pathways—a G-protein coupled receptor pathway, and a second involving a two-component phosphorelay system based on a membrane-localized histidine kinase (Estelle, 1998; D’Agostino and Kieber, 1999; Urao et al., 2000). Both systems provide an extracellular ‘‘sensory’’ mechanism. However, the linkages to translocated CK ‘‘signals’’ on the one hand and the many instances where CK have been shown to interact with regulatory genes (Rupp et al., 1999) or regulate cellular enzyme activities on the other (Ehness and Roitsch, 1997; Chen, 1997; Kakimoto, 1998), have yet to be established. Activation tagging has identified a series of mutants of Arabidopsis (Kakimoto, 1996) that show typical CK response in the absence of CK. The phenotype is due to overproduction of a hybrid histidine kinase (CKI1) that has the features expected of a sensor. Further, a number of the response regulators of the two-component systems (the A-type regulators—Urao et al., 2000) are induced by exogenous CK (Taniguchi et al., 1998; Kiba et al., 1999). Initially identified from etiolated Arabidopsis seedlings as the IBC6 and IBC7 genes (induced by CK) by Brandstatter and Kieber (1998), these genes (now called ARR4 and ARR5) are apparently induced in all tissues, including roots.

Roots and Cytokinins

A few CK binding proteins were isolated from the membrane fraction of cultured Arabidopsis cells (Brault et al., 1999). However, soluble high-affinity CK binding proteins have also been purified and characterized (Kobayashi et al., 2000). It thus seems likely that the many and diverse effects of CK on developmental processes in plants might be the result of a diversity of CK signaling pathways (Brault and Maldiney, 1999). Moreover, a number of specific ‘‘paracrine’’ roles within cells might be separate from those of signaling (Faiss et al., 1997). Yoon et al. (1999) have isolated and characterized a cDNA encoding a Ca2+-dependant protein kinase from tobacco (NtCDPK1). It is transcribed in roots, stems, and flowers but at very low levels in leaves. Application of a number of PGRs, including CK, stimulated transcription in leaves. The protein is expressed in a membrane fraction. CK and sucrose differentially regulate the transcription of another protein kinase (WPK4) belonging to the SNF1-related protein kinase family expressed specifically in the photosynthetic tissues of wheat seedlings (Ikeda et al., 1999). The physiological role of WPK4 in vivo has not been defined but it is likely to be involved in regulation of C metabolism through protein phosphorylation. The upregulation by CK and downregulation by sucrose does, however, represent the first instance in which CK are directly linked to what might be described as an element determining ‘‘assimilate supply.’’ Given this evolving picture, it is likely that similar relationships at the molecular level will be revealed for root functions. While there is little doubt that CK function as a central regulatory component(s) in plant development, our knowledge of CK biology is further hampered by the lack of definitive evidence for their de novo synthesis in higher plants (cf. Prinsen et al., 1997). There is no doubt that plants synthesise the purine ring de novo, but attempts to unequivocally demonstrate the activity of DMAPP:AMP isopentenyl transferase (IPT), or the presence of an ipt gene similar to that found in bacteria, have not succeeded. Alternative schemes, such as the release of CK from the turnover of tRNA (Murai, 1994), have been proposed, but none has proven to be the obvious solution. These considerations, along with an array of supporting circumstantial evidence, have led Holland (1997) to put forward the provocative hypothesis that all CK found in plants is due to synthesis by bacteria living symbiotically on or as endophytes within plant organs. Whether or not bacteria contribute to the plant’s CK

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complement, these considerations raise considerable uncertainty about the site(s) of synthesis and impact directly our understanding of the relationships between this group of regulators and roots. II.

EFFECTS ON ROOT GROWTH

A.

Reduced Root Mass and Elongation

The sensitivity of root growth to inhibition by CK is best exemplified by efforts to transform plants with the ipt gene cloned from Agrobacterium tumefaciens (Klee, 1994). Once transformed, shoots that have elongated sufficiently from callus or from tissue explants in culture are transferred to a root-inducing medium. A recent exception is described by Soh et al. (1998), who found that callus from cowpea hypocotyls only exhibited rhizogenic potential with media containing CK. Media containing CK and auxin or auxin alone were not effective in inducing adventitious roots. Generally, however, root induction in dicotyledons requires a high auxin-to-cytokinin ratio, and even low levels of ipt expression can completely suppress root formation. Thus, whole plants are not obtained easily from transgenic explants that carry the ipt gene with a ‘‘leaky’’ promoter and grafting transformed shoots to a rootstock may be needed to recover seed (Braun and Wood, 1976; Pigeaire et al., 1997). This problem was overcome by transforming plants with constructs of the ipt gene under the control of tightly regulated heat shock promoter (hsp70, Smigocki, 1991; Rupp et al., 1999) or tetracycline-dependent CaMV35S elements (Faiss et al., 1997) so that CK overproduction was conditional. The reduced root mass in these transgenics indicated that CK not only inhibit root initiation, but also root growth in general. Furthermore, reduced root mass (also observed by Roeckel et al., 1998) may contribute to pleiotropic effects, such as reduced stature and leaf size, observed in a number of CK-overproducing transgenics (Medford et al., 1989; Smigocki, 1991; Smigocki et al., 1993; Harding and Smigocki, 1994; Thomas et al., 1995). The inhibition of root growth by CK apparently interacts strongly with effects of other hormones. Studies on auxin-resistant mutants that are also crossresistant to CK (tomato, diageotropica [dgt] mutant) indicate that CK possibly inhibits root growth by increasing active auxin pools to inhibitory levels (Coenen and Lomax, 1998). Likewise, studies using Arabidopsis mutants ckr1 and ein concluded that inhibition of root growth, following exogenous application of

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CK, may be mediated by increased production of ethylene (Cary et al., 1995). Control of adventitious rooting likely involves auxin and CK interplay. Very little is known about the branching of roots, but it is thought to proceed like apical dominance in shoots except that the nature of the response to CK and auxin is reversed (see also chapter by Lloret and Casero in this volume). For example, exogenous CK or increasing endogenous CK with a CK oxidase inhibitor, 4PU-30, can strongly inhibit lateral root formation in Lactuca sativa seedlings (Zhang and Hasenstein, 1999). CK is envisaged to be a root tip–derived inhibitor that counters auxin arriving from the shoot for lateral root stimulation. Dewitte et al. (1999) have applied a highly sensitive and specific immunocytochemical method to reliably detect CK in aldehyde-fixed sections of shoot apices of tobacco. This method of in situ localization coupled with HPLC-tandem MS analysis of extracts has provided, for the first time, both qualitative and quantitative information on the cellular distribution of individual CK forms. Their studies were directed specifically at the transition of shoot apices from vegetative to floral stage. They showed unequivocally that free CK bases essentially disappeared during the reorganization of the meristem just prior to flowering, bringing into question the long-held view that CK are positive regulators in the switch from a vegetative to a reproductive meristem. The immunocytochemical analyses were sufficiently sensitive to show, for example, that dihydrozeatin (DHZ) and isopentenyladenine (iP) were cytoplasmic and perinuclear while trans-zeatin (Z) was localized within the nucleus. These tools used in tandem hold promise of identifying CK-driven events in other tissues including those of root meristems and in the lateral roots and nodule primordia induced by other signals. B.

Promotion of Nodulation

CK applied to roots of various legumes induce cell division in the cortex and formation of nodulelike structures (pseudonodules) that are devoid of rhizobia (Dart, 1977). This is also the case for roots of the actinorhizal plants of the genus Alnus (RodriguezBarrueco and Bermudez de Castro, 1973). These findings, as well as a number of other studies (cf. Hirsch, 1992), led to the concept that a gradient of plant growth regulators derived from the vascular system of the root interacts with bacterial signals to initiate nodule development (Libbenga and Bogers, 1974). This idea is supported in the unfolding molecular understanding of the early events of nodule initiation.

Recent evidence has revealed a complex exchange of plant and bacterial signal molecules that interact in establishing the symbiosis and account, at least in broad terms, for the specificity between the host and bacterium. The root produces a suite of specific flavonoid molecules that not only serve as chemo attractants but also induce the expression of nodulation (nod) genes in the bacteria (Phillips, 2000). The nod genes encode a suite of enzymes that lead to the synthesis of nod factors, lipochitoligosaccharides (LCOs), that precede bacterial entry and cause dedifferentiation of root cortical cells to establish a new primordium (Long, 1996; Day et al., 2000). One of the host genes expressed early in nodule organogenesis, Enod2, was found to be induced by auxin transport inhibitors (Hirsch et al., 1989) and CK application (Hirsch and Fang, 1994). Enod2 encodes a putative hydroxyproline-rich cell wall protein and is expressed exclusively in the nodule peripheral parenchyma (Chen et al., 1998). Recently Jimenez-Zurdo et al. (2000) demonstrated CK-induced expression of seven Msnod transcripts in alfalfa roots, four of which were also regulated by auxin. CK-mediated enhancement of expression appears to occur posttranscriptionally but whether parenchyma-specific and CK upregulation share the same regulatory elements in the transcript is unknown. The development of cortical and/or pericycle cells to establish the site for a nodule is controlled by positional information in the form of a signal from the stele that determines the susceptibility of these cells to mitogens like CK (Pawlowski and Bisseling, 1996). Uridine has been identified as a possible ‘‘signal’’ (Smit et al., 1995), but its source and how it interacts with CK/auxin and LCOs to establish the site for a nodule primordium are unknown. In addition to serving as a factor in the establishment of nodules, CK produced by Rhizobium bacteroids (Torrey, 1986) may also influence development of nodules and their supporting roots, or even the shoot (Upadhyaya et al., 1991), once a symbiosis is established. III.

SYNTHESIS OF CYTOKININS BY ROOTS

A.

Are Roots Always the Primary Site of CK Synthesis?

The obvious associations between roots and their response to CK, coupled with the observation of CK in the transpiration stream, have perpetuated the frequently unverified assumption that roots are the pri-

Roots and Cytokinins

mary source of CK in plants. Even very recent studies assume that ‘‘CK are predominantly root-borne phytohormones distributed in the shoot via the xylem stream’’ (Lopez-Carbonnell et al., 1998). Such a generalization should be considered cautiously. Letham (1994) has reviewed data supporting the idea that roots are a major source of CK. Briefly, the lines of evidence include exudation of CK from aseptic cultures of roots and root tips, marked CK accumulation following adventitious root formation and formation of lateral root primordia, pronounced increase in root CK levels in response to shoot decapitation, presence of CK in the xylem of a diverse range of species, and the ability of exogenous CK to substitute for roots in inducing or maintaining growth and/or physiological responses of the shoot. One critical piece of evidence missing from the list is an unambiguous demonstration of conversion of isotopically labeled precursors into CK in excised roots. The studies that have addressed this question, and reported affirmative results, appear to be significantly flawed owing to inadequate purification of labeled products, unrealistic claims of label incorporation rates, or inconsistent conclusions drawn from product characterization protocols (Letham, 1994). B.

CK Production in the Shoot and the Importance of Translocation to the Roots

Roots are unlikely to be the sole source of CK, and, thus far, developing seeds, cambial tissues, and the shoot apex have been implicated as possible sites of synthesis (Letham, 1994). Developing seeds, in particular, are likely to be CK sources since, in some instances, CK accumulation in situ far outweighs the amount that might be furnished by translocation from roots (Davey and Van Staden, 1979; Brenner and Cheikh, 1995; Morris, 1997; Emery et al., 2000). Calculations based on estimates for the water economy of developing Lupinus albus fruits predicted that the accumulated CK could not possibly be supplied by the CK content of xylem sap leaving the root system (Zhang and Letham, 1990). A more recent study incorporating estimates of CK delivery in both xylem and phloem supports this conclusion (Emery et al., 2000); the combined fluxes of CK in the xylem and in the phloem contributing <1% of CK accumulating in the fruit. Emery et al. (2000) concluded that, at this site of intense CK accumulation, in situ synthesis is the major contributor. In developing fruits of cowpea (Vigna

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unguiculata [L.] Walp), diurnally reverse xylem flow to leaves has been documented (Pate et al., 1985; Peoples et al., 1985), providing a possible mechanism that would result in CK produced in seeds being eventually loaded onto phloem in vegetative organs. In this way the seed would be not only a major site of CK synthesis but also a major source of these regulators for the plant as a whole, potentially exerting some regulatory ‘‘influence’’ of the developing seeds on events elsewhere, possibly even in roots. Finally, most studies have neglected the potential for phloem to xylem exchange of CK in roots and their recirculation to the shoot. This type of circulation has been clearly demonstrated in Lupimus albus for nitrogenous solutes (Layzell et al., 1981), inorganic cations (Jeschke et al., 1987), and ABA (Wolf et al., 1990), and in Ricinus communis for solutes of N and S (Clarkson and Saker, 1990). There are no experimental data that have addressed this possibility. But, given the apparent mobility of CK in phloem (Komor et al., 1993; Taylor et al., 1990; Emery et al., 2000), it seems unwise to assume that the level of CK in the transpiration stream is an accurate reflection of synthesis in the root. The importance of this point cannot be overemphasized in the interpretation of studies trying to establish which organs are sources of CK or that attempt to establish the occurrence of chemical signaling from root to shoot and vice versa. Beveridge et al. (1997) have interpreted the influence of the shoot on CK content of xylem in the rms mutant of pea as an indication of a signal transferred to the roots where synthesis is altered. The alternative, of reduced phloem translocation of shoot synthesized CK, is not considered. Also related to the question of shoot to root CK recirculation is the potential for shoots to signal roots through CK. This has not been tested, but given new data that in certain phenological stages shoot tissues, such as developing seeds, may be a major source of CK, this possibility could be explored by paying more attention to CK in phloem exudates sampled at the stem base. Until the mid-1990s, only partial CK profiles for phloem were established from a handful of bioassay studies, and from only three papers that used more rigorous identification, such as GC-MS or immunoassay (Hoad, 1995). The reason for CK delivery to roots being largely ignored is the difficulty in identifying and quantifying CK in phloem. For most plant species the only method of sampling phloem is by making incisions on stems, petioles, or fruit tips to expose the vasculature and soaking the cut ends of the organ in a buffer containing a chelating agent like EDTA. There

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are many potential problems with such an approach since it is likely to abstract solutes from the apoplast of adjacent tissues including the xylem. It is also not quantitative, since there were no consistent relationships between phloem metabolite quantity and soaking time (Weibull et al., 1990). Despite these shortcomings the method has been used in a semiquantitative manner to study circulation of CK in Sinapsis alba and Chenopodium rubrum (Lejeune et al., 1994, Macha´c˘kova´ et al., 1996). Uncompromised quantitative data exist only for Lupinus sp. and Ricinus communis, species that exude phloem sap spontaneously after sieve tubes are severed. Even for these species there are few rigorous analyses. Our own observations (Atkins, 1999; Emery et al, 2000, unpublished results) and those of Komor et al. (1993) indicate that both the forms that are present and the quantity of CK in phloem are highly variable. In our experience, even GC-MS analysis of exudates collected in milliliter-range volumes, with excellent recoveries of labeled internal standards, may detect no CK in particular samples. On the other hand, we have occasionally measured quantities of [9R]iP or CK-nucleotides that far exceed that of any CK form ever observed in the xylem. Although incomplete, another definitive record of CK occurrence in the phloem provided GC-MS full scans of Z and [9R]Z in R. communis (Kamboj et al., 1998). For this species, comparison of the various profiles between xylem and phloem collected from stems supported the idea that there may be vascular recirculation of CK through the root. Future work to quantify the contribution of rootproduced CK to the plant should take into consideration the potential of transfer or exchange of CK between xylem and phloem, recirculation, and phloem delivery of shoot-produced CK to roots.

C.

Localization of Root CK Synthesis and the Role of Microorganisms

There is a widely held view that CK biosynthesis in roots is localized at the tips (Letham, 1994). This is based on studies that show a gradient in CK concentration, which is greatest at the tips and decreases toward more proximal sections of primary roots. However, root cap removal causes a dramatic decrease in CK content in the tip, suggesting localized synthesis in cells surrounding the meristem. In mature roots CK levels may increase again over regions of lateral root primordia.

There is little doubt that roots contain substantial levels of CK and that they load CK onto the transpiration stream. In addition to the possibility of circulation from the shoot via phloem, microbes associated with the root or in the rhizosphere could contribute to the CK in xylem. Apparently a wide range of microbes associated with plants in the rhizosphere, in the soil, or in a symbiotic association produce CK (Greene, 1980; Costacurta and Vanderleyden, 1995; Frankenberger and Arshad, 1995; Upadhyaya et al., 1991). Indeed, in one study up to 90% of bacteria isolated from the rhizosphere of various crop plants were shown to be potential producers of CK (Barea et al., 1976). Given the intimate relationship of rhizosphere organisms with the root surface, this represents a potentially significant exogenous CK supply. Considerably more is understood of the molecular processes of CK biosynthesis in microbes (Gaudin et al., 1994) than in plants (Prinsen et al., 1997). For example, the enzyme for the reaction that controls the addition of an isopentenyl side chain to the purine ring, which forms the earliest occurring CK (either [9RMP]iP or [9R]iP), is considered crucial for controlling levels of CK found in plants. Genes for this step are only known from bacteria and include isopentenyl tranferase, (ipt), trans-zeatin sythetase (tzs), and Pseudomonas trans-zeatin synthetase (ptzs). Failure to clone a plant ipt gene has questioned assumptions about biosynthetic pathways (Prinsen et al., 1997) and even placed their occurrence in plants into doubt (Holland, 1997). On the other hand, there has been recent progress in our understanding of the metabolism of CK (Rinaldi and Comandini, 1999a,b). CK oxidase (Morris et al., 1999) has been cloned and sequenced from maize, and both O-glucosyltransferase (ZOG1; Martin et al., 1999a) and O-xylosyltransferase (ZOX1; Martin et al., 1999b) have been described. It is believed that glycosyl conjugates of CK are important as temporary stored forms of CK that are resistant to degradation by CK oxidase. ZOX1 is highly expressed in developing seeds of Phaseolus vulgaris, but Northern analysis detected only a low level of expression in vegetative tissues of this species and no transcripts in roots (Martin et al., 1999b). What remains undetermined is the extent to which CK synthesized by microorganisms contribute to the effective complement of the plant CK profile and, further, to what degree these control plant growth. At one extreme is the hypothesis, proposed by Holland (1997), that plants are incapable of manufacturing their own CK, and rely totally on that produced by microbial symbionts. It is an intriguing idea, but

Roots and Cytokinins

one that is very hard to prove or disprove. Microbes are ubiquitous on or in all plant organs and perhaps are even present in small numbers in tissue culture systems thought to be axenic (Holland and Polacco, 1994). In the absence of a technique that could definitively show the bacterial contribution of CK separate from that of the plant, it would be most prudent, especially in studies of roots, to assume that bacteria contribute at least part of what is detected in plant tissues and the transpiration stream. IV.

CK METABOLISM IN ROOTS

A.

Endogenous CK in Root Tissue

Given the strong associations between CK and roots, and their synthesis in roots in many circumstances, surprisingly few researchers have analyzed CK directly from root tissues. Most of what we know about their occurrence in roots is inferred from measurements of xylem exudates. As noted above, the potential for phloem-to-xylem transfer complicates such inferences since xylem sap CK could be potentially shoot derived. To better understand how CK synthesis in roots might occur, one should therefore consider what forms and quantities are present in root tissues, especially the actively dividing root tips. Surprisingly, there has been no comprehensive and definitive study of CK form and quantity during root development. This knowledge gap is significant when coupled to the slow progress in isolating enzymes of the CK biosynthetic pathway and cloning of corresponding genes. Consequently there is great uncertainty over just how CK biosynthesis occurs (Prinsen et al., 1997). In particular, there is no convincing evidence that demonstrates which forms predominate early in the synthetic pathway. As discussed above, the effects of CK on root development have been fairly well studied and they undoubtedly influence events in roots. However, questions about how this group of compounds regulates root growth and what forms are active in the regulatory processes remain to be answered. Definitive identification of CK present in root tissues is sparsely scattered across a variety of species and developmental stages. The most valuable records come from two studies (Wagner and Beck, 1993, Dieleman et al., 1997). As part of a whole-plant CK inventory and using immunoassay after HPLC separation, Wagner and Beck (1993) checked for an impressive array of 20 CK in Urtica dioica, including the (often ignored) cis-isomers. The roots had similar quantities of CK as

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shoot tissues, but the profiles were different. Roots were dominated by [9R]Z and, surprisingly, a CK rarely even looked for, cis-(OG)[9R]Z. Z, [9R-MP]Z, [9R]iP, and [9R-MP]iP were present in minor amounts. Unlike the shoots, the CK profile in the root was sensitive to nitrate supply, and significant changes in ribosides and nucleotides were reported. Interestingly, the profile in roots did not directly correspond to that of the xylem, which consisted mainly of [9R]Z, a low quantity of Z, and about six other forms that were assumed to be contaminants because of their very low concentrations. The best developmental analysis of CK in roots was also part of a complete plant CK inventory (Dieleman et al., 1997). The researchers used a battery of exploratory and quantitative techniques including immunoaffinity chromatography, HPLC, GC-MS, LC-MS, and enzyme immunoassay to describe what happened to the CK complement during bud break and lateral shoot outgrowth in Rosa hybrida. Throughout the study the root CK forms did not change. [9R]Z, [9RMP]Z, and an unidentified CK were the major forms, with [9R-MP]iP, [9R]iP, and Z present in minor amounts. However, the relative quantities of all but Z and the unknown CK decreased substantially in roots with plant age following bud break and lateral shoot extension. Other important studies used Phaseolus vulgaris (Scott and Horgan, 1984), Pseudotsuga menziesii (Doumas et al.,1989), Nicotiana tabacum (Faiss et al.,1997), and Chicorium intibus seedlings (Vuylsteker et al., 1998). The work of Scott and Horgan (1984) is frequently cited in discussions of root CK identity. Their results indicated, like those of Wagner and Beck (1993), that the major CK present in roots was an O-glucosyl conjugate, (OG)Z, which was not found in the xylem. However, this was the only CK for which a mass spectrum was obtained. All others, including Z, [9R]Z, and (OG)[9R]Z, were only tentatively identified by HPLC retention time. Furthermore, any CK having low activity in the Amaranthus betacyanin bioassay (e.g., any cis-CK) would have been overlooked, since this test was used as a preliminary identification. The results of Doumas et al. (1989) reinforce the importance of glucosyl conjugates in roots since they found that for six CK identified by RIA (Z, DHZ, [9R]Z, [9R]DHZ, [9R]iP, and iP), the corresponding glucosides were present in much greater amounts. Vuylsteker et al. (1998) also found O-glucosides accounted for one-third to one-half (of a suite of 10 CK), and Faiss et al. (1997) reported (OG)[9R]Z to account for 45% of a total of 16 CK. These studies

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also report isopentenyl-type CK, [9R-MP]iP, and [9R]iP in roots. If indeed there is CK synthesis in roots through a mechanism similar to the activity of IPT, then addition of DMAPP to the N6 position of AMP would yield [9R-MP]iP and by phosphatase hydrolysis, [9R]iP. A host of other work has involved some CK analysis in root tissue, but it all suffers from limitations in the range of CK forms investigated. Much of the problem is related to the lack of availability of mass spectrometry equipment and therefore necessary reliance on immunoassay. While immunoassays can be just as effective as mass spectrometry in covering a range of previously identified CK, they must be used with care, taking into consideration the cross-reactivities of antibodies, and should only be used on highly purified samples after thorough HPLC separation. Even so, information is lost when levels of CK ‘‘groups’’ (i.e., free bases, ribosides, or nucleotides) rather than individual CK are reported (Kuiper et al., 1989; Kudoyarova et al., 1998). Many other studies, using a limited choice of antibodies or purification techniques, have mainly concentrated on quantifying one or more of four classically regarded CK, including Z, [9R]Z, iP, and [9R]iP (Udomprasert et al., 1995; Kwak et al., 1996; Nan et al., 1999; Reiber and Neuman, 1999a,b; Yang et al., 2000). The latter type of study usually seeks to correlate total root CK quantity with the sensing of root environmental change or root-to-shoot signaling. This would only be valid if the major CK are known (and are all included in analyses) and if one can assume that sensing of an environmental change does not implicate a change in CK form. Since we know so little about such matters, studies to date must be regarded as preliminary. Moreover, they are unlikely to contribute to elucidating the importance of CK form and function in roots. At this point it appears that few generalizations can be made about root CK synthesis and metabolism. However, some crude similarities among studies can be pointed out, bearing in mind that they are based on few species and usually randomly chosen developmental stages. 1. There is an apparent lack of dihydro-CK (i.e., DHZ, [9R]DHZ, [9R-MP]DHZ), which may implicate an important role for CK-oxidase activity as a means of tightly regulating root CK. This stems from the finding that dihydro-CK are the most resistant to oxidase breakdown (Jones and Schreiber, 1997). In turn, tight regulation of CK levels in the root may play a central role in regulating levels in the whole plant considering

Emery and Atkins

the results of Motyka et al. (1996) obtained with ipttransformed tobacco. Endogenous CK was increased conditionally, following derepression of tetracylcine dependent ipt transcription. When measured in intact plants, only the roots showed a substrate-induced increase (about 100-fold) in CK oxidase activity. 2. In the variety of root tissues measured to date, the ribosyl-CK are ubiquitous, with [9R]Z as one of the predominant forms. [9R]Z is highly active in all known bioassays and is frequently identified in root xylem exudate, indicating that it is also highly xylem mobile. 3. The occurrence of isopentenyl-CK is confusing, since they were absent in the majority of investigations that searched for them, but present as predominant forms in two studies. This may have been due to the high turnover of isopentenyl-CK, as has been observed in other plant tissues (Prinsen et al., 1997), or due to differences in plant development among studies. When these forms have been detected in high concentrations, it has been in extracts from either roots of young seedlings or actively growing root tips (i.e., Doumas et al., 1989; Vuylsteker et al., 1998). 4. Nucleotide CK are always present, and, in cases where careful analytical methods have been used, as the predominant forms. It is highly probable that they are more common than most of the literature indicates. Many studies find that the major CK in roots are ribosides. However, Hammerton et al. (1996) analysed Phaseolus roots and determined that, if special care was taken to limit nucleotide breakdown during extraction, [9R-MP]Z and [9R-MP]iP dominated the CK profile. Thus nucleotide CK may not be late-formed conjugates; a nucleotide pathway possibly precedes the appearance of free bases or ribosides. It is not clear whether hydroxylation of isopentenyl CK takes place at the base, riboside, or ribotide level (Prinsen et al., 1997). However, a recent in vivo labeling study in which plants were incubated in media enriched with D2O, found a higher enrichment in [9R-MP]Z compared with the corresponding nucleoside, consistent with the idea that CK nucleotides are initial products of the synthetic pathway (Astot et al., 2000). 5. O-glucosyl CK are often found in large quantities in roots, and accumulate in particularly high concentrations in roots of ipt-transformed tobacco (Faiss et al., 1997). They are thought to be storage forms of CK that can be mobilized by reversing their conjugation with sugar, and do not seem to be very mobile in xylem (for an exception see Badenoch-Jones et al., 1996). This may mean that CK synthesized in excess

Roots and Cytokinins

by roots are stored as O-glucosides and -oxidized when needed to modulate root growth or to send a signal to the shoot. However, this is by no means clear since the glucosides appear to fluctuate independently of free CK in response to treatments like reduced-N or exogenous auxin (Wagner and Beck, 1993; Vuylsteker et al., 1998). They may therefore have an independent role in root growth. The occurrence of cis-(OG)[9R]Z as the major form of CK in Urtica roots is particularly curious, raising the question of whether the occurrence of cis-CK in roots has in many cases simply been overlooked. These forms are the main CK in potato tubers, for example (Suttle, 2000), and can be nonenzymatically isomerized by exposure to light (M. Mok, personal communication), leaving the possibility open for an upregulation of CK activity during transfer from root to shoot (or in the sprouting of tubers). B.

CK in Xylem

There is a vast literature on the movement of CK in xylem sap from the root to the shoot. Like other root tissues, the forms of CK in xylem are not always adequately assessed because of limitations in the analytical methods used. Furthermore, it is often assumed that their spectrum is unchanging in response to developmental status or environmental variations. It appears as though the ribosides, [9R]Z or [9R]DHZ, are most often found as the predominant forms in xylem perhaps qualifying them as ‘‘transport’’ CK (Grayling and Hanke, 1992; Wagner and Beck, 1993; Letham, 1994; Dieleman et al., 1997). However, there are many exceptions, and such a generalization may be misleading. The importance of determining the form of CK has been emphasized by numerous reports of complex CK profiles in xylem from a variety of species (cf. Letham, 1994). Twelve different CK were identified in the xylem of wheat seedlings with similar concentrations among free bases, ribosides, and O-glucosides (BadenochJones et al., 1996). They found similar results for oat, and at no point did [9R]Z or [9R]DHZ predominate for either species. In other cases, such as rice, chickpea, and white lupin, the nucleotide-CK are the primary xylem-mobile forms (Murofushi et al., 1983; Emery et al., 1998, 2000). As is the case for root tissues (Hammerton et al., 1996), it seems likely that CK nucleotides would be more commonly identified if more care were taken to prevent their breakdown to ribosides. With developmental change, profiles of CK forms in the xylem sap change. In soybean, in addition to

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the ribosides, considerable quantities of free base, Oglucoside, and nucleotide CK were found (Nooden and Letham, 1993), but at some stages a free base DHZ predominated. Similarly, analysis of xylem sap of lupin at a single stage of development implicated [9R]Z and [9R]DHZ as the major transport forms (Jameson et al., 1987). However, our study of lupin xylem exudates found 18 possible forms of CK and uncovered sharp changes in their profile during fruit development (Emery et al., 2000). Following abscission, and during early pod set on the mainstem inflorescence, cis-[9R-MP]Z was the major xylem form; the only others were minor quantities of the ribosides (cis-[9R]Z, [9R]Z, [9R]DHZ). However, later during seed filling, cis-[9R-MP]Z all but disappeared and there was a switch to high concentrations of [9RMP]DHZ, [9R]Z, and [9R]DHZ and the appearance of O-glucosides. The processes that lead to these relatively rapid changes of composition in xylem are unknown and there is no information about the site(s) where changes occur. V.

CYTOKININS AS AGENTS FOR COMMUNICATION BETWEEN ROOTS AND SHOOTS

A.

Confirming a Translocational Role for Cytokinins

From the above discussion it is apparent that xylem export of CK to the shoot may be more complex biochemically than was generally assumed. CK are versatile enough to encode signals sent to the shoot to communicate numerous root environmental stresses and to elicit many separate, or a general syndrome of, physiological changes. Consequently, distinguishing among the various roles of CK in root-to-shoot communication is a formidable, but important, task. Jackson (1993) critically reviewed data available up to the early 1990s. In essence, there were thorough physicochemical analyses that implicated xylem CK in the control of a number of physiological processes. These were leaf senescence, stomatal opening, root-toshoot partitioning, seed set, and other morphological changes in the shoot in response to a variety of stresses sensed by the roots, such as mineral deficiencies, flooding, and drought. Despite the rigorous nature of the analyses, the studies were considered problematic, as they failed to account for dilution factors, xylem flow rates, and, hence, actual root-to-shoot delivery rates. Since sap collection usually necessitates decapitating plants, a procedure that drastically lowers flow com-

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pared to intact transpiring plants, CK concentrations do not necessarily reflect delivery rates. The tone of Jackson’s (1993) review was one of disappointment since so much experimentation had not revealed the physiological importance of seemingly considerable and environmentally-sensitive alterations of xylem CK. Advancements have been made that to some extent overcome the shortcomings of earlier studies. However, other concerns have evolved. Most studies consider the CK concentration or delivery rate exiting the roots in the xylem sap as correlative evidence of root-to-shoot signaling. For example, in studies of plant response to a drying soil, attempts were made to address the concept of delivery rates. Previously two reports concluded differently; one that drought made little difference (Masia et al., 1994), the other that drought significantly reduced concentrations of CK in xylem sap (Bano et al., 1993). In response to this apparent conflict, Shashidhar et al. (1996) used a root pressure chamber to approximate the rate of transpirational flow in intact sunflower plants and calculated CK delivery rates from the analysis of xylem sap. They found that CK delivery declined markedly by subjecting the roots to drought. However, the CK concentrations were based on an ELISA assay of unpurified sap. No separations of CK were attempted, and the analyses used a single antibody that crossreacted well with only [9R]Z and Z. Thus, it is possible the quantification overlooked significant changes in CK that were not reactive toward the antibodies used. Furthermore, to recreate water potential differences, and hence flows in intact plants, the pressures employed to drive the sap upward were logistically quite high (up to 1.5 MPa). Moreover, it was recently shown that pressure-xylem flow curves for detopped plants saturated at much lower pressures, even in highly water-conductive plants like tomato (Emery and Salon, 2001). Depending on the species, saturation of flow could occur at much lower rates than those of transpiration. This means that water uptake dynamics of detopped plants might differ from those that occur in situ. Better physiological techniques will be required to determine delivery rates of xylem solutes. The actual cellular uptake of CK offered by the transport fluids may be the controlling factor in shoot response to potential root signals (Kaminek et al., 1997). However, the CK profile observed exiting from detopped plants at the base of the stem may well be altered on its passage upward in the stem and petioles. For example, radioactive CK supplied through a wick into the xylem of derooted shoots indi-

Emery and Atkins

cated that large amounts, perhaps even the majority, undergo rapid radial transfer within the stem (Jameson et al., 1987). CK may be loaded into the phloem and translocated further before being transferred back to the xylem. Consistent with this idea, a progressive enrichment from the stem base to developing inflorescences has been observed in white lupin (Taylor et al., 1990; Emery and Atkins, unpublished). What is more, once in the phloem CK could circulate back to the roots, perhaps as a shoot-to-root signal. B.

CK Form and Signaling

Despite the longstanding idea that translocated CK constitutes a signal, the chemical form of CK that represents the signal molecule(s) has not been defined. The importance of assessing CK form in relation to separate plant responses was highlighted in a study of CK in xylem sap from oats and wheat (BadenochJones et al., 1996). This study demonstrated the potential complexity of CK profiles in xylem, the complement comprising free bases, ribosides, nucleotides, and O-glycosides including dihydro-derivatives and both cis- and trans-isomers. In a strategy of profile matching, the CK identified from the xylem were tested at multiples of their endogenous concentrations in sap in senescence and transpiration bioassays. The sampled CK concentrations were considered relevant in terms of shoot delivery since sap collections were only made from the first 5 mm3 of root pressure exudate. This first sampling is thought to quantitatively represent the sap in transit at the time of cutting the shoot (although this sap may also be artificially concentrated from the cutting process; Else et al., 1994). The results are remarkable because they demonstrated that different entities of a complex CK xylem profile may elicit different physiological changes in the shoot. For example, Z was consistently the most active form that increased transpiration, although ribosides and O-glucosides also had a promotive effect. Most surprisingly, however, was the finding that O-glucoside CK had a potent senescence-retarding effect, which was much greater than any similar effect of the free base or riboside forms. This places a group of CK that had been previously dismissed as xylem immobile front and center in potential root control of shoot senescence. Other findings indicated that nucleotide- and cis-CK were relatively inactive. It may turn out that the more inclusive studies of xylem CK become, the more is learned about CK form and shoot signaling. Other correlative evidence comes from our experience with changes in xylem CK form

Roots and Cytokinins

with development (Emery et al., 2000). Quantitatively it is possible that the root may completely supply newly fertilized ovaries of Lupinus albus with all the CK they require, which, at anthesis, is mainly composed of cis-isomers. After pod set and during seed maturation, the CK complement in xylem is compo sed mainly of trans-CK. Taken together, this may indicate developmentally specific roles for different forms of CK in xylem. C.

Communicating the Root Environment to the Shoot

Nitrogen availability influences the relative sink strengths of the shoot and root, and therefore is a major determinant of shoot:root ratio (Beck 1996). In a series of studies, the Urtica dioica model plant was used to overcome most of the concerns that had hampered previous efforts to establish CK as important root-to-shoot signaling agents. U. dioica was used at a constant preflowering developmental stage and cultured under controlled growth conditions for which the xylem CK content had been rigorously characterized (Wagner and Beck, 1993). The compounding problems of xylem dilution and calculations of shoot delivery rates were circumvented by adopting an apparatus that permitted collection of xylem sap from intact plants at actual transpiration rates. Manipulations of root N supply indicated that daily CK gain by the shoot via the xylem correlated positively with shoot:root ratio. Confirmation that CK delivery represented an effective signal was provided by observing the significant reversal of 14C-labeled photosynthate flux from roots to shoots following [9R]Z feeds into the xylem. Although less complete, other investigations corroborate the conclusion that CK communicate aspects of nutrient availability to the shoot. Moreover, studies of root CK signaling in response to different stresses are starting to converge. Ma et al. (1998) related xylem delivery of CK with that of amino acids and found that relative CK delivery decreased with transient N deficiency and subsequent declines in yield of narrow-leafed lupin (Lupinus angustifolius L.). In terms of mechanistic ties between N availability and CK production, there are some hints that there is localized increase in CK by roots actually in contact with high levels of nutrients (Ivanov et al., 1998). There is some molecular evidence for CK mediating the response of both Arabidopsis and maize to increased NO3 supply (Taniguchi et al., 1998; Sakakibara et al., 1999; Kiba et al., 1999) through differential expression of genes for response regulators.

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As yet, however, the relationship of CK content or form to such signal transduction pathways is not clear. A logical, but yet untested, model has proposed that CK and the mass flow of sugars are closely linked and that this might explain the opposite growth patterns of roots and shoots following a phase of N deprivation (Van der Werf and Nagel, 1996). Accordingly, soon after a decline in N supply, CK production and its delivery to shoots are reduced. Because lower CK affects shoots and roots differentially, cell division and expansion, and thus demand for sugar, would increase in roots and decrease in shoots. This would lead to greater osmotic and pressure gradients in roots and, as a consequence, relatively higher rates of delivery of sugar. Nitrogen deficiency also appears to be linked to CK involvement in root response to temperature stress. Ali et al. (1996) observed that during periods when root temperatures were low enough to reduce yields of tomato, nitrate delivery to the shoot declined, although apparent root nitrate acquisition remained stable. Coincidentally, delivery of Z and [9R]Z to the shoot declined, as indicated by a constant xylem CK concentration but significantly lower transpiration rates. In a similar way, yields of inbred Zea mays sensitive to chilling stress may be limited by root to shoot CK supply (Lejeune et al., 1998). Transpiration and concentrations of two CK, [9R]Z, and [9R]iP declined with root chilling, leading to a severe reduction in their delivery to shoots. Interestingly, CK apparently do not play an important role in signaling a root response to heat stress to the shoot. In two Phaseolus species with contrasting heat tolerance, Udomprasert et al. (1995) reported that the heat tolerant P. acutifolius had considerably higher [9R]Z and [9R]iP in roots and leaves than two genotypes of the less tolerant P. vulgaris. Furthermore, high shoot or root temperatures decreased root [9R]Z levels in all plants tested. However, heat stress to either roots or shoots caused very little change in quantities of the CK assayed in the shoots of any of the genotypes, and exogenous [9R]Z pretreatment or the use of grafted heat-sensitive shoots to tolerant rootstocks, did not alleviate the injurious effects of heat on photosynthetic activity. Similarly, delivery of CK from roots to shoots was not implicated in regulating stomatal response to high root temperature (Dodd et al., 2000), nor was xylem [9R]Z concentration correlated with rising temperature effects on bud break of Rosa hybrida (Dieleman et al., 1998). While the importance of CK root-to-shoot signaling is still not completely clear, there appears to be

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some compelling evidence for an important role in some shoot responses. However, the opposite conclusions reached from research of root cooling versus heating serve as a caution. Different scenarios of root environments, species, and developmental stages will have to be closely considered for effects on welldefined physiological shoot responses before a general model for CK control of shoot response (like that proposed by Van der Werf and Nagel, 1996) can be validated. D.

Shoot Control of Root CK

Even if the effects of root-derived CK on shoots remain unclear, it is obvious from studies of the ramosus mutants of pea (rms1, rms2, rms3, and rms4) that the shoot may elicit considerable control over export of CK from roots (Beveridge, 2000). Hyperbranching phenotypes led to the speculation that rms mutations were CK related. In fact, three of the four mutants have a very reduced concentration of [9R]Z in xylem exiting the root. Reciprocal grafting studies between mutant and wild-type (WT) seedlings have questioned traditional thinking that the root employs CK to pass on information of the soil environment to the shoot. In this system, it appears that the shoot, rather than the root, has primary control over CK production. For example, grafts using rms4 scions retained the characteristically low xylem [9R]Z of the mutant, whereas mutant rootstocks with WT shoots had normal xylem [9R]Z concentrations (Beveridge et al., 1997). Since CK are known to increase branching, the results with rms mutants are more consistent with the hypothesis that the process of shoot branching brings about a negative feedback signal to the root to diminish CK export (Beveridge, 2000). Even increased branching induced by BAP application to the branch meristems reduced xylemborne [9R]Z in WT plants. No speculation of the nature of the shoot-to-root signal has been made, but it has been referred to as novel. However, neither the implications of CK form nor shoot-to-root cycling has been given much consideration. In terms of form, with the exception of [9R]Z, exploration of the CK complement comprised tentative identifications using antibodies of [9R]iP and [9R]Z following a single-step reverse-phase HPLC. Dihydro forms, O-glycosides, and cis-isomers would have been largely overlooked. The experience of Badenoch-Jones et al. (1996) suggests that one cannot be entirely sure that all important CK possibly involved in branching control have been described. With respect to shoot-to-root cycling, if the CK impor-

tant to branching is produced in the branch meristems, then lowered xylem [9R]Z may indicate more local retention and less circulation of shoot-produced [9R]Z. Identification of the phloem CK profile, or even the affirmation of the presence of [9R]Z, could be a valuable first step before the search for a novel signal becomes necessary. If needed, a subsequent whole-plant, quantitative CK budget could confirm if phloem circulation of CK to roots is important in the mutant response. If the shoot-to-root signal in peas is not a CK, then it may well be a sugar. Very recently Havelange et al. (2000) used stem girdling and sucrose replacement assays to show that increased sucrose delivery to roots raises [9R]Z delivery to shoots in Sinapsis alba. What sets their results apart from the model of Van der Werf and Nagel (1996) is that they propose that sucrose acts first as a signal, stimulating root CK production and sugar demand (sink intensity), and, second, as a substrate to satisfy its consequent increased demand. Root [9R]Z output was linked to floral induction. When flowering was inhibited in normally induced plants, either stem girdling or transpiration arrest (under high humidity) diminished [9R]Z delivery to shoots. BAP application restored floral induction. Supporting results have been reported for Chenopodium rubrum using root removal and BAP applications (Vondrakovaet al., 1998). However, the link is still uncertain. Immunocytochemical detection that showed that CK disappeared before floral induction in shoot apices of tobacco (Dewitte et al., 1999) and earlier work with S. alba indicated that shoot apices were supplied with early pathway forms of CK (iP or [9R]iP). Supply was largely through the phloem, whereas the xylem contained mostly [9R]Z. This might be expected to be synthesised downstream from the forms found in the phloem (Lejeune et al., 1994). Once again, there may be confusion due to incomplete CK profiles since the analytical approach relied on immunoassays limited to crossreactivities of two antibodies. Molecular evidence also points to sugar signaling. In cell suspension cultures of Chenopodium rubrum supplied with sucrose, invertase and a hexose transporter protein are coordinately expressed (Ehness and Roitsch, 1997). Expression of both genes was specifically induced by CK, raising the possibility that hexose mediates the role of CK through a sugar-sensing mechanism (Smeekens, 1998). The above discussion of CK as messengers between root and shoot indicates that their importance is likely to be process specific, and perhaps downplays

Roots and Cytokinins

the role of root produced CK from that perceived a decade ago. However, evidence continues to accumulate that transported CK are essential, in selected cases, for environmentally sensitive and integrated whole-plant growth. A more negative viewpoint comes from a study that overloaded root CK production using ipt-transformed tobacco (controlled by a tetracycline-dependent 35S promoter) and studied the effects on sequential leaf senescence and apical dominance (Faiss et al., 1997). Although levels of [9R]Z in xylem increased 10-fold, no decrease of senescence or release of apical dominance resulted. These studies together with those of rms pea mutants (Beveridge et al) are powerful reminders that even reliable and complete analyses of xylem-mobile CK and correctly assessed rates of delivery, without causal reference to the target process in the shoot, will not greatly advance our understanding of potential long-distance signaling.

VI.

FUTURE DIRECTIONS

The CK hormone group is closely connected with root development and, as such, plays a key role in the integration of root growth with that of the shoot. The challenge is to find out just how this integration takes place. Molecular biological technology will eventually provide critical breakthroughs. However, their successful application requires new information and unique genetic tools. The most important piece of information needed is clear definition of the source(s) of CK (i.e., plants or their associated microbial flora). If indeed plants have a unique synthetic pathway, the proteins involved and their encoding genes will provide the most powerful means for progress. However, if bacteria are a significant source of these regulators, then altering their activity will also become a relevent target for genetic analysis. Despite the lack of clear evidence for a significant or even dominant role of bacteria in furnishing the plant with CK or their precursors, the possibility of their direct role cannot be ignored. Given the difficulty in screening out CK mutants, the approach by Martin et al. (1999a,b), whereby the isolation of the gene follows that of the protein, may be a more rewarding approach. It is still unknown how widespread or important CK synthesized in the root are to the plant’s CK ‘‘budget.’’ Physiological efforts should be directed at CK budgets that focus on the inputs of individual organs and track whole-plant circulation of each chemical form of CK. This means there is a need for more intensive study of

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the role of phloem, which in turn will lead to more meaningful studies of root–shoot signaling. Despite recent progress in identifying mechanisms that appear to represent the first steps in signal transduction pathways sensitive to CK, the relationship of these to the chemical forms of CK in translocation channels is obscure. Considerably more attention should be paid to the forms of CK in tissue and transport fluids before a realistic approach to the molecular biology of CK signaling can be developed.

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Emery and Atkins Macha´c˘kova´ I, Eder J, Motyka V, Hanus˘ J, Krekule J. 1996. Photoperiodic control of cytokinin transport and metabolism in Chenopodium rubrum. Physiol Plant 98:564–570. Martin RC, Mok MC, Mok DWS. 1999a. Isolation of a cytokinin gene, ZOG1, encoding zeatin O-glucosyltransferase from Phaseolus lunatus. Proc Natl Acad Sci USA 96:284–289. Martin RC, Mok MC, Mok DWS. 1999b. A gene encoding the cytokinin enzyme zeatin O-xylosyltransferase of Phaseolus vulgaris. Plant Physiol 120:553–557. Masia A, Pitacco A, Braggio L, Giulivo C. 1994. Hormonal responses to partial drying of the root system of Helianthus annuus. J Exp Bot 45:69–76. Medford JI, Horgan R, El-Sawi Z, Klee HJ. 1989. Alterations of endogenous cytokinins in transgenic plants using a chimeric isopentenyl tranferase gene. Plant Cell 1:403–413. Morris RO. 1997. Hormonal regulation of seed development. In: Larkins BA, Vasil IK, eds. Cellular and Molecular Biology of Plant Seed Development. Dordrecht, Netherlands: Kluwer Academic, pp 117–149. Morris RO, Bilyeu KD, Laskey JG, Cheikh NN. 1999. Isolation of a gene encoding a glycosylated cytokinin oxidase from maize. Biochem Biophys Res Commun 255:328–333. Motyka V, Faiss M, Strnad M, Kaminek M, Schmulling T. 1996. Changes in cytokinin content and cytokinin oxidase acitivity in response to derepression of ipt gene transcription in transgenic tobacco calli and plants. Plant Physiol 112:1035–1043. Murai N. 1994. Cytokinin biosynthesis in tRNA and cytokinin incorporation into plant RNA. In: Mok DWS, Mok MC, eds. Cytokinins Chemistry, Activity and Function. Boca Raton, FL: CRC Press, pp 87–100. Murofushi N, Inoue A, Watanabe N, Ota Y, Takahashi N. 1983. Identification of cytokinins in root exudate of the rice plant. Plant Cell Physiol 24:87–92. Nan R, Carman JG, Salisbury FB. 1999. Low irradiances affect abscisic acid, indole-3-acetic acid and cytokinin levels of wheat (Triticum aestivum L.) tissues. J Plant Physiol 155:556–560. Nooden LD, Letham DS. 1993. Cytokinin metabolism and signalling in the soybean plant. Aust J Plant Physiol 20:639–653. Pate JS, Peoples MB, Van Bel AJE, Kuo J, Atkins CA. 1985. Diurnal water balance of the cowpea fruit. Plant Physiol 77:148–156. Pawlowski K, Bisseling T. 1996. Rhizobial and actinorhizal symbioses: what are the shared features? Plant Cell 8:1899–1913. Peoples MB, Pate JS, Atkins CA, Murray DR. 1985. Economy of water, carbon and nitrogen in the developing cowpea fruit. Plant Physiol 77:142–147.

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26 Abscisic Acid in Roots—Biochemistry and Physiology Eleonore Hose, Angela Sauter, and Wolfram Hartung Julius von Sachs Institut, University of Wu¨rzburg, Wu¨rzburg, Germany

I.

INTRODUCTION

ABA content of roots occurs in a great variability within plant species (Table 1). Concentrations range from
50 up to 1890 pmol (g DW)1, with plants from dry and extreme habitats having high endogenous ABA concentrations. Under stress conditions, concentrations were substantially higher, ranging from 120 pmol (g DW)1 to >7570 pmol (g DW)1. Hartung and Turner (1997) have compared ABA concentrations of roots of seven lupin species and found that Lupinus digitatus, a species that occurs at the edge of the Sahara Desert, showed the highest response to water deficit (+465% increase of ABA after 50% water loss). The roots of the mesophytic species L. atlanticus exhibited only 54% increase after the same stress treatment. ABA concentrations also vary within an individual plant root. Maize and runner bean roots show the highest concentrations in the first 1–3 mm of the root tips. This is very likely a consequence of the lack of vacuoles in the root tip cells. As cells become more vacuolated toward the root base, ABA concentrations decrease. According to the anion trap concept (Behl et al., 1981), ABA accumulates in the slightly alkaline cytosol of the root cells. This results in the longitudinal ABA gradient of individual roots (Hartung et al., 1999). Similar longitudinal gradients of radioactivity after incubation of barley roots with ABA have been detected by Behl et al. (1981). No significant radial ABA gradients between cortex and stele were detected in roots of runner beans (Hartung et al., 1999).

Abscisic acid (ABA) is now well established to play a significant role as stress hormone in higher and lower plants. Usually, its biosynthesis is increased under stress conditions. After transport to target cells it improves water relations of terrestrial plants by closing their stomata and affecting their meristems. When ABA is externally applied, similar phenomena can be observed (Trewavas and Jones, 1991; Hartung et al., 1999). Attempts to produce ABA-free mutants or transgenic plants failed. Such plants would not be able to exist in a terrestrial environment with all its natural stress stimuli. It is therefore not surprising that ABA can be detected universally in all plants and that it must play an important role in roots as well. Hence, root systems are often those tissues that experience first the emergence of a stress situation.

II.

ABA FORMATION AND CONTENT IN ROOTS

Early evidence that plant roots synthesize and accumulate ABA has been presented by Hartung and AbouMandour (1980). They observed ABA accumulation in root cultures of Phaseolus coccineus after at least seven passages. Such cultured roots were not in contact with shoot tissues for several cell generations. This shows unequivocally that roots are able to synthesize ABA. 435

436

Hose et al.

Table 1 ABA Content of Roots Under Different Stress Factors in Different Plant Species Plant Species Zea maysa Zea maysb Anastatica hie¨rochunticac Chamaegigas intrepidusd Xerophyta dasylirioidesc Lycopersicon esculentume Chamaegigas lanceolatumd Myrothamnus moschatad Simondsia chinensise Helianthus annuusf

ABA content of unstressed roots

Stress factor

ABA content of stressed roots

7.4 pmol (g FW)1 9.7 pmol (g FW)1 21 pmol (g FW)1 56 pmol (g DW)1 90 pmol (g DW)1 110 pmol (g DW)1 200 pmol (g DW)1 483 pmol (g DW)1 780 pmol (g DW)1 950-1890 pmol (g DW)1

PEG Salt Salt Drought Drought Salt Drought Drought Salt PEG

39.2 pmol (g FW)1 34.9 pmol (g FW) 301 pmol (g FW)1 1266 pmol (g DW)1 297 pmol (g DW)1 210 pmol (g DW)1 6261 pmol (g DW)1 2808 pmol (g DW)1 120 pmol (g DW)1 5680–7570 pmol (g DW)1

Sources: aZeier, (1998); bSauter, unpublished data; cKettemann, unpublished data; dSchiller et al. (1997); eWaisel and Hartung, unpublished data; f Ludewig et al. (1988).

III.

CELLULAR COMPARTMENTATION OF ABA IN ROOTS

To study the intracellular compartmentation of ABA in cells of barley and maize roots, efflux compartmental analysis was performed (Behl et al., 1981; Behl and Hartung, 1984; Hose, 2000). Assuming a cytosol/ vacuole ratio of 1/10, cytosolic concentration proved to be 10 times higher than the vacuolar concentration. This finding together with results from uptake experiments of labeled ABA showed that intracellular ABA distributes in alkaline compartments of root cells following exactly the Henderson-Hasselbalch equation (Hartung and Dierich, 1983). ABA carriers are not involved in ABA compartmentation in most of the root cells. Such carriers were only detected in the extreme root tips under very acid conditions (Astle and Rubery, 1983) and are not expected to be present in the root hair zone under normal physiological conditions.

IV.

PERMEABILITY COEFFICIENTS AND REFLECTION COEFFICIENTS

Keeping in mind that diffusion is the dominant transport mechanism for ABA within cells, permeability of root membranes determines the velocity of ABA distribution within root cells and root cell compartments. Hartung and Gratzer (unpublished) and Daeter et al. (1993) have determined permeability coefficients of root cortex membranes of maize and runner bean (Table 2). The tonoplast permeability was lower by a factor of 2–3 than that of the plasmalemma. This is different from mesophyll cells where plasma mem-

branes are at least 10 times more permeable than tonoplasts. Mesophyll plasma membranes proved to be more permeable to ABA than plasma membranes of root cortical cells by a factor of 6–10. This results in substantially slower redistribution of ABA within root tissues. The loss of ABA into a surrounding aqueous medium would also be significantly slower than the loss from leaf cells. During radial transport of ABA into the xylem elements, the cortex and its apoplastic barrier, the endodermis, have to be penetrated. The barrier properties of the cortex and of the endodermis for a substance such as ABA can be quantified with the reflection coefficient . In the case of  = 1.0, the apoplastic barrier would be impermeable for ABA; if  = 0, no barrier would exist. Freundl et al. (2000) were the first to determine reflection coefficients for ABA using maize and sunflower roots, under physiological conditions (Table 3). They showed  to be always <1, indicating that the endodermis is not a perfect barrier for ABA. However,  is not constant; it varies with plant species, external ABA concentration, apoplastic pH, and radial water flow that can be induced by transpiration. As a result of this, ABA can be translocated directly via an apoplastic bypass flow across the endodermis to the xylem elements. V.

INTERNAL FACTORS THAT REGULATE ABA ACCUMULATION IN ROOTS

A.

ABA Biosynthesis

ABA is formed in green tissues by the indirect pathway via zeaxanthin, violaxanthin, and xanthonal

Abscisic Acid

437 Table 2 Permeability Coefficients of ABAH for Plasmalemma and Tonoplast of Different Cell Types Permeability coefficient PS [109 ms1] Plasmalemma a

Tonoplast

12.9 3.30 0.26c 0.78d 0.50e 0.70f 2.17g

Guard cell Mesophyll cellb Root cortical cell

1.26 0.54 0.14c 0.30d 0.15e 0.36f 5.49 102g

Sources: (coefficients were determined by efflux compartmental analyses) a Valerianella locusta, Baier et al. (1988); bValerianella locusta, Daeter and Hartung (1990); c, dZea mays, Jovanovic et al. (1992); ePhaseolus vulgaris, Jovanovic et al. (1992); fZea mays, Gratzer and Hartung, unpublished data; gPhaseolus vulgaris, Gratzer and Hartung, unpublished data.

(=xanthoxin). Parry and Horgan (1992) have shown that roots contain all the precursors, which are necessary to form ABA via carotenoids. Taylor (1991) admitted that in roots carotenoid content is sometimes very low and becomes a rate-limiting factor. However, in intact plants ABA can also be delivered in amounts from the shoot to the root. Saab et al. (1990) have germinated maize seedling with the inhibitor fluridone to produce ABA-deficient roots. Fluridone prevents the formation of pigments including that of zeaxanthin and violaxanthin. ABA content of the roots was never reduced to zero, which raises the question how such a residual ABA may be synthesized. It may be interesting in this context to cite the work of Okamoto et al. (1988), who demonstrated that plant tissues contain all the enzymes necessary to synthesize ABA by the direct pathway via farnesyldiphosphate. All enzymatic steps of ABA biosynthesis require oxygen. Consequently, ABA biosynthesis is retarded by water logging (Jackson, 1991).

Recently, it has been shown that besides mevalonic acid deoxyxylulose-5-phosphate can serve as a precursor for isopentenylpyrophosphate (Lichtentaler, 1999). Whether this alternative early step in isoprenoid (and ABA) synthesis applies also for roots remains to be investigated. Aldehyde oxidase (AO) is a key enzyme required for the last step of ABA biosynthesis, the oxidation of abscisic aldehyde to ABA (Walker-Simmons et al., 1989). Lee and Milborrow (1997) have demonstrated that in their cell-free avocado systems it is the xanthoxal that is oxidized by AO to xanthoxic acid. An important component of AO is the molybdenum cofactor (MoCo), as is also the case for nitrate reductase. Peuke et al. (1994) have shown that an optimal nitrate supply stimulates ABA biosynthesis in the roots of castor beans. Nitrate treatment induces nitrate reductase, which includes the MoCo and consequently the oxidation of the ABaldehyde or xanthoxal.

Table 3 Apparent Reflection Coefficient of Abscisic Acid ( ABA) for Maize Roots Cultivated Either Aeroponically or Hydroponically and for Roots of Helianthus annuus Grown in a Hydroponic Culturea Apparent reflection coefficient— ABA External ABA (nM) 100 100 a

Pressure gradient (MPa) 0.02 0.06

Zea mays aeroponic

Zea mays hydroponic

Helianthus annuus hydroponic

0.68  0.06 0.54  0.04

0.65  0.10 0.52  0.11

0.97  0.02 0.97  0.02

After a suction experiment endogenous ABA flow through the roots have been determined and with the given external ABA concentration apparent reflection coefficients were calculated. Source: Freundl et al. (2000).

438

B.

Hose et al.

ABA Degradation and Conjugation

There is no evidence that ABA may be metabolized in roots differently than in other plant organs. The use of the specific inhibitors of P450 cytochrome monoxigenases such as tetcyclacis or paclobutrazol indicated that also in roots ABA is degraded to the 8 0 -hydroxymethyl ABA, and then to phaseic acid (PA) and dihydrophaseic acid (DPA). ABA conjugates, predominantly ABA-glucose ester (ABA-GE), are also formed by root tissues, especially under conditions of inhibited oxidative metabolism to PA (Sauter and Hartung, 2000). The occurrence of five unidentified ABA conjugates (Hansen and Do¨rffling, 1999) and PA-glucose ester (Bano et al., 1994) in the xylem sap of sunflowers indicates that these conjugates can be formed in root tissues.

Root ABA content can also be decreased by exudation into the soil solution. Slovik et al. (1995) concluded from computer simulations that significant amounts of ABA are lost to the rhizosphere, especially when the soil solution is alkaline. This model also predicted that ABA should be present in the rhizosphere in the low nanomolar range to prevent ABA loss to the soil solution. This is especially important under nontranspiring conditions. Indeed, ABA could be detected in the soil in a concentration range of 0.8–6 nM under different crops. Highest concentrations were found in acid soils of coniferous forests (Table 4). Such external ABA can be taken up by the roots and translocated into the shoot, especially when a plant is transpiring.

VII. A.

VI.

LONG-DISTANCE TRANSPORT AND ABA HOMEOSTASIS AND ACCUMULATION

Abscisic acid accumulation in roots depends on import from the shoot via phloem vessels and on export to the shoot in the xylem. In castor bean (Ricinus communis) seedlings, the cotyledons export under water deficiency substantial amounts of ABA via the phloem to the roots (Zhong et al., 1996). ABA transport from the shoot to the roots is particularly significant under the following conditions: (1) salt stress in Lupinus albus (Wolf et al., 1990); (2) phosphate deficiency in Ricinus communis (Jeschke et al., 1997a); (3) under conditions where ammonium is the only nitrogen source, in Ricinus (Peuke et al., 1994); and (4) in maize plants that are supplied by their seminal roots only (Jeschke et al., 1997b).

ENVIRONMENTAL FACTORS Properties of Drying Soils from Extreme Habitats

Zhang and Davies (1987, 1989) have convincingly proven that soil drying increases the ABA concentrations in root tissues. However, the relative water content of the roots has to be reduced drastically, at least down to 50%, to stimulate ABA production (Cornish and Zeevaart, 1985; Cornish and Radin, 1990; Hartung and Turner, 1997; Hartung et al., 1999). The question is whether drought-induced ABA biosynthesis in roots is sufficiently sensitive to play a physiological role in stress response. There are, however, characteristics of soil drying that may be responsible for ABA accumulation and may also increase the sensitivity of the root tissues to ABA when exposed to the drying soil. Soils from extreme habitats like deserts exhibit, besides reduced water content, high salt concentrations, relatively high pH values, and high soil strength. These questions have been discussed in detail in two recent

Table 4 ABA Concentrations in the Soil Solutions Under Various Plants Depend on Soil pH, Soil Water Content, and the Distance to Roots pH of the soil solutiona 3.5–4.5 5.5–7 7.5–8 a

ABA (nM)a 3–8 0.8–3 0.3–2.5

Soil water content (%)b 15 20 40 55

ABA (nM)b 0.5–3 0.8–1.7 0.4–0.6 0.25

Coniferous forest, garden soil, barley, Phacelia tanacetifolia (Hartung et al., 1996). Different crops (Hartung et al., 1996). c Maize (Mu¨ller et al., 1989). b

Distance to roots (mm)c

ABA [pmol (g DW)1]c

<2 2–20 20–200

70–500 25–60 10–40

Abscisic Acid

book chapters (Hartung and Jeschke, 1999; Hartung et al., 1999) and will therefore be dealt in a shorter manner here. Salt stress increases ABA production in roots of several species of lupins (Wolf et al., 1990) and castor bean (Peuke et al., 1994; Jeschke et al., 1997a). Accumulation of ABA in the roots results from increased biosynthesis and from import from the shoot via the phloem. When plants are salt affected, significant amounts of ABA can be loaded into the root xylem, so ABA xylem concentration is elevated up to 10-fold. Alkaline conditions that very often accompany saline soils cause large problems, especially for growth and development of roots of Fabaceae species. As mentioned above, ABA was predicted to be lost to an alkaline soil solution according to the anion trap concept. Degenhardt et al. (2000) investigated ABA relations of different species grown in a solid alkaline, desalted, and fertilized substrate. Under such growing conditions, roots of Zea mays developed an exodermis, a tissue that may prevent ABA loss to the surrounding medium. Roots of lupins and of broad beans did not form Casparian bands in their hypodermis. In such cases, ABA was lost to soil. However, this loss was compensated by an increased ABA synthesis. B.

Nutrient Supply

Nitrate nutrition has only weak effect on ABA concentration in roots of castor bean (Peuke et al., 1994). Generally, a tendency for reduced ABA amounts under nitrate deficiency was observed. This is in contrast to earlier reports on slightly increased ABA in roots of nitrate-deficient plants. The chemical form of N is much more important for the ABA accumulation in roots than its content in the nutrient medium. If NH4+ is the only nitrogen source of castor bean plants, ABA concentration in roots is increased fourfold and in xylem sap threefold (Peuke et al., 1994). However, if the N source is nitrate, ABA concentrations in tissues and transport fluids are only slightly affected. As pointed out above, soils of extreme habitats can be alkaline. Alkaline conditions reduce the availability of phosphate to roots as long as roots are not able to acidify their rhizosphere. In castor bean plants cultivated under phosphate deficiency, ABA biosynthesis in the roots is increased drastically. All this ABA is fed into the xylem vessels, causing xylem sap ABA to raise sixfold without an additional deposition of ABA in the root tissue (Jeschke at al., 1997a).

439

When root tips penetrated a compacted soil, a transient increase of ABA in the roots and in the xylem sap was observed (Hartung et al., 1994; Mulholland et al., 1996a,b). This was accompanied by an increased radial growth of the tip, number of root hairs, and size of the root cap. Very similar morphological changes can be observed when seedlings were cultivated in 0.1–1.0
M ABA. The advantage of having dense root hairs results from the fact that they can act as an anchor to facilitate the penetration of the compacted soil.

VIII.

ABA AS A ROOT-TO-SHOOT STRESS SIGNAL

It is now clear that stomatal behavior can be closely related to changes in the soil moisture even when root water relations are not affected by such changes. Using split-root experiments with maize, in which one part of the root was well watered and the other suffered from water shortage, Blackman and Davies (1985) showed that stomata close despite the maintenance of a high water potential in the leaves. Their and other data (Davies and Zhang, 1991) strongly indicate that roots sense some aspects of soil water status. They synthesize a chemical messenger, which unequivocally has been shown to be ABA, and send this messenger to the shoot. As mentioned above, factors that are characteristic for drying soils induce the accumulation of ABA in root tissues. Acting together they may have a synergistic effect on the enzymes of ABA biosynthesis, resulting in a high sensitivity of the ABA-synthesizing system. However, until recently only little information was available regarding the transport paths and transport mechanisms of ABA in roots. Hartung and Behl (1975) have shown that ABA can be transported laterally across the endodermis of bean roots into the stele. It was further assumed that radial ABA transport occurs exclusively in the symplast (Else et al., 1994, 1995; Jackson et al., 1996). In this case the intensity of the ABA signal in the xylem depends strongly on the lateral water flow through roots. ABA would be diluted drastically when transpirational water flow is increased. Computer simulations of Slovik et al. (1995) support such assumptions. Modeling of a symplastic ABA transport pathway of the root-to-shoot signal has indicated dramatic changes in the ABA concentration of the xylem sap. Even a short period of shading may change the transpiration and induce considerable fluctuations of ABA in the vicinity of the guard cells.

440

Steudle and coworkers (e.g., Frensch and Steudle, 1989; Steudle, 1993, 1994; Steudle and Peterson, 1998) have suggested that water and solutes, including ions, pass through the root tissue apoplast. This resulted in a complex model, the composite transport model, which is fulfilling the various physiological demands of roots. ABA could be translocated to the xylem through the apoplast by solvent drag with the transpirational water stream. Such a bypass flow reduces the dilution that might have been caused by increased water flow. A small contribution (1%) of such an apoplastic bypass flow has been predicted by the computer model of Slovik et al. (1995) to buffer the ABA dilution caused by symplastic transport (Hartung et al., 1998). Experimental evidence for the presence of an apoplastic ABA bypass flow was provided by Freundl et al. (1998, 2000). They applied ABA in the physiological concentration range of 5 nM up to 100 nM to media of excised root systems of Zea mays. The xylem sap was then collected by application of subatmospheric pressure to the cut surface of the mesocotyl (Freundl et al., 1998). An intensified radial water flow (JVr), enforced by the subatmospheric pressure gradient, did not reduce the ABA concentration of the xylem sap but even increased it (Fig. 1). The endogenous ABA content of maize roots can be increased by inhibition of the oxidative ABA breakdown following preincubation with tetcyclacis (Rademacher et al.,

Figure 3 ABA concentration in the xylem sap of Zea mays at an external ABA concentration of 100 nM in root media of different pH values (*, pH 5.5; &, pH 4.8; ~, pH 3.8).

Hose et al.

Figure 2 ABA concentrations in the xylem sap of Zea mays at different external ABA concentrations (- - -, 70 nM ABA; ____ , 100 nM ABA) and after preincubation with tetcyclacis (*). Increasing the subatmospheric pressure gradient even raised the ABA concentrations in the xylem. Mean  SD, n = 3 root systems.

1987). ABA concentrations in the xylem sap are increased in a comparable way after enhancing the water flow. A similar rise in xylem ABA concentration can be observed after application of 70–100 nM ABA to the medium of the maize roots (Fig. 2). Such ABA concentrations of xylem sap were reported repeatedly for stressed plants (Zhang and Davies, 1989; Hartung and Davies, 1991; Hartung and Heilmeier, 1993)

IX.

FACTORS THAT INFLUENCE APOPLASTIC ABA TRANSPORT

A.

Apoplastic pH

Being a weak acid (pka = 4.8), ABA is distributed within the apoplast and the symplast of root tissues according to the anion trap concept and the Henderson-Hasselbalch equation. Acidification of the apoplast, as occurs in ammonium-supplied roots (Roberts et al., 1982; Marschner, 1995; Mu¨ller et al., 1990; Gerenda´s and Ratcliffe, 2000), should redirect ABA to the cytosol of root cortical cells. ABA should then be translocated predominantly in the symplast. An increased water flow Jvr should therefore dilute the signal. Indeed, as shown by Peuke et al. (1994), the concentration of ABA in the xylem sap is significantly higher in the plants supplied by NH4+ (Fig. 3). However, the ABA is diluted whenever Jvr is increased.

Abscisic Acid

Figure 1 ABA concentration in the xylem sap of Zea mays dependent on the external ABA concentration and the applied pressure gradient. An increase of the subatmospheric pressure gradient resulted in a stimulated radial water flow (JVr). The increased JVr did not dilute the ABA concentration in the xylem.

B.

Apoplastic Barriers

In maize roots, two cortical layers can build up Casparian bands in their radial cell walls (Peterson, 1988). Cultivated in hydroponics, only an endodermal Casparian band can be detected in maize roots. Growing Zea mays in moist air (aeroponics) leads to formation of a second Casparian band in the hypodermis, a so-called exodermis (Enstone and Peterson, 1998; Zimmermann et al., 2000). A comparison of the radial ABA flows (JABA) of maize roots containing either one (endodermis) or two (endodermis and exodermis) cell layers with Casparian bands showed a clear reduction of JABA through exodermal roots, especially under high water flow rates (Fig. 4). On the other hand, roots that were preincubated with radioactive ABA lost significantly smaller amounts of ABA to the surrounding media when a Casparian band was present in the hypodermis. Hose (2000) compared the efflux of ABA from exodermal and nonexodermal maize roots using the compartmental efflux analysis as described by Behl et al. (1981). The half-time of isotope exchange from the apoplast of exodermal roots was two to three times higher than in the nonexodermal controls (Fig. 5). This indicates that the Casparian band of the maize exodermis is an effective barrier for ABA slowing down ABA efflux. Lower efflux results in an increased

441

Figure 4 Radial water flows (JABA) through maize roots containing 1 Casparian band in the endodermis (&, nonexodermal roots) or 2 Casparian bands in the cortex (&, exodermal roots) at different external ABA concentrations.

ABA concentration in the root apoplast. Such an ‘‘extra ABA’’ can stimulate the hydraulic conductivity of root cortical plasma membranes (Hose et al., 2000). It is also available for apoplastic translocation

Figure 5 Half-times of ABA exchange of the apoplastic compartment in exodermal (&) and nonexodermal (&) maize roots.

442

Hose et al.

by solvent drag directly into the xylem vessels. Experiments of Degenhardt et al. (2000) support this conclusion. They observed an increased ABA efflux to an alkaline substrate only from roots lacking an exodermis. X. ABA CONJUGATES AS ROOT-TOSHOOT STRESS SIGNALS It has been postulated (Munns and King, 1984; Netting et al., 1992; Munns and Sharp, 1993) that an ABA conjugate, the ABA adduct, can act as an additional root-to-shoot stress signal. Until now, the chemical structure of this compound could not be elucidated. Bano et al. (1993, 1994) identified enhanced concentrations of glucose esters of ABA, PA, and DPA in the xylem sap of stressed sunflower and rice plants (Table 5). The concentrations of free and bound ABA decreased again after relief of the stress condition. In comparison, Dietz et al. (2000) found a significantly lower ABA-GE concentration in the xylem sap of barley. However, this was stimulated under salt stress by four- to fivefold. Five conjugates of ABA were detected in the xylem sap of well-watered sunflower plants (Hansen and Do¨rffling, 1999). Under stress a sixth conjugate appeared, and the concentration of the others was enhanced significantly. The conjugates were alkali hydrolyzable. Drought stress increased the amount of ABA conjugates with -glucosidic linkages, predominantly ABA glucose ester. Abscisic acid glucose esters have also been detected in the xylem sap of castor beans, Zea mays (Jeschke et al., 1997a,b), and in the winter annual desert plant Anastatica hierochuntica

(Grimmer, 1993). In Anastatica xylem sap the ratio of ABA/ABA-GE was 15; it decreased to 1 when the plants were stressed by a mixture of NaCl and CaCl2. It was pointed out by Baier et al. (1988) that the permeability coefficient of plasma membranes for ABA-GE is extremely low. The question arises how ABA conjugates are transported from their site of synthesis, the cytosol of cortical root cells, to the xylem vessels. ABAGE may also originate in the soil solution. According to Sauter and Hartung (2000), the origin of ABA-GE is in the root symplast rather than in the soil, although it was detected in soil solutions with concentrations up to 30 nM (Table 6). External conjugated ABA cannot be dragged with the water flow across the hydrophobic endodermis, as is the case for free ABA. Especially, aeroponically cultivated maize roots with a complete exodermis are not able to take up external ABA-GE. Both tissues, the exodermis and the endodermis, are good barriers for ABA conjugates. However, hydrolytic enzymes of the root cortex apoplast are able to cleave the conjugate (Sauter and Hartung, 2000). The released free ABA can then be translocated in the apoplast to the xylem vessels. Translocation of endogenous ABA-GE must occur within the root symplast. Once arrived at the xylem parenchyma cells, the conjugate can be released into the xylem elements since the plasma membranes of stelar cells show a fivefold higher permeability than those of the cortical cells (Fig. 6). Formation and release of ABA conjugates are highest under conditions that inhibit oxidative ABA degradation. However, the mechanisms of membrane transport of ABA-GE are still unknown. Since ABA-GE can be loaded into the xylem elements and transported even to a higher extent under stress conditions, the conju-

Table 5 Content of Free and Bound ABA in Xylem Sap of Well-Watered, Drought-Stressed, and Rewatered Rice Seedlingsa; Barley Seedlings Were Grown at Varying Salt Concentrations and Xylem Sap of Excised Roots Was Investigated for ABA-GE Rice

Well-watered Drought-stressed Rewatered a

Barley

Free ABA (nM)

Bound ABA (nM)

NaCl (mM)

ABA-GE (nM)

7.2  1 92  3 52  12

73  7.5 499  75 87  5.7

0 50 100

0.32  0.09 0.50  0.26 1.31  0.30

Sources: Bano et al., 1993,1994.

Abscisic Acid

443

Table 6 ABA-GE Concentration (nM) in Soil Water Samples Collected from Two Different Types of Soil from Agricultural Areas and from Various Soil Depths Sandy soil Depth (cm) 2.5 25.0 45.0

Calcareous soil

Sunflower

Maize

Rye

Sunflower

Wheat

Potato

0.7 0.5 0.4

5.2 4.0 2.8

0.9 1.1 0.6

34.9 14.1 3.0

5.4 3.6 10.3

16.1 13.7 0.5

Source: Sauter and Hartung, 2000.

gate can be considered as a long-distance signal. Dietz et al. (2000) have investigated the fate of the ABA-GE after having arrived in the leaf apoplast, and results show that esterases in the leaf apoplast release free ABA from its conjugates.

XI.

THE FUNCTION OF ROOT ABA

A.

Growth and Development

Sinnott (1960) has described morphological and physiological responses of roots to drought, and Trewavas and Jones (1991) showed that ABA mimics more or less accurately these responses. Indeed, this applies for the root/shoot ratio, for the formation of root hairs, and for lateral root development, which can be stimulated by ABA applications (Simonis, 1947; AbouMandour and Hartung, 1980; Biddington and Dearman, 1982). A stimulated development of root systems, of maize plants growing in drying soils, was

reported many years ago (Weaver, 1926). Studies of root growth under reduced soil water potential showed that maize root tips continued growing although shoot development was already inhibited. This may occur provided root ABA has increased substantially. Maintenance of root growth at low soil water potential and increased internal ABA concentrations are associated with a substantial deposition of proline, with an increased activity of xyloglucan endotransglycosylase, and with expansins (Sharp et al., 1988; Saab et al., 1990; Sharp, 1990). B.

Drought Tolerance

ABA increases and maintains drought and desiccation tolerance in embryos of ripening seeds and in leaf tissues of poikilohydric angiosperms (Ingram and Bartels, 1996; Hartung et al., 1998). Data proving the induction of desiccation tolerance in roots by ABA are rare. Studies of root desiccation tolerance of the resur-

Figure 6 (A) ABA-GE content of cortical and stelar tissue segments after preincubation for 24 h with 5 nM ABA (1), 50 nM ABA (2), and 50 nM ABA plus tetcyclacis (3). (B) Release of ABA-GE from stelar and cortical segments. After preincubation the segments were transferred to a hormone-free nutrient solution and over 120 min the concentration of ABA-GE in the medium was measured.

444

Hose et al.

rection plant Chamaegigas intrepidus have shown drastic (20-fold) accumulation of ABA in drying roots (Schiller et al., 1997). This was accompanied by the formation of polypeptides that showed similarities with ABA-inducible dehydrins of Craterostigma plantagineum. The physiological stress responses of dehydrins and their functions remain obscure.

XII.

ABA EFFECT ON HYDRAULIC CONDUCTIVITY OF ROOTS

Direct effects of ABA on hydraulic conductivity of roots have been reported repeatedly. When maize plants were supplied with water only by their seminal roots, they compensated for the reduction in root uptake area by an increased hydraulic conductance (Lpr) (Jeschke et al., 1997b). The authors concluded that it was shoot-derived ABA that stimulated Lpr. Other authors have applied hydrostatic or osmotic pressure gradients to intact root systems in order to elucidate the effects of ABA on the pressure-gradient driven water flow. Results are summarized in Table 7. In most cases a stimulation of Lpr by ABA was observed. In some cases ABA had no effect on Lpr or even inhibited it. Freundl et al. (2000) and Hose et al. (2000) investigated whole root systems using the suction method (Freundl et al., 1998), isolated root tips, using the root pressure probe, and individual cells, using cell pressure probe. Results have indicated that ABA acts directly on root cortex plasmalemma.

The ABA effect is transient. One hour after application of ABA the stimulation of Lpr was sevenfold. After another hour Lpr decreased again and reached its control value. Such ABA effect was observed at the expected concentration of the apoplast of stressed roots (100 nM according to Hose et al., 2000). This effect was highly specific for undissociated (+)-cistrans-ABA both on the cellular and the root levels. In accordance with the composite transport model of water movement in roots (Steudle, 1993), effects have been more pronounced at the cell than at the root level. When radial water flow was largely apoplastic, the effect was negligible. The stress hormone ABA facilitates water uptake into roots as the soil starts drying and ABA is accumulated in root tissues. While the apoplastic water transport path is mainly used in transpiring plants under a hydrostatic pressure gradient, ABA contributes to the regulation of water uptake by affecting the cell-to-cell path. With this mechanism, plants are able to adapt to conditions of water shortage. As stress develops the roots adapt by varying the cell-to-cell component of water transport.

XIII.

ABA AND PLANT STRESS FOLLOWING TRANSPLANTATION

It is a common observation of gardeners and horticulturists that plants moved from high-humidity conditions to the more stressful environment of a greenhouse or field have a tendency to wilt despite an

Table 7 Hydraulic Conductivities (Lpr) of Various Plants Before and After ABA Applicationa Reference Hydrostatic pressure gradient Glinka, 1973 Markhart et al., 1979 Fiscus, 1981 Davies et al., 1982 Glinka and Abir, 1989 Ludewig et al., 1988 Freundl et al., 2000 Hose et al., 2000 Osmotic pressure gradient Cram and Pitman, 1972 Quintero et al., 1999 a

Technique

Plant species

ABA [M]

Lpr

Factor 2 0.9–0.2

Suction Pressure chamber Pressure chamber Pressure chamber Pressure chamber Pressure chamber Suction Root pressure probe

Helianthus annuus Phaseolus vulgaris Phaseolus vulgaris Triticum aestivum Helianthus annuus. Helianthus annuus Zea mays Zea mays

4106 510 – 2104 3107b 106 4106 10 10 –104 5107 1107

" # # # " " " "

Osmotic exudation

Hordeum vulgare Zea mays Helianthus annuus

0.4105–1.9105

no effect

4106

"

Osmotic exudation

5

0.5–0.3 2 1.4–2 1.7–3.2 3–4

1.4

Depending on the technique of measuring Lpr and the applied ABA-concentration both stimulating (") and inhibiting (#) effects as well as no effect on root hydraulic conductivity have been found. b ABA applied in [mol cm2] leaf area.

Abscisic Acid

optimal supply of water. The situation is more severe when regenerated plantlets are transferred from their cultivation flasks to the soil. Plantlets of Ruta divaricata did not survive transplanting to soil because they were extremely low in ABA (Hartung and AbouMandour, 1996). When pretreated with ABA or tetcylclacis (that increase the endogenous ABA content), all the regenerates survived the transplantion. A similar response was reported for rice seedlings (FlorezNimedez et al., 1995). The beneficial role of ABA is very likely a result of ABA effects on the water conductivity of the roots and having been transported to the shoot on reducing the water loss by transpiration. XIV.

CONCLUDING REMARKS

ABA, the universal stress hormone of higher plants, plays a significant role in enabling plant survival in a terrestrial environment by controlling their water relations. It improves the stress tolerance of the whole plant, and influences root growth and development as well as the plant’s water uptake. Roots are an important site of ABA biosynthesis and accumulation and therefore a source of this long-distance stress signal. REFERENCES Abou-Mandour AA, Hartung W. 1980. The effect of abscisic acid on growth and development of intact seedlings, root and callus cultures and stem and root segments of Phaseolus coccineus. Z Pflanzenphysiol 100:25–34. Astle MC, Rubery PH. 1983. Carriers for abscisic acid and indole-3-acetic acid in primary roots: their regional localisation and thermodynamic driving forces. Planta 157:53–63. Baier M, Gimmler H, Hartung W. 1988. Permeability of guard cell plasma membrane and tonoplast. J Exp Bot 41:351–358. Bano A, Do¨rffling K, Bettin D, Hahn H. 1993. Abscisic acid and cytokinins as possible root-to-shoot signals in xylem sap of rice plants in drying soil. Aust J Plant Physiol 20:109–115. Bano A, Hansen H, Do¨rffling K, Hahn H. 1994. Changes in the content of free and conjugated abscisic acid, phaseic acid and cytokinins in the xylem sap of droughtstressed sunflower plants. Phytochemistry 37:345–347. Behl R, Hartung W. 1984. Transport and compartmentation of abscisic acid in roots of Hordeum distichon under osmotic stress. J Exp Bot 36:1433–1440. Behl R, Jeschke WD, Hartung W. 1981. A compartmental analysis of abscisic acid in roots of Hordeum distichon. J Exp Bot 32:889–897.

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446 Fiscus EL. 1981. Effects of abscisic acid on the hydraulic conductance and the total ion transport through Phaseolus root systems. Plant Physiol 68:169–174. Flores-Nimedez AA, Do¨rffling K, Vergara BS. 1995. Amelioration of drought-induced transplanting shock in rice by an abscisic acid analog in combination with the growth retardant tetcyclacis. J Agron Crop Sci 3:145–150. Frensch J, Steudle E. 1989. Axial und radial hydraulic resistance to roots of maize (Zea mays L.). Plant Physiol 91:719–726. Freundl E, Steudle E, Hartung W. 1998. Water uptake by roots of maize and sunflower affects the radial transport of abscisic acid and the ABA concentration in the xylem. Planta 209:8–19. Freundl E, Steudle E, Hartung W. 2000. Apoplastic transport of abscisic acid through roots of maize: effect of the exodermis. Planta 210:222–231. Gerenda´s J, Ratcliffe RG. 2000. Intracellular pH regulation in maize root tips exposed to ammonium at high external pH. J Exp Bot 51:207–219. Glinka Z. 1973. Abscisic acid effect on root exudation related to increased permeability to water. Plant Physiol 51:217–219. Glinka Z, Abir N. 1989. Abscisic acid promotes passive fluxes of vacuolar solutes in excised sunflower roots. In: Longhman BC, Gasparikova O, Kolek J, eds. Developments in Plant and Soil Sciences, Structural and Functional Aspects. Dordrecht, Netherlands: Kluwer, pp 97–99. Grimmer C. 1993. Hormonelle Streßsignale aus der Wurzel der winteranuellen Wu¨stenpflanze Anastatica hie¨rochuntica L. Diploma thesis, University of Wu¨rzburg, cited in Hartung W, Jeschke WD (1999). Hansen H, Do¨rffling K. 1999. Changes of free and conjugated abscisic acid and phaseic acid in xylem sap of drought-stressed sunflower plants. J Exp Bot 50:1599– 1605. Hartung W, Abou-Mandour AA. 1980. Abscisic acid in root cultures of Phaseolus coccineus L. Z Pflanzenphysiol 97:265–270. Hartung W, Abou-Mandour AA. 1996. The beneficial role of abscisic acid for regenerates of Ruta graveolens spp divariacata Tenore (Gams) suffering from transplant shock. Angew Bot 70:221–223. Hartung W, Behl R. 1975. Lokalisation des akropetalen Tansports von 2-[14C] Abscisinsa¨ure in Wurzeln von Phaseolus coccineus L. und Hinweise fu¨r einen Radialtransport von ABA zwischen Zentralzylinder und Rindenzylinder. Planta 122:53–59. Hartung W, Davies WJ. 1991. Drought-induced changes in physiology and ABA. In: Davies WJ, Jones HG, eds. Abscisic Acid, Physiology and Biochemistry. Oxford, U.K.: BIOS Scientific, pp 63–79.

Hose et al. Hartung W, Dierich B. 1983. Uptake and release of abscisic acid by runner bean root tip segments. Z Naturforsch 38c:719–723. Hartung W, Heilmeier H. 1993. Stomatal responses to abscisic acid in natural environments. In: Jackson MB, Black CR, Eds. Interacting stresses on plants in a changing climate. Berlin; Springer-Verlag, pp 525–542. Hartung W, Jeschke WD. 1999. Abscisic acid: A long-distance stress signal in salt-stressed plants. In: Lerner HR, ed. Plant Responses to Environmental Stresses. New York; Marcel Dekker, pp 333–348. Hartung W, Turner NC. 1997. Abscisic acid relations in stressed roots. In: Altman A, Waisel Y, eds. Biology of Root Formation and Development. New York; Plenum, pp 125–132. Hartung W, Zhang J, Davies WJ. 1994. Does abscisic acid play a stress physiological role in maize plants growing in heavily compacted soil? J Exp Bot 45:221–226. Hartung W, Sauter A, Turner NC, Fillery I, Heilmeier H. 1996. Abscisic acid in soils: what is its function and which factors and mechanisms influence its concentration? Plant Soil 184:105–110. Hartung W, Wilkinson S, Davies WJ. 1998. Factors that regulate abscisic acid concentrations at the primary site of action at the guard cell. J Exp Bot 49 (special issue):361–367. Hartung W, Peuke AD, Davies WJ. 1999. Abscisic acid - a hormonal long distance stress signal in plants under drought and salt stress. In: Pessarakali M, ed. Handbook of Crop Stress. 2nd ed. New York; Marcel Dekker, pp 731–747. Hose E. 2000. Untersuchungen zum radialen Abscisinsa¨ureund Wassertransport in Wurzeln von Helianthus annuus L. und Zea mays L. Thesis, University of Wu¨rzburg. Hose E, Steudle E, Hartung W. 2000. Abscisic acid and hydraulic conductivity of maize roots: a root cell and pressure probe study. Planta 211:874–882. Ingram J, Bartels D. 1996. The molecular basis of dehydration tolerance in plants. Annu Rev Plant Physiol Plant Mol Biol 47:377–403. Jackson MB. 1991. Regulation of water relationships in flooded tomato plants by ABA from leaves, roots and xylem sap. In: Davies WJ, Jones HG, eds. Abscisic Acid Physiology and Biochemistry. Oxford, U.K.: Bios Scientific, pp 217–226. Jackson MB, Davies WJ, Else MA. 1996. Pressure-flow relationships, xylem solutes and root hydraulic conductance in flooded tomato plants. Ann Bot 77:17–24. Jeschke WD, Peuke AD, Pate JS, Hartung W. 1997a. Transport, synthesis and catabolism of abscisic acid (ABA) in intact plants of castor bean (Ricinus communis L.) under phosphate deficiency and moderate salinity. J Exp Bot 48:1737–1747.

Abscisic Acid Jeschke WD, Holobrada M, Hartung W. 1997b. Growth of Zea mays L. plants with their seminal roots only. Effects on plant development, xylem transport, mineral nutrition and the flow and distribution of abscisic acid (ABA) as a possible shoot to root signal. J Exp Bot 48:1229–1239. Jovanovic L, Daeter W, Hartung W. 1992. Compartmental analysis of abscisic acid in root segments of two maize lines differing in drought susceptibility. J Exp Bot 43(suppl):37. Lee HS, Milborrow BV. 1997. Endogenous biosynthesis precursors of (+)-abscisic acid. V. Inhibition by tungstate and its removal by cinchonine shows that xanthoxal is oxidised by a molybdo-aldehyde oxidase. Aust J Plant Physiol 24:727–732. Lichtenthaler HK. 1999. The 1-deoxy-D-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants. Annu Rev Plant Physiol Plant Mol Biol 50:47–65. Ludewig M, Do¨rffling K, Seifert H. 1988. Abscisic acid and water transport in sunflowers. Planta 175:325–333. Markhart AH, Fiscus EL, Naylor AW, Kramer PJ. 1979. Effect of abscisic acid on root hydraulic conductivity. Plant Physiol 64:611–614. Marschner H. 1995. Mineral Nutrition of Higher Plants. 2nd ed. London; Academic Press. Mu¨ller M, Deigele C, Ziegler H. 1989. Hormonal interactions in the rhizosphere of maize (Zea mays L.) and their effects on plant development. Z Pflanz Bodenkunde 152:247–254. Mu¨ller R, Steigner W, Gimmler H, Kaiser WM. 1990. Effect of ammonium on dark-CO2 fixation and on cytosolic and vacuolar pH values in Eremosphaera viridis de Bary (Chlorococcales). J Exp Bot 41:441–448. Mulholland BJ, Black CR, Taylor OB, Roberts JA, Lenton JR. 1996a. Effect of soil compaction on barley (Hordeum vulgare L.) growth I. J Exp Bot 47:539–550. Mulholland BJ, Black CR, Taylor OB, Roberts JA. 1996b. Effect of soil compaction on barley (Hordeum vulgare L.) growth II. J Exp Bot 47:551–556. Munns R, King RW. 1988. Abscisic acid is not the only stomatal inhibitor in the transpiration stream. Plant Physiol 88:703–708. Munns R, Sharp RE. 1993. Involvement of ABA in controlling plant growth in soils at low water potential. Aust J Plant Physiol 20:425–437. Netting AG, Willows RD, Milborrow BV. 1992. The isolation of the prosthetic group released from a bound form of abscisic acid. Plant Growth Regul 11:327–339. Okamoto M, Hirai N, Koshimizu K. 1988. Biosynthesis of abscisic acid. Mem Coll Agric Kyoto Univ 132:79–115. Parry AD, Horgan R. 1992. Abscisic acid biosynthesis in roots. I. The identification of potential abscisic acid precursors, and other carotenoids. Planta 187:185–191.

447 Peterson CA. 1988. Exodermal Casparian bands, their significance for ion uptake by roots. Physiol Plant 72:204–208. Peuke AD, Jeschke WD, Hartung W. 1994. The uptake and flow of C, N and ions between roots and shoots in Ricinus communis L. III. Long-distance transport of abscisic acid depending on nitrogen nutrition and salt stress. J Exp Bot 45:741–747. Quintero JM, Fournier JM, Benlloch M. 1999. Water transport in sunflower root systems: effects of ABA, Ca2+ status and HgCl2. J Exp Bot 50:1607–1612. Rademacher W, Fritsch H, Graebe JE, Sauter H, Jung J. 1987. Tetcyclacis and triazole-type plant growth retardants: their influence on the biosynthesis of gibberellins and other metabolic processes. Pestic Sci 21:241– 252. Roberts JKM, Wemmer D, Ray P, Jardetzky O. 1982. Regulation of the cytoplasmic and vacuolar pH in maize root tips under different experimental conditions. Plant Physiol 69:1344–1347. Saab IN, Sharp RE, Pritchard J, Voetburg GS. 1990. Increased endogenous abscisic acid maintains primary root growth and inhibits shoot growth of maize seedlings at low water potential. Plant Physiol 93:1329– 1336. Sauter A, Hartung W. 2000. Abscisic acid conjugates—do they play a role as long-distance stress signal in the xylem? J Exp Bot 51:929–936. Schiller P, Heilmeier H, Hartung W. 1997. Abscisic acid (ABA) relations in the aquatic resurrection plant Chamaegigas intrepidus under naturally fluctuating conditions. New Phytol 136:603–611. Sharp RE. 1990. Comparative sensitivity of root and shoot growth at low water potential. BSPGR Monogr 21:29– 44. Sharp RE, Silk WK, Hsiao TC. 1988. Growth of primary maize roots at low water potential. I. Spatial distribution of expansive growth. Plant Physiol 87:50–57. Simonis W 1947. U¨ber den Einfluß des Bodenwassergehaltes auf die Kohlensa¨ureassimilation und den Ertrag bei Einja¨hrigen. Ber Dtsch Bot Ges 59:52–68. Sinnott EW. 1960. Plant Morphogenesis. New York; McGraw-Hill. Slovik S, Daeter W, Hartung W. 1995. Compartmental redistribution and long-distance transport of abscisic acid (ABA) in plants as influenced by environmental changes in the rhizosphere—a biomathematical model. J Exp Bot 46:881–894. Steudle E. 1993. Pressure probe techniques: basic principles and application to studies of water and solute relations at the cell, tissue and organ level. In: Smith JAC, Griffith H, eds. Water Deficits: Plant Responses from Cell to Community. Oxford, U.K.: Bios Scientific, pp 5–36.

448 Steudle E. 1994. The regulation of plant water at the cell, tissue, and organ level: role of active processes and of compartmentation. In: Schulze ED, ed. Flux Control in Biological Systems. From Enzymes to Populations and Ecosystems. San Diego, CA: Academic Press pp 237–299. Steudle E, Peterson CA. 1998. How does water get through roots? J Exp Bot 49:775–788. Taylor IB. 1991. Genetics of ABA synthesis. In: Davies WJ, Jones HG, eds. Abscisic Acid Physiology and Biochemistry. Oxford, U.K.: Bios Scientific, pp 23–37. Trewavas AJ, Jones HG. 1991. An assessment of the role of ABA in plant development. In: Davies WJ, Jones HG, eds. Abscisic Acid Physiology and Biochemistry. Oxford, U.K.: Bios Scientific, pp 169–188. Walker-Simmons M, Kudran D, Warner R. 1989. Reduced accumulation of ABA during water stress in a molybdenum cofactor mutant of barley. Plant Physiol 90:728–733. Weaver JE. 1926. Root Development of Field Crops, New York; McGraw-Hill.

Hose et al. Wolf O, Jeschke WD, Hartung W. 1990. Long distance transport of abscisic acid in NaCl-treated intact plants of Lupinus albus. J Exp Bot 41:593–600. Zeier J. 1998. Pflanzliche Abschlussgewebe der Wurzel: Chemische Zusammensetzung und Feinstruktur der Endodermis in Abha¨ngigkeit von Entwicklung und a¨ußeren Einflu¨ßen. Thesis, University of Wu¨rzburg. Zhang J, Davies WJ. 1987. Increased synthesis of ABA in partially dehydrated root tips and ABA transport from roots to leaves. J Exp Bot 38:2015–2023. Zhang J, Davies WJ. 1989. Abscisic acid produced in dehydrating roots may enable the plant to measure the water status of the soil. Plant Cell Environ 12:73–81. Zhong W, Hartung W, Komor E, Schobert C. 1996. Phloem transport of abscisic acid in Ricinus communis L. seedlings. Plant Cell Environ 19:471–477. Zimmermann HM, Steudle E. 1998. Apoplastic transport across young maize roots: effect of the exodermis. Planta 206:7–19. Zimmermann MH, Hartmann K, Schreiber L, Steudle E. 2000. Chemical composition of apoplastic transport barriers in relation to radial hydraulic conductivity of corn roots (Zea mays L.). Planta 210:302–311.

27 Role of Ethylene in Coordinating Root Growth and Development Ahmed Hussain and Jeremy A. Roberts University of Nottingham, England

I.

INTRODUCTION

Plant growth and development are coordinated by chemicals known as phytohormones. Anton Nicolyovitch Nelubjov (1901) first proposed that ethylene had the capacity to influence plant behavior a century ago, and since that time it has been shown to be responsible for profoundly affecting growth and development, even at concentrations as low as 0:1
L L1 (Osborne, 1989). However, unlike the other phytohormones, ethylene is a simple volatile hydrocarbon (C2 H4 ) that is active in the gaseous form. A diverse range of physiological processes are influenced by ethylene. These include the senescence and abscission of leaves and flowers, the ripening of fruit, and the germination of seeds (Abeles et al., 1992). Moreover, dicotyledonous seedlings exposed to ethylene display a characteristic syndrome of responses that are known collectively as the triple response. This diagnostic phenomenon comprises a reduction in stem elongation, an increase in the radial expansion of the stem, and a diagravitropic orientation of the aerial tissues. However, ethylene also plays a fundamental role in coordinating the growth and development of roots. In this chapter the importance of ethylene in the development of root morphology is described, and the role of the gas in the regulation of plant responses to the rooting environment is evaluated. Finally, the exciting opportunities of generating desirable root characteristics by the manipulation of ethylene biosynthesis and/or perception are discussed.

II.

ETHYLENE AND ROOT DEVELOPMENT

A.

Primary Root Development and Growth

The growth of the primary root begins during seed germination. Ethylene has long been recognized as having the capacity to stimulate germination in some seeds and thus initiate the process of primary root development (Vacha and Harvey, 1927). Indeed, the ethylene-insensitive mutant of Arabidopsis thaliana Etr1 exhibits reduced germination rates relative to wild-type plants (Bleeker et al., 1988). It has been proposed that ethylene may be directly involved in generating the force within the embryo necessary to breach the seed coat, although the precise mechanism is unclear. One possibility is that the gas contributes to the weakening of the endosperm cap by increasing the activity of hydrolytic enzymes such as polygalacturonases and expansins (Bradford et al., 1999; Chen and Bradford, 2000). However, an alternative explanation is that ethylene may have an indirect role by interacting with known inhibitors of germination such as abscisic acid (ABA) (Abeles et al., 1992). Production of ethylene in dormant seeds is generally low; however, the development of the embryo is accompanied by greater rates of ethylene production (Corbineau et al., 1989). The exact role of ethylene in coordinating embryo development has yet to be defined. Ethylene has been shown to have an inhibitory effect on cell division in the roots of peas and maize, possibly by reducing DNA polymerase activity or the 449

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size of the meristematic region (Burg, 1973). Therefore, an inhibitory role for ethylene in root development may be anticipated. However, recent studies using the ethylene-insensitive Never ripe (Nr) mutant in tomato have demonstrated that under unstressed conditions root development occurs in a manner equivalent to wild-type plants and no morphological differences between the genotypes are observed (Clark et al., 1999). Indeed, detailed analysis of root growth actually suggested that root growth was greater in the Nr mutants. The same study observed that under suboptimal rooting conditions, Nr plants exhibited an inability to penetrate sand. This resulted in greatly elongated roots running along the surface, relative to wild-type plants which were able to penetrate the sand and exhibit normal root growth (Clark et al., 1999). In similar studies with tomato seedlings grown on agar of different concentrations, Zacarias and Reid (1992) suggested that ethylene may be necessary for the development of the root cap which facilitates penetration of the media. The role of ethylene in mediating the development of the primary roots as a whole appears uncertain. Mudge (1988) suggested that ethylene is not the principal regulatory factor and is only indirectly involved in the early stages of root development. By fusing the promoter of a gene encoding the ethylene biosynthetic enzyme ACC synthase (ASC1) of Arabidopsis to the -glucuronidase (GUS) reporter gene, Rodrigues-Pousada et al. (1993) were able to study the expression of this gene during the development of primary roots. It was observed that expression was highest in the root tip but only between 4 and 12 days after germination. Expression of the ACS1 gene may be indirectly stimulated by the auxin indoleacetic acid (IAA) in this zone, or ethylene may have an inhibitory effect on the formation of root branching since lateral root development correlated with reductions of ACS1 gene expression in the root tip. Rodrigues-Pousada et al. (1993) considered that the observed concentration of expression in vascular tissue in primary roots could be associated with ethylene-mediated root growth and vascular development, low ‘‘basal’’ levels of ethylene promoting elongation with higher levels of the hormone inhibiting the process. Ethylene has been shown to correlate with the formation and lignification of the xylem vessels (Abeles et al., 1992). This observation is consistent with the hypothesis that ethylene may be important in promoting elongation of primary roots by assisting in the development of the vascular system.

Hussain and Roberts

B.

Adventitious Rooting

Ethylene is generally considered to be a promoter of adventitious rooting (Robbins et al., 1983). The process of adventitious root formation comprises, initially, the initiation of root primordia followed by their activation. Later those primordia emerge and elongate. The application of an inhibitor of ethylene biosynthesis, aminoethoxyvinylglycine (AVG), has been shown to reduce the number of adventitious roots in mung bean cuttings (Jusaitis, 1986). Similar responses were observed following treatment with Ag2þ or 2,5-norbornadiene, both inhibitors of ethylene perception (Robbins et al., 1985). More recently, reduced adventitious rooting has been reported in ethylene-insensitive mutants of tomato and petunia, and a role for ethylene in the formation of adventitious roots in these species has been proposed (Clark et al., 1999). Although auxin has the capacity to induce adventitious rooting (see Chapter 21 by De Klerk in this volume), the use of ethylene-insensitive mutants, coupled with the use of compounds that impair ethylene perception or biosynthesis, clearly demonstrates that ethylene is directly involved in stimulating adventitious root formation. However, crosstalk between ethylene and auxin perception and response pathways may be fundamental in the initiation or development of adventitious roots. Exposure of developing primary or adventitious roots to ethylene typically results in rapid inhibition of root elongation and promotion of its radial expansion, responses that may be reversed by the removal of the gas or the blocking of its action (Abeles et al., 1992). Although ethylene is known to influence cell division and elongation, it is considered that its effect on cell walls is the primary cause of reduced elongation and increased radial expansion of roots. Analysis of cells in pea roots following treatment with ethylene suggested that there was an alteration in cellulose deposition with a shift from a transverse to a longitudinal pattern (Vian et al., 1982). This change in cell wall structure resulted in the altered direction of cell expansion. C.

Ethylene and Responses to Gravity

Gravitropism in roots and shoots may be partly regulated by differing mechanisms (Smalle and Van der Straeten, 1997). In roots, gravity is thought to be perceived by sedimenting amyloplasts in cells within the central columella of the root cap (see Chapter 30 by Pilet and Chapters 20 and 29 by Porterfield in this volume). An involvement of ethylene in gravitropism was considered to be the consequence of elevated auxin

Role of Ethylene

concentrations on the lower side of horizontally stimulated roots with the increased production of the gas, inhibiting elongation and initiating a downward curvature of the root (Burg and Burg, 1968; Evans, 1991). The auxin-insensitive mutants of Arabidopsis (axr1, axr2, axr3, and aux1) exhibit an agravitropic root response (Estelle and Klee, 1994) and are also insensitive to ethylene. Moreover, AUX1 gene expression is elevated at the root tip (Bennett et al., 1996), the region of the root that is known to control gravitropism. This substantiates claims that auxin-induced ethylene production could be contributing to the downward curvature of roots. Ethylene is an important component of the gravitropic machinery (Roman et al., 1995). These investigators showed that the ethylene root-insensitive eir1 mutant of Arabidopsis displays an agravitropic phenotype despite being responsive to auxin. Other studies examining the role of ethylene in gravitropism have employed the use of ethylene biosynthesis inhibitors, and revealed reductions in the rate of bending in various species (Abeles et al., 1992). One possible explanation for the effects of ethylene is a consequence of the ability of the gas to block auxin transport (Lee et al., 1990; Roman et al., 1995). On balance, there is good evidence to support a role for ethylene in mediating gravitropic responses in roots, but it is not clear whether its primary function is in the transduction of an auxin-mediated event. D.

Lateral Root Development

Lateral root formation is apparently mediated by auxin (see Chapter 23 by Gaspar et al. in this volume). As IAA possesses the capacity to increase ACC synthase activity and therefore stimulate ethylene production, the gas may act as an ‘‘intermediate’’ in the effects of auxin on lateral root development (Abel and Theologis, 1996; Rodrigues-Pousada et al., 1999). Indeed, Rodrigues-Pousada et al. (1993) demonstrated that increased activity of ACS1 correlated with the initiation of the lateral root meristem in Arabidopsis, agreeing with previous work detecting an ‘‘indirect,’’ or auxin-mediated, correlation of ethylene and lateral root initiation (Torrey, 1976). However, RodriguesPousada et al. (1993) argued that ethylene may play an additional role in the elongation process because the development of lateral roots was associated with an increase in expression of ACS1. In support of this hypothesis, the ethylene- and auxin-insensitive mutant of Arabidopsis, axr1, has been shown to display reduced lateral root formation (Lincoln et al., 1990). However, a critical role for the gas seems unlikely as

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ethylene-insensitive mutants of Arabidopsis exhibit normal lateral root development (Bleeker et al., 1988). Laskowski et al. (1995) suggested that although hormones mediate the development of lateral root primordia, once induced, the roots grow without ethylene involvement. Therefore, the association of ethylene production with lateral root development may be related not to the development of the root per se but with the wounding of cells as the lateral root forces its way through the cortex. Moreover, Charlton (1996) reported that there is an increase in the activity of hydrolytic enzymes such as polygalacturonase (PG) in front of the lateral root primordia which is involved in the breakdown of the cortical cells (Bonfante and Peretto, 1993). The activity of PG is known to be promoted by ethylene during such processes as fruit ripening and abscission (for review see Roberts et al., 2000) and therefore, this hormone may play an important role in facilitating lateral root development by permitting their extension through the cortex and the epidermis. E.

Root Hair Development

Root hairs are important structures that serve to absorb water and minerals, facilitate the penetration of the root tip through the soil matrix, and anchor the root (see Chapter 5 by Ridge and Katsumi in this volume). Ethylene was first shown to increase the growth of root hairs in pea, fava bean, and lupin > 60 years ago (Abeles et al., 1992). Root hair development and growth were investigated using Arabidopsis as a model plant. Tanimoto et al. (1995) showed that the constitutive ethylene mutant of Arabidopsis (ctr1) produced root hairs that were longer than those of the wild-type, whereas the normal formation of such trichoblasts in wild-type plants failed to occur when treated with inhibitors of ethylene biosynthesis (AVG) or action (Ag2þ ). Treatment of roots of wild-type plants with ethylene or ACC invoked analogous root hair morphology to that observed in an untreated ctr1 mutant. However, Schneider et al. (1997) suggested that the influence of ethylene on root hair formation acts downstream from the root-hair-less genes RHL1, RHL2, and RHL3 since the reduced trichoblast development, as a result of mutations in these genes, is only weakly restored following ethylene treatment. The ROOT HAIR DEFECTIVE (RHD6) gene also appears to be involved in root hair development since the mutation of this gene results in a reduced number of root hairs but an increase in the incidence of multiple root hairs on each epidermal cell. Intriguingly this response is

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also reproduced following treatment of wild-type plants with AVG (Masucci and Schiefelbein, 1994). The RHL1-3 genes are considered to promote root hair development constitutively in both trichoblasts and atrichoblasts roots; however, their action in atrichoblasts is impaired by the function of the genes GLABRA 2 (GL2) and TRANSPARENT TESTA GLABRA (TTG). These genes appear to regulate root hair formation negatively in atrichoblasts (Galway et al., 1994; Masucci and Schiefelbein, 1996), with the result that mutations in either of these genes (GL2 or TTG) results in ectopic root hair formation. It has been shown that both GL2 and TTG prevent root hair development in atrichoblasts by negatively regulating ethylene and auxin activity, both of which act downstream in the process of root hair formation (Masucci and Schiefelbein, 1996). The action of both hormones converges at the AUXIN RESISTANT 2 (AXR2) gene which when suppressed by mutation results in insensitivity of roots to both ethylene and auxin whereas exogenous ACC or IAA does not revert the phenotype (Masucci and Schiefelbein, 1996). Mutants of Arabidopsis that are insensitive to either ethylene or auxin (Etr1 and aux1) exhibit normal root hair morphology whereas double mutants, plants insensitive to both ethylene and auxin, display reductions in root hair formation (Smalle and Van der Straeten, 1997). The observed reduction in root hair formation in double mutants may be overcome by application of IAA but not ACC, implying that ethylene may act through more than one pathway (Masucci and Schiefelbein, 1996). Ethylene is reported to promote the elongation of root hairs (Pitts et al., 1998). Analysis of two mutants of Arabidopsis that either are insensitive to ethylene (Etr1) or overproduce ethylene (eto1-1) revealed that root hair elongation was enhanced in eto1-1 plants whereas elongation was impaired in the Etr1 mutant relative to wild-type plants. Ethylene may therefore influence a number of events associated with the differentiation and development of root hairs although its precise role in coordinating these processes in vivo is unclear. III.

IMPORTANCE OF ROOT ETHYLENE IN RESPONSE TO ENVIRONMENTAL STRESS

A.

Waterlogging and Hypoxia

Excessive amounts of water in the rooting environment can become deleterious for normal plant function. This

is due to the greatly reduced diffusion of gases when air spaces within the soil matrix are filled with water, leading to hypoxia or anaerobic conditions (see Chapter 42 by Armstrong and Drew in this volume). Ethylene plays an important role in mediating the responses in nonwetland species to periods of flooding or hypoxia (Jackson, 1985; Drew, 1997). Under these conditions, the promotion of stress ethylene is observed in many species and the gas has been proposed to mediate plant responses such as aerenchyma development that assist in overcoming hypoxic stress by improving the flow of O2 from the shoot to the root and reducing the number of cells that consume O2 (Drew et al., 2000; Chapter 42 by Armstrong and Drew in this volume). ACC synthase and ACC oxidase activity are stimulated when maize and tomato plants are subject to hypoxic conditions (Wang and Arteca, 1992; Olson et al., 1995; He et al., 1996), and these increases are correlated with an elevation in ethylene production. Aerenchyma was induced in maize by exposure of well-oxygenated plants to low concentrations of ethylene, whereas aerenchyma formation in hypoxic roots was reduced following treatment with inhibitors of ethylene action (Drew, 1997). In species such as rice, ethylene also promotes the suberization and lignification of cells in or close to the hypodermis, and these events reduce the diffusion of O2 out of the root and into the soil environment (Drew et al., 2000; Chapter 56 by Beyrouty in this volume). The activity of the cell wall degrading enzymes, such as cellulases, is stimulated under hypoxic conditions or in response to ethylene (He et al., 1994). Furthermore, cell wall metabolism can be regulated by the enzyme xyloglucan endotransglycosylase (XET). Increased expression of a gene encoding XET was found when maize plants were subjected to flooding or ethylene treatment, and it has been proposed that this enzyme may contribute to the formation of aerenchyma (Saab and Sachs, 1996). Thus, lysigenous aerenchyma development may involve the enzymically mediated breakdown of cortical cells by ethylene. It should be noted that aerenchyma formation may also occur when maize plant roots experience periods of nitrogen or phosphorus deficiency (He et al., 1992). This process is mediated by ethylene, as with responses to hypoxia, but may be the consequence of increased sensitivity to low levels of constitutive ethylene rather than increases in production of the gas. A second response exhibited by plants that may help them to avoid hypoxic stress under flooded conditions is a stimulation of adventitious rooting. Morgan and Drew (1997) proposed that replacing existing roots

Role of Ethylene

that have been damaged by hypoxic conditions with new roots that possess larger air spaces within the cortex facilitates function and growth. Ethylene has the capacity to stimulate the formation of adventitious roots, as previously described, and there is evidence from studies on Rumex palustris that this might be a consequence of an increase in sensitivity of the rootforming tissues to endogenous IAA (Visser et al., 1996). B.

Soil Impedance and Water Stress

As roots develop, they inevitably encounter the mechanical impedance of the soil (see Chapter 45 by Masle in this volume). Ethylene production has long been known to increase when roots encounter a physical barrier and is recognised to play an important role in the penetration of roots through soil of high bulk density (Kays et al., 1974). The involvement of ethylene in increasing the ability of tomato and Arabidopsis roots to penetrate high-resistance media was demonstrated by Sarquis et al. (1991) and Zacarias and Reid (1992). Indeed, Zacarias and Reid (1992) grew Arabidopsis plants on media containing Ag2þ , to impair ethylene binding, and observed that penetration into the media was greatly reduced relative to plants grown in the absence of silver. Hussain et al. (1999) demonstrated that ethylene was important for tomato roots that encountered compacted soil. Penetration was impaired in transgenic plants with a reduced capacity to synthesize the hormone. Furthermore, Clark et al. (1999) reported that the ethylene-insensitive mutant of tomato Nr exhibited a reduced capacity to penetrate a sandy media relative to wild-type plants. Exogenous ethylene (0:01
L L1 ) promotes the radial expansion of roots (Osborne, 1989), and it is this response that aids root penetration into strong soils (Hettiaratchi, 1990). The capacity of ethylene to stimulate root hair production has been discussed. By stimulating root hair growth, ethylene can increase the ability of roots to penetrate strong soils since root hairs provide anchorage. Ethylene is also suggested to mediate helical movement of the root cap, which may facilitate penetration (Woods et al., 1984). Increased impedance of soils is generally associated with increased bulk density. As the density increases, the availability of O2 to roots may become reduced. He et al. (1996) described how ethylene promoted aerenchyma formation when maize roots encountered high mechanical impedance. Therefore, aerenchyma formation may be important for root function when impeded (Drew et al., 2000). Indeed, the process of penetrating

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strong soils may demand greater rates of respiration, and thus an efficient oxygen supply may also be important. Ethylene plays a role in regulating the growth of roots under water-stressed conditions. Low water potentials promote an increase in ethylene production in the primary root of the vp5 mutant of maize that has an impaired capacity to synthesize ABA (Spollen et al., 1997). Furthermore, inhibitors of ethylene biosynthesis or action restored root growth in vp5 plants exposed to low water potentials. It was suggested that a relationship between ethylene and ABA regulates root growth under water-stressed conditions, since inhibition of ABA biosynthesis with fluridone promoted an increase in ethylene biosynthesis and associated reductions in root growth were observed (Spollen et al., 1997; Chapter 26 by Hose et al. in this volume). Waterstressed roots of woody plants are often considered to be responsible for the observed abscission of leaves upon rewatering. Gomez-Cadenas et al. (1996) suggested that water stress results in an ABA-mediated increase in ACC production and, upon rewatering, ACC is transported in the transpiration stream to the shoot where it can be converted to ethylene to stimulate leaf abscission. However, a general role for stress ethylene in regulating responses to water stress has yet to be established, and the speed with which the stress is imposed may explain why much of the data appear equivocal (Morgan and Drew, 1997). C.

Nutrient Availability

Chapin (1990) suggested that plant responses to nutrient starvation are mediated by changes in the balance of phytohormones, with the involvement of both ABA and cytokinins. Ethylene, however, was proposed to have an important role as a localized stress signal, coordinating responses to transient changes in nutrient availability (Lynch and Brown, 1997). The reduced availability of nitrogen and phosphorus did not stimulate ethylene production. Instead, it appeared to increase the sensitivity of specific cortical cells to constitutive levels of the gas, although this effect may be species specific. Phosphorus starvation of pea plants also induced aerenchyma formation (Eshel et al., 1995). The formation of aerenchyma under low-nitrogen or low-phosphorus conditions is believed to reduce the number of active cells within the root demanding essential nutrients for growth and development (Eshel et al., 1995). Furthermore, lysis of aerenchyma-forming cortical cells permits the nitrogen and phosphorus from such cells to be mobilized and sent toward the

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root apices to maintain growth. It is also hypothesized that reduced gravitropism as a consequence of phosphorus deprivation-induced ethylene production may result in the development of a shallow but widespread root system (Lynch and Brown, 1997). This seems an effective adaptive strategy, considering that phosphorus availability is generally greatest at the surface (Bonser et al., 1996). Another ethylene-mediated response to nutrient deficiency is the promotion of root hair elongation. These structures greatly increase the surface area of the roots, and thus their elongation will improve nutrient acquisition. Indeed, root hair production was shown to be stimulated in the ethylene/auxin insensitive mutant of Arabidopsis, axr2, under low-phosphorus conditions (Wilson et al., 1990). Bates and Lynch (1996) argued that changes in root hair elongation are a consequence of local availability of nutrients rather than a generalized response to stress. Therefore, it is appealing to put forward ethylene as a candidate for regulating elongation of root hairs in response to local nutrient availability (Lynch and Brown, 1997). Ethylene may also result in root hair formation in regions of the root where their development is not typical (Abeles et al., 1992; Tanimoto et al., 1995). Thus, it appears that ethylene is important in mediating adaptive strategies to overcome the effects of reduced nutrient availability, particularly of phosphorus.

IV.

ETHYLENE AS A ROOT-GENERATED SIGNAL

The growth and development of plants are mediated by signals produced by environmental stimuli. Frequently the source of these signals originates from the roots and is transported to the aerial tissues in the transpiration stream. Indeed, hormonal signals from stressed roots that moderate shoot growth are widely reported (see Chapter 28 by Bacon et al. in this volume). As a gaseous compound, ethylene may move from roots to shoots within intercellular air spaces (Eklund et al., 1992). However, not only is this form of transport potentially slow, but movement is difficult to regulate and therefore it seems unlikely that ethylene may act as a root signal in its gaseous form. Moreover, under waterlogged conditions the reduced availability of O2 inhibits the oxidation of ACC to ethylene (Jackson, 1993). Wang and Arteca (1992) observed increases in ethylene production in the shoots of water-

logged tomato plants despite reduction in ethylene production in roots. Since ACC is water soluble and may be transported in the transpiration stream, it was proposed that this ethylene precursor might constitute the root signal in such cases. Under this scheme, ACC arriving in the shoot is converted into ethylene and thus influences shoot growth and development. Jackson (1993) substantiated this hypothesis by reporting that inhibitors of ACC synthesis supplied to waterlogged plants resulted in reduced leaf epinasty, a classical symptom of exposure to ethylene. Measurement of ACC in the xylem sap of waterlogged tomato plants and the calculation of the delivery rates of ACC to the shoots have unequivocally demonstrated that waterlogging stimulates ACC synthesis in roots and an elevated amount of this ethylene precursor is then exported to the shoot (Else et al., 1995). English et al. (1995) reported increases in the expression of an ACC oxidase gene in the leaves of flooded tomato plants in response to increased delivery of ACC. A similar root-generated ACC signal may also be involved in coordinating leaf growth in tomato plants grown in compacted soil (Hussain et al., 2000). Hussain et al. (1999) used a split-root approach to examine root-sourced signals that mediate shoot responses to soil compaction. The experiments comprised plants that were grown with their root systems divided between uncompacted and compacted soil. Reductions in leaf growth were attributed to increases in ethylene production. However, a recovery in leaf growth and a reduction in ethylene production were observed after severing the part of the root system growing in soil of high bulk density. Increased ACC oxidase activity was discovered in leaves of compaction-stressed tomato plants whereas no upregulation in ACC synthase activity was detected, implying that ACC synthesized in roots may control ethylene production in shoots in an analogous manner to that reported for flooded plants (Hussain, unpublished).

V.

ETHYLENE AND THE RHIZOSPHERE

An efficient system for extracting essential nutrients and water from the rhizosphere is necessary for the establishment and growth of plants. To exploit the soil for these resources, plants frequently exhibit associations with microorganisms within the rhizosphere. Ethylene is an important component within the mechanisms of initiating fungal and bacterial associations and in mediating responses to biotic stress within the rhizosphere (Lynch and Brown, 1997).

Role of Ethylene

The symbiotic association between rhizobaceria and roots occurs in structures known as nodules, especially those in leguminous plants (see Chapter 47 by Vance in this volume). In most legumes it has been reported that exogenous ethylene inhibits nodule formation (Lee and LaRue, 1992; Van Workum et al., 1995). Moreover, applications of AVG has been shown to increase nodule formation in alfalfa (Peters and Crist-Estes, 1989), and mutants of pea that exhibit poor nodulation displayed normal rates of nodulation following treatment with inhibitors of ethylene production (Guinel and LaRue, 1992). Inoculation of Vicia sativa with Rhizobia resulted in increased production of ethylene, relative to unnodulated plants (Zaat et al., 1989). Increases in ethylene production were accompanied by reductions in root elongation and by proliferation of root hair formation, responses that were not displayed following treatment with AVG. Increased production of ethylene was also reported for nodulated roots of soybean, a response attributed to greater ACC levels in the host tissue (Hunter, 1993). Therefore, ethylene may be regarded as being involved in nodule formation. Lynch and Brown (1997) postulated that ethylene is involved in several stages of legume–Rhizobium symbiosis, including the initial response of Rhizobia to Nod factors. Van Workrum et al. (1995) suggested that ethylene production, following the initial infection, might be important in regulating the number of subsequent nodules formed. Indeed, higher rates of nitrogen fixation were associated with greater production of ethylene whereas instances of low nitrogen fixation resulted in reduced ethylene production (Ligero et al., 1987). Glick et al. (1999) reported that growth-promoting rhizobacteria produced ACC deaminase, an enzyme that reduces ethylene production by cleaving ACC into ammonia and -ketobutyrate (Honma and Shimomura, 1978). Indeed, bacterial mutants impaired in their capacity to synthesize ACC deaminase failed to stimulate root elongation of canola plants (Glick et al., 1994) whereas the promotion of root elongation by infection with ACC deaminase-producing bacteria could be mimicked by applications of AVG (Hall et al., 1996). Upon infection, growth-promoting rhizobacteria synthesize and secrete IAA into the roots of host plants, resulting in stimulation of cell proliferation and thus in the development of the nodule. In addition, the elevated levels of auxin may lead to an upregulation of ACC synthase activity (Glick et al., 1999). Extra ACC produced by the host plant as a consequence of rhizobacterial infection will be exuded from the roots, providing a unique source of nitrogen that may only be

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utilized by rhizobacteria. This permits these organisms to proliferate in an environment in which others cannot. Once established within the host, rhizobacteria utilize ACC deaminase to lower ACC within the host plant and thus promote root elongation.

VI.

MANIPULATION OF ETHYLENE BIOSYNTHESIS AND ACTION IN THE ROOTS TO PROMOTE DESIRED CHARACTERISTICS

The recent cloning of genes involved in the synthesis and perception of ethylene will permit the manipulation ethylene-mediated developmental processes. This may be achieved at several levels. Firstly, the biosynthesis and catabolism of ethylene may be altered, and secondly, ethylene perception may be modulated. The ability to manipulate ethylene production or action may be of benefit in promoting desirable changes in root development. The biosynthetic pathway of ethylene is well characterized and well described. Ethylene biosynthesis is regulated by the transcription of ACC synthase and ACC oxidase genes, each encoded by multigene families. Indeed, distinct ACC synthase and ACC oxidase genes are considered to respond to specific developmental and environmental stimuli (Theologis, 1992). Biosynthesis of ethylene has been reduced by using antisense technology for an ACC oxidase gene in tomato (LE-ACO1) (Hamilton et al., 1990) and ACC synthase (Oeller et al., 1991). Furthermore, by incorporating a bacterial gene encoding ACC deaminase, Klee et al. (1991) were able to reduce ethylene production by limiting the availability of ACC. Genes encoding ethylene receptors in Arabidopsis (Klee and Tieman, 1996) and tomato (Lanahan et al., 1994) have now been characterized, and their attenuation offers an additional strategy by which responses to the hormone can be manipulated. The technologies responsible for reduced ethylene biosynthesis or perception have already proved of value to the agricultural and horticultural industry. However, ethylene is important in promoting many desirable aspects in the development of the root system, particularly in mediating responses to abiotic and biotic stresses. It is therefore unlikely that plants impaired in ethylene biosynthesis or perception would provide commercially desirable phenotypes unless enhanced root elongation or delayed senescence of root structures is achieved. Ethylene is generally considered to have an inhibitory role in the develop-

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ment of bacterial and fungal symbioses; thus, reductions in the ethylene biosynthesis may result in an increase in the frequency of nodule formation or mycorrhizal development. The nutritional advantages of these relationships may promote increased growth and yield. Increased production of ethylene can be achieved by applications of ethephon. Owing to the characterization of an increasing number of constitutive and tissuespecific promoters, it may be possible in the near future to generate transgenic plants that overproduce ethylene at a given time or location. Alternatively, manipulation of homologs of the CTR1 gene product of Arabidopsis could lead to constitutive ethylene responses in some root tissues. Increased ethylene action may promote many desirable characteristic responses in plants. For example, ethephon can promote the initiation of adventitious root primordia. However, it also impairs the elongation of adventitious roots. Similarly, ethylene is considered to be involved in the development of lateral roots, in determining root thickness, in formation of root hairs, and in the formation of an aerenchyma. VII.

CONCLUSION

In this chapter the effects of ethylene on the growth and development of roots have been discussed. Ethylene stimulates or impairs many physiological and developmental processes in roots either directly or as an intermediate in the action of auxin. The gas seems to be particularly important in mediating appropriate adaptive responses to environmental stress and pathogen attack. With an increasing number of components of the ethylene signal transduction pathway now being identified (Bleeker, 1999), it seems likely that in the near future the precise role of the hormone in coordinating root development and providing a root–shoot signaling system will be fully elucidated. ACKNOWLEDGMENTS The authors wish to thank the Biotechnology and Biological Science Research Council for financial support. REFERENCES Abel S, Theologis A. 1996. Early genes and auxin action. Plant Physiol 111:9–17.

Abeles FB, Morgan PW, Saltveit ME. 1992. Ethylene in Plant Biology. 2nd ed. San Diego, CA: Academic Press. Bates TR, Lynch JP. 1996. Stimulation of root hair elongation in Arabidopsis thaliana by low phosphorus availability. Plant Cell Environ 19:529–538. Bennett MJ, Marchant A, Green HG, May ST, Ward SP, Millner PA, Walker AR, Schulz B, Feldmann K. 1996. Arabidopsis AUX1 gene: a permease-like regulator of root gravitropism. Science 273:948–950. Bleeker AB. 1999. Ethylene perception and signalling: an evolutionary perspective. Trends Plant Sci 4:269–274. Bleeker AB, Estelle MA, Somerville C, Kende H. 1988. Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana. Science 241:1086– 1089. Bonfante P, Peretto R. 1993. Cell-wall separation during the outgrowth of lateral roots in Allium porrum L. Acta Bot Neerl 42:187–197. Bonser AM, Lynch J, Snapp S. 1996. Effect of phosphorus deficiency on growth angle of basal roots in Phaseolus vulgaris. New Phytol 132:281–288. Bradford KJ, Chen F, Cooley MB, Dahal P, Downie B, Fukunaga KK, Gee OH, Gurusinghe S, Mella RA, Nonogaki H, Wu C-T, Yang H, Yim K-O. 1999. Gene expression prior to radicle emergence in imbibed tomato seeds. In: Black M et al., eds. Seed Biology. Oxon, U.K.: CABI Publishing, pp 231–253. Burg SP. 1973. Ethylene in plant growth. Proc Natl Acad Sci USA 70:591–597. Burg SP, Burg EA. 1968. Ethylene formation in pea seedlings; its relation to the inhibition of bud growth caused by indole-3-acetic acid. Plant Physiol 43:10691074. Chapin FS. 1990. Effects of nutrient deficiency on plant growth: evidence for a centralised stress-response system. In: Davies WJ, Jeffcoat B, eds. Importance of Root to Shoot Communication in the Response to Environmental Stress. Monograph 21. British Society for Plant Growth Regulation, pp 135–148, Charlton WA. 1996. Lateral root initiation. In: Waisel Y, Eshel A, Kefkafi U, eds. Plant Roots: The Hidden Half. 2nd ed. New York; Marcel Dekker, pp 149–175. Chen F, Bradford KJ. 2000. Expression of an expansin is associated with endosperm weakening during tomato seed germination. Plant Physiol 124:1265–1274. Clark DG, Gubrium EK, Barrett JE, Nell TA, Klee HJ. 1999. Root formation in ethylene-insensitive plants. Plant Physiol 121:53–59. Corbineau F, Rudnicki RM, Come D. 1989. ACC conversion to ethylene by sunflower seeds in relation to maturation, germination and thermodormancy. Plant Growth Regul 8:105–115. Drew MC. 1997. Oxygen deficiency and root metabolism: injury and acclimation under hypoxia and anoxia. Annu Rev Plant Physiol Plant Mol Biol 48:223–250.

Role of Ethylene Drew MC, He C-J, Morgan PW. 2000. Programmed cell death and aerenchyma formation in roots. Trends Plant Sci 5:123–127. Eklund L, Cienciala E, HS˘llgren J-E. 1992. No relation between drought stress and ethylene production in Norway spruce. Physiol Plant 86:297–300. Else MA, Hall KC, Arnold GM, Davies WJ, Jackson MB. 1995. Export of abscisic acid, 1-aminocyclopropane-1carboxylic acid, and nitrate from roots to shoots of flooded tomato plants. Plant Physiol 107:377–384. English PJ, Lycett GW, Roberts JA, Jackson MB. 1995. Increased 1-aminocyclopropane-1-carboxylic acid oxidase activity in shoots of flooded tomato plants raises ethylene production to physiologically active levels. Plant Physiol 109:1435–1440. Eshel A, Nielsen K, Lynch J. 1995. Responses of bean root systems to low level of P. In: Plant Roots from Cells to Systems. 14th Long Ashton International Symposium, Bristol, U.K. Estelle M, Klee HJ. 1994. Auxin and cytokinin in Arabidopsis. In: Meyerowitz EM, Somerville CR, eds. Arabidopsis, Vol 27. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, pp 555–578. Evans ML. 1991. Gravitropism: interactions of sensitivity modulation and effector redistribution. Plant Physiol 95:1–5. Galway ME, Masucci JD, Lloyd AM, Walbot V, Davis RW, Schiefelbein JW. 1994. The TTG gene is required to specify epidermal cell fate and cell patterning in Arabidopsis root. Dev Biol 166:740–754. Glick BR, Jacobson CB, Schwarze MMK, Pasternak JJ. 1994. 1-Aminocyclopropane-1-carboxylic acid deaminase mutants of the plants of the plant growth promoting rhizobacterium, Pseudomonas putida GR12-2 do not stimulate canola root elongation. Can J Microbiol 40:911–915. Glick BR, Li J, Shah S, Penrose DM. 1999. ACC deaminase is central to the functioning of plant growth promoting rhizobacteria. In: Kanellis AK, Chang C, Klee H, Bleecker AB, Pech JC, Grierson D, eds. Biology and Biotechnology of the Plant Hormone Ethylene II. Dordrecht, Netherlands: Kluwer, pp 293–298. Gomez-Cadenas A, Tadeo FR, Talon M, Primo-Millo E. 1996. Leaf abscission induced by ethylene in waterstressed intact seedlings of Cleopatra mandarin requires previous abscisic acid accumulation in roots. Plant Physiol 112:401–408. Guinel FC, LaRue TA. 1992. Ethylene inhibitors partly restore nodulation to pea mutant E107 (brz). Plant Physiol 99:515–518. Hall JA, Peirson D, Ghosh S, Glick BR. 1996. Root elongation in various agronomic crops by the plant growth promoting rhizobacterium Pseudomonas putida GR122. Isr J Plant Sci 44:37–42.

457 Hamilton AJ, Lycett GW, Grierson D. 1990. Antisense gene that inhibits synthesis of the hormone ethylene in transgenic plants. Nature 346:284–287. He C-J, Morgan PW, Drew MC. 1992. Enhanced sensitivity to ethylene in nitrogen- or phosphate-starved roots of Zea mays L. during aerenchyma formation. Plant Physiol 98:137–142. He C-J, Drew MC, Morgan PW. 1994. Induction of enzymes associated with lysigenous aerenchyma formation in roots of Zea mays during hypoxia and nitrogen starvation. Plant Physiol 105:861–865. He C-J, Finlayson SA, Drew MC, Jordan WR, Morgan PW. 1996. Ethylene biosynthesis during aerenchyma formation in roots of Zea mays L. subjected to mechanical impedance and hypoxia. Plant Physiol 112:1679–1685. Hettiaratchi DRP. 1990. Soil compaction and plant root growth. Phil Trans R Soc Lond B 329:3343–355. Honma M, Shimomura T. 1978. Metabolism of 1-aminocyclopropane-1-carboxylic acid. Agric Biol Chem 42:1825–1831. Hunter WJ. 1993. Ethylene production by root nodules and effect of ethylene on nodulation in Glycine max. Appl Environ Microbiol 59:1947–1950. Hussain A, Black CR, Taylor IB, Roberts JA. 1999. Soil compaction: a role for ethylene in regulating leaf expansion and shoot growth in tomato (Lycopersicon esculentum Mill.)? Plant Physiol 121:1227–1239. Hussain A, Black CR, Taylor IB, Roberts JA. 2000. Does an antagonistic relationship between ABA and ethylene mediate shoot growth when tomato (Lycopersicon esculentum Mill.) plants encounter compacted soil? Plant Cell Environ 23:1217–1226. Jackson MB. 1985. Ethylene and responses of plants to soil waterlogging and submergence. Annu Rev Plant Physiol 36:145–174. Jackson MB. 1993. Are plant hormones involved in root to shoot communication? Adv Bot Res 19:104-187 Jusaitis M. 1986. Rooting of intact mungbean hypocotyls stimulated by auxin, ACC and low temperature. HortScience 21:1024–1025. Kays SJ, Nicklow CW, Simons DH. 1974. Ethylene in relation to the response of roots to physical impedance. Plant Soil 40:565–571. Klee HJ, Tieman D. 1996. Potential applications of controlling ethylene synthesis and perception in transgenic plants. In: Kanellis AK, Chang C, Klee H, Bleecker AB, Pech JC, Grierson D, eds. Biology and Biotechnology of the Plant Hormone Ethylene. Dordrecht, Netherlands: Kluwer, pp 289–299. Klee HJ, Hayford MB, Kretzmer KA, Barry GF, Kishore GM. 1991. Control of ethylene synthesis by expression of a bacterial enzyme in transgenic tomato plants. Plant Cell 3:1187–1193. Lanahan MB, Yen H-C, Giovannoni JJ, Klee HJ. 1994. The Never ripe mutation blocks ethylene perception in tomato. Plant Cell 6:521–530.

458 Laskowski MJ, Williams ME, Nusbaum HC, Sussex IM. 1995. Formation of lateral root meristems is a twostage process. Development 121:3303–3310. Lee JS, Chang W-K, Evans ML. 1990. Effects of ethylene on the kinetics of curvature and auxin redistribution in gravi-stimulated roots of Zea mays. Plant Physiol 94:1770–1775. Lee KH, LaRue TA. 1992. Pleiotropic effects of sym-17-A mutation in Pisum sativum L. cv. Sparkle causes decreased nodulation, altered root and shoot growth and increased ethylene production. Plant Physiol 100:1326–1333. Ligero F, Lluch C, Olivares J. 1987. Evolution of ethylene from roots and nodulation rate of alfalfa (Medicago sativa L.) plants inoculated with Rhizobium meliloti as affected by the presence of nitrate. J Plant Physiol 129:461–467. Lincoln C, Britton JH, Estelle M. 1990. Growth and development of the axr1 mutants of Arabidopsis. Plant Cell 2:1071–1080. Lynch J, Brown KM. 1997. Ethylene and plant responses to nutritional stress. Physiol Plant 100:613–619. Masucci JD, Schiefelbein JW. 1994. The rhd6 mutations of Arabidopsis thaliana alters root-hair initiation through an auxin- and ethylene-associated process. Plant Physiol 106:1335–1346. Masucci JD, Schiefelbein JW. 1996. Hormones act downstream of TTG and GL2 to promote root hair outgrowth during epidermis development in Arabidopsis root. Plant Cell 8:1505–1517. Morgan PW, Drew MC. 1997. Ethylene and plant responses to stress. Physiol Plant 100:620–630. Mudge KW. 1988. Effects of ethylene on rooting. In: Davis TD, Haissig BE, Sankhla N, eds. Adventitious Root Formation in Cuttings. Portland, OR: Dioscorides Press, pp 150–161. Neljubov D. 1901. Ueber die horizontale nutation der stengel von Pisum sativum und einiger anderen pflanzen. Beih Bot Centralbl 10:128–139. Oeller PW, Min-Wong L, Taylor LP, Pike DA, Theologis A. 1991. Reversible inhibition of tomato fruit senescence by antisense RNA. Science 254:437–439. Olson DC, Oetikr JH, Yang SF. 1995. Analysis of LE-ACS3, a 1-aminocyclopropane-1-carboxylic acid synthase gene expressed during flooding in the roots of tomato plants. J Biol Chem 270:14056–14061. Osborne DG. 1989. The control role of ethylene in plant growth and development. In: Clijster H, de Proft M, Marcelle R, Van Poucke M, eds. Biochemical and Physiological Aspects of Ethylene in Lower and Higher Plants. Dordrecht, Netherlands: Kluwer, pp 1–11. Peters N, Crist-Estes D. 1989. Nodule formation is stimulated by the ethylene inhibitor aminoethoxyvinylglycine. Plant Physiol 91:690–693.

Hussain and Roberts Pitts RJ, Cernac A, Estelle M. 1998. Auxin and ethylene promote hair elongation in Arabidopsis. Plant J 16:553–560. Robbins JA, Reid MS, Paul JL, Rost TL. 1985. The effects of ethylene on adventitious root formation in mung bean (Vigna radiata) cuttings. J Plant Growth Regul 4:147– 157. Robbins JA, Kays SJ, Dirr MA. 1983. Enhanced rooting of wounded mung bean cuttings by wounding and ethephon. Hortsci 108:325–329. Roberts JA, Whitelaw CA, Gonzalez-Carranza ZH, McManus MT. 2000. Cell separation in plants—models, mechanisms and manipulation. Ann Bot 86:223– 235. Rodrigues-Pousada RA, De Rycke R, Dedonder A, Van Caeneghem W, Engler G, Van Montagu M, Van der Straeten D. 1993. The Arabidopsis 1-aminocyclopropane-1-carboxylate synthase gene 1 is expressed during early development. Plant Cell 5:897–911. Rodrigues-Pousada RA, Van Caeneghem W, Van Chauvaux N, Van Onckelen H, Van Montagu M, Van der Straeten D. 1999. Hormonal cross-talk regulates the Arabidopsis thaliana 1-aminocyclopropane-1-carboxyate synthase gene 1 in a developmental and tissuedependent manner. Physiol Plant 105:312–320. Roman G, Lubarsky B, Kieber JJ, Rothenberg M, Ecker JR. 1995. Genetic analysis of ethylene signal transduction in Arabidopsis thaliana: five novel mutant loci integrated into stress response pathway. Genetics 139:1393–1409. Saab IN, Sachs MM. 1996. A flooding-induced endo-transglycosylase homolog in maize is responsive to ethylene and associated with aerenchyma. Plant Physiol 112:385–391. Sarquis JI, Jordan WR, Morgan PW. 1991. Ethylene evolution from maize (Zea mays L.) seedling roots and shoots in response to mechanical impedance. Plant Physiol 96:1171–1177. Schneider K, Wells B, Dolan L, Roberts K. 1997. Structural and genetic analysis of epidermal cell differentiation in Arabidopsis primary roots. Development 124:1789– 1798. Smalle J, Vand der Straeten D. 1997. Ethylene and vegetative development. Physiol Plant 100:593–605. Spollen WG, LeNoble ME, Sharp SE. 1997. Regulation of root ethylene production by accumulation of endogenous ABA at low water potentials. Plant Physiol 105(s uppl):25. Tanimoto M, Roberts K, Dolan L. 1995. Ethylene is a positive regulator of root hair development in Arabidopsis thaliana. Plant J 8:943–948. Theologis A. 1992. One rotten apple spoils the whole bushel: the role of ethylene in fruit ripening. Cell 70:181–184. Torrey JG. 1976. Root hormones and plant growth. Annu Rev Plant Physiol 27:435–459.

Role of Ethylene Vacha GA, Harvey RB. 1927. The use of ethylene, propylene and similar compounds in breaking the rest period of tubers, bulbs, cuttings and seeds. Plant Physiol 2:187– 193. Van Workum WAT, Van Brussel AAN, Tak T, Wijffelman CA, Kijne JW. 1995. Ethylene prevents nodulation of Vicia sativa ssp. nigra by exopolysaccharide-deficient mutants of Rhizobium leguminosarum bv. viciae. Mol Plant-Microbe Interact 8:278–285. Vian B, Mosiniak M, Reis D, Roland J-C. 1982. Dissipative process and experimental retardation of the twisting in the growing plant cell wall. Effects of ethylene-generating agent and colchicine: a morphogenetic revaluation. Biol Cell 46:301–310. Visser EJW, Cohen JD, Barendse GWM, Blom CWPM, Voesenek LACJ. 1996. An ethylene-mediated increase in sensitivity to auxin induces adventitious root formation in flooded Rumex palustris Sm. Plant Physiol 112:1687–1692. Wang TW, Arteca RN. 1992. Effects of low O2 root stress on ethylene biosynthesis in tomato plants (Lycopersicon

459 esculentum Mill cv Heinz 1350). Plant Physiol 98:97– 100. Wilson AK, Pickett FB, Turner JC, Estelle M. 1990. A dominant mutation in Arabidopsis confers resistance to auxin, ethylene and abscisic acid. Mol Gen Genet 222:377–383. Woods SL, Roberts JA, Taylor IB. 1984. Ethylene induced root coiling in tomato seedlings. Plant Growth Regul 2:217–225. Zaat S, Van Brussel A, Tak T, Lugtenberg B, Kijne J. 1989. The ethylene-inhibitor aminoethoxyvinylglycine restores normal nodulation by Rhizobium leguminosarum biovar viciae on Vicia sativa subsp. nigra by suppressing the ‘thick short roots’ phenotype. Planta 177:141–150. Zacarias L, Reid MS. 1992. Inhibition of ethylene action prevents root penetration through compressed media in tomato (Lycopersicon esculentum) seedlings. Physiol Plant 86:301–307.

28 Root Signals Mark A. Bacon, William J. Davies, Darren Mingo, and Sally Wilkinson Lancaster University, Lancaster, Lancashire, England

I.

INTRODUCTION

often distinctions between them are arbitrary and unrealistic. There are now many new genetic tools available to allow us to test our ideas on the roles played by chemical signals moving between roots and shoots. Used in combination with some of the technologies mentioned above, these tools give us the capacity to investigate the complex chemical responses which help fit plants for survival, growth, and yielding in very hostile environments

Restriction of water supply from the soil can result in shoot water deficit and this will limit shoot growth and functioning. Nevertheless, shoot water deficits do not always result from soil drying; indeed in some circumstances, drought-induced stomatal closure and growth limitation can even result in an increase in shoot water potential relative to that of a well watered plant (Jones et al., 1983). It is necessary to ask what is limiting the reopening of stomata and the growth of the shoots under such circumstances. Some ingenious experimental manipulations such as the root pressure vessel (Munns and Passioura, 1985) and the split-root technique (Blackman and Davies, 1985; Gowing et al., 1990) have allowed several groups to conclude that chemical signaling between roots and shoots can limit shoot growth and functioning even when the water relations of shoots of plants that grow in drying soil are comparable to those of plants in well-watered soil. Of course in nature, chemical and hydraulic regulation of growth occur together, with the relative importance of the two mechanisms varying between genotypes and with different soil conditions and with the extent of the drought conditions. The literature has concentrated on hydraulic limitations on plant functioning (e.g., Nonami et al., 1997) in drying soil, so it is important to put the case for chemical regulation. We should not forget, however, that the two systems depend upon each other for effective functioning and that

II.

GENERATION OF ROOT SIGNALS

Plant water loss reduces the water potential and turgor of roots. Water uptake from the soil limits the changes in root water status, but we have reason to believe that quite subtle changes in root cell water relations can have important effects on signaling (see Spollen et al., 1993). Unfortunately, while we now have quite detailed information on root water status in response to environmental stress, with spatial variation in water relations provided by the micropressure probe, the great majority of these data have been collected from roots growing in artificial media or in nutrient solutions (e.g., Fig. 1). Such data are difficult to collect with roots in soil, but it is important that we realize the importance of physical and chemical influences generated in drying soil may not be replicated in artificial media. 461

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Figure 1 Typical cellular turgor profile in the apical 10 mm of a maize root growing at high (&) or low (*) water potential.

If water removed from soil by plants is not replenished, then soil and root water potentials and turgors can reach very low values (Fig. 1). Cell water deficits will influence cell metabolism and we know that the synthesis of several plant growth regulators can be affected (Wright, 1977). These changes contribute to root signaling and we could imagine, for example, that the production and export from roots of the plant hormone abscisic acid (ABA) can be related to soil water status (Fig. 2) (Zhang and Davies, 1989) because root water status can also be related to the water status of the soil. Hormonal root signals will not vary solely as a result of a change in root water status. The pH relations of different compartments in the root will also be affected by soil drying (Slovik et al., 1995), and these changes have big effects on the circulation of hormones around the plant. Hartung and coworkers have shown that a drought-induced alkalinization of the apoplast enhances the remobilization of ABA arriving in the root from the shoot with the result that a variable proportion of the ABA moving from the root to the shoot (the ‘‘root signal’’) will actually be root sourced (Slovik et al., 1995). The intensity of the ABA signal will be increased at reduced soil water

potential as a result of extra root synthesis and enhanced mobilization (see also Chapter 26 by Hose et al. in this volume). Soil water status, which may vary significantly over short distances across the rhizosphere, affects both growth and physiology of roots. Substantial rates of water uptake can result in localized drying around the root, and the effects of this resistance on water uptake and plant water deficit can be amplified as roots can shrink as plant water deficits develop. Root resistance to water uptake varies among species because of the variations in the pathway for water movement into roots. Moreover, the pathway can vary as the fluxes of water change (Steudle and Peterson, 1998). It has recently been shown that water channels in roots (aquaporins) can fine-tune water fluxes, perhaps in response to transpiration demand (Tyerman et al., 1999). All of these variables influence root water status in drying soil and therefore modify the chemical signalling process. As soil dries, uptake of mineral nutrients can be modified and soil strength will also be increased. Changes in plant growth and functioning as soil dries may be at least in part attributable to changes

Root Signals

Figure 2 Typical relationship between root ABA content and bulk soil water content for maize plants growing in drying soil columns. (Modified from Zhang and Davies, 1989.)

in the physical properties of the soil or to limited nutrient uptake, but the extent of these effects is largely undefined. Increasing mechanical impedance of soil as water potential declines limits root penetration, and this change will feed back on root water status and the production of signals (Hartung and Heilmeier, 1993). There may be some direct physical interaction between roots and soil of high mechanical impedance or aeration. The roots may be restricted as the soil hardens and this change will also influence the synthesis of plant growth regulators in roots (e.g., Jackson, 1993; Chapter 45 by Masle in this volume). Signals may also be hormones and other organic molecules taken up from the soil. Hartung et al. (1996) have shown that there can be quite a lot of the hormone abscisic acid in the rhizosphere but have suggested that the importance of this hormone in this location may be to prevent substantial amounts of plant-sourced ABA from diffusing out of roots. The soil-based hormone therefore has an important influence on the signaling process. There is much metabolic activity in the rhizosphere (see Chapter 26 by Hose et al. in this volume) and it seems likely that the production of hormones here will greatly affect root growth and functioning and could affect root signalling either directly or indirectly.

463

III.

WHAT CONSTITUTES A SIGNAL AND WHAT ARE THE LONG-DISTANCE CHEMICAL SIGNALS UNDER DROUGHT?

A.

The Signaling Process

Many chemical signals move from roots to shoots in the transpiration stream, and, at least for plants in drying soil, they may provide shoots with a measure of the access that roots have to soil water (Tardieu et al., 1992). This will not be just a measure of the soil water status but rather an amalgam of the effects of this variable and the distribution of the roots. Shoot growth and functioning may then be modified as a function of the intensity of this signal. The transpiration stream moves rapidly in plants, but in very tall trees the arrival of a root sourced signal may still take days. Root signaling alone is clearly not an appropriate explanation for many of the dynamic responses to stress that are exhibited by the leaf. We discuss below how partitioning of hormones arriving in the leaf can have an important impact on the extent of the leaf’s response to the signal and that this partitioning can be a function of many different variables. On very hot and dry days, water movement through the soil–plant–air continuum is substantial, unless the stomata close to limit water flux. Else et al. (1995) and others have discussed the effect of variation in water flux on the delivery of chemical signals to the shoot. They have argued that high transpiration rates reduce concentrations of chemical signals but that delivery of chemical signals to the site of action in the shoot may still be high. It may be necessary to calculate the quantitative delivery of a signal (concentration flux) if we are to convincingly demonstrate that signaling is affected by a change in the edaphic environment. Jackson (1993) has also suggested that it may be necessary to include an estimate of the size of the source tissue and the sink tissue for the signal if we are to assess the precise nature of signaling. Hose et al. (Chapter 26 in this volume) have discussed the existence of apoplastic bypass in roots of different species. This means that a variable proportion of the xylem signal passes through the plasma membrane and that the dilution effects of transpiration may be unpredictable. Freundl et al. (1998) have suggested that high apoplastic bypass reduces the dilution effect of high transpiration rate. This may mean that concentrations of hormones in the xylem of such plants do not vary with transpiration through the day. Tardieu et al. (1992) have reported field measurements which con-

464

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firm this view. However, we should also remember that there is considerable debate (Schurr, 1997) over methods of sampling xylem sap and the difficulty of assessing concentrations of solutes in the xylem of nontranspiring plant parts. B.

Abscisic Acid

There is strong evidence from a number of different studies that ABA plays an important role in the regulation of stomatal behaviour and growth in droughted plants (cf. Dodd and Davies, 1996). This hormone may also have a role in the root-to-shoot signaling process (Zhang and Davies, 1990a,b). But it is not clear whether there is always enough ABA in the xylem stream to explain the stomatal and growth responses that are generated by soil drying (e.g., Munns and Sharp, 1993; Munns and Cramer, 1996). This is a difficult question to answer, but recent experiments where xylem pH was manipulated (e.g., Wilkinson et al., 1998; Bacon et al., 1998) and experiments with transgenic plants and mutants (e.g., Hussain et al., 1999; Mullholland et al., 1999) seem to support the case for a central role of ABA in the regulation of gas exchange and growth of at least some plant species. Even a well-watered plant contains a significant concentration of ABA in the transpiration stream. This means that the delivery of this hormone into the leaf over the course of the day can be very substantial. If all of this ABA would have reached sites of action on the guard cells then the stomata would nearly always be fully closed. Clearly this is not the case, which tells us that most ABA delivered to the leaf in the xylem is intercepted in some way before it can reach the stomata. ABA is a weak acid and will thus distribute through the leaf compartments according to the Henderson-Hassalbach equation. Relatively alkaline compartments will accumulate ABA, which then becomes trapped there. Under most circumstances, the symplast of the leaf is alkaline relative to the apoplast, so in the well-watered plant most xylem-delivered ABA enters the symplast and has no access to binding sites on the stomata, at least some of which face outward into the apoplast. Hartung and coworkers have shown using elegant experiments and detailed modeling that small reductions in the water potentials of leaves can alkalinize the apoplast of the leaf and that ABA trapped in the symplast may be released to gain access to sites of action around the plant. Interestingly, however, modeling suggests that ABA released from the leaf sym-

plast may travel to the roots in the phloem (a very alkaline compartment) and return to the leaf in the xylem before it reaches the guard cells (Slovik et al., 1995). Thus, while stress-induced redistribution of ABA can be an efficient means of control of water loss, the signaling pathway may be a rather tortuous one. Our own work (Wilkinson and Davies, 1997) suggests that a drought-induced alkalinization of the xylem stream allows a greater proportion of a rootsourced ABA signal to penetrate to sites of action within the leaf. Even mild soil drying can increase xylem pH, and this change will be enough to limit stomatal conductance and limit transpiration (Fig. 3) even without the production of extra ABA. This is an important observation because it suggests that there is always enough ABA in the plant to close stomata, even when the plant is well watered. There are many reports in the literature of drought-induced xylem alkalinization, and Wilkinson et al. (1998) reported that many other environmental perturbations also change xylem pH (Table 1). These authors have suggested that xylem pH may have an important intermediary role in the regulation of plant responses to many stresses. We have confirmed the involvement of ABA in the pH responses of stomata using flacca, the ABA mutant

Figure 3 Effect of gravimetric soil water content on the pH of xylem sap expressed within 2 min from 30-mm shoot stumps cut from 6- to 9-week-old wild-type tomato plants. A linear regression with 95% confidence limits is shown. (From Wilkinson et al., 1998.)

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465

Table 1 pH Changes That Occur in Plant Xylem or Apoplastic Sap Under Various Conditions Species

Source of sap

pH change

Conditions of change

Reference

Phaseolus coccineus Anastatica hierochuntica

6.5–7.0 6.5–7.1 6.5–7.6 5.8/6.6–7.1

Wet soil–2-day drought Wet soil–dry soil

Hartung and Radin (1989) Hartung and Radin (1989)

Helianthus annuus

Root xylem Root xylem Shoot xylem Shoot xylem

Gollan et al. (1992)

Commelina communis

Shoot xylem

6.0–6.5/6.7

Lycopersicon esculentum Ricinus communis Helianthus annuus

Root xylem Shoot xylem Leaf apoplast

2.3 M–0.69 M Hþ 6.0–6.6 5.7–6.4

Samanea pulvinus Robinia wood

Extensor apoplast Shoot xylem

6.2–6.7 6.0–5.0–5.5

Actinidea chinensis Betula pendula

Shoot xylem Shoot xylem

5.3–6.2 7.5/8.0–5.7

Hordeum vulgare

Leaf apoplast

6.6–7.3

Helianthus annuus Helianthus annuus

Leaf apoplast Leaf apoplast

6.8–7.4 6.0–6.5 6.2–7.0

High soil water–below 0.13 g/g Wet soil–4/5-day drought Drained soil–flooded soil End of night–end of day Light–dark(þ2:5 mM nitrate) White light–dark Jan–April/May–Nov/ Dec Spring–rest of year Rest of year – catkin bud break Control–brown rust infected Low nitrate–high nitrate NH4 NO 3–  NO3 NH4 NO 3 –HCO3

tomato. Against a low ABA background, the pH response of the stomata disappears and is only restored when low concentrations of ABA are added to the xylem stream (Fig. 4). These are concentrations of the hormone found in well-watered plants and do not, themselves, limit stomatal opening. Interestingly, alkaline xylem sap applied to leaves containing a low ABA background actually opens stomata (Fig. 4). This observation suggests that ABA may have a role even in the well-watered plant to limit excessive stomatal opening which may lead to excessive rates of water loss and the risk of plant water stress. While many herbaceous plants show an alkalinization of xylem sap in response to soil drying, our more recent work suggests that this response is not always seen in woody plants. Recent work has suggested that much ABA may move through the plant in a complexed form and assessment of concentrations of the free hormone in the xylem may therefore underestimate the role that this chemical species may play in signaling to guard cells (Hartung et al., 1998; Netting, 2000). If this is the case, then the location and activity of enzymes that might act to break down complexed ABA is important information if we are to fully understand the long distance signaling processes. Hartung et al.

Wilkinson and Davies (1997) Else (1996) Schurr and Schulze (1995) Hoffmann and Kosegarten (1995) Lee and Satter (1989) Fromard et al. (1995) Ferguson et al. (1983) Sauter and Ambrosius (1986) Tetlow and Farrar (1993) Dannel et al. (1995) Mengel et al. (1994)

Figure 4 Effect of artificial xylem sap pH on the transpiration rate of detached leaves of the flacca mutant of tomato in the light in the presence and absence of 0:03
M (þ) ABA. Leaves were preincubated in artificial sap for 1 h in the dark. The transpiration rate was calculated for each leaf every 30 min and expressed as a mean (n ¼ 4–6)  SE. (From Wilkinson et al., 1998.)

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(1998) have investigated the apoplastic fluids washed from plants and reported significant levels of the glucose ester of ABA and the enzymes that will release the free hormone from this complex.

C.

Ethylene

Recently there has been considerable interest in the possibility that ethylene which arises as a result of root stress can regulate growth of plants in drying or compacted soil (e.g., Hussain et al., 1999; Spollen et al., 2000). We have known for some time that soil flooding stimulates the synthesis of ACC, the immediate precursor of ethylene and that this compound moves to the shoots with the transpiration stream of plants (e.g., Else et al., 1995). Once in the leaf, ethylene is synthesized from ACC and this potent molecule will influence many of the plant’s physiological and developmental processes. It has been known for many years that plant water deficit substantially increases the production of ethylene (Wright, 1977), and it is possible therefore that the effects of soil drying and soil compaction stress on plant growth and development may be mediated through the effects of such an increase. Evidence in the literature suggests that ethylene has little or no effect on stomatal behavior, but ethylene can act as both a promoter and an inhibitor of growth. Leaf growth of nearly all terrestrial plants is inhibited by ethylene. In response to compaction stress imposed on roots, enhanced delivery of ACC to shoots has been reported (Hussain et al., 1999) and we might therefore argue that ACC can act as a root signal of the effects of compaction. Hussain et al. (1999) have confirmed the role of ethylene in the regulation of growth of tomato plants growing in compacted soil. To do this they used transgenic plants with a reduced capacity to produce ethylene. Shoot growth of transgenics and wild types was comparable in uncompacted soil and in uniformly compacted soil. Plants with roots split between two containers (one compacted and the other uncompacted soil) showed different responses. The wild-type genotype showed reduced growth while AC01AS (the low ethylene transgenic) showed growth rates comparable to those of control plants growing in uncompacted soil. In the wild type, enhanced accumulation of ethylene was accompanied by reduced growth rates, suggesting that the ability of the transgenic to sustain growth was related to its lower ethylene production. When roots of wild types in compacted soil were excised from the plants, shoot growth rate was restored. Treatment of half-compacted plants with

silver ions had a similar effect. This is a treatment known to block ethylene action. These results suggest that ethylene has a key role as a root signal when roots of tomato plants encounter compacted soil. Of course, soil drying results in increasing soil strength and so we cannot rule out a limitation of shoot growth in plants in drying soil that is promoted by the enhanced synthesis of ethylene. This possibility makes it most important that we investigate the effects of soil stress on plant signaling in media that generate the appropriate physical and chemical influences on the plant. While we may be able to investigate the effects of a reduced water potential per se on roots in solution culture, we cannot generate the physical effects of soil drying in such a system. It seems likely that these effects will be an important component of the plant’s drought stress response. To unpick these potentially complicated interactions we need a very precise definition of the stress that is affecting plants. In addition, the new genetic tools that are increasingly available will allow us increased insight into hormone effects against a background of water and chemical and physical relationships that can be carefully controlled. Recent work by Sharp and colleagues (Sharp et al., 2000) highlights an apparently important role for ABA in plants in limiting the effects of runaway ethylene synthesis under stress. One of the effects of this is that in very specialized situations ABA applications may actually increase leaf growth under stress by reducing the ethylene-induced growth limitation (Hussain et al., 2000; Chapter 27 by Hussain and Roberts in this volume). This implies that in plants in drying soil ABA may sustain shoot growth when ethylene accumulation is substantial. This suggestion is in contrast to the generally held view that ABA is an inhibitor of shoot growth of plants in drying soil (e.g., Zhang and Davies, 1991). Bacon et al. (1998) recently reported a very sensitive limitation of leaf growth of barley which is apparently attributable to ABA. This limitation can be promoted by ABA levels that are found in wellwatered plants, provided the pH relations of the leaf allow the hormone to reach sites of action for growth regulation in the leaf. Taken together, these results suggest that ABA can act as both an inhibitor and a promoter of shoot growth depending on a variety of other variables, of which apoplastic pH and ethylene status of the plant are two that may be important. We should not be surprised that the balance between hormones has an important influence on the growth and functioning of stressed plants or that changes in hormone ratios can cause an inhibitive effect to become a

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467

promotive effect. Most work on root signaling has concentrated on the effects of single hormones, but this has been necessary to generate enough precision to convince skeptics that hormonal signaling is indeed a reality. As we have more genetic tools and new technology at our disposal, we can investigate the interactions among hormones that are undoubtedly important in this complex field. Stoll et al. (2000) reported the important effects of changes of ABA/ cytokinin ratios have on the development of grapevines growing in drying soil in the field in Australia.

IV.

EXPLOITATION OF THE PLANTS STRESS SIGNALING SYSTEM

Ten years ago, Loveys (1991) suggested that it may be possible to exploit the plant’s chemical signaling system and dry part of a root system of a vegetatively vigorous plant and thereby restrict shoot growth under circumstances where excessive vigor may be a disadvantage. One such circumstance is the production of a fruit crop on a plant producing more leaves than are required to supply carbohydrate to developing reproductive plant parts. Many grapevines are pruned to reduce excessive vigor, and Loveys and colleagues have successfully used partial root drying (PRD) to reduce the need for pruning in this crop (Dry et al., 1996). By watering alternately on the two sides of rows of grapevines, partial root drying can be achieved and much water can be saved. Of particular significance in this work is that fruit yield is not reduced by the PRD treatment while leaf growth is very significantly lim-

ited. Most importantly, the quality of the fruit produced is greatly increased by the PRD treatment (Table 2). More conventional deficit irrigation with the same amount of water used in PRD results in a reduced amount of low-quality fruit. Our own work with tomato has used a similar irrigation system and resulted in the production of a high-quality crop of fruit. The volume of production of the tomato crop is only slightly reduced by the PRD treatment with the result that water use efficiency of production is greatly increased (Davies et al., 2000). It is of interest to determine why PRD reduces leaf growth of grapevines and tomatoes but has no influence on fruit growth and development. One hypothesis is that root-sourced chemical signals travel to the shoots in the xylem but that delivery to the fruit is limited (Davies et al., 2000). It has long been known that in expanding fruit of many plant species a large proportion of water is transported into the fruit via the phloem. For example, Greenspan et al. (1994) demonstrated that in grape the majority of water reaches the fruit via the xylem before veraison (first appearance of color in the grapes) but that phloem transport predominated after veraison. This tendency is also particularly extreme in tomato and in the later stages of growth of fruit of this crop considerably > 90% of water may be transported to the fruit by the phloem (Ho et al., 1987). In recent experiments we have applied reduced amounts of water to split root tomato crops in two different ways. Water is either applied alternately to the two halves of the root system (PRD) or distributed evenly between the two sides of the divided roots

Table 2 Comparison of Various Yield Characteristics of a Tomato Crop Grown Under Control or PRD Irrigation Regimens Parameter Fruit weight (g) Fruit diameter (mm) No. of fruit per plant FW yield per plant (g) DW yield per plant (g) % Ripeness % Damage Brix value Fruit pH Freq. blossom end rot % Fruit DW content WUE (g FW dm3 ) WUE (g DW dm3 )

n

Control

2000 þ fruit 200 þ fruit 55 plants 55 plants 55 plants 2000 þ fruit 2000 þ fruit 64 fruit 32 fruit 1599 fruit 64 fruit 55 plants 55 plants

78.9 55.3 35.5 2700 156.6 76 14 3.9 4.4 2.6 5.8 26.5 1.5

s.e.

PRD

s.e.

0.6 0.3 2.1 170 3.24 9 2 0.6 0.2 – 0.12 2.12 0.03

66.8 49.8 37.2 2300 149.5 82 12 4.7 4.3 1.6 6.5 45.1 2.9

0.6 0.3 1.7 870 4.14 7 4 0.1 0.2 – 0.18 4.1 0.08

Significance P < :0001 P < :0001 ns P < :05 ns ns ns P < :0001 ns – P < :005 P < :0001 P < :005

% Dif. 15 10 5 15 4:5 8 14 21 2 38 12 70 93

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Bacon et al.

(RDI). The effects of these treatments were compared with twice as much water applied evenly to the two halves of the root system (full irrigation). PRD and RDI had very different effects on stomatal behavior (Fig. 5) and vegetative growth (Fig. 6) even though the water relations of the two crops were comparable, suggesting that the generation of root signals by partial drying of the roots can have an important effect on plant physiology and development.

V. CONCLUSIONS The results discussed above suggest that if we are to understand the nature and significance of root signaling in plants and subsequently exploit this knowledge for more effective plant production, we need to understand the effects of soil drying on both chemical and hydraulic relations of plants. The synthesis of growth regulators in roots in drying soil is not well understood, but we can speculate on its importance. In addition, the pathways of water and solute movement in the root and the volume flux of water will influence the signaling process. Variables such as the pH of the different compartments in the shoot and the hydraulic architecture will greatly influence the efficacy of given signals arriving in the shoot. All of these processes and

Figure 6 Leaf size as a % of control (&) in PRD (*) and RDI (~) treatments (see text for details).

more will combine to provide the plant with sensitive regulation of gas exchange and growth. Other processes will also be affected, but there has been little work on anything but the effects of long-distance signaling on stomatal behavior and cell expansion processes. We are now beginning to see the first fruits of the commercial exploitation of our increased understanding of the physiological processes regulating plant growth and functioning under drought. In < 20 years we have moved from the first recognition of the importance of chemical drought signaling to its manipulation in a multimillion-dollar plant production industry in a dryland environment.

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Figure 5 Stomatal conductance (plotted as % of control (&) in PRD (*) and RDI (~) treatments (see text for details).

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469 which factors and mechanisms influence its concentration? Plant Soil 184:105–110. Hartung W, Wilkinson S, Davies WJ. 1998. Factors that regulate abscisic acid and its concentration in the xylem. J Exp Bot 49:361–367. Ho LC, Grange RI, Picken AJ. 1987. An analysis of the accumulation of water and dry matter in tomato fruit. Plant Cell Environ 10:157–162. Hoffmann B, Kosegarten H. 1995. FITC-dextran for measuring apoplast pH and apoplastic pH gradients between various cell types in sunflower leaves. Physiol Plant 95:327–335. Hussain A, Black CR, Taylor LB, Roberts JA. 1999. Soil compaction. A role for ethylene in regulating leaf expansion and shoot growth in tomato? Plant Physiol 121:1227–1237. Hussain A, Black CR, Taylor IB, Roberts JA. 2000. Does an antagonistic relationship between ABA and ethylene mediate shoot growth when tomato (Lycopersicon esculentum Mill.) plants encounter compacted soil? Plant Cell Environ 23:1217–1226. Jackson MB. 1993. Are plant hormones involved in root-toshoot communication. Adv Bot Res 19:103–187. Jones HG, Luton MT, Higgs KH, Hamer PJC. 1983. Experimental control of water status in an apple orchard. J Hort Sci 58:301–316. Loveys BR. 1991. What use is a knowledge of ABA physiology for crop improvement? In: Davies WJ, Jones HG, eds. Abscisic Acid. Oxford, U.K.: Bios Scientific Publishers, pp 245–259. Lee Y, Satter RL. 1989. Effects of white, blue, red light and darkness on pH of the apoplast in the Samanea pulvinus. Planta 178:31–40. Mengel K, Planker R, Hoffmann B. 1994. Relationship between leaf apoplast pH and iron chlorosis of sunflower (Helianthus annuus L.). J Plant Nutr 17:1053– 1065. Mullholland B, Hussain A, Black CR, Taylor IB, Roberts JA. 1999. Does root-sourced ABA have a role in mediating growth and stomatal responses to soil compaction in tomato (Lycopersicon esculentum L.)? Physiol Plant 107:267–276. Munns R, Cramer G. 1996. Is co-ordination of leaf and root growth mediated by abscisic acid? Plant Soil 185:33– 49. Munns R, Passioura JB. 1985. Effect of prolonged exposure to NaCl on the osmotic-pressure of leaf xylem sap from intact, transpiring barley plants. Aust J Plant Physiol 11:497–507. Munns R, Sharp RE. 1993. Involvement of abscisic acid in controlling plant growth in soils of low water potential. Aust J Plant Physiol 20:425–437. Netting AG. 2000. pH, abscisic acid and the integration of metabolism in plants under stressed and non-stressed conditions: cellular responses to stress and their implication for plant water relations. J Exp Bot 51:147–158.

470 Nonami H, Wu YJ, Boyer JS. 1997. Decreased growthinduced water potential—primary cause of growth inhibition at low water potential. Plant Physiol 114:501–509. Sauter JJ, Ambrosius T. 1986. Changes in the partitioning of carbohydrates in the wood during the bud break in Betula pendula Roth. J Plant Physiol 124:31–43. Schurr U. 1997. Xylem sap sampling-new approaches to an old topic. Trends Plant Sci 3:293–298. Schurr U, Schulze ED. 1995. The concentration of xylem sap constituents in root exudate, and in sap from intact, transpiring castor bean plants (Ricinus communis L.). Plant Cell Environ 18:409–420. Sharp RE, LeNoble ME, Else MA, Thorne ET, Gherardi F. 2000. Endogenous ABA maintains shoot growth in tomato independently of effects on plant water balance. Evidence for the involvement of ethylene. J Exp Bot 51:1575–1584. Slovik S, Daeter W, Hartung W. 1995. Compartmental redistribution and long-distance transport of abscisic acid (ABA) in plants as influenced by environmental changes in the rhizosphere—a biomathematical model. J Exp Bot 46:881–894. Spollen WG, Sharp RE, Saab IN, Wu Y. 1993. Regulation of cell expansion in roots and shoots at low water potential. In: Smith JAC, Griffiths H, eds. Water Deficits, Plant Responses from Cell to Community. Oxford, U.K.: Bios Scientific Publishers, pp 37–52. Spollen WG, LeNoble ME, Samuels TD, Bernstein N, Sharp, RE. 2000. Abscisic acid accumulation maintains primary root elongation at low water potential by restricting ethylene production. Plant Physiol 122:967–976. Steudle E, Peterson CA. 1998. How does water get through roots? J Exp Bot 49:775–788. Stoll M, Loveys B, Dry P. 2000. Improving water use efficiency of irrigated horticultural crops. J Exp Bot 51:1627–1634.

Bacon et al. Tardieu F, Zhang J, Katerji N, Bethenod O, Palmer S, Davies WJ. 1992. Xylem ABA controls the stomatal conductance of field-grown maize subjected to soil compaction or soil drying. Plant Cell Environ 15:185–191. Tetlow IJ, Farrar JF. 1993. Apoplastic sugar concentration and pH in barley leaves infected with brown rust. J Exp Bot 44:929–936. Tyerman SD, Bohnert HJ, Maurel Y. (1999). Plant aquaporins: their molecular biology, biophysics and significance for plant water relations. J Exp Bot 50:1055– 1071. Wright STC. 1977. The relationship between leaf water potential (leaf) and the levels of abscisic acid and ethylene in excised wheat leaves. Planta 134:183–189. Wilkinson S, Davies WJ. 1997. Xylem sap pH increase: a drought signal received at the apoplastic face of the guard cell which involves the suppression of saturable ABA uptake by the epidermal symplast. Plant Physoil 113:559–573. Wilkinson S, Corlett JE, Oger L, Davies WJ. 1998. Effects of xylem sap pH on transpiration from wild-type and flacca mutant tomato leaves: a vital role for abscisic acid in preventing excessive water loss from wellwatered plants. Plant Physiol 117:703–709. Zhang J, Davies WJ. 1989. Abscisic acid produced in dehydrating roots may enable the plant to measure the water status of the soil. Plant Cell Environ 12:73–81. Zhang J, Davies WJ. 1990a. Changes in the concentration of ABA in the xylem sap as a function of changing soil water status can account for changes in leaf conductance and growth. Plant Cell Environ 13:277–285. Zhang J, Davies WJ. 1990b. Does ABA in the xylem control the rate of leaf growth in soil-dried maize and sunflower plants? J Exp Bot 41:765–772. Zhang J, Davies WJ. 1991. Antitranspirant activity in xylem sap of maize plants. J Exp Bot 42:317–321.

29 Environmental Sensing and Directional Growth of Plant Roots D. Marshall Porterfield University of Missouri-Rolla, Rolla, Missouri

I.

INTRODUCTION

tion to gravity, plant roots respond tropically to H2O (Mambani and Lal, 1983: Jafe et al., 1985), oxygen (Porterfield and Musgrave, 1998), and touch (Millet and Pickard, 1988; Ishikawa and Evans, 1992; Ito et al., 1995). These responses are referred to as hydrotropism, oxytropism, and thigmotropism, respectively. The interaction of gravitropism, hydrotropism, oxytropism, and thigmotropism (GHOT) and how these contribute in controlling directional growth through the soil is referred to as the GHOT integrated model of tropic root growth. While different mechanisms for sensing these various environmental stimuli do exist, tropic growth responses are thought to be mediated through a shared signal transduction pathway and response mechanism. This interaction allows for integration of environmental stimuli in determining the ultimate direction of root growth.

The growth of plant roots through the soil is a complex and misunderstood process. Roots acquire water and mineral nutrients from the soil while providing a mechanically sound base that anchors and supports the shoot system as it grows up and outward toward light. The soil system is complex and challenges the root system with numerous biotic and abiotic stresses. Abiotic challenges include salt stress and mineral nutrient deficiency. Water can be either limiting (water stress) or excessively available to the level where it actually reduces or depletes oxygen (hypoxia and anoxia respectively) in the soil. Soil compaction can limit the flux of water and oxygen through the bulk soil to the rhizosphere as well as mechanically impede the growth of the roots. A typical soil also contains numerous impenetrable obstacles such as stones and rocks. In response to these challenges, biochemical and physiological response mechanisms have evolved to both avoid and tolerate these different types of abiotic stress. Many of the important aspects of molecular and biochemical processes that impart stress tolerance have been described, but stress avoidance by differential tropic curvature is not well understood. Despite a body of classical work that was begun as far back as the 18th century, very little attention has been paid to these important mechanisms. Now, root biologists are beginning to explore how plant roots can sense and tropically respond to these types of stimuli using modern research tools. In addi-

II.

TROPIC RESPONSE AND MECHANISMS OF DIFFERENTIAL GROWTH

Various environmental stimuli, acting through hormones, modulate important aspects of plant growth and development. One important class of responses to external stimuli is that of the tropisms. Tropisms are responses that cause a change in the direction of growth of a plant organ as occurs in response to light (phototropism) and gravity (gravitropism, commonly referred to as geotropism in older writings). Charles Darwin first demonstrated in 1881 that the growing 471

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coleoptile of monocot seedlings bend toward a light source. The first demonstration of gravitropism was by T.A. Knight in 1811. Gravitropism is easily observed during seed germination when the young root turns downward regardless of the way in which the seed is planted. This bending, known as positive gravitropism, enables a plant to anchor itself in the soil. The young stem, which turns upward away from the earth, is said to be negatively geotropic. Later it was shown that the tropic response of both roots and shoots involved the action of the plant hormone auxin during phototropism and gravitropism. Since the physiological basis of tropic curvature in plant roots is best understood during gravitropism, this will be briefly discussed here. This will also facilitate discussion of integration of mechanisms for tropic response later in this chapter. All life evolved in the presence of gravity, so organisms have developed mechanisms to both counteract and use this force. Plants, in particular, have developed the capacity to perceive gravity and respond by differential growth. This gravity-determined growth (gravitropism) assists the roots of plants by orienting growth down into the soil to acquire both water and mineral nutrients. The process of plant curvature in response to gravity has been studied for almost 200 years (Knight, 1811). While the underlying mechanisms remain unclear, a basic model of gravitropism has been established (Fig. 1). Reorientation of the root within the gravita-

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tional field induces a differential in the movement of signaling molecules from the root cap to the elongation zone. Both calcium and auxin (IAA) are thought to be involved in this signal transduction pathway (Filner and Hertel, 1970; Lee et al., 1983a, b;1984; Young et al., 1990). This radial asymmetry in growth regulator concentrations within the root produces differences in cell expansion in the zone of elongation, which induces differential growth. The physiology of gravitropism is commonly studied by separating the process into three distinct stages (Fig. 2). Initially, plants sense the direction of the gravitational vector (sensing) in specialized cells in the root cap called statocytes. Statocytes contain starch-filled plastids (statoliths) that mediate gravity sensing either directly, by movement of the statoliths (starch statolith theory), or indirectly, by contributing to the total density of the protoplast within the cell wall (gravitational pressure model). The role of the amyloplasts has been demonstrated by studies reporting that starchless and reduced-starch mutants containing nonsedimenting amyloplasts do respond to gravity, although at a reduced level of sensitivity (More, 1987, 1989; Caspar and Pickard, 1989). Furthermore, statolith-free gravity responsive single-cell systems have been described for characean algae (Staves et al., 1992) and Ceratopteris fern spores (Chaterjee et al., 2000). Experiments to test and exclude one of these hypotheses are difficult to design, but we do know that the statoliths are involved.

Figure 1 Classical model of the gravitropic response in plant roots. When the root is oriented with the gravitational vector, the transported levels of IAA promote cell expansion in the two sides of the zone of elongation equally. Actual curvature is thought to result from uneven auxin transport on both sides of the root. During gravitropism IAA transport is redirected toward the lower side of the root to inhibit cell expansion in the zone of elongation, causing the root to curve downward. Calcium has also been implicated, but the actual role of calcium in the response is not clear.

Environmental Sensing

Figure 2 occur.

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The three defined steps of the model gravitropic response in plant roots and the regions of the root where these steps

Regardless of the mode of gravity sensing, rotation of a root within the gravitational field changes the movement of signaling molecules (auxin, calcium, and/or possible others as well) from the root cap to the zone of elongation (signal transduction). Such molecules then induce differential growth through lateral changes in longitudinal cell expansion mediated either by both inhibition or by stimulation of cell expansion (response). This classic model of gravitropism has remained essentially unchanged for decades and literally hundreds of studies have attempted to clarify the details. Despite questions regarding the exact role of the starch statolith in gravity sensing, it is generally agreed that the root cap acts as the site of gravity perception. Gravitropism has been studied almost exclusively in plant roots as they respond strongly to gravity during almost all phases of growth and development. This strong root response in combination with the consistency of gravity on earth commonly obscures other tropic responses, and hindered past attempts to design experiments to study these responses. Today, using a new array of genetic mutants and armed with new technologies, researchers are starting to understand other types of tropic responses in plant roots. Like gravitropism, such responses are thought to be initiated by sensing in the root cap and to be mediated by the same system involving radial asymmetry in signaling molecules in the zone of active elongation. III.

HYDROTROPISM IN PLANT ROOTS

A.

Background and History

Positive hydrotropism is defined as the bending, by differential growth, of roots toward soil regions containing increasing moisture, and is dependant on the ability to sense a moisture gradient. Hydrotropism was

first considered by Dodert (1700), who suggested that roots grow downward as a function of both soil water content and soil surface warming by the sun. Because of the significant biological and technical hurdles in designing experiments that faced early investigators studying hydrotropic responses in roots, research in this area has been difficult and slow. Biologically there is the problem of studying hydrotropism with interference from gravitropism, and technically, creating an appropriate moisture gradient to induce root hydrotropism can be challenging. Because of such difficulties most early researchers simply did not believe that hydrotropism existed. During this early period of hydrotropism research there was much controversy, and many conflicting statements were made about the existence of hydrotropism (Dodart, 1700; Dutrochet, 1824; Johnson, 1829; Keith, 1815; Knight, 1811; Lefebure, 1801; Van Tieghem, 1869). For example, Dutrochet (1824) mounted Vicia seeds under the surface of a sponge completely saturated with water. This arrangement was probably ineffective at creating a significant moisture gradient, and as a result the roots did not bend up toward the sponge. Dutrochet’s (1824) conclusion was that hydrotropism did not exist. The earliest clear demonstration of the existance of hydrotropism was made by Knight (1811). Sachs (1872) reinvestigated this topic and using a simple and elegant experimental design demonstrated hydrotropic responses by roots of Pisum, Phaseolus, Vicia, Zea, Helianthus, Tropaeolum, and Ipomoea plants. He used a hanging sieve with zinc sides and a cloth bottom to reproducibly create moisture gradients to study hydrotropic responses (Fig. 3). The apparatus was filled with moist sawdust that was sown with seed, and hung at an angle of 45 C. By hanging the sieve at that angle, the projecting root was subjected to a difference of moisture on the two sides. The side of the

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hydrotropic responses have also been documented and studied in other plant organs including rhizoids and pollen tubes (Molisch, 1883; Voechting, 1902). Sporangiophores were also studied and exhibited either positive or negative hydrotropism depending on conditions (Errera, 1892; Mioshi, 1894; Molisch, 1883). Despite the efforts of the early investigators, many questions were left unanswered. These include the physiological basis of sensing, signal transduction, growth response, and the nature of interaction of hydrotropism with gravitropism. Modern studies of hydrotropism are being aided by the use of agravitropic mutants. Current knowledge and research efforts will be discussed below. B.

Figure 3 Experimental apparatus and approach used by Sachs (1872) to demonstrate and study hydrotropic reactions in plant roots. The metal sieve was filled with moist sawdust, which was rested on a cloth layer at the bottom of the sieve. This arrangement allowed for uneven dispersion of water vapor from the cloth layer into the still air where the root was suspended. The water vapor gradient is represented by a gradation of gray tone emanating from the cloth layer.

root nearest the lower portion of the sieve would be exposed to higher concentrations of moisture than the side nearest the higher side of the sieve. Because of this moisture gradient the root would bend to the lower side of the sieve bottom that was nearest it. Sachs also used this arrangement to test for hydrotropic responses in shoots and found them to be hydrotropically insensitive. Later Darwin (1881) used Sachs’ methods and showed positive hydrotropism of roots in Phaseolus, Vicia, Avena, and Triticum. Molisch (1883) also published a comprehensive work on hydrotropism. He studied the morphological and anatomical structure of hydrotropically responding roots and demonstrated that hydrotropic bending resulted from bilateral differences in cellular expansion in the zone of elongation of Zea and Pisum roots. He also noted that adventitious roots were especially sensitive to hydrotropism. Hooker (1915) overcame gravitropic interference with hydrotropism by using a clinostat to study positive root hydrotropism in Lupinus albus. Loomis and Ewan (1936) completed an exhaustive study showing hydrotropic root sensitivity in 7763 seedlings representing 29 genera and 14 families. Besides roots,

Hydrotropic Sensing

Hydrotropic responses are mediated by an active sensor and are not artificially induced by lateral differences in water potential within the moisture gradient (Fig. 4). Sensing thresholds for the hydrotropic response were first studied in clinostat rotated Lupinus roots under moisture levels of between 80% and 100% relative humidity (RH) (Hooker, 1915). This was also the moisture range used for the study of hydrotropism by the agravitropic pea variety ageotropum (Jaffe et al., 1985; Takahashi and Suge, 1991) and by maize (Takahashi and Scott, 1991). Hooker (1915) showed that lupin roots responded to a gradient of 0.4% RH/cm, but estimated that they could sense and respond in a gradient as low as 0.2% RH/cm. In ageotropum peas, the roots bent to a gradient of 1.0% RH/cm (Jaffe et al., 1985). All these values should, however, be considered cautiously, as Takahashi and Suge (1991) have described how difficult it is to provide a constant moisture gradient and to measure the precise humidities within an experimental gradient. In their experiments the roots showed different intensities of hydrotropic responses within the experimental gradient, ultimately compromising the ability to measure the presentation time for the response (Takahashi and Suge, 1991). Detailed quantitative study of the sensitivity of root hydrotropism will require the development of a sophisticated humidity-controlled chamber. The root cap is the site of hydrotropic sensing. Darwin (1881) and Molisch (1883) both reported that wrapping the root tips (approximately the apical 1.5–2.0 mm) with a hydrophobic mixture of olive oil and lampblack (partially burned lamp oil) or a wet tissue paper blocked the hydrotropic response of Phaseolus, Vicia, Pisum, Avena, Zea, and Triticum

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475

mental standardization, it can be difficult to relate such results back to parameters that are measurable in the rhizosphere. Results obtained so far suggest that the region of root cap may include an apparatus not only for sensing the gravistimulus, but also for sensing the hydrostimulus. The ageotropum pea mutant was also used to show that the perception of hydrostimulation in the root cap is very rapid (Stinemetz et al., 1996). Hydrotropic response occurs in < 2 min following the hydrostimulation of the root cap, and 5 min of hydrostimulation is sufficient to fully induce a complete hydrotropic response. These very short threshold times for perception of root hydrotropism are similar to those times reported for root gravitropism.

C.

Figure 4 Diagram demonstrating the difference between an active sensor-based tropic response and an induced artifact. In the latter, curvature of the root might arise from differentially altered turgor potential of the root cells on either side of the root by the moisture gradient. If the turgor potential of the root cells were to increase on the side of the root with a higher water potential, these cells could expand more than the cells on the side with the lower water potential. This would lead to curvature of the root toward the drier soil. Instead we see the root curvature is in the opposite direction, signifying the process is an active sensor-mediated phenomenon.

roots. Hooker (1915) also came to the conclusion that the hydrotropic sensitivity resides chiefly in the tip of Lupinus roots but also somewhat in the immediate region above the root tip. In experiments using agravitropic pea roots (cv. Ageotropum), the root cap was removed as was done previously for gravitropism studies (Juniper et al., 1966). The removal of the tip completely blocked the hydrotropic bending of pea and maize roots (Jaffe et al., 1985; Takahashi and Suge, 1991; Takahashi and Scott, 1991, 1993; Takano et al., 1995). Another approach that has been used to study hydrotropism is based creating a water potential gradient across the root cap by asymmetrical application of sorbitol-containing agar blocks (Takano et al., 1995). Using this technique the magnitude of the hydrotropic responses is more reproducible. Responses have been observed for a water potential gradient across the root cap of only 0.5 Mpa. While this approach does allow for experi-

Hydrotropic Signal Transduction

Transduction and transmission of a hydrotropic stimulus require 90–120 min for movement from the root cap to more basal tissues involved in differential growth leading to root curvature (Stinemetz et al. 1996). Both auxin and calcium are implicated as being the transmitted signals (Takahashi and Suge; 1991; Takahashi, 1994; Takano et al., 1997) as pretreatment with either the auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) or calcium chelator ethyleneglycol-bis-(beta-amino-ethylether) N,N,N 0 N 0 tetraacetic acid (EGTA) inhibited root hydrotropism in ageotropum pea. Both inhibitors inhibited root gravitropism and hydrotropism in Alaska pea. The hydrotropic curvature could be recovered in EGTAtreated roots when EGTA was subsequently replaced with a 10 mM CaC12 solution prior to hydrostimulation (Takano et al. 1997). The calcium channel blocker lanthanum (LaCl3 ) also inhibited hydrotropic curvature of ageotropum roots but was unaffected by nifedipine and verapamil, i.e., by compounds known to block transmembrane calcium movement. Application of a calcium ionophore (A23187) promoted hydrotropic curvature of ageotropum roots and was accelerated by water stress and was significantly inhibited by lanthanum. These results indicate that apoplastic calcium and its influx through the plasma membrane are involved in the induction of hydrotropism in roots. A gradient of water potential in the root cap may cause a physiological change that is mediated by calcium, which ultimately leads to the curvature in the elongation region associated with the hydrotropic response.

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D.

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Hydrotropic Response Mechanism

Molisch (1883) demonstrated that hydrotropic bending resulted from bilateral differences in cellular expansion in the zone of elongation in both Zea and Pisum. Modern studies using computer-assisted image analysis showed that the hydrotropic curvature in the roots of ageotropum pea was chiefly due to inhibition of elongation on the humid side of the roots whereas cells on the dry side elongated at a normal rate (Takahashi and Suge, 1990). In analyzing this it is important to remember that the region of elongation is modulated as a function of water potential of the rhizosphere (Sharpe et al., 1988). Takano et al. (1995) considered this when concluding that the zone of hydrotropic bending was similar to that for gravitropism (Pilet and Nougarede, 1970). The effects of hydrostimulation on the water status of root tissues, cell wall growth, and the hydraulic properties of the elongating tissues were studied by Hirasawa et al. (1997). They found no bilateral differences in water potential or in osmotic potential in the elongating region of hydrotropically responding roots. Plastic extensibility of the tissue was higher on the side associated with lower moisture. However, no differences in turgor pressure or yield threshold were measured. Therefore, the extensibility of the cell wall is thought to mediate differential cell expansion rates of tissues during hydrotropic responses. The causes of these changes in cell wall extensibility and in cell elongation rates were studied in detail in relation to the endoxyloglucan transferase gene (PsEXGT1) expression in the roots of ageotropum (Takano et al., 1999). The Ps-EXGT1 gene was strongly expressed in elongating roots and was most abundant in the rapidly growing region. When root elongation was inhibited by water stress, Ps-EXGT1 transcription was repressed. The roots curved hydrotropically owing to differential growth of the cortical cells in the elongation zone when the root cap was exposed to a water potential gradient. In this case the cells on the side of lower water potential were much longer than those on the side of higher water potential. The expression pattern of Ps-EXGT1 in the hydrotropically responding roots showed that the accumulation of Ps-EXGT1 mRNA was much greater on the side of lower water potential than on that of higher potential just prior to commencement of a positive hydrotropic response. These results suggest that the transcription of Ps-EXGT1 is involved in regulation of modification of cell wall properties during differential expansion and subsequent root curvature in hydrotropically responding roots.

IV.

OXYTROPISM IN PLANT ROOTS

A.

Background and History

Oxytropism can be considered to be a specific type of chemotropism. When research into this was begun the term aerotropism was used, as researchers did not acknowledge which component of the atmosphere was actually mediating the observed response. The first report of oxytropism in plant roots (Molisch, 1884) described the growth and curvature of roots grown in water. He observed that when roots were partially submerged in water, near the water/air interface, they responded by growing upward toward the interface. This led to experiments to test if roots could sense and respond to gases. In such experiments a chamber containing the test gas was employed, which was closed by a rubber stopper containing several slits. Seedlings were fastened to this chamber with their roots in close proximity to the slits through which the gas was applied. The findings of Molisch were contested by Bennett (1904) who conducted experiments and published a paper refuting the idea that roots can respond to atmospheric gases. This report cited possible uneven wetting in Molisch’s experimental design and hydrotropic responses as the source of Molisch’s observations. It turns out it was Bennett’s experiments were compromised owing to the use of pure CO2 as an atmospheric diluent, as it was not presumed to be ‘‘metabolically poisonous.’’ We now know that highly elevated CO2 levels lead to metabolic problems (Chang et al., 1983; Longhurst et al., 1990; Nobel, 1990) that ultimately compromised Bennett’s experiments. Sammett (1905) also did some early work on oxytropism and was able to demonstrate oxytropic responses using an apparatus that arranged roots growing in soil around a central aerated core. In this apparatus the roots were deflected away from growing downward (gravitropism) toward the aerated core (oxytropism), thereby supporting Molisch’s original conclusions that plants roots can sense and respond to air. Pfeffer (1906) coined the term oxytropism and hypothesized that the roots were responding specifically to oxygen. This early work on oxytropism received little attention and remained largely unknown until researchers began investigating the relationship between root system growth and soil flooding (Wiersum, 1960; Tackett and Pearson, 1964; Waddington and Baker, 1965; Huck, 1970; Jackson, et al., 1991). While most of these investigators attributed such changes in root growth to a modification of the gravitropic set point

Environmental Sensing

(Digby and Firn, 1980), Wiersum (1967) rediscovered Molisch’s original work and conducted experiments to test for oxytropism in plant roots. In these experiments he grew roots in soil contained in asbestos tubes whose lower portions were placed in water. This resulted in an increase in water content with depth and, as a consequence, a decrease in aeration. Wiersum reported that roots grew down to a layer that corresponded with a minimal required oxygen concentration. In plants of Brassica napus and potato, the roots were allowed to grow down in the tube to a minimal layer before the water level was raised, thereby initiating a tropic response that caused the roots to turn upward, toward better-aerated layers. Despite this evidence, oxytropism remained out of the mainstream of root biology, and flooding-induced changes in root orientation were attributed only to gravity sensing. As previously discussed in the section on hydrotropism, work on oxytropism has been protracted by biological and technical difficulties. Technically, it is difficult to create experimental oxygen gradients, and gravitropic responses always manifest themselves strongly. Using both genetic (agravitropic mutants) and physiological (clinostat rotation) approaches to nullify gravity sensing, progress has been made in studying hydrotropic responses. In recent years access to microgravity (104 to 106 g) conditions has been made possible by NASA funded spaceflight experimentation programs and has allowed researchers to study plant growth under the influence of minimal gravity. Because much of this work has been focused on studying gravity sensing and gravitropism, the orientation of roots in the absence of gravity has been described as being ‘‘random’’ by investigators (Merkys et al., 1981; Cowles et al., 1984; Merkys et al., 1984; Slocum et al., 1984; Perbal et al., 1986; Schulze et al., 1992; Perbal and Driss-Ecole, 1993; Dutcher et al., 1994; Perbal and Driss-Ecole, 1994; Johnsson et al., 1996), who have failed to consider other tropic responses in interpreting the observed growth patterns. To illustrate this, the roots of spaceflight-grown roots have commonly been shown growing away from water and nutrients into the air (for a good example of this phenomena see Cowles et al., 1984). The most recent work on oxytropism was the direct result of results obtained from microgravity experimentation. During two space shuttle flights (Porterfield et al., 1997b) metabolic changes in the roots of Arabidosis thaliana consistent with adaptation to low O2 conditions were documented. Root tissue samples were analyzed from space flight and ground

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control roots and revealed significant increases in the activity and gene expression of the fermentative enzyme alcohol dehydrogenase (ADH) associated with the space flight materials. These metabolic changes are thought to be mediated by inhibition of gravity-driven convective transport of oxygen from the bulk medium into the root tissue. These metabolic changes have also been measured in wheat and Brassica rapa in recent microgravity experiments (Porterfield et al., 2000a). In association with these metabolic changes the investigators also observed the altered root growth patterns—namely, the reorientation of the roots and growth of the roots away from the source of water and mineral nutrients and into the air—that in the past have been associated solely with the loss of gravitropism, as described above. The metabolic evidence of microgravity inhibition of oxygen bioavailability and the associated growth of the roots away from the rooting medium and into the air, suggests that root orientation in the absence of gravity is not random, but is actually influenced by oxygen. Indeed, later experimental work has demonstrated oxygen-mediated tropic responses in gravity-sensing and agravitropic pea roots (Porterfield and Musgrave, 1998) and has shown that this response is mediated by an active sensor, not the result of a not a metabolic artifact (Fig. 5). Porterfield (1997) also has shown that motile unicellular freshwater alga (Euglena gracilis) can sense and respond to oxygen (oxytaxis) in the dark when the photosynthetic apparatus is not active and oxygen is not being produced by the chloroplasts. These experiments with Euglena further demonstrate that the ability to sense and respond to oxygen is not limited to higher plants, but is also present in unicellular motile algae. This suggests that oxygen-sensing mechanisms are not recent evolutionary advancements associated with terrestrial plants, but may have been present before in algal protist ancestors. B.

Oxytropism and Root Metabolism

As compared to knowledge of tropic mechanisms in gravitropism and hydrotropism, very little is known about oxygen sensing, signal transduction, and differential growth responses of roots. To date there have been no investigations of the physiological and biochemical basis of oxytropic sensing, signal transduction, and responses or plant roots. Instead, investigations have described how oxytropic responses relate to hypoxic and anoxic responses of plant roots as commonly occur in association with soil waterlog-

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Figure 5 Diagram demonstrating the difference between an active sensor-based tropic response and an induced artifact. In the latter, curvature of the root might arise from differentially altered metabolic activity of the cells on either side of the root by the oxygen gradient. If the metabolic activity of the root cells were higher on the side of the root exposed to higher oxygen concentration, these cells could expand more than the cells on the side with the lower oxygen concentrations. This would lead to curvature of the root toward the side with lower oxygen concentrations. Alternatively, we see the root curvature is in the opposite direction signifying the process is an active sensor-mediated phenomenon, not a metabolic artifact.

ging. Waterlogging displaces soil gases and decreases oxygen availability owing to the limited solubility and diffusibility of oxygen in water as compared to air. As a result of these limitations the oxygen that is dissolved in the soil solution can be reduced to below critical levels (Saglio et al., 1984) or completely depleted in a matter of hours owing to the metabolism of roots or of the soil organisms (Drew and Lynch, 1980; Meek et al., 1983). The reduced capacity for metabolic activity that ensues can ultimately inhibit root activity and wholeplant mineral nutrition (Trought and Drew, 1980; Porterfield et al., 1997a, 2000b). The changes that occur when roots are subjected to low-oxygen stress have been well documented and include biochemical, anatomical, and morphological adaptations for stress tolerance (for a complete review see chapter 42 by Armstrong and Drew in this volume). Short-term bio-

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chemical stress tolerance of hypoxia and anoxia involves continued production of ATP through the fermentation of ethanol by the marker enzyme alcohol dehydrogenase (ADH). In the studies that have looked at localization of ethanolic fermentation in the root system, activity has been shown to be associated primarily with the root tips of maize (Wignarajah and Greenway, 1976; Saglio et al., 1988; Hole et al., 1992), Nicotiana plumbaginifolia (Rousselin et al., 1990), wheat (Porterfield et al., 2000a), and Arabidopsis (Porterfield et al., 1997b). How do oxytropic responses relate to growth and metabolic responses associated with hypoxia and anoxia? Using a microrhizotron apparatus (Porterfield and Musgrave, 1998) to deliver and maintain specific oxygen gradients, this question has been studied using garden pea (Pisum sativum L. cv. Weibul’s Apollo) and the agravitropic pea mutant (cv. Ageotropum). Based on the distance between the gas ports of this apparatus, the microrhizotron produced a linear oxygen concentration gradient of
0:8 mmol mol1 mm1 . Oxytropic curvature was shown to occur all along this gradient (Fig. 6A). After 48 h of growth in the gradient all roots reached the same final angle of curvature regardless of the absolute oxygen concentration. This demonstrates that the absolute oxygen concentration is not as important as the spatial oxygen concentration differences within the gradient. However, the kinetics of root curvature was slower in regions associated with lower oxygen concentrations. This was correlated with a decrease in root elongation associated with metabolic oxygen limitations as indicated by analysis of ADH activity (Fig. 6B). The analysis showed that significant changes in the amount of ADH activity were associated with slower curving and growing roots. Oxytropic curvature occurred even in roots exposed to O2 concentrations that were not low enough to induce hypoxically responsive protein. Oxytropic orientation of the roots is a stress avoidance tropism (SAT), allowing the growing root tip to avoid soil regions where oxygen limitations would require induction of metabolically costly fermentation. That oxytropic curvature occurred in roots before ADH was induced also suggests that the O2 sensor associated with oxytropism may not be involved with the induction of hypoxic metabolism. What, then, is the oxygen sensor? Hemoglobins were first identified in plants in soybean root nodules and are referred to as symbiotic hemoglobins because they are thought to function in transporting oxygen in nitrogen-fixing root nodules (Appleby, 1992;

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Figure 6 Tropic, growth, and metabolic responses of agravitropic roots of Pisum sativum cv. Ageotropum growing in an oxygen concentration gradient. Tracking curvature and reorientation of the roots, within the oxygen concentration gradient that ranges from air equilibrium to anoxia, shows that roots respond to the oxygen concentration gradient by reorienting and growing toward the regions of higher oxygen concentrations (A). While all of the roots respond, the rate of curvature decreases with absolute oxygen concentration within the gradient. This decrease in the reorientation rate was correlated (B) with a decrease in root elongation and the activity of alcohol dehydrogenase. Values are means  SE. (From Porterfield and Musgrave, 1998.)

Arredodondo-Peter et al., 1998). Numerous different nonsymbiotic hemoglobins have also been identified and some have been shown to be expressed specifically in the meristemic region of the root tip (Jacobsen-Lyon et al., 1996). Root-tip-localized hemoglobins could act as oxygen sensors and induce

tropic responses in a manner that is analogous to that described for gravitropism and hydrotropism. The importance of a tip-localized sensing system will be discussed in subsequent sections. More work on the possible role of hemoglobins as oxygen sensors is required to test this important hypothesis.

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V. THIGMOTROPISM IN ROOTS A.

History and Background

Plant responses to touch and other forms of mechanical stimulation have been most often studied in relationship to aerial plant organs. Touch-mediated morphological adaptations (thigmomorphogenesis) have been studied in relation to wind, rain, and experimentally induced stimulations (Jaffe, 1973, 1976; Biddington, 1986). Such responses include shortening of the internodal length in herbaceous annuals like Arabidopsis (Braam and Davis, 1990), and inhibition of stem and leaf growth and development of reaction wood (Telewski and Jaffe, 1986) in trees like Pinus taeda and Abies fraseri. Touch responses are also well documented in relation to the thigmonsatic movements (Siboaka, 1969) of the carnivorous Venus fly trap (Dionaea muscipula) and of a sensitive plant (Mimosa pudica) as well as thigmomorphogenic activity of tendrils of various climbing vines (Huberman and Jaffe, 1986). Roots also respond to touch stimulus, and this response is an important mechanism of growth of roots in the soil. Roots commonly encounter rocks, stones, and regions of highly compacted soil that can restrict or alter root growth through the bulk soil. Given that these obstacles can potentially mechanically impede root extension, thigmotropism is an important mechanism determining the direction of growth of roots within the soil. We have already referenced the work of Charles Darwin in relation to gravitropism, phototropism, and hydrotropism. The first real definitive demonstrations of touch-mediated growth responses in roots were described by Darwin (1881) in his book The Power of Movement in Plants. He used a relatively simple approach to thigmostimulate roots to such a degree as to actually overwhelm gravity-sensing responses. First Darwin studied the activity of roots as they came into contact with flat planar surfaces. He grew the roots of Vicia faba so that gravity would direct the roots down onto a glass or wood surface. The contact of the root cap with that surface initiated a tropic response following the subtle deformation of the shape of the root cap. The tropic response resulted in deflection of the root away from the gravitational vector into a direction that was parallel with the planar surface. In other words, the root cap made contact with the surface and this resulted in the modification of the direction of the growth of the root axis to match the planar direction of the surface. If the surface topography was modified or irregular,

the root responded to match the surface. Darwin also applied touch stimulus by attaching a mica chip to one side of the root cap. In using this approach the root cap was constantly stimulated no matter how much the root curved in retreat from the stimulus. When stimulated in such a manner the roots showed continuous tropic curvature and curved first up and away from gravity, then continually around forming circles and coils. B.

Thigmotropic Sensing by the Root Cap Cells

Mechanical sensing in the root cap has been most often studied in gravity sensing, where the mechanical stimulation is a result of the force of gravity acting on the cell or cellular organelles. Thigmotropic stimulation originates from the mechanically inductive process of root growth, elongation, and penetration in the soil, and, as in gravity sensing, the root cap has been shown to act as the primary site of thigmosensing. Darwin’s experiments (1881) with Vicia faba showed that constant stimulation of the root cap was sufficient to overwhelm gravity sensing and redirect the growth of the stimulated root into circular patterns. Millet and Pickard (1988) have described the use of small glass probes to unilaterally touch and stimulate the root cap of individual maize roots and induce thigmotropic responses that were described to be comparable to gravitropic responses (Millet, 1991). Furthermore, such touch stimulation was enhanced by removal of the mucilage. Thigmostimulation has also been applied to maize roots by unilateral application of agar blocks (Ishikawa and Evans, 1992). This approach was used to test for localized thigmosensing in the root cap. The investigators removed the root cap before applying the stimulus. Obviously the act of removing the root cap can be considered to be a severe and extreme form of thigmostimulation, so the investigators waited 6 h before testing for thigmotropic responses. Although the decapped roots did respond to subsequent stimulation at the root tip, this does not disprove the hypothesis that the root cap acts as the primary site to thigmosensing, because the surgically modified root tips could have regenerated some portions of the root cap during the 6-h recovery period. Given that the root cap is the first part of a root to come in contact with new regions of the soil, and the mechanical compression forces associated with root elongation are focused there, the root cap remains the most probable site for thigmosensing. The touch response of Arabidopsis

Environmental Sensing

thaliana roots was studied recently by Massa and Gilroy (1999) and was shown to be mediated by root cap sensing as laser ablation of peripheral cap cells inhibited the normal touch response in roots. The idea that mechanical stimulation could be sensed at the cellular level in plant systems through the activity of stretch-activated ion channels in the plasma membrane was first promoted by Barbara Pickard (Pickard, 1985; Edwards and Pickard, 1987; Pickard and Ding, 1988). This idea was originally discussed in the context of gravity sensing but was later expanded to cover other forms of mechanical stimuli. The hypothesis is that stretch-activated ion channels (Pickard and Ding, 1993) that are interlinked either through the cytoskeleton and/or the cell wall would allow the movement of ions like calcium into the cell that would mediate the touch-induced response. The interaction of the channels with the cell wall and/or with the cytoskeleton could focus the mechanical force that the cells were to receive. C.

Thigmotropic Signaling and Differential Root Growth

The role of calcium in endogenous signaling in association with touch-mediated morphological responses is best understood in Arabidopsis. Touch-mediated changes in gene expression are rapid and have been shown to occur within 10 min of the touch stimulus (Braam and Davis, 1990). Four of these Arabidopsis touch genes (TCH) have been identified and include calmodulin and calmodulinlike proteins (Braam et al., 1997). This suggests that calcium-mediated signaling might be involved with propagation and/or regulation of other touch-induced changes in plant systems including thigmotropism in the root system. The calcium binding THC3 gene is expressed in roots (Antosiewicz et al., 1995) and shown to correlate strongly with the process of cellular expansion. All four subclasses of wheat calmodulin genes are expressed in the root tip including the root cap, meristemic regions, and the zone of elongation (Yang et al., 1998). Ishikawa and Evans (1992) used an agar block thigmostimulation method to show that the thigmotropic responses of intact roots were stronger when the applied agar block contained CaCl2 . Rapid changes in cytosolic calcium concentrations have been shown to occur in thigmostimulated root cells (Legue et al., 1997) using confocal calcium ratio imaging. Thigmotropic bending was shown in Arabidopsis thaliana to be mediated by a growth response in the elongation zone (Massa and Gilroy, 1999). Lateral dif-

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ferences in cytosolic calcium in the distal part of the elongation zone (Ishikawa and Evans, 1995) can induce bilateral differences in cell expansion. Ishikawa and Evans (1992) applied calcium-containing agar blocks to the distal region of the zone of elongation, causing inhibition of cell elongation in this responsive region. The fourth touch gene, TCH4 (Xu et al., 1995), identified from Arabidopsis suggests that modifications to the mechanical properties of cell walls may facilitate differential growth responses. The TCH4 gene encodes a xyloglucan endotransglycoslyase enzyme that may be involved in modifying the extensibility of cell walls. Lateral differences in cell wall extensibility could also develop bilateral differences in expansion in the zone of elongation that could lead to curvature and modification of the direction of root growth.

VI.

TROPIC INTERACTIONS IN CONTROLLING DIRECTIONAL GROWTH

Growing roots sense gravity and grow toward it. At the same time they obtain information about soil water, oxygen, and mechanical obstacles. Separate specific systems for sensing these environmental stimuli must exist together with the ability to integrate all of this information to determine the ultimate direction of growth. To study hydrotropism and oxytropism researchers had to find ways to nullify gravity sensing without dramatically altering the physiology of the root. This has been accomplished by using both genetic (agravitropic mutants) and physiological (clinostat and space shuttle) approaches, and has shown that disruption of gravity-mediated responses is not a total disruption of other tropic mechanisms. Hooker (1915) placed Lupinus roots on a horizontal clinostat which showed that when gravity perception was disrupted during clinorotation, the roots where still able to bend hydrotropically. When agravitropic pea roots were placed in different positions in either a water potential (Takahashi et al., 1992) or an oxygen gradient (Porterfield and Musgrave, 1998), the root bends toward the tropic stimulus. Again showing that the genetic lesion associated with disruption of gravity sensing does not alter or effect sensing and signal transduction associated with other tropisms. Genetic and physiological approaches to nullify gravity sensing have allowed us to study these tropic responses individually, and to compare these to responses observed when gravity sensing is left intact. These comparative

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studies do offer the insight in understanding how and to what degree plant roots integrate tropic sensing in modulating root responses. Interaction of gravitropism and hydrotropism is known to occur in roots, but the nature of that interaction is incomplete, as differences have been noted in different species. In comparison to the agravitropic pea mutant, wild-type gravity-sensing pea roots responded weakly to a moisture gradient (Jaffe et al., 1985; Takahashi and Suge, 1991) unless gravity sensing was disrupted (Takahashi et al., 1996). This suggests that gravity and hydrotropic sensing and responses are active simultaneously. The interaction of gravity and hydrotropic responses has also been noted in maize (Takahashi and Scott, 1991). Here the investigators utilized a novel characteristic of a cultivar in which gravitropic responses do not manifest themselves until the roots are stimulated with light. Dark-grown roots showed hydrotropism while light-grown roots responded to gravity when exposed to light and oriented at an angle that is 45 below the horizon. This is not just a simple matter of light-mediated switching between hydro- and gravitropism as light-exposed roots do respond to the moisture gradient when light-stimulated, at an angle of 70 below horizontal. Therefore the interactions of these responses are complex and not easily determinable as the exact intensity of hydrostimulus has never been determined in those experiments. Unlike like gravity stimulus, the intensity of hydrostimulus may not be easily determined because of technical difficulties making it hard to stimulate both systems in an equivalent manner. Another important clue comes from studies where gravitropic and hydrotropic interactions in 12 different wheat cultivars were examined (Oyanagi et al., 1995). No strong correlations between the levels of graviresponsiveness and hydroresponsiveness were noted. This suggests that although gravity and hydrotropism are able to interact, they are definitely physiologically separated from one another. The interaction of gravity and oxygen-sensing tropic responses was addressed by Porterfield and Musgrave (1998). In the experimental apparatus gravity and oxygen gradient vectors were 90 out of phase with one another. The roots of the agravitropic pea mutant responded to the oxygen gradient by reorienting a full 90 from the gravity vector toward the direction of the oxygen gradient (Fig. 7). This was shown compared to the response of a gravity-sensing variety Weibul’s Apollo to the oxygen gradient. The gravitysensing variety curved to a constant 45 , which is

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Figure 7 Comparison of oxytropic reorientation of both agravitropic (Ageotropum) and wild-type gravity-sensing (WT) pea roots in an artificial oxygen gradient. The vector of the oxygen gradient was out of phase with the gravity vector by 90 and the roots were initially oriented downward, parallel to the gravitational vector (0 ). While the agravitropic mutant reoriented a full 90 with regard to the oxygen gradient, the wild-type gravity-sensing roots curved to only 45 . This suggests that gravity- and oxygen-sensing tropism are interacting equally in determining the final angle of root growth. (From Porterfield and Musgrave, 1998.)

approximately one-half of the vector gradient difference between gravity and oxygen within the system. This suggests that roots equally integrate both the gravity and oxygen signals. This would explain flooding-induced diagravitropic or plagiogravitropic root growth that is associated with lodging of crop plants (Wiersum, 1960, 1967; Waddington and Baker, 1965; Huck, 1970). In a flooded field situation we would expect that the gravity and oxygen gradient vectors would be out of phase by 180 as the source of the oxygen gradient would be the soil surface. Given that changes in root growth patterns are associated with waterlogging and result from integration of gravity and oxygen sensing, it may be more appropriately referred to as oxygravitropic growth. This term better defines the environmental parameters that lead to the observed changes and distinguishes this phenomenon from true diagravitropic and plagiogravitropic growth that result from a modification of the gravitropic set point. Thigmotropic and gravitropic responses are related in that they are both mechanically based responses. Both require physiological systems for mechanically transducing the applied forces into the appropriate signals. Darwin’s (1881) original work clearly showed that thigmotropic responses of roots could clearly

Environmental Sensing

overwhelm gravitropism, and this was later confirmed for other species (Wilson, 1967; Millet and Pickard, 1988; Ishikawa and Evans, 1992). Okada and Shimura (1990) first showed how a combination of gravity and thigmostimulation induces a wavy growth pattern in Arabidopsis roots. This was accomplished by growing the plants on flattened inclined blocks of agar that was concentrated enough to block penetration of the roots into the gel. The humidity of the aerial environment was maintained so as to prevent significant hydrostimulation from occurring. So, as roots grow down along the gel in response to gravity, they constantly receive unilateral thigmostimulation by the agar. When the agar slabs are oriented vertically, these patterns of root growth disappear. Other investigators have noted that such an arrangement that provides constant gravity and touch stimulation will also induce slanting or deflected growth of the roots at a fixed angle away from gravity as well as root waving (Simmons, 1995; Mullen et al., 1998; Rutherford and Masson, 1996). These patterns of growth are best explained by a combination of thigmostimulation and endogenous circumnutation growth patterns in the roots. Reduced gravitropism mutants show a pattern that switches from waving to coiling (Mullen et al., 1998). However, it is important to remember that this is an artificial system. In a natural environment roots receive thigmostimulation in encountering rocks and other obstacles, in the normal course of their downward growth. Indeed, studies that have looked at root responses in this type of experimental system have shown that the thigmotropic component of the root growth response can function without gravity sensing, although gravity sensing is required for normal bend formation (Massa and Gilroy, 1999).

VII.

SUMMARY AND DISCUSSION

In growing through the bulk soil, roots sense and respond to environmental stimuli while interacting with the soil. By doing so they alter the physical and chemical properties of some of that soil. This zone of interaction is referred to as the rhizosphere and varies both temporally and spatially along and around the root axis. Typically the majority of the rhizosphere activity is associated with the growing root tip. This region is also known to be the region where tropic curvature is manifested by lateral differences in cell elongation rates in the zone of elongation. Calcium,

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hydrogen ions, and the plant hormone auxin have all been implicated in mediating this differential growth response (see Chapters 30 by Pilet and 31 by Poovaiah et al. in this volume). The first portion of the root to encounter a new unaffected soil region is the root cap, which has been shown to be the primary site for sensing gravity, water, and touch and probably also oxygen. As the root tip grows and penetrates though the soil, it is sensing and tropically responding to environmental stimuli and in doing so may avoid soil regions where conditions would elicit a stress response. Gravitropism defines the default direction for root growth whereas hydrotropism, oxytropism, and thigmotropism can be considered to be stress avoidance tropisms (SATs). This is analogous to an explorer using a compass to go West but having to alter his path along the way to avoid obstacles and dangers. These different forms of environmental stimuli must all be sensed through distinctly different sensory systems. Whereas gravity and touch responses are based on mechanical sensory transduction, water and oxygen sensing must be based on a protein receptor ligandbinding activator. Interactions between gravitropism and each of the other tropic responses covered here have been noted and to some level investigated. We know that separate systems for sensing each of these stimuli exist in the root cap, but do not understand how these tropisms all interact in determining the ultimate direction of root growth. In analyzing the interaction of gravitropism and tropic responses to the other stimuli (water, oxygen, and touch), it is clear that these tropic responses are not exclusive of one another and integrated responses are observed when differential stimuli are applied. In other words, while each tropic response system is separate in sensing and responding to the stimuli, this does not exclude the possibility of a shared transduction pathway and response mechanism. Therefore the best hypothesis to explain interaction of these tropisms is an integrated model where gravity, oxygen, touch, and water (GOTW) sensing modulate a shared signal transduction and response mechanism. When one of these environmental signals is removed, as occurs in the microgravity environment of space flight, the root would be expected to simply orient growth in regard to the remaining environmental clues. Therefore it may be possible to control the orientation of growth of plant roots in a microgravity-based bioregenerative life support systems using generated water, or oxygen gradients within the rooting module.

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486 Pickard BG, Ding JP. 1993. The mechanosensery calciumselective ion channel: key component of a plasmalemma control center? Aust J Plant Physiol 20:439– 459. Pillet PE, Nougarede A. 1970. RNA, structure, infrastructure et geotropisme radiculares. Physiol Veg 8:277–300. Porterfield DM. 1997. Orientation of motile unicellular algae to oxygen: oxytaxis in Euglena. Biol Bull 193:229–230. Porterfield DM, Musgrave ME. 1998. The tropic response of plant roots to oxygen: oxytropism in Pisum sativum L. Planta 206:1–6. Porterfield DM, Crispi ML, Musgrave ME. 1997a. Changes in soluble sugar, starch, and alcohol dehydrogenase in Arabidopsis thaliana exposed to N2 diluted atmospheres. Plant Cell Physiol 38:1397–1401. Porterfield DM, Matthews SW, Daugherty CJ, Musgrave ME. 1997b. Spaceflight exposure effects on transcription, activity, and localization of alcohol dehydrogenase in the roots of Arabidopsis thaliana. Plant Physiol 113:685–693. Porterfield DM, Barta DJ, Ming DW, Morrow RC, Musgrave ME. 2000a. Astroculture root metabolism and cytochemical analysis. Adv Space Res 26:315–318. Porterfield DM, Dreschel TW, Musgrave ME. 2000b. A ground-based comparison of nutrient delivery technologies originally developed for growing plants in the spaceflight environment. Hort Technol 10:179– 185. Rousselin P, Lepingle A, Faure JD, Bitoun R, Caboche M. 1990. Ethanol-resistant mutants of Nicotiana plumbaginifolia are deficient in the expression of pollen and seed alcohol dehydrogenase activity. Mol Gen Genet 222:409–415. Rutherford R, Masson PH. 1996. Arabidopsis thaliana sku mutant seedlings show exaggerated surface-dependent alteration in root growth vector. Plant Physiol 111:987–998. Sachs J. 1872. Abelenkung der Wurzeln von ihrer normalen Wachstumrichtung durch feuchte Korper. Arb D Bot Inst Wurzburg 1:209–222. Saglio PH, Rancillac F, Bruzan E, Pradet A. 1984. Critical oxygen for growth and respiration of excised and intact roots. Plant Pysiol 76:151–154. Saglio PH, Drew MC, Pradet A. 1988. Metabolic acclimation to anoxia by low (2–4 kPa partial pressure) oxygen pretreatment (hypoxia) in root tips of Zea mays. Plant Physiol 86:61–66. Schulze A, Jensen PJ, Desrosiers M, Buta JG, Bandurski RS. 1992. Studies on the growth and indole-3-acetic acid and abscisic acid content of Zea mays seedlings grown in microgravity. Plant Physiol 100:692–698. Sharpe RE, Kuhn Silk W, Hsiao TC. 1988. Growth of maize primary root at low water potentials. I. Spatial distribution of expansive growth. Physiol Plant 87:50–57. Siboaka T. 1969. Physiologuy of rapid movements in higher plants. Annu Rev Plant Physiol 20:165–184.

Porterfield Simmons C, Soll D, Migliaccio F. 1995. Circumnutation and gravitropism cause root waving in Arabidopsis thaliana. J Exp Bot 46:143–150. Slocum RD, Gaynor JJ, Galston AW. 1984. Cytological and ultrastructural studies on root tissues. Ann Bot 54:65– 76. Staves MP, Wayne R, Leopold AC. 1992. Hydrostatic pressure mimics gravitational pressure in characean cells. Protoplasma 168:141–152. Stinemetz C, Takahashi H, Suge H. 1996. Characterization of hydrotropism: the timing of perception and signal movement from the root cap in the agravitropic pea mutant Ageotropum. Plant Cell Physiol 37:800–805. Tackett JL, Pearson RW. 1964. Oxygen requirements of cotton seedling roots for penetration of compacted soil cores. Soil Sci Soc Am Proc 28:600–605. Takahashi H. 1994. Hydrotropism and its interaction with gravitropism in roots. Plant Soil 165:301–308. Takahashi H, Scott TK. 1991. Hydrotropism and its interaction with gravitropism in maize roots. Plant Physiol 96:558–564. Takahashi H, Scott TK. 1993. Intensity of hydrostimulation for the induction of root hydrotropism and its sensing by the root cap. Plant Cell Environ 16:99–103. Takahashi H, Suge H. 1991. Root hydrotropism of an agravitropic pea mutant, ageotropum. Physiol Plant 82:24– 31. Takahashi H, Brown CS, Dreschel TW, Scott TK. 1992. Hydrotropism in pea roots in a porous-tube water delivery system. Hort Sci 27:430–432. Takahashi H, Takano M, Fujii N, Yamashita M, Suge H. 1996. Induction of hydrotropism in clinorotated seedling roots of Alaska pea, Pisum sativum J Plant Res 109:335–337. Takano M, Takahashi H, Hirasawa T, Suge H. 1995. Hydrotropism in roots: sensing of a gradient in water potential by the root cap. Planta 197:410–413. Takano M, Takahashi H, Suge H. 1997. Calcium requirement for the induction of hydrotropism and enhancement of calcium-induced curvature by water stress in primary roots of pea, Pisum sativum L. Plant Cell Physiol 38:385–391. Takano M, Fujii N, Higashitani A, Nishitani K, Hirasawa T, Takahashi H. 1999. Endoxyloglucan transferase cDNA isolated from pea roots and its fluctuating expression in hydrotropically responding roots. Plant Cell Physiol 40:135–142. Telewski FW, Jaffe MJ. 1986. Thigmomorphogenesis: the role of ethylene in the response of Pinus taeda and Abies fraseri to mechanical perturbation. Physiol Plant 66:227–233. Trought MCT, Drew MC. 1980. The development of waterlogging damage in young wheat plants in anaerobic solution cultures. J Exp Bot 31:1573–1585. Voechting H. 1902. Ueber die Keimung der Kartoffeknollen. Bot Zeit 10:87.

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30 Root Growth and Gravireaction: A Critical Study of Hormone and Regulator Implications Paul-Emile Pilet University of Lausanne, Lausanne, Switzerland

I.

approaches into the research of root growth and gravitropism (Lee et al., 1984). Elongation and gravireaction of roots are consequences of an irreversible increase in the volume of their cells. This is essentially due to the changes in (1) the extensibility of their walls, (2) their permeability to water, (3) their osmotic potential (Taiz 1984; Cosgrove, 1987; French and Hsiao, 1994; Maruyama and Boyer, 1994), (4) their proton extrusion (Cleland, 1982; Rayle and Cleland, 1992; Kutschera, 1994), and (5) the calcium availability (Evans et al., 1991). A few other processes that could also be implicated in root graviresponses were critically presented by Evans and Ishikawa (1997).

INTRODUCTION

The topics considered in this chapter have given rise to numerous reviews (Torrey, 1976; Audus, 1975, 1983; Pilet, 1977, 1996, 1998a; Jackson and Barlow, 1981; Feldman, 1984; Pilet and Barlow, 1984, 1987; Zeevaart and Creelman, 1988). For a long time after the publication in 1926 of the two papers by Cholodny and Went, auxin control of the growth and gravireaction of roots was accepted as being a reality (see Thimann, 1977; Pilet, 1994). Gradually, other endogenous regulators have been detected in growing roots (i.e., cytokinins, gibberellins, and ethylene). Combining the effects of such regulators with those of indole-3yl-acetic acid (IAA) and some new auxins and IAA conjugates had complicated the action of the implicated endoregulators. The discovery of growth inhibitors formed or released by the root cap gave rise to a new line of thought showing their possible action in the control of differential growth, which determines gravitropism (Pilet, 1977; Wilkins, 1979). The occurrence of abscisic acid (ABA) in growing roots indicated that ABA could be one of those growth inhibitors (Audus, 1983; Chapter 26 by Hose et al. in this volume). Proton secretion may also be an important characteristic of the growing roots and of their gravireacting zone (Mulkey et al., 1983; Pilet et al., 1983). Finally, transport and accumulation of calcium have induced new

II.

GROWTH

It should be remembered that growth of roots is an irreversible increase of their mass and, very generally, of their length (axial elongation). Classically, the results of growth measurements are expressed by the average values obtained for a group of roots that are as homogeneous as possible. More recently, root extension of a large population of roots has been analyzed and characterized by a certain number of classes, each defined by a particular value of initial growth rate (Pilet, 1986, 1996). 489

490

A.

Pilet

Mean Data for Individual Roots

Results related to the elongation rate (given in mm h1 ) of primary maize roots is discussed briefly for roots in vertical and horizontal positions kept in darkness or in white light. The growth rate of vertical roots in the dark followed an endogenous rhythm, which was reduced by white light and suppressed by gravity (Fig. 1). Thus, the inhibitory effect of light alone on growth was found to be weaker but of longer duration than that observed when only gravity had acted. Moreover, growth inhibition due to gravity was observed between 1 and 2 h after the gravistimulus, and it was reinforced by light. When the roots in vertical position were placed in darkness after being illuminated, growth has recovered.

Figure 1 Growth rate of intact maize (cv. LG 11) primary roots maintained 4 h in a vertical position and then 6 h vertically (A, D) or horizontally (E, H). Roots were kept 10 h in darkness (A, E), 10 h light (B, F), 2 h in darkness and 4 h in light (C, G), and 2 h in light and 4 h in the dark (D, H). Two regrouped series of experiments are presented (A, B, C, and D with A as the control and E, F, G, and H with E as the control). (From Beffa and Pilet, 1982.)

B.

Population Analysis

Relatively large changes in elongation rates were found when a group of roots was measured despite the care taken in the selection of these roots (Pilet and Saugy, 1985). Thus, various populations of roots can be classified into a number of categories defined by the magnitude of their elongation rate. The importance of each class is emphasized by the number of roots. In the present series of data, 17 classes were selected according to growth rate, each class having a range of 0.09 mm h1 (Fig. 2).

Figure 2 Growth rate (cv. LG 11) measured over 8 h of intact primary roots of maize attached to their caryopses. A total of 2508 roots were tested (12 sets of determinations with 210  25 roots each). Values for individual roots are reported for a given number of classes (in the present experiment, 17 growth rate classes, each with the range of 0.09 mm h1 ). After being selected, roots were maintained vertically for 8±0.25 h in the dark. The mean total root lengths h1 at zero time and after 8 h were 15:3  0:9 mm and 21:2  1:4 mm, respectively. The total numbers of roots in the three groups used for the measurement of the cap length (see Fig. 3) were 244 (A), 321 (B), and 208 (C). (From Pilet, 1986.)

Root Growth and Gravireactivity

Later in this chapter, in connection with the discussion of growth inhibitors, the role that cap cells may play in the formation or the release of these regulators is considered. The results just presented are used for discussion of the influence that the cap may have on the control of root elongation. Three groups of approximately equal size were selected from a total population of 2508 roots for their low (A), medium (B), and high (C) growth rates (Fig. 2). For each group, the percentage of roots reported having different sizes of cap length (eight classes, counted for each 0.09 mm) was presented (Fig. 3). Cap length was mea-

Figure 3 Length (cv. LG 11) of the root cap of intact primary roots of maize attached to their caryopses given by the relative number of roots in each cap length class. Three groups of roots were selected according to their mean growth rate (see Fig. 2): 0.20–0.39 (A), 0.60–0.69 (B), and 1.10–1.29 (C). The numbers of root caps measured in each group were 221 (A), 232 (B), and 203 (C). Mean values obtained from four sets of measurements with 45  4 roots each were separated into eight classes (each with a range of 0.09 mm). As in the growth rate study, roots were kept in a vertical position for 8  0:25 h in the dark. Mean lengths (in mm) of the root caps (distribution of the data not being normal, the standard errors cannot be given) were 0.645 (A), 0.450 (B), and 0.384 (C). (From Pilet, 1986.)

491

sured along the central axis of each root from the tip to where the calyptrogen cells join the quiescent center (Barlow, 1975). The mean length of the caps for the three groups selected is given in the legend: cap length is related with the rate of root elongation. At least for the cv. LG 11 of maize, slow-growing roots had long caps; faster-growing roots had shorter caps.

III.

GRAVIREACTION

Root gravitropism results from alterations in local rates of cell extension and a consequent development of a differential elongation between its upper and lower sides. The curvature of primary roots that are placed in a horizontal position is due to the faster rate of growth by their upper than by their lower part (Fig. 4). Several possibilities may account for such differences in growth. For example, if elongation is stimulated in the upper part, growth can be arrested, slowed, or unchanged in the lower part (Table 1). The promotion of cell extension within the upper half of gravireactive roots could be the main cause of bending (Pilet and Nougare`de, 1970, 1974; Iversen, 1973; Jackson and Barlow, 1981). Growth of 2-day-old horizontal lentil roots (Pilet and Nougare`de, 1970) indicates that cell length of the lower part increased by only 7.5% but by 59% for the upper part. Cortical parenchymatous cells of the elongating zone of maize roots increased by 60% in length and by 73% in the upper side, whereas no significant change occurred in the lower side (Pilet and Nougare`de, 1974). Nevertheless, a decrease in the extension rate of cells of the lower half of such roots was reported. For such cases, the redistribution of growth inhibitors and hormones (Audus, 1975; Pilet and Barlow, 1988) will be discussed later. It is obvious

Table 1 Root Gravireaction (Positive Curvature) Occurs When the Initially Upper Side Elongates Faster Than the Lower Sidea;b Side

1

2

3

4

5

6

Upper Lower

þ 0

þ —

þ 

0 —

0 

0 

Six ways (1; 2; . . . ; 6) in which this could arise are given. Elongation rate may be: increased (þ), decreased (—), unchanged (0), or arrested (). For instance, in the first possibility (1), the positive gravitropism of root was due to both the increase (þ) in the elongation of the upper part of the horizontal roots and to the unchanged (0) elongation of the lower part. Sources: Lackson and Barlow, 1981; Pilet, 1996. a b

492

Pilet

Roots may attain a stable graviposition at any angle (A) and must have a gravitropic set point (Firn and Digby, 1997). A single mechanism might be sufficient to account for all forms of root gravibending. Such a unifying model proposes that the ability to change the angle of roots, with respect to those with the vertical, is part of the basic gravimechanism. IV.

HORMONE IMPLICATIONS

All known hormones are potentially capable of influencing root growth and gravireaction (Firn, 1983; Pilet and Barlow, 1987). Growth hormones never act separately in vivo (Bourquin and Pilet, 1990). However, to analyze the role of these hormones in the regulation of extension of root cells, it is necessary if only for methodological reasons to investigate the effects of only one hormone at a given time. In this chapter, we examine two of the hormones, indole-3yl-acetic acid (IAA) and abscisic acid (ABA). Distinction has to be made between the use of externally applied hormones and that of endogenous hormones. However, it is quite clear that application of external IAA or ABA to tissues may change the level and the metabolism of their endogenous IAA (Pilet, 1998b) or ABA (Pilet, 1998a). A. Figure 4 (A) Changes (cv. LG 11) in positive gravicurvature with time (h), and growth rate (measured every 20 min) of the upper (B) and lower (C) sides of the primary roots (initial length: 15  3 mm) of maize, first kept 3 h in vertical position, and then maintained horizontally for 6 h. Data represent the mean of 10 roots. (From Pilet and Ney, 1981.)

that the rate at which root bends is related to the magnitude of the differential elongation. In another series of experiments (Barlow and Rathfelder, 1985), it was reported that when using primary roots of maize (cv. LG 11) a difference in the extension rate of 12:7  103 mm min1 between the upper and lower sides resulted in a 1 min1 rate of bending. The implication of the cap cells in the elongation and gravireaction has been critically discussed (Pilet, 1986, 1998a,b, 1996; Blancaflor et al., 1998). White clover roots, submitted to microgravity, space, or two-axis clinostat, showed ultrastructure changes of the cap columella cells in which the amyloplast redistribution was analyzed (Smith et al., 1999).

Applied Hormones

The effects of applied IAA and ABA on root growth were investigated in various plant species (Scott, 1972; Pilet and Chanson, 1981; Pilet and Elliot, 1981; Audus, 1983). The reactivity of growing roots to applied hormones depends on several factors that are not often discussed. For example, the initial growth rate was found to be an essential parameter on which the effect of applied IAA and ABA depends strongly (Pilet and Saugy, 1983; Pilet, 1998a). Elongation of the slowgrowing roots was inhibited by IAA as well as by ABA, with an effect being positively correlated with the concentrations of applied hormones. On the contrary, growth of the fast-growing roots was stimulated by IAA and ABA when applied at a low concentration (5  109 M). At a higher concentration (1  106 M), both hormones were inhibitory. This indicates, as discussed later, that the endogenous content of IAA and ABA is not the same in slow- and fast-growing roots. Obviously, other factors that affect growth also need to be known. For instance, possible variations in uptake of IAA and ABA along the root axis are not known. Moreover, endogenous IAA and ABA may be released into the immersion buffer with more ABA than IAA

Root Growth and Gravireactivity

being lost (Pilet and Rebeaud, 1983). It is accepted that if IAA and ABA are released from roots, other substances must also be lost, and consequently, the entire osmotic equilibrium of such roots can be affected. It is also clear that the hormones that are taken up will be metabolized and redistributed within a 6-h period. Nevertheless, such a pulse method of exposure to hormones offers the advantage of leaving the osmotic and endogenous hormones’ equilibrium unchanged together with an effect that continues throughout the growth period (Pilet, 1998a). Immersion of roots for 30 min in buffered IAA or ABA solutions, followed by a rinse in the buffer (6 h), indicates that the effect of IAA on the root elongation is stronger than that of ABA (Fig. 5). The greater part of the observations on the effects of applied hormones on root growth were done with roots on filter papers containing water solutions of IAA or ABA. Data obtained for such a technique have to be evaluated very carefully. A water gradient inside the roots, owing to the contact of only one part of it with the wet filter paper, may inverse the distribution (related to gravity) of an endogenous hormone such as ABA.

493

Immersion of roots in a buffered solution containing IAA or ABA is a better method. Agar blocks have often been used (Pilet, 1964; Thimann, 1977), but the sizes of the blocks cause some difficulties when application is made to the roots and the location of the IAA uptake site is not very clear. This method can be employed with good quantitative results when IAA or ABA is applied on cut sections (Pilet and Barlow, 1981). The use of resin beads loaded with IAA offers another useful technique for the study of the effects of local IAA applications on intact growing roots (Meuwly and Pilet, 1986, 1987, 1991b). Amberlite IRA beads (diameter 0.45±0.05 mm) applied to primary roots of cv. LG11 maize (Meuwly and Pilet, 1987) were used both as growth markers and as IAA donors (Pilet 1998b). A strong curvature toward and above the bead occurred when IAA was applied near the tip (2.2 mm). No curvature developed after applications of the beads at a greater distance from the tip. When using resin beads according to our technique, Legue´ et al. (1994) reported that IAA enhanced gravireaction of Brassica roots. They also found that the curvature was higher for normal plants than for transgenic ones (Pilet, 1998a). Quantification (gas chromatography–mass spectrometry [GC-MS]) of IAA in maize roots was done for applied [13 C]IAA and compared to that of endogenous [12 C]IAA. Apical, elongation, and differentiation zones were tested (Meuwly and Pilet, 1991a). Here IAA was applied by immersion, and this method induced significant disturbances. The use of resin beads loaded is surely less injurious. Growth responses to applied IAA was studied for roots of wild-type Arabidopsis and of their mutants (Evans et al., 1994a). Roots from mutants showed less inhibition for applied IAA than did roots of wild type. But IAA at 1011 M induced a promotion of root elongation (see Pilet, 1998a,b). B.

Figure 5 Elongation of intact primary roots pretreated 30  5 min in buffered solution (3,3-dimethyl-glutaric acid at 103 M: pH 6:0  0:1) containing IAA (A) or ABA (B) at different concentrations. Roots of 12  1 mm in length (at zero time) were partly (the distal 10  1 mm) immersed in a vertical position. Following washing in a buffer, they were kept for 6 h in the dark (19:0  0:5 C). Data with SE are the mean of five experiments, each with 40  5 roots. (From Pilet and Saugy, 1987.)

Endogenous Hormones

The culture techniques used for growing roots, the mode of IAA and ABA application, and applied concentrations of these hormones may modify the level of endogenous IAA and ABA significantly. Only one case of changes in IAA and ABA levels in relation to root growth is discussed below (see Fig. 2). In this case, a large population of maize roots was used and the IAA and ABA contents were determined by GC-MS. The population of roots was divided into six (for the IAA determination) or nine (for ABA) growth rate

494

classes in order to quantify the endogenous IAA and ABA content in the elongation zone (2.5–5.0 mm from the tip). A clear negative correlation was found between the endogenous level of these hormones and their elongation rates (Fig. 6). The endogenous level of both hormones was similar in the elongation zone of the fast-growing roots. More IAA was found in the elongation zone of the slow-growing roots, and the ABA level increased dramatically when compared to the fast-growing roots. The shape of the curve for endogenous ABA indicates that a small difference in the growth of the slow-growing roots corresponds to a large change in ABA. On the other hand, fast-growing roots contain only relatively small amounts of ABA. This attests also to the variations in the endogenous ABA level in maize roots grown under similar conditions and to the difference between IAA and ABA in their relation to axial elongation.

Pilet

The two forms of endogenous IAA, the free and the ester-linked IAA (Bandurski et al., 1977) take part in the regulation of the growth rate of maize roots. Some data indicate that all six classes of 2-day-old maize roots contain larger amounts of ester IAA in the elongation zone than of free IAA. There is an inverse correlation between the level of ester-linked IAA and growth rate (Saugy and Pilet, 1987). This does not necessarily imply that the control of growth rate is assured only by hydrolysis of the ester. Roots containing large amounts of IAA also contain a high level of the ester. Thus, the levels of the two forms of IAA seem to be related to growth in a similar manner. Although IAA conjugates may be a major source of IAA, static measurements of their levels are not sufficient to explain their interaction with growth processes. The available data show that the relative content of free and of ester-linked IAA depends on the age and the growth rate of the root cells. To better understand the roles of free and ester-linked IAA in root growth, studies of their compartmentation, of local differences in the rate of hydrolysis of ester IAA, and of their transport in the root are also necessary. C.

Figure 6 Variation in the endogenous level of IAA and ABA as a function of the elongation rate (growth classes; see Fig. 2) during an 8-h period. The content of IAA and ABA is only given for the elongation zone (segments of 2.5– 5.0 mm). Each sample contained 30 segments. Bars indicate SE. (From Pilet and Saugy, 1987.)

Hormone Biosynthesis and Accumulation

It has been clearly observed that most IAA in roots is due to IAA conjugates coming from seeds and caryopses (Cohen and Bandurski, 1982). The de novo formation of IAA had been demonstrated in maize roots. Incorporating of 2 H from 2 H2 O into IAA molecules was shown to occur in intact plantlets and excised primary roots cultured in vitro (Ribaut et al., 1993). On the other hand, it was established that water stress alters the ABA level in roots (see Ribaut and Pilet, 1991) and this is correlated with some metabolic changes (Morgan, 1990) including IAA content. Ribaut and Pilet (1994) reported that with increasing stress (by using mannitol solution from 0 to 0.66 M), a decrease in growth, correlated with an increased IAA content, was obtained. The use of transformed plants may give some information about the biosynthesis of endogenous hormones. For example, roots from pea seedlings, transformed by several strains of Agrobacterium rhizogenes, are characterized by a low IAA level in comparison to normal roots (Schaerer and Pilet, 1993; Pilet, 1998a). It has been frequently reported that the sensitivity to applied IAA and ABA of roots from transgenic plants is significantly higher (Shen et al., 1990; Pilet, 1998a).

Root Growth and Gravireactivity

D.

495 Table 2 IAA Content in Three Parts of the Elongation Zone of Horizontal Maize Roots Dissected Longitudinallya

Hormone Sensitivity

The change in sensitivity of the tissues may explain the root responses toward IAA (Hejnowicz, 1961; Trewavas, 1982) and toward ABA. Firn (1986) and Weyers et al. (1987) attempted to clarify this point by considering physicochemical differences in hormone uptake efficiency, metabolic capacity, and affinity for receptors. All those may lead to further changes in the nature and amplitude of the growth responses (Pilet, 1994). A model based on ‘‘sensitivity threshold’’ distribution and proportional rate reactions to regulatory factor levels was proposed by Breadford and Trewavas (1994). It may account for a wide range of growth processes.

E.

IAA content Part b

Upper Middlec Lowerb

ng (gFWÞ1

ng ðgDWÞ1

%

27:8  0:9 35:1  1:3 32:1  1:1

221:1  5:8 264:3  10:2 242:8  6:8

21.4 54.7 23.9

a

Each value is the mean of 12 samples of 100 roots. Cortex only. c Stele and cortex. Source: Saugy and Pilet, 1984. b

1987). ABA also controls the cell cycle in the root quiescent center (QC) of maize (Mu¨eller et al., 1993). Excision and culture of the QC resulted in a dramatic activitation of cell division when compared to that in QC from intact roots, where cap cells are produced and ABA is moving basipetally through the QC (Pilet, 1975a, 1986). Applied ABA on the QC of cultured roots significantly decreased the rate of mitosis. To come back to the hormone regulation of root gravireactivity, something has to be said about the original ‘‘forum’’ organized by T. Trewavas (1992). The general topic of the meeting was, ‘‘What remains of the Cholodny-Went theory?’’ Although some results seem to be in accordance with that hypothesis, some critical comments can be retained (Pilet, 1992). If one only takes IAA into account, this hypothesis raises more problems than it solves and some crucial questions have yet to be answered. Although a significant, but slight, IAA redistribution in the gravireacting roots is observed, neither the differential growth nor the growth distribution is really explained. Moreover, it is unknown whether this asymmetrical IAA distribution is the cause or

Hormone Redistribution in Gravireacting Roots

Whether an asymmetrical redistribution of IAA occurs during root gravireaction and thereby causes the differential growth is of prime interest. Only a few of the data available for maize roots are presented and discussed here (Table 2). IAA, measured by GC-MS, seems to be located predominantly in the stele. When roots were placed for 2 h in a horizontal position, the IAA content of the cortex of the lower part in the elongation zone increased slightly but significantly. Endogenous ABA is also asymmetrically redistributed after 2 h of gravipresentation (Table 3). Thus, it is clear that a positive gravireaction is related to a higher ABA level in the lower half of the roots than in the upper half. Whether ABA plays any direct role in the endogenous regulation of root growth is still an open question, and recent reports that deny such a role for ABA have to be considered inconclusive (Pilet and Barlow,

Table 3 ABA Content (in ng per 100 Segments and in Relative Values) in the 4  0:1 mm Root Tip Segment of Root from Which Tip Cap and Apex Have Been Removeda 1. Positive gravireaction Part Upper Lower

2. No gravireaction

3. Negative gravireaction

ng

%

ng

%

Ng

%

16:2  1:4 20:0  1:8

44 56

19:1  1:4 20:9  0:5

48 52

18:9  0:7 17:1  1:5

53 47

a The root tips 10 mm long were maintained for 2 h in a horizontal position (in white light) prior to excision. Data for the three kinds of root gravireaction (1, 2, and 3) are shown. Source: Pilet and Rivier, 1981.

496

the consequence! Besides this, the Cholodny-Went hypothesis does not take into account the availability or the affinity of the IAA receptors, and it does not consider the differential ‘‘sensitivity’’ of root tissues to IAA.

F. IAA Transport The preferential direction of IAA movement in roots vertically and horizontally kept, has prompted much research (Section I) and discussion. This is a crucial and unsolved problem particularly related to the root growth and gravireaction (Pilet, 1994). Recent schematic representations, summarized in Fig. 7, show that a preferential IAA transport occurs in the stele in the acropetal direction. Nevertheless, a basipetal movement, localized in the cortical part, is essential for controlling the differential elongation and, consequently, the graviresponses of the roots. When roots are placed in a horizontal position, a lateral transport of IAA occurs. It was observed inside the root cap and corresponds to downward movement. Consequently, more IAA is transported in the lower part of the cortex.

Pilet

V.

GROWTH INHIBITORS

Very early in the history of root studies, it appeared that growth was the consequence of a ‘‘balance’’ between growth-promoting and growth-inhibiting agents (Thimann, 1977; Audus, 1983; Pilet, 1996). Some interactions of regulators that brought about changes in root extension have been explained through the involvement of endogenous inhibitors. A potent inhibitor was first demonstrated in plant root extracts and christened as  inhibitor (Bennet-Clark and Kefford, 1953; Cartwright et al., 1956; Pilet, 1958). Later it was shown that the main component of the ‘‘complex’’ corresponding to this inhibitor was ABA (Cornforth et al., 1965; Pilet, 1998a). In later years, the use of microsurgery of root tips has been extended, using decapped and half-decapped roots, decapitated and half-decapitated roots. Such treatments were given before growth and gravireaction analyses (Wilkins, 1975, 1979; Pilet, 1977). Removal of the root cap largely eliminated gravicurvature, although its excision may have caused some injury effects (Jackson and Barlow, 1981). This is partly due to the generation of ethylene in response to wounding (Yang and Pratt, 1978). One of the reasons why decap-

Figure 7 Proposed model of maize root tip and the types of IAA transport. (A) Root tip (Pilet, 1977, 1986, 1998b). Diagram not to scale. Ca, cap; dc, desquamant cells; qc, quiescent center; a, apex; s, stele; c, cortex; ez, elongation zone. (B, C) IAA transport. (B) Vertical root. A preferential acropetal transport (from the base to the cap) (Pilet, 1964, 1975a; Scott and Wilkins, 1968; Wilkins, 1971) in the stele (Saugy and Pilet, 1984). A basipetal transport (from the cap to the base), limited in distance inside the cortex (Davies et al., 1976; Tsumuri and Ohkavi, 1978; Pernet and Pilet, 1979). (C) Horizontal root. The two types of IAA transport reported for vertically placed roots, more IAA moving basipetally in the cortical cells. A downward lateral transport in the cap (Young et al., 1990; Pilet and Meuwly, 1995; Pilet, 1996, 1997, 1998a,b) is suggested.

Root Growth and Gravireactivity

Figure 8 Curvature of horizontally (A, B) and vertically (C, E) kept apical maize (cv. Kelvedon 33) root segments. Roots were decapitated at zero time (C, E) and caps from 6-h gravireactive roots were placed on the cut sections. Curvature measured after 6 h (B) or 14 h (C, D, E). (From Pilet, 1976b.)

Figure 9 Curvature of horizontally maintained apical maize (cv. Kelvedon 33) root segments measured after 14 h (A, C) and 6 h (D,E). Segments half-decapitated (A, C) with or without a mica barrier inserted inside the half apex (Pilet, 1973). Segments into which a mica barrier was inserted vertically (D) or horizontally (E) inside the cap or the cap and the apex (Pilet, 1976b). Outline of root cap (rc), apex (a), and elongation zone (ez) of the maize root tip. Each mean value is given in degrees  SE.

497

itation or decapping delays or prevents gravireaction is that such treatment removes the site of gravity perception (Audus, 1979). A few properties of these inhibitors are summarized in Figs. 8 and 9. Endogenous inhibiting regulators are transported basipetally from the cap cells to the extension zone of the growing roots. When roots are placed horizontally, the inhibitors are accumulated in the lower part of the root axis contributing to the positive gravicurvature (Fig. 8). Production of such growth factors in horizontal roots is larger in the lower part of the cap and they move laterally, from the upper to the lower sides, inside the apex. This is shown by experiments where a mica barrier had been inserted into half-decapitated roots (Fig. 9). It is not excluded that microsurgery may change in some way the real nature of the transit and the main direction of the inhibiting substances (Pilet, 1977, 1986). A similar behavior was reported for IAA transport in decapped roots (Hagengenstein and Evans, 1988b; Young et al., 1990). Growth inhibitors not only affect cell extension and hence control root growth and gravireaction, but also inhibit cell differentiation (Darbelley and Perbal, 1984). In the lower half of the horizontally placed root, maturation of some organelles (mitochondria) appeared to be delayed, whereas in the upper half it was faster than in control vertical roots. More analyses of the extractable growth inhibitors are required to ascertain their chemical characteristics. Ether extracts of maize root caps contain several different compounds separable by thin-layer chromatography (TLC) (Wilkins and Wain, 1974). One of the most important compounds is ABA (Pilet, 1975a). ABA is present in the cap (Rivier et al., 1977) and may induce an inhibition of root growth (Pilet and Chanson, 1981). Other inhibiting substances found in the root tip may also regulate the differential growth of gravireacting roots. Judging by their physiological properties as revealed by the microsurgical experiments, such substances may include ABA derivatives as well as other compounds (Suzuki et al., 1979; Pilet, 1996). The effects of some herbicides on growth and gravitropism may be interpreted in terms of their action on the IAA and ABA levels (Pilet, 1998a,b). For instance, the uptake of the dinitroaniline herbicide, the 14 Clabeled trifluraline, when applied on maize and pea roots, was higher in the elongation zone. It induced an increase of ABA content, which may explain the growth-inhibiting action of this herbicide (Locher and Pilet, 1995).

498

Pilet

Light may inhibit root elongation and by that imply the presence of some inhibiting substances (Wilkins and Wain, 1974; Pilet and Ney, 1978). Such a mode of photoinhibition seems generally to be accepted (Audus, 1983; Feldman, 1984). Illumination of total maize roots leads to an increase in their ABA level (Pilet and Rivier, 1980).

VI.

PROTON EFFLUX AND SURFACE pH

Proton pumps play a crucial role in the transport of ions across the membranes of root cells (Churchill et al., 1983). It is well established that growing roots are capable of extruding Hþ (Gabella and Pilet, 1979; Gepstein, 1982). Proton efflux is an active process driven by some plasmalemma- and tonoplast-bound ATPases (Chanson and Pilet, 1987; Gavin et al., 1993). The pH of an incubation medium, which decreases when containing elongating roots, influences IAA uptake by tissues, cells, and membrane vesicles (Rubery and Sheldrake, 1974; Davies and Rubery, 1978; Rubery, 1978). It should be noted that IAA uptake occurs particularly in the apical part of the root and declines in the more basal parts (Martin and Pilet, 1986). Such uptake is dependent on the proton gradient across the plasmalemma. The high level of proton extrusion in the elongating zone of maize roots enhances nonsaturable and saturable absorption of IAA (Martin and Pilet, 1987). Qualitative measurements (Mulkey and Evans, 1981) and quantitative ones (Pilet et al., 1983; Versel and Pilet, 1986) show that maize roots develop a higher proton efflux in their growing part. When roots were placed in a horizontal position, the acidification ability of their upper side was stronger than that of their lower side (Fig. 10). Local growth rates and proton fluxes were measured simultaneously using Sephadex (G25) beads containing a pH indicator (Bromocresol Purple). Proton extrusion, which was greatest in the elongation zone of the horizontal roots, was strongly reduced on the lower side and slightly increased on their upper side compared with that of vertical roots. Thus, the differential elongation of the gravireacting maize roots also has to be related to the asymmetry of their proton fluxes. The observed changes in Hþ extrusion probably induce differences in the acidification of the walls of epidermis and cortex cells. An increase in the elongation rate of such cells in the upper side may result from a stimulation of proton extrusion across the plasmalemma (Rayle and Cleland, 1992; Lu¨then and Bo¨ttgers,

Figure 10 Variation along the root of the local relative growth rate (A, C) and the proton flux (B, D) of maize (cv. LG 11) roots maintained vertically (A, B) and horizontally (C, D). Bars indicate SE. Growth data: Values corresponding to results grouped together for two periods of time (1–2 h and 2–3 h after experiment start) and for a 0.6 mm distance between each bead. pH surface data: Hþ flux measured by using beads containing Bromocresol Purple (0.7 mM at pH 6.8). Each result reported is the mean of at least 50 measurements. (From Versel and Pilet, 1986.)

1993; Kutschera, 1994; Pilet, 1996; Evans and Ishikawa, 1997). Some interesting results related to the gravity induced changes in bioelectric parameters have been reported and critically discussed by Weisenseel and Meyer (1997).

VII.

CALCIUM IN GROWTH AND GRAVIPERCEPTION

Calcium ions were shown to control growth and gravireaction and to regulate several physiological processes in plant cells (Hepler and Wayne, 1985; Poovaiah and Reddy, 1987). Calcium asymmetries develop in hypocotyls and coleoptiles before gravireaction, suggesting that Ca2þ redistribution may be correlated with the differential growth rather than with their

Root Growth and Gravireactivity

consequent results (Goswami and Audus, 1976). More Ca2þ ions are found in the lower side of gravireactive maize roots (Miyazaki et al., 1986), and it seems clear that the curvature develops toward the side having higher Ca2þ content (Lee et al., 1983a; Poovaiah et al., 1987). Changes in cytoplasmic levels of Ca2þ are induced by light and gravity (Williamson and Ashley, 1982) and trigger a cascade of biochemical reactions that result in a growth response (Poovaiah et al., 1987). Manipulation of Ca2þ gradients in the root cap may modify subsequent growth and gravitropic responses (Lee et al., 1983b). A polar transport of labeled Ca2þ across the tips of horizontal roots was obtained with preferential movement toward the lower side (Lee and Evans, 1985). Such a movement of Ca2þ is significantly slower in roots treated with auxin transport inhibitors (Lee et al., 1984). When Ca2þ was applied to only one side of the cap of vertical roots, a curvature toward this side developed. When caps of intact roots were treated by Ca2þ chelating substances, the roots did not respond to gravity. However, replacing these chelators with calcium restored the gravireaction (Lee et al., 1983b). Only slight polar movement of Ca2þ was observed across the elongation zone of gravistimulated dark-grown roots, whereas substantial redistribution of labeled calcium across the growing zone of gravistimulated light-grown roots occurred. As discussed previously, roots of some cultivars of maize exhibit normal gravireaction when grown in light, whereas in darkness they show little or no bending (Pilet, 1975b, 1976a, 1998a). The effects of Ca2þ show a strong interaction with the pH. Acidification of low Ca2þ roots resulted in transient growth stimulation followed by a gradual decline in the growth rate, whereas exposure of such roots to high pH promoted growth. Growth of highCa2þ roots was fast at either low or high pH (Hasenstein and Evans, 1988a). Thus Ca2þ is necessary for the acid growth of roots (Pilet, 1994). The elevation of free Ca2þ brought about the interaction of Ca2+ with H+ in the apoplast of root tips and should be involved in transmission of the gravisignal (Suzuki et al., 1994). The initial event of it is probably associated with an increase in cytosolic Ca2þ in the columella of the root cap. It is generally accepted that cap amyloplasts may act as gravireceptors (Audus, 1979) and sediment within the presentation time (Sack et al., 1985). This should be defined as the minimum time of gravistimulation required to elicit a detectable bending reaction. Amyloplasts of the root columella appear to have high Ca2þ levels (Chandra et al., 1982). However, it

499

should be noticed that amyloplasts are only one part of the system. Roots of starchless mutants show a similar gravitropsim to controls having amyloplasts (Caspar et al., 1985). On the other hand, roots having their caps removed were temporarily unable to exhibit gravitropism. Decapping caused stimulation of amyloplast development in the quiescent center and in the apex prior to the regeneration of new caps. This indicates that starch grains may also function as statoliths (gravireceptors) in cells other than those of the columella (Barlow, 1974; Barlow and Grundwag, 1974). Thus, the gravity-induced sedimentation of starch grains in the cap cells can still be accepted as the first step of gravireaction, and it seems quite possible that the graviperception may result from the pressure that these statoliths exert on the endoplasmic reticulum of the statocytes (Sievers et al., 1984; Pilet, 1998). Gravistimulation may also be followed by alterations in the symmetry of the electric cell potential (Behrens et al., 1985). The resting potential of statocytes in vertical roots is about 118 mV. Upon gravistimulation, the membrane potential of the statocytes, located in the lower flank of the cap, dropped to 93 mV whereas that in the upper flank fell to 13 mV. Such a drop in potential gives a good indication that ion fluxes across the plasmalemma were altered by gravistimulation. When the reticulum of these cells was isolated and incubated with Ca2þ (at micromolecular concentrations), Ca2þ had accumulated in the vesicular membrane fraction (Sievers et al., 1984). Such an accumulation of Ca2þ is ATP dependent. Cytosolic Ca2þ may also affect gene expression, a change that is essential for root graviresponses. It was shown (Raghothama et al., 1987) that reversible protein phosphorylation regulated by Ca2þ and by Ca2þ -calmodulin complex (Marme´ and Dieter, 1983) seem to be a crucial regulatory process involved in the transduction of gravity signals in roots. Changes in protein phosphorylation and Ca2þ -calmodulin-dependent protein kinase may control root gravitropism (Poovaiah et al., 1987). This is implied by the faster gravireaction of horizontally placed maize roots after illumination (Pilet, 1975b). Such roots form rapidly new proteins, and the level of their specific mRNAs is enhanced (Feldman, 1986). Measurements of labeled methionine incorporation have shown that significant calcium-dependent changes in protein formation appear after a very short light treatment, which initiated root curvature under gravity (Poovaiah et al., 1987). The use of roots of transgenic plants may also yield some interesting indications on root growth

500

Pilet

and gravireaction regulated by IAA and ABA (Legue´ et al., 1994; Pilet, 1998a).

VIII.

CONCLUSIONS

1. An elongation gradient exists in growing roots from their tips to their bases with a maximal extension rate near the apex. This zone of fast extension is considered to be the site of gravireaction, whereas the root cap is that of graviperception. The downward bending of roots due to gravity is the consequence of differential growth rates between the upper and lower sides of this elongating zone. Such roots have to be placed in a horizontal position, at least during the presentation time. There is also some evidence that cell extension is promoted within the upper half and inhibited on the lower half of horizontally kept roots. Nevertheless, more detailed information is required about root growth patterns. Cytological analyses of parenchyma cells and precise determinations of surface extension (e.g., by using resin beads, etc.) will make it possible to elucidate such different questions. 2. Growth and gravitropism can also be analyzed for a large population of selected roots grouped into several classes that are characterized by their initial elongation rate. With this new methodological approach, it has been possible to compare and characterize the growth properties of elongating and gravireacting roots having a low or a high extension rate. Further experiments should be based on kinetic analyses of the changes in the elongating rate of roots from several growth classes under different conditions of pH, light, and temperature. 3. Special attention was given in the past to two of the main growth hormones, IAA and ABA, and their role in the establishment of the elongation gradients within the expanding zone of growing roots. The effects of those hormones on the differential elongation of such roots were also investigated. It is clear that some data are sometimes conflicting and the classic concepts are not completely satisfactory. New data about the effects of IAA and ABA metabolites and conjugates as well as for other growth hormones are now necessary for an understanding of their role in the regulation of root elongation and gravireaction. 4. The responses of the roots to applied IAA, ABA, other hormones, and Ca2þ may change with the sensitivity of the tissues for such ‘‘effectors.’’ Of course, this concept of sensitivity must be quantified. It could be related to some modifications—for exam-

ple, to the uptake efficiency, to metabolic capacity, and to affinity of specific receptors. 5. It seems clear now that the root cap does not play a role only in graviperception. At first, IAA, which moves acropetally in the stele, may be accumulated in the cap cells. But this IAA is then transported from the cap, inside the cortex, to the root base. Such basipetal movement may affect the IAA redistribution from upper to lower cells of the cortex, inducing the differential elongation. Finally, a lateral IAA transport could occur inside the cap and may contribute to the asymmetrical IAA distribution in the elongating zone. The cap may also be the source of growth inhibitors that are basipetally transported to the growing and gravireactive zone of the root tip. Microsurgical experiments, based on decapping and decapitation of roots, removal of the total cap or just half of it, support the hypothesis that a basipetal migration of such growth inhibitors, formed in the cap cells or released by them, may also induce the gravitropism. Such regulators are produced mostly by the base of the cap when roots are kept horizontally. Light strongly reduces the growth of roots that were previously kept in the dark. This could depend on the level of some growth inhibitors (such as ABA) present in the root tip, whose content is enhanced by light treatment. 6. Elongating roots secrete protons owing to the activity of plasmalemma ATPases. The highest Hþ extrusion occurs in the growing part of roots, and it seems clear that Hþ efflux enhances both nonsaturable and saturable IAA uptake. When roots were turned from a vertical to a horizontal position, their pH surface due to the Hþ extrusion changed. During the first steps of gravireaction, the Hþ efflux was stronger in the upper side than in the lower side. Such proton flow and the related surface potential changes raise a large number of questions: What are the real functions of Hþ pump? What are the IAA and ABA implications? What are the Hþ and Ca2þ interactions? What are the interferences with the cell wall which may absorb Hþ ? and so on. 7. Cytosolic Ca2þ is also involved in the control of root elongation and gravitropism. Calcium level is not uniform, being higher in the lower part of the growing zone of gravireactive roots. Applied calcium to the cap may change the elongation of roots and their responses to gravity. Ca2þ movement is reduced in roots treated with some inhibitors of IAA transport. Apparently, calcium changes are also involved in transduction of the gravity signal. 8. Several biomolecular techniques should be used to clarify some unsolved problems related to root

Root Growth and Gravireactivity

growth and gravireactivity. Various hormones and Ca2þ , which control root elongation and gravitropism, may affect the expression of some genes. Roots from various plant transformants are characterized by some modifications of their sensitivity to IAA. Their axial extension, graviperception, and gravireaction are fairly often different when compared to those of ‘‘normal’’ roots. However, as experimental results of such comparisons are conflicting, much remains to be done to clarify the contradictions.

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Pilet Legue´ V, Tepfer M, Perbal G. 1994. Gravitropic behaviour and response to auxin of rape roots transformed by Agrobacterium rhizogenes A.4. Proceedings of 5th European Symposium Life Research in Space. ESA, pp 127–131. Locher R, Pilet P-E. 1995. Trifluralin uptake and its effect on ABA content in growing maize and pea roots. J Plant Physiol 146:569–571. Lu¨then H, Bo¨ttger M. 1993. The role of protons in the auxininduced root growth inhibition. A critical reexamination. Bot Acta 106:58–63. Marme´ D, Dieter P. 1983. Role of Ca2þ and calmodulin in plants. In: Cheung WY, ed. Calmodulin and Cell Function. London; Academic Press, pp 263–311. Martin H, Pilet P-E. 1986. Saturable uptake of indol-3ylacetic acid by maize roots. Plant Physiol 81:889–895. Martin H, Pilet P-E. 1987. Effect of pH on IAA uptake by maize root segments. Plant Physiol 83:262–264. Maruyama S, Boyer SJ. 1994. Auxin action on growth in intact plants: threshold turgor is regulated. Planta 193:44–50. Mengel K, Schubert S. 1985. Active extrusion of protons into deionized water by roots of intact maize plants. Plant Physiol 79:344–348. Meuwly P, Pilet P-E. 1986. Local application of indol-3ylacetic acid, by resin beads to intact growing maize roots. Planta 169:16–22. Meuwly P, Pilet P-E. 1987. Maize root growth and localized indol-3yl-acetic acid treatment: a new methodological approach. Plant Physiol 84:1265-1269. Meuwly P, Pilet P-E, 1991a. Simultaneous GC-MS quantification of endogenous [12 C] and applied ½13 C] indole3yl-acetic acid levels in growing maize roots. Plant Physiol 95:170–183. Meuwly P, Pilet P-E. 1991b. Local treatment with indole-3ylacetic acid induces differential growth responses in Zea mays roots. Planta 185:58–64. Miyazaki A, Katsumi K, Ishizaka S, Fujii T. 1986. Redistribution of phosphorus, sulfur, potassium and calcium in relation to light-induced gravitropic curvature in Zea roots. Plant Cell Physiol 27:693–700. Morgan PV. 1990. Effects of abiotic stresses on plant hormone systems. In: Alscher RB, Cummings JB, eds. Stress Responses in Plants. New York; Wiley-Liss, pp 113–146. Mulkey TJ, Evans ML. 1981. Geotropism in corn roots: evidence for its mediation by differential acid efflux. Science 212:70–71. Mulkey TJ, Evans ML, Kuzmanoff KM. 1983. The kinetics of abscisic acid action on root growth and gravitropsim. Planta 157:150–157. Mu¨ller ML, Pilet P-E, Barlow PW. 1993. An excision and squash technique for analysis of the cell cycle in the root quiescent centre of maize. Physiol Plant 87:305– 312.

Root Growth and Gravireactivity Pernet JJ, Pilet P-E. 1979. Importance of the tip on the [53 H]-indole-3yl-acetic acid transport in maize root. Z Pflanzenphysiol 94:273–279. Pilet P-E. 1958. Analyse biochromatographique des auxines radiculaires. Rev Ge´n Bot 65:605–634. Pilet P-E. 1964. Auxin transport in roots. Nature 204:560– 561. Pilet P-E. 1973. Growth inhibitors from the root cap of Zea mays. Planta 111:275–278. Pilet P-E. 1975a. Abscisic acid as a root growth inhibitor: physiological analyses. Planta 122:299–302. Pilet P-E. 1975b. Effect of light on the georeaction and growth inhibitor content of roots. Physiol Plant 33:94–97. Pilet P-E. 1976a. The light effect on the growth inhibitors produced by the root cap. Planta 130:245–249. Pilet P-E. 1976b. Effects of gravity on the growth inhibitors of geostimulated roots of Zea mays. Planta 131:91– 93. Pilet P-E. 1977. Growth inhibitors in growing and geostimulated maize roots. In: Pilet P-E, ed. Plant Growth Regulation. Berlin; Springer-Verlag, pp 115–128. Pilet P-E. 1986. Importance of the cap in maize root growth. Planta 169:600–602. Pilet P-E. 1992. What remains of the Cholodny-Went theory? IAA in growing and gravireacting maize roots. Plant Cell Environ 15:779–780. Pilet P-E. 1993. Plant hormones in action. Hum Environ Sci 10:89–93. Pilet P-E. 1994. Les auxines: e´mergence et diversification d’un concept. La vie des sciences. C R Acad Sci Paris 11:149–161. Pilet P-E. 1996. Hormones implications in root gravity: a reexamination. In: Suge H, ed. Plant in Space. Sendai, Japan: Tohoku University Press, pp 61–72. Pilet P-E. 1998a. Some cellular and molecular properties of abscicic acid: its particular involvement in growing plant roots. Cell Mol Life Sci 54:851–865. Pilet P-E. 1998b. Applied indol-3yl-acetic acid on the cap and auxin movements in gravireacting maize roots. J Plant Physiol 152:135–138. Pilet P-E, Barlow PW. 1987. The role of abscisic acid in root growth and gravireaction. Plant Growth Regul 6:217– 265. Pilet P-E, Chanson, A. 1981. Effect of abscisic acid on maize root growth: a critical examination. Plant Sci Lett 21:99–106. Pilet P-E, Elliott MC. 1981. Some aspects of the control of root growth and georeaction: the involvement of indole acetic acid and root growth and abscisic acid. Plant Physiol 67:1047–1050. Pilet P-E, Meuwly R. 1986. Local application of indol-3ylacetic acid, by resin beads to intact growing maize roots. Planta 169:16–22. Pilet P-E, Ney D. 1978. Rapid, localized light effect on root growth in maize. Planta 144:109–110.

503 Pilet P-E, Ney D. 1981. Differential growth of georeacting maize roots. Planta 151:146–150. Pilet P-E, Nougare`de A. 1970. RNA: Structure, infrastructure et geotropisme radiculaire. C R Acad Sci Paris 272:418–421. Pilet P-E, Nougare`de A. 1974. Root cell georeaction and growth inhibition. Plant Sci Lett 3:331–334. Pilet P-E, Rebeaud JE. 1983. Effect of abscisic acid on growth and indoly-3-acetic acid levels in maize roots. Plant Sci Lett 31:117–122. Pilet P-E, Rivier L. 1980. Light and dark georeaction of maize roots and endogenous levels of abscisic acid. Plant Sci Lett 18:201–206. Pilet P-E, Rivier L. 1981. Abscisic acid distribution in horizontal maize root segments. Planta 153:453–458. Pilet P-E, Saugy M. 1985. Effect of applied and endogenous indol-3yl-acetic acid on maize root growth. Planta 164:254–258. Pilet P-E, Saugy M. 1987. Effect on root growth of endogenous and applied IAA and ABA: a critical reexamination. Plant Physiol 83:33–38. Pilet P-E, Versel JM, Mayor G. 1983. Growth distribution and surface pH patterns along maize roots. Planta 158:398–402. Poovaiah BW, Reddy ASN. 1987. Calcium messenger system in plants. CRC Crit Rev Plant Sci 6:47–103. Poovaiah BW, McFadden JJ, Reddy ASN. 1987. The role of calcium ions in gravity signal perception and transduction. Physiol Plant 71:401–407. Raghothama KG, Reddy ASN, Friedmann M, Poovaiah BW. 1987. Calcium-regulated in vivo protein phosphorylation in Zea mays L. root tips. Plant Physiol 83:1008–1013. Rayle DL, Cleland RE. 1992. The acid-growth theory of auxin-induced cell elongation is alive and well. Plant Physiol 99:1271–1274. Ribaut JM, Pilet P-E. 1991. Effects of water stress on growth, osmotic potential and abscisic acid content of maize roots. Physiol Plantarum 81:156–162. Ribaut JM, Pilet P-E. 1994. Water stress and indole-3ylacetic acid content of maize roots. Planta 193:502–507. Ribaut JM, Schaerer S, Pilet P-E. 1993. Deuterium-labeled indol-3yl-acetic acid neo-synthesis in plantlets and excised roots of maize. Planta 189:80–82. Rivier L, Milon H, Pilet P-E. 1977. Gas chromatography– mass spectrometric determinations of abscisic acid levels in the cap and the apex of maize roots. Planta 134:23–27. Rubery PH. 1978. Hydrogen ion dependence of carriermediated auxin uptake by suspension-cultured crown gall cells. Planta 142:203–206. Rubery PH, Sheldrake AR. 1974. Carrier-mediated auxin uptake transport. Planta 118:101–121. Sack FD, Suyamoto MM, Leopold SC. 1985. Amyloplast stimulation kinetics in gravistimulated maize roots. Planta 165:576–300.

504 Saugy M, Pilet P-E. 1984. Endogenous indol-3yl-acetic acid in stele and cortex of gravistimulated maize roots. Plant Sci Lett 37:93–99. Saugy M, Pilet P-E. 1987. Changes in the level of free and ester indol-3yl-acetic in growing maize roots. Plant Physiol 85:42–45. Schaerer S, Pilet P-E. 1993. Quantification of indol-3yl-acetic acid in untransformed and Agrobacterium rhizogenes– transformed pea roots using GC-MS. Planta 189:55– 59. Scott TK. 1972. Auxins and roots. Annu Rev Plant Physiol 23:235–258. Scott TK, Wilkins MB. 1968. Auxin transport in roots. II. Asymmetric flux of IAA in Zea roots. Planta 83:328– 334. Shen WH, Davioud E, David C, et al. 1990. High sensitivity to auxin is a common feature of hairy roots. Plant Physiol 94:554–560. Sievers A, Behrens HM, Buckhout TJ, Gradmann D. 1984. Can a Ca2þ pump in the endoplasmic reticulum of the lepidium root be the trigger for rapid changes in membrane potential after gravistimulation? Z Pflanzenphysiol 114:195–200. Sievers A, Hensel W. 1982. The nature of graviperception. In: Wareing PF, ed. Plant Growth Substances. London; Academic Press, pp 479–506. Smith JD, Staehelin LA, Todd P. 1999. Early root cap development and graviresponse in white clover (Trifolium repens) grown in space and on a two-axis clinostat. J Plant Physiol 155:543–550. Suzuki T, Kondo N, Fujii T. 1979. Distribution of growth regulators in relation to light-induced geotropic responsiveness in Zea roots. Planta 145:323–329. Suzuki T, Takeda C, Sugawara T. 1994. The action of gravity in agravitropic Zea primary roots: effect of gravistimulation on the extracellular free-Ca2þ content in 1-mm root tip in the dark. Planta 192:379–383. Taiz L. 1984. Plant cell expansion: regulation of cell wall mechanical properties. Annu Rev Plant Physiol 35:585–657. Thimann KV. 1977. Hormone Action in the Whole Life of Plants. Boston; University of Massachusetts Press. Torrey JG. 1976. Root hormones and plant growth. Annu Rev Plant Physiol 27:435–459.

Pilet Trewavas AJ. 1982. Growth substance sensitivity: the limiting factor in plant development. Physiol Plant 55:60– 72. Trewavas AJ. 1992. What remains of the Cholodny-Went theory? Introduction. A summing up. Plant Cell Environ 15:761, 793–794. Tsurumi S, Ohwaki Y. 1978. Transport of 14 C-labeled indoleacetic acid in Vicia root segments. Plant Cell Physiol 19:1195–1206. Versel JM, Pilet P-E. 1986. Distribution of growth and proton efflux in gravireactive roots of Zea mays. Planta 167:26–29. Weisenseel MH, Meyert AJ. 1997. Bioelectricity, gravity and plants. Planta 203:98–106. Weyers JDB, Paterson NW, Brook R. 1987. Toward a quantitative definition of plant hormone sensitivity. Plant Cell Environ 10:1–10. Wilkins H, Wain RL. 1974. The root cap and control of root elongation in Zea mays seedlings exposed in white light. Planta 121:1–8. Wilkins MB. 1971. Hormone movement in geotropism. In: Gordon SA, Cohen MJ, eds. Gravity and the Organism. Chicago; University of Chicago Press, pp 107–112. Wilkins MB. 1975. The role of the root cap in root geotropism. Curr Adv Plant Sci 13:317–328. Wilkins MB. 1979. Growth-control mechanisms in gravitropism. In: Haupt W, Feinleib ME, eds. Encyclopedia of Plant Physiology, Vol 3 (Physiology of Movements). Berlin; Springer-Verlag, pp 601–626. Williamson RE, Ashley CC. 1982. Free Ca2þ cytoplasmic streaming in the alga Chara. Nature 296:647–651. Yang SF, Pratt HK. 1978. The physiology of ethylene in wounded plant tissues. In: Kahn G, ed. Biochemistry of Wounded Plant Tissues. Berlin; Walter de Gruyter, pp 595–622. Young LM, Evans ML, Hertel R. 1990. Correlations between gravitropic curvature and auxin movement across gravistimulated roots of Zea mays. Plant Physiol 92:792–796. Zeevaart JAD, Creelman RA. 1988. Metabolism and physiology of abscisic acid. Annu Rev Plant Physiol 39:439–373.

31 Calcium and Gravitropism B. W. Poovaiah and Tianbao Yang Washington State University, Pullman, Washington

A. S. N. Reddy Colorado State University, Fort Collins, Colorado

I.

INTRODUCTION

recognized that the root cap is the probable site of signal perception. He discovered that the removal of the root cap eliminates the ability of roots to respond to gravity. Other investigators have since confirmed Darwin’s observation (Konings, 1968; Evans et al., 1986). Increased emphasis on space exploration has resulted in a renewed interest in understanding how plants respond to gravity (Halstead and Dutcher, 1987). Studies on the mechanisms involved in perception and transduction of the gravity signal by roots would ultimately enable us to understand gravitropism. Furthermore, an understanding of gravitropism would allow scientists to control the growth and development of plants under microgravity conditions in space. In this chapter, we restrict ourselves to the role of calcium and calcium-modulated proteins in the transduction of the gravity signal. In recent years, genetic approaches have proven to be very effective in elucidating gravity signal transduction pathways. These recent advances are also summarized in this chapter. Detailed reviews on various aspects of gravitropism (Scott, 1972; Torrey, 1976; Pilet, 1979, 1983; Wilkins, 1979; Firn and Digby, 1980; Feldman, 1985; Pickard, 1985a,b; Moore, 1985; Moore and Evans, 1986; Halstead and Dutcher, 1987; Poovaiah et al., 1987; Masson, 1995; Ranjeva et al., 1999; Chen et al., 1999; Chapter 30 by Pilet in this volume) and on the

Plant organs respond to different environmental signals such as gravity and light. Gravity gives plants proper orientation, resulting in the proper shape and form that we take for granted; the roots grow down into the soil and the shoots grow up toward the light. Under microgravity conditions, as in space, plant growth patterns lack a clear sense of direction. The understanding of gravitropism has important agricultural implications. Gravitropism allows plants to compete for the available resources in their immediate environment, which helps to maximize their growth and development under varying conditions. In higher plants, the directional growth of an organ in response to stimuli such as gravity and light is considered a tropic movement (see Chapter 29 by Porterfield in this volume). Such growth responses could be either positive or negative with respect to a specific stimulus. In general, stems show a positive response to light and a negative response to gravity. In contrast, most roots show a positive response to gravity and a negative response to light. Investigations on plant tropism date back more than a century when Darwin studied the phototropic responses of maize seedlings (Darwin, 1880). Although the precise mechanism of signal perception and transduction in roots is not understood, Darwin 505

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role of calcium as a messenger in signal transduction in general have been published (Hepler and Wayne, 1985; Poovaiah and Reddy, 1987, 1993; Roberts and Harmon, 1992; Bowler and Chua, 1994; Gilroy and Trewavas, 1994; Sinclair et al., 1996; Sinclair and Trewavas, 1997; Trewavas and Malho, 1998; Trewavas, 1999). Plant roots have been widely used to study the transduction of gravity and light signals (Poovaiah et al., 1987; Roux and Serlin, 1987). Most roots show a positive gravitropic response after either dark or light treatment. However, roots of some varieties of plants (e.g., Zea mays cv. Merit, and Zea mays cv. Golden Cross Bantam 70) show positive gravitropic response only in light (Feldman, 1983; Miyazaki et al., 1986; Lu and Feldman, 1997). Calcium acts as a messenger in transducing gravity and light signals in plant roots (Pickard, 1985a,b; Evans, 1991; Evans et al., 1986; Poovaiah et al., 1987; Lu and Feldman, 1997; Sinclair and Trewavas, 1997; Chen et al., 1999; see also in this volume Chapter 30 by Pilet and 20 and 29 by Porterfield).

II.

CALCIUM SIGNALING IN GRAVITROPISM

Free cytosolic calcium is the most common signal transduction element in both plant and animal cells (Poovaiah and Reddy, 1993; Clapham, 1995; Trewavas and Malho, 1998; Reddy, 2001). Calcium is essential for survival, yet prolonged high intracellular calcium levels can cause cell death. Hence, cells stringently control intracellular calcium levels through numerous calcium-binding proteins. Several reviews have appeared on this topic (Poovaiah and Reddy, 1993; Gilroy and Trewavas, 1994; Bowler and Chua, 1994; Reddy, 1994; Trewavas and Malho, 1998; Reddy, 2000). The development of fluorescent and luminescent indicators to monitor changes in free calcium in living cells has led to the realization that changes in cytoplasmic calcium could mediate diverse plant responses, and has made calcium one of the bestcharacterized secondary messengers in plants (Poovaiah and Reddy, 1993; Gilroy and Trewavas, 1994; Trewavas and Malho, 1998). Many environmental and hormonal signals cause an elevation in cytosolic calcium concentration (Reddy and Reddy, 2001). This increase in cytosolic calcium initiates a cascade of biochemical events. The gravitropic response is separated into three phases: signal perception, transduction, and response.

The primary event that takes place in roots subjected to gravity is the initial perception of the signal. This initial perception takes place in the root cap. The root cap is composed of short-lived, parenchymalike cells, and new cells are continuously being added to it (see also this volume Chapter 3 by Sievers et al.). Hence, the root cap persists throughout the life of a growing root. The events that take place between stimulus perception and the final growth response are grouped under the term ‘‘transduction.’’ The third phase is the final response of altered growth pattern in the elongation zone of the roots, which leads to curvature. Evidence of the importance of calcium in gravitropism is obtained by simple but elegant experiments using calcium chelators (e.g., EGTA) and calcium ionophores (e.g., A23187). Manipulation of calcium gradients in the root cap can change the gravitropic response (Lee et al., 1983b). Depletion of calcium in the root cap, using calcium chelators, results in the loss of gravisensitivity (Fig. 1, A1), and subsequent replenishment of calcium to depleted roots restores gravisensitivity (Fig. 1, A2). Furthermore, root curvature can also be induced by creating calcium gradients across the root cap using calcium or calcium chelators such as EGTA (Fig. 1 A3, A4). The root tip contains four times more calmodulin, a calcium-binding protein, than the root base (Poovaiah et al., 1987). Amyloplasts in the columella cells of the root cap contain high concentrations of calcium (Chandra et al., 1982) and high amounts of calmodulin as compared to other surrounding cells in the roots (Dauwalder et al., 1986). Calmodulin inhibitors inhibit gravitropism without inhibiting the growth rate of the roots (Biro et al., 1982). Inhibitory effects of KN-93, an inhibitor of calcium/calmodulin-dependent protein kinase II on light-regulated maize root gravitropism, have also been observed (Lu and Feldman, 1993). Moreover, calmodulin antagonists inhibit polar transport of calcium in roots (Stinemetz and Evans, 1986), suggesting that establishment of the calcium gradient, which is essential for gravitropism, is a calmodulin-dependent process. It has been demonstrated that gravity induces a rapid increase in cytosolic calcium in the elongating cells on the growing side of gravistimulated maize coleoptiles (Gehring et al., 1990). Attempts to measure changes in cytoplasmic-free calcium concentration in roots in response to gravity have failed to detect gravity-induced changes (Legue et al.,1997). It is possible that short calcium transients may have occurred but were not

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Figure 1 Diagrammatic representation of the effect of calcium on gravitropic response in maize roots. (A) Effect of calcium or EGTA applied in agar blocks on gravitropism: (1) EGTA applied to the root cap prevents gravitropic response; (2) calcium application following EGTA pretreatment restores gravitropic response; (3) calcium application to one side of the cap causes bending towards the calcium; (4) EGTA application to one side of the cap causes bending away from EGTA. (Adapted from Lee et al., 1983b.) (B) Effect of calcium manipulation on light-induced gravitropic response in maize roots (cv. Merit): (1) dark-grown roots nonresponsive to gravity; (2) dark-grown roots exposed to light; (3) dark-grown roots treated with EGTA þ A23187 (calcium ionophore) prior to light exposure; (4) dark-grown roots treated with EGTA þ A23187 and transferred to calcium þ A23187 prior to light exposure. (From Poovaiah et al., 1987).

captured by the images that were collected at 1-min intervals. Primary roots of a mutant maize (cv. Merit) do not exhibit gravitropism in the dark, but become gravisensitive after a brief exposure to light (Fig. 1B). Roots of maize (cv. Merit) that were depleted of calcium by EGTA and A23187 (calcium ionophore) prior to light treatment did not show any gravitropic curvature (Reddy et al., 1987). However, replenishment of calcium to depleted roots restored sensitivity to gravity. Treatments such as heat shock and cold shock that cause an influx of calcium can substitute for light, causing a positive gravitropic response in the dark (Perdue et al., 1988). Furthermore, the calcium channel blocker verapamil was found to inhibit the light response. Calmodulin antagonists such as calmidazolium and compound 48/80 were shown to inhibit lightinduced gravitropism in maize (cv. Merit) roots (Bjorkman and Leopold, 1987). Stinemetz et al. (1987) observed increases in calmodulin levels following light treatment in Merit corn roots. These studies suggest that the effects of calcium could be mediated through calmodulin. The discovery of various components of the calcium messenger system in roots such as calmodulin and calcium-regulated protein kinases, and the observation of an increase in cytosolic calcium in

response to gravity, have led researchers to propose a messenger role for calcium in gravitropic bending. Gravistimulation of sunflower hypocotyls leads to asymmetric redistribution of calcium in these tissues (Bode, 1959). Since Bode’s study, calcium asymmetry in gravistimulated roots has been reported by several investigators using different systems (Goswami and Audus, 1976; Roux and Serlin, 1987). In gravistimulated roots, calcium accumulated preferentially on the lower side, which is growing slower. Studies have shown that calcium redistribution occurs prior to gravicurvature, suggesting that asymmetrical distribution of calcium could lead to the development of differential growth (Roux and Serlin, 1987). Using 45 Ca it has been shown that calcium moves from the upper to the lower side of horizontally oriented maize roots (Lee et al., 1983a). Measurements of calcium levels in gravi- and light-stimulated maize roots, using proton-induced xray emission, showed higher levels of calcium in the lower half than in the upper half, both in root caps and in the elongation zone (Miyazaki et al., 1986). In both stems and roots, curvature is toward the side with higher calcium levels. Exogenous calcium application can reduce the growth rate rapidly, and cell wall extension is greatly affected by apoplastic calcium (Cleland and Rayle, 1977; Slocum and Roux, 1983).

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Furthermore, Sakai-Wada and Yagi 1993) have observed changes in the calcium localization in the dividing cells of the maize root tip.

III.

CALCIUM-BINDING PROTEINS

Elevation of cytosolic calcium initiates a cascade of events resulting in a physiological response. The changes in cytosolic calcium are sensed by a group of calcium-modulated proteins that are believed to be involved in cellular regulation. These binding proteins have a structural feature called an EF hand which is present in multiple copies and binds calcium with high affinity (Heizmann and Hunziker, 1991). Many calcium-binding proteins have been identified and characterized in plants and animals (Moncrief et al., 1990; Heizmann and Hunziker, 1991; Roberts and Harmon, 1992; Poovaiah and Reddy, 1993; Zielinski, 1998). Calcium-binding proteins are inactive in the absence of bound calcium, but when the concentration of cytosolic calcium increases, the calcium-binding proteins such as calmodulin (CaM) and calcium-dependent protein kinases bind to calcium and become active. Once bound to calcium, these proteins become active and interact with other proteins in the cell and alter their activity. In this chain of events, calcium acts as a switch conveying the signal from cell surface to the metabolic machinery, eventually resulting in a physiological response. A.

Calmodulin

CaM is one of the most abundant calcium-binding proteins in eukaryotes. It is a highly conserved protein and is considered to be a multifunctional protein because of its ability to interact and regulate the activity of a number of other proteins (Roberts et al., 1986; Poovaiah and Reddy, 1987; Snedden and Fromm, 1998; Zielinski, 1998). The properties of plant CaM are very similar to those of animal CaM. In recent years, considerable progress has been made in studying CaM gene expression and the organization of CaM genes in plants. cDNAs or genomic clones that code for CaM were isolated from a number of plant systems (Roberts and Harmon, 1992; Poovaiah and Reddy, 1993; Zielinski, 1998). Analysis of cDNA and genomic clones suggests the presence of multiple CM genes in plants (Ling et al., 1991; Perera and Zielinski, 1992; Takezawa et al., 1995; Lee et al., 1995; Yang et al., 1996). Some CaM isoforms and CaM-related genes are responsive to a

variety of physical and chemical signals. Exposure of dark-grown Merit corn root tips to light increased the level of CaM mRNA (Jena et al., 1989). In Arabidopsis, Braam and Davis (1990) have shown rapid (10–30 min) induction of mRNAs corresponding to four cDNAs (TCH1, TCH2, TCH3, and TCH4) in response to a variety of stimuli such as touch, wind, rain, and wounding. Of these four genes, TCH1 was identified as a CaM gene, and TCH2 and TCH3 were identified as CaM-related genes. Other studies have confirmed the induction of calmodulin genes by touch and wounding (Perera and Zielinski, 1992; Takezawa et al., 1995; Ito et al., 1995; Bergey and Ryan, 1999). These studies suggest that physical and chemical signals induce the expression of CaM. The expression of some of the touch genes was found to be regulated by calcium (Braam, 1992). The touch and wind signals elevate cytosolic calcium (Knight et al., 1991, 1993; Legue et al., 1997). Hence, the probable sequence of events in touch signal transduction is elevation of cytosolic calcium, which in turn regulates the expression of the specific genes, including those that encode for its own receptor. In addition to CaM, there are indications that CaM-like proteins and additional calcium-binding proteins could be involved in sensing cytosolic calcium changes and mediating calcium action in plants (Krause et al., 1989; Braam and Davis, 1990; Clark et al., 1992; Poovaiah and Reddy, 1993; Zielinski, 1998). A cDNA for a CaM-like protein (p21) that shares 65% amino acid similarity with other CaM sequences has been isolated from Arabidopsis (Ling and Zielinski, 1993). From the same system Braam and Davis (1990) isolated two partial cDNAs, TCH2 and TCH3, that code for CaM-related proteins. These showed 44% and 70% amino acid identities, respectively, with CaM. The p21 protein has several unique structural features, including a 45-amino acid carboxy terminal extension with no homology to any known proteins. Another CaM-related cDNA was isolated from Petunia which contained an extra domain of 35 amino acids at the carboxy terminal end. A CTIL CaaX-box motif in the C-terminal is required for efficient prenylation of the protein (RodriguezConcepcion et al., 1999). B.

Calcium- and Calcium/CalmodulinRegulated Enzymes

Calcium either directly or through calmodulin regulates the activity of a number of enzymes and the function of structural proteins that play a key role in cellular regulation (Klee, 1991; Meador et al., 1992).

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In recent years, considerable progress has been made in identifying and characterizing CaM-binding proteins in plants. Identification of these proteins has greatly increased our understanding of how calcium and CaM regulate various biochemical and molecular processes that eventually lead to a physiological response in animal cells. 1.

Protein Kinases

Protein phosphorylation which is catalyzed by protein kinases is one of the major mechanisms of signal integration in eukaryotic cells. Protein kinases play a pivotal role in the majority of the signal transduction pathways (Nishizuka, 1986, 1988; Asaoka et al., 1992; Cohen, 1990, 1992; Ranjeva and Boudet, 1987; Veluthambi and Poovaiah, 1984, 1986; Poovaiah and Reddy, 1990; Schulman et al., 1995; Newton, 1997; Plowman et al., 1999, Cross et al., 2000). Extracellular signals, either directly or through second messengers, regulate the activity of protein kinases, which in turn regulate the activity of their substrates by phosphorylation. The diverse actions of various signals and amplification of signals are largely achieved through protein kinases (Cohen, 1992). Extensive studies in animal systems indicate that CaM-dependent protein kinases are central to calcium-mediated signal transduction pathways (Colbran and Soderling, 1990; Schulman et al., 1995; Newton, 1997; Plowman et al., 1999, Cross et al., 2000). In plants, three distinct calcium and calcium/CaM-dependent protein kinases have been cloned (Harper et al., 1991; Patil et al., 1995; Lu and Feldman, 1997). a.

Calcium-Regulated Protein Phosphorylation in Roots

In vivo protein phosphorylation studies have shown that calcium-dependent protein phosphorylation occurs in roots (Raghothama et al., 1987). To study the role of calcium-dependent protein phosphorylation in light-dependent gravitropism of maize root tips, we performed in vivo protein phosphorylation studies with dark-grown and with light-treated roots by manipulating tissue calcium levels (McFadden and Poovaiah, 1988). Exposure of dark-grown roots to 7 min of light resulted in the promotion of phosphorylation of specific polypeptides corresponding to 94,000, 92,000 and 48,000 kDa (Fig. 2). In later studies we were able to detect light-dependent changes in protein phosphorylation within 1 min. Interestingly, the light-dependent changes in protein phosphoryla-

Figure 2 Rapid changes in protein phosphorylation associated with gravity perception in roots of maize (cv. Merit). Apical segments of dark-grown roots were preloaded with 32 P for 1 h, then washed in buffer. (A) Roots were left in buffer for 15 min in the dark (control). (B) Roots were exposed to light for 7 min after 8 min of dark incubation. (C) Light treatment was the same as in (B), but EGTA and A23187 were present for 15 min. Proteins were extracted and separated by two-dimensional gel electrophoresis as described earlier (Raghothama et al., 1987). Arrows indicate the phosphoproteins that are affected by light. (From McFadden and Poovaiah, 1988).

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tion were observed only in the root tips, the site of light and gravity signal perception. Light had no effect on the phosphoprotein pattern in the root base, suggesting the specificity of light-dependent changes and the physiological significance of these changes in light-induced gravitropism. Depletion of calcium by addition of EGTA and the calcium ionophore A23187 prior to light treatment decreased lightinduced promotion of the phosphorylation of these polypeptides. Replenishment of calcium to depleted root tips restored the light effect on protein phosphorylation. These results strongly suggest that light induces rapid and specific changes in protein phosphorylation and that these changes are mediated by calcium. b. Calcium-Dependent and CalmodulinIndependent Protein Kinases Calcium-dependent protein kinases are one of the best characterized and most widely distributed protein kinases in plants. Since the discovery of a calciumdependent and calmodulin-independent protein kinase (CDPK) in soybean, such protein kinases have been purified and characterized from a number of plant systems (Harmon et al., 1987; Putnam-Evans et al., 1990; Roberts and Harmon, 1992; Harmon et al., 2000). CDPK is activated by micromolar concentrations of calcium and is not dependent on CaM for its activity. Harper et al. (1991) isolated a cDNA (SK5) from soybean that codes for a CDPK. The deduced amino acid sequence of soybean CDPK contains a catalytic domain and a CaM-like region with four calcium-binding domains at the carboxy terminal end. The presence of these calcium-binding domains explains the direct calcium activation of CDPK. The kinase domain showed highest homology (39%) with the catalytic domain of the -subunit of CaM KII. So far, this new type of protein kinase, where the kinase domain is fused to a CaM-like region, has been found in plants and protozoans. However, in the case of calpain, a calcium-activated protease, the catalytic domain is fused to the CaMlike regulatory domain (Suzuki and Ohno, 1990). Using a variety of approaches, the presence of CDPK-like enzymes was shown in a number of plant species, indicating the ubiquitous nature of these enzymes in plants (Roberts and Harmon, 1992; Harmon et al., 2000). cDNAs that encode for CDPKs have been isolated from carrot (Choi and Suen, 1991) and corn root tips (Takezawa et al., 1996a).

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c.

Calcium/Calmodulin-Dependent Protein Kinases

Several types of calcium/CaM-dependent protein kinases (CaM kinase I, CaM kinase II, CaM kinase III, phosphorylase kinase, and myosin light chain kinase) have been well characterized in mammalian systems (Fujisawa, 1990; Colbran and Soderling, 1990; Klee, 1991). All of these CaM-dependent protein kinases, except CaM KII, have limited substrate specificity. CaM KII phosphorylates a wide range of substrates, and it is therefore considered to be a multifunctional protein kinase. CaM KII is present in different species of vertebrates, invertebrates, yeast, and other fungi (Colbran and Soderling, 1990; Pausch et al., 1991). cDNAs that code for five different polypeptides of CaM KII have been isolated and characterized (Tobimatsu and Fujisawa, 1989). Plant scientists have also identified CaM-dependent protein kinases (Roberts et al., 1986; Poovaiah and Reddy, 1987, 1993; Zielinski, 1998; Reddy, 2000). In recent years, two types of calmodulin-dependent protein kinases in plants have been cloned and characterized. One type (CCaMK) is a chimeric calcium/ calmodulin-dependent protein kinase with a neural visininlike calcium-binding domain (Patil et al., 1995; Takezawa et al., 1996b). The other type (CaMK) has an association domain on the C-terminal end and lacks a calcium-binding domain (Watillon et al., 1993; Lu et al., 1996; Lu and Feldman, 1997). It has been suggested that calcium/calmodulin-dependent protein kinase plays a role in light-regulated root gravitropism (Lu and Feldman, 1997). However, these two kinases share similar kinase- and calmodulin-binding domains. Plants, unlike animal systems, seem to have both calcium-dependent protein kinases (CDPK) and calcium/ CaM-dependent protein kinases (CCaMK and CaMK). Having these different types of protein kinases would explain how calcium might regulate diverse physiological processes in plants. 2.

Other Calmodulin-Target Proteins

Calmodulin-binding proteins that are specific to metabolically active plant parts have been detected (Poovaiah and Reddy, 1993; Snedden and Fromm, 1998; Zielinski, 1998). There are many CaM-binding proteins in plants and some of them are specific to a particular tissue or cell type. To better understand the mode of calcium/CaM action, it is essential to identify and characterize all CaM-binding proteins in plants. In recent years, labeled calmodulin has been widely used to isolate calmodulin target proteins (Asselin et al.,

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1989; Widada et al., 1989; Fromm and Chua, 1992; Fordham-Skelton et al., 1994; Stirling et al., 1994). Two cDNAs (CBP-1 and CBP-5) that code for CaMbinding proteins have been isolated from a maize root tip cDNA library using 35 S-labeled calmodulin (Reddy et al., 1993). Some of the examples of calmodulin-binding proteins that have been cloned and characterized are: a homolog of the multidrug resistance gene (Wang et al., 1996a) kinesin (Reddy et al., 1996; Wang et al., 1996b); glutamate decarboxylase (Baum et al., 1993); elongation factor 1 (Durso and Cyr, 1994; Wang and Poovaiah, 1999); basic leucine zipper protein (Szymanski et al., 1996), calcium ATPase (Malmstrom, 1997; Harper et al., 1998); ion transporter (Schuurink et al., 1998; Arazi et al., 1999); small auxin-up RNA (Yang and Poovaiah, 2000a); ethylene upregulated gene (Yang and Poovaiah, 2000b); and others (see Table 1). 3.

Interactions Between Calcium/Calmodulin and Auxin-Regulated Genes

Gravitropic curvature is a consequence of differential cell elongation between two sides of the stimulated organ. For many years, it has been assumed that such differential elongation is regulated by an auxin gradient across the tissue, as originally proposed by

Cholodny (1927) and Went (1928). Auxin plays a central role in growth and development by controlling cell division, cell elongation, and cell differentiation (see also Chapter 23 by Gaspar et al., in this volume; Went and Thimann, 1937). It appears that auxin regulates cellular elongation by modulating the activity of the plasma membrane proton pump that affects the cell wall extensibility, and by regulating the expression of a number of auxin-responsive genes (Jones, 1994; Chen et al., 1999). A correlation between the gravitropic response and the redistribution patterns of exogenously applied radiolabeled IAA across gravistimulated organs was reported by Young et al. (1990). In the last decade, the role of auxin in gravitropism has been further confirmed and strengthened by the following two approaches. One approach is to isolate auxin-responsive genes and study their expression pattern during gravitropism (McClure and Guilfoyle, 1989; Gee et al., 1991; Abel and Theologis, 1996). Another approach is the isolation and characterization of auxin and gravitropic mutants. There is a close relationship between the mechanism of auxin action and calcium signaling (Kubowicz et al., 1982; Raghothama et al., 1985; Drobak and Ferguson, 1985; Reddy et al., 1988; Ettlinger and Lehle, 1988). However, only recently Yang and Poovaiah (2000a)

Table 1 Calmodulin-Binding Proteins in Plants Functional group

Protein

Reference

Metabolism

Glutamate decarboxylase NAD kinase Apyrase (nuclear NTPase) Kinesin heavy-chain-like protein

Baum et al., 1993 Anderson et al., 1980 Hsieh et al., 1996 Reddy et al., 1996; Wang et al., 1996 Durso and Cyr, 1994; Wang and Poovaiah, 1999 Malmstrom et al., 1997 Harper et al., 1998 Schuurink et al., 1998; Arazi et al., 1999 Patil et al., 1995

Cytoskeleton

Elongation factor 1 Ion transport

Vacuolar Ca2þ -ATPase ER Ca2þ -ATPase Plasma membrane transporter

Protein phosphorylation

Chimeric Ca2þ /CaM-dependent protein kinase (CCaMK) Ca2þ /CaM-dependent protein kinases

Transcription factor Hormone action Heat-shock protein Others

Basic leucine zipper protein (TGA3) Small auxin-up RNAs encoded protein Ethylene upregulated protein Heat-shock-repressed protein Multidrug-resistant protein homolog Root tip protein

Watillon et al., 1993 Lu and Feldman, 1997 Szymanski et al., 1996 Yang and Poovaiah, 2000a Yang and Poovaiah, 2000b Lu and Harrington, 1994 Wang et al., 1996 Reddy et al., 1993

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have provided direct evidence indicating that calcium/ calmodulin interact with SAUR, an auxin-regulated protein. Earlier studies by McClure and Guilfoyle (1989) indicated that SAURs are involved in gravitropism and are differentially regulated during bending. SAURs belong to one group of the early auxin response genes (Abel and Theologis, 1996). Initially isolated from soybean (McClure and Guilfoyle, 1987), SAUR genes have also been characterized from mung bean (Yamamoto et al., 1992), Arabidopsis (Gil et al., 1994), apple (Watillon et al., 1998), and maize (Yang and Poovaiah, 2000a). In all cases examined, SAUR genes encode short transcripts, with highly conserved open reading frames, that accumulate rapidly and specifically after auxin treatment. During gravitropism SAUR transcripts were more abundant on the bottom side of hypocotyls that were undergoing gravitropic curvature (McClure and Guilfoyle, 1989). Interestingly, the asymmetric induction of SAURs in the organ coincides with auxin asymmetric distribution leading to differential elongation. The finding that the SAUR gene family encodes for Ca2þ /CaM-binding proteins has important implications for understanding the ‘‘cross-talk’’ between calcium/CaM messenger system and auxin signal transduction. First, the expression of SAURs, and CaM in some cases is rapidly induced by auxin (Jena et al., 1989; Okamoto et al., 1995). The free calcium level in the cell rapidly increases following auxin treatment (Gehring et al., 1990). Secondly, calcium binds to CaM and the Ca2þ /CaM complex binds to the SAUR protein and modulates its functions in the cell, for example, cell elongation (Guilfoyle et al., 1992; Gee et al., 1991). Thus, understanding the role of SAURs in gravitropism would be a major step in the overall understanding of auxin-mediated growth and the role of Ca2þ /CaM-mediated signaling in plants.

IV.

GENETIC APPROACHES TO UNDERSTAND GRAVITY SIGNAL TRANSDUCTION

Auxins, calcium, and statoliths play an important role in gravity signal transduction (Sack, 1991; MacCleery and Kiss, 1999). Genetic studies have confirmed some of these earlier observations and allowed identification of several genes involved in gravity signal transduction (Chen et al., 1999; Ranjeva et al., 1999; Rosen et al., 1999; see also Chapters 30 by Pilet and 29 by Porterfield in this volume).

A large number of Arabidopsis mutants have been isolated that exhibit altered or no gravitropic response (Bell and Maher, 1990; Hobbie and Estelle, 1994; Chen et al., 1999). A number of auxin-resistant mutants have been isolated that show defective gravitropic responses (Hobbie and Estelle, 1994). Mutants that are insensitive to hormones such as ethylene and auxin display defects in gravitropism. For example, auxin-resistant mutants (axr1, axr2, axr3, axr4, and aux1) and ethylene-insensitive root (eir1) show defects in gravitropic responses. In some mutants the defects are minor (e.g., axr1 and axr4); in others (axr2, axr3, aux1, and dwf), gravitropic responses are significantly affected (Lincoln et al., 1990; Hobbie and Estelle, 1995; Estelle, 1996). axr1 is an auxin response mutation. Auxin synthesis, uptake, and metabolism are not affected in this mutant. axr4 and aux1 mutants show defects only in root gravitropism (Hobbie and Estelle, 1995). axr2 mutants have agravitropic hypocotyl shoots and roots (Wilson et al., 1990). The AXR1 encodes a novel protein related to ubiquitin–activating enzyme (E1) that catalyzes the first step in the biosynthesis of ubiquitin–protein conjugates (Leyser et al., 1993). AXR1 is found primarily in the nucleus of dividing and elongating cells, suggesting that it may be involved in degradation of one or more nuclear proteins (Pozo et al., 1998). The induction of small auxin-upregulated mRNAs is reduced in the axr1 mutant. These genetic studies indicate that ubiquitin-mediated protein degradation plays a central role in the auxin response (Gray et al., 1999; Gray and Estelle, 2000). AXR3 is shown to be a member of the auxin-inducible genes (Rouse et al., 1998). In another auxin-resistant mutant (aux1), roots are agravitropic (Maher and Martindale, 1980; Hobbie and Estella, 1994). In addition to altered gravitropism, aux1 shows decreased growth sensitivity to auxin, ethylene, and cytokinin. Bennett et al. (1996) cloned the AUX1 gene which encodes a membrane protein similar to plant amino acid permeases (Bennet et al., 1996; Fischer et al., 1998), indicating that it is likely to be involved in the uptake of amino acid–like signaling molecules such as IAA. Recently, it has been shown that AUX1 is involved in carrier-mediated auxin uptake and that it regulates root gravitropism by facilitating auxin uptake in the apical tissues of roots (Marchant et al., 1999). The expression of AUX1 was shown to overlap with the auxin efflux carrier (PIN2), another gene necessary for gravitropism (Bennett et al., 1998). Agravitropic mutants (agr1, agravitropic 1; eir1, ethylene-insensitive root 1; and pin2, pin-formed 2) that were isolated independently define a single locus.

Calcium and Gravitropism

In the agr1 mutant, root growth sensitivity to auxin is increased, whereas sensitivity to auxin transport inhibitor is decreased. The eir1 mutant has reduced sensitivity to ethylene, and the roots of this mutant are agravitropic (Luschnig et al., 1998). Cloning of AGR/ EIR/PIN2 has revealed that it encodes a component of the auxin efflux carrier and belongs to a member of a family of plant genes with similarities to bacterial membrane transporters (Chen et al., 1998; Galweiler et al., 1998; Luschnig et al., 1998; Muller et al., 1998; Utsuno et al., 1998). In yeast cells, AGR1 causes increases in the efflux of IAA from the cells and increases resistance to fluoro-IAA, a toxic IAA-derived compound (Chen et al., 1998; Luschnig et al., 1998). In gravistimulated cells, AGR1 expression is localized to a region undergoing a curvature (Chen et al., 1998). The expression of this gene is confined to the root, suggesting a root-specific role in transporting auxin (Luschnig et al., 1998). The expression of AGR1/ EIR1/PIN2 and AUX1 is confined to distal and central elongating zones of Arabidopsis roots and is consistent with their involvement in gravitropism (Chen et al., 1998; Muller et al., 1998). In arg1 (altered response to gravity) mutant, roots and hypocotyls show altered gravitropic response and do not show pleiotropic phenotypes found in other gravitropic mutants. Hence the protein coded by ARG1 is implicated in graviperception. ARG1 encodes a DnaJ-like protein with potential transmembrane domains. Based on its structural features, it was speculated that ARG1 may be involved in either connecting some aspects of gravity signal transduction to the cytoskeleton or targeting proteins involved in gravity signal transduction (Sedbrook et al., 1999). The rgr1 mutation confers resistance to auxin and reduces root and hypocotyl gravitropic response (Simmons et al., 1995). In another mutant, rcn1 (roots curl in NPA), the transport of auxin is affected. The RCN1 gene encodes a protein similar to the regulatory subunit A of protein phosphatase A and rescues a PP2A-A mutation in yeast. These results suggest that RCN1 is a part of protein phosphatase 2A and it is likely that it regulates auxin transport (Garbers et al., 1996). To elucidate the molecular mechanism underlying shoot gravitropism, Tsaka’s group isolated several shoot gravitropic (sgr) mutants of Arabidopsis thaliana (sgr1, sgr2 and sgr3, sgr4, sgr5 and sgr6). The inflorescence stems of these mutants showed abnormal gravitropic responses. The sgr1, sgr2, and sgr4 mutants showed severe defects in response to gravitropic stimulation in the inflorescence stems, and hypocotyls, but

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not in the roots. The sgr3, sgr5, and sgr6 mutants showed altered responses to gravitropism only in the inflorescence stems, but were normal in both the hypocotyls and roots. These results support the hypothesis that some mechanisms of gravitropism are genetically different in these three organs of A. thaliana. In addition, these mutants showed normal phototropic responses, suggesting that these genes are specifically involved in gravity perception and/or gravity signal transduction for the shoot gravitropic response (Fukaki et al., 1996; Yamauchi et al., 1997). Analysis of two mutants of shoot gravitropism (sgr1, which is allelic to scarecrow, and sgr7, which is allelic to shortroot), suggests an important role for the endodermis in gravity perception in shoots (Tasaka et al., 1999). In the rhg mutant (root and hypocotyl gravitropism), the inflorescences showed normal responses to gravitropic stimulation, whereas roots and hypocotyls showed impaired responses (Fukaki et al., 1997). These results further support the hypothesis that the mechanisms of gravitropism are genetically distinct in different plant parts. Genetic evidence with starch mutants supports the involvement of statoliths and also the presence of other gravity sensing mechanisms (Ranjeva et al., 1999). Roots of starchless mutants are less sensitive to gravity (Caspar and Pickard, 1989; Kiss et al., 1989, 1997; Vitha et al., 2000). Analysis of gravitropic response of a wild-type, two reduced starch mutants (ACG 20 and ACG 27), and a starchless mutant (ACG 21) of Arabidopsis supports a statolith hypothesis for gravity perception in plants (Kiss et al., 1997; Chapter 3 by Sievers et al. in this volume). Following the initial perception, calcium and auxin appear to play a central role in the growth response (Sack, 1991; Sinclair and Trewavas, 1997). In wild-type Arabidopsis calmodulin mRNA levels increase upon gravistimulation. However, in the agr3 mutant that showed a reduced gravitropic response, gravitropic stimulation decreased the level of calmodulin transcripts (Sinclair et al., 1996). Recent cloning of several mutant genes that affect gravitropism indicates that the cytoskeleton proteins (ARG1, DnaJ-like protein) (Ingber, 1999; Sedbrook et al., 1999) are involved in polar auxin transport (AGR1/EIR/PIN and AUX1) (Bennet et al., 1996; Chen et al., 1998; Galweiler et al., 1998; Luschnig et al., 1998; Muller et al., 1998; Utsuno et al., 1998), and that protein degradation (AXR1) (Leyser et al., 1993) plays an important role in gravity signal transduction. Studies with gravitropic mutants indicate that the molecular mechanisms of the gravitropic responses in roots, hypocotyls, and inflorescence

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stems are different, although some of the components of the regulatory mechanisms for gravitropic responses are shared by these organs (Fukaki et al., 1996, 1997; Yamauchi et al., 1997; Tasaka et al., 1999). Further functional analysis of the cloned genes involved in gravitropism, and identification of other mutant genes that display defects in gravitropism, should provide significant insights in understanding the role of these hormones in gravity signal perception and transduction. Although many physical signals such as light and temperature are known to regulate expression of specific genes, it is not known if any specific genes in plants are turned on/off by gravity. Global analysis of gene expression in plants grown under microgravity conditions (in space flight) and on Earth (1 g) using microarray analysis should permit the identification of gravity-regulated genes (Reddy et al., 1998; Chapter 29 by Porterfield in this volume). The sequence of biochemical events that could occur in gravitropism are illustrated in a schematic diagram (Fig. 3). According to this model, the initial event in gravity perception is the localized increase in cytosolic calcium in root cap cells. Although the mechanism by which gravity induces the increase in cytosolic calcium is unclear, it is likely that phosphoinositide hydrolysis could be involved in light-induced gravity response. Increases in cytosolic calcium activate calmodulin, leading to stimulation of calciumand calcium/CaM-dependent enzymes such as CaATPase and protein kinases, ultimately leading to the creation of both intra- and extracellular calcium gradients. This asymmetric calcium distribution could differentially modify cytoskeletal proteins, microtu-

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bule orientation, cell wall synthesis, and deposition. As a result, a growth gradient is created with more growth on the nonstimulated side, resulting in bending. ACKNOWLEDGMENTS This work was supported by grants from the NSF and NASA to B.W.P; the NSF; NASA; and from the Agricultural Experiment Station to A.S.N.R. REFERENCES Abel S, Theologis A. 1996. Early genes and auxin action. Plant Physiol 111:9–17. Anderson JM, Charbonneau H, Jones HP, McCann RO, Cormier MJ. 1980. Characterization of the plant nicotinamide adenine dinucleotide kinase activator protein and its identification as calmodulin. Biochemistry 19:3113–3120. Arazi T, Sunkar R, Kaplan B, Fromm H. 1999. A tobacco plasma membrane calmodulin-binding transporter confers Ni2þ tolerance and Pb2þ hypersensitivity in transgenic plants. Plant J 20:171–182. Asaoka Y, Nakamura S, Yoshida K, Nishizuka Y. 1992. Protein kinase C, calcium and phospholipid degradation. Trends Biochem Sci 17:414–417. Asselin J, Phaneuf S, Watterson DM, Haiech J. 1989. Metabolically 35 S-labeled recombinant calmodulin as a ligand for the detection of calmodulin-binding proteins. Anal Biochem 178:141–147. Baum G, Chen YL, Arazi T, Takatsuji H, Fromm H. 1993. A plant glutamate decarboxylase containing a calmodu-

Figure 3 A schematic diagram illustrating the involvement of calcium in gravitropism in roots. (Modified from Poovaiah et al., 1987.)

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32 Respiratory Patterns in Roots in Relation to Their Functioning Hans Lambers University of Western Australia, Crawley, Western Australia, Australia

Owen K. Atkin University of York, York, England

Frank F. Millenaar Utrecht University, Utrecht, The Netherlands

I.

INTRODUCTION

Finally, both genetic and environmentally induced variations in root respiration are discussed in the light of the functioning of the whole plant.

A significant portion of the carbohydrates that are produced daily in photosynthesis are respired in the roots. The magnitude varies with age of the plants, growth conditions, and the species under investigation. The rate of root respiration, measured as O2 consumption or CO2 release, also varies between species, even when plants are grown under very similar conditions. This chapter discusses factors that are responsible for the differences in root respiration. An important feature of the respiration in higher plants is the participation of an alternative, nonphosphorylating electron transport path, which decreases the efficiency of ATP production in respiration (Lambers, 1985). Since the nonphosphorylating path can be a major sink for carbohydrates, the regulation of the activity of this path warrants an extensive discussion. Respiratory energy is the driving force for biosynthetic reactions, as well as for maintenance and transport processes. In recent years more information has become available on the quantitative significance of the various sinks for respiratory energy. Methods that are employed in this area of research and data that have been obtained in this manner will receive attention.

II.

QUANTITATIVE SIGNIFICANCE OF ROOT RESPIRATION

Between one-quarter and two-thirds of all the photosynthates produced per day are respired in the same period (Poorter et al., 1990; Van der Werf et al., 1994). A major portion of this respiration occurs in the roots, connected with their growth and maintenance and with the absorption of ions (see Section V.B). A. Root Respiration as a Fraction of the Photosynthates Translocated to the Roots Between one-third and two-thirds of all the carbohydrates translocated to the roots are used in respiration (Table 1). This fraction tends to increase with increasing plant age, as a consequence of a decreased translocation of assimilates to the roots and is partly due to the proportionally greater amount of maintenance energy required when root growth slows down (see Section V). 521

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Table 1 Utilization of Carbohydrates for Root Respiration as a Percentage of Those Translocated to the Rootsa Utilization for root respiration

Species

Z. mays

Reference

Decreases upon tap root formation

high

29–53 54 38 67 35–44 41 15 19 32

high

60

Low N supply

Van der Werf et al. (1992a)

low

51

High nutrient supply

Van der Werf et al. (1992a)

low

54

Low N supply

Van der Werf et al. (1992a)

Daucus carota Festuca ovina Glycine max Helianthus annuus H. annuus Hordeum distichum H. vulgare Monocotyledons with a potential growth rate Monocotyledons with a potential growth rate Monocotyledons with a potential growth rate Monocotyledons with a potential growth rate Nardus stricta Pisum sativum Triticum aestivum T. aestivum Zea mays

Special remarks

50 52–60 67 52 44–57

High N Low N High nutrient supply

NO 3 -fed Seedlings, pregrown in CaSO4 Sand grown, nutrient-limited Excised root tips, cultivated in vitro

49

Steingro¨ver (1981) Atkinson and Farrar (1983) Harris et al. (1985) Hatrick and Bowling (1973) Szaniawski and Kielkiewicz (1982) Farrar (1985) Johansson (1992) Van der Werf et al. (1992a)

Atkinson and Farrar (1983) De Visser (1985) Barneix et al. (1984) Lambers et al. (1982) Kandler (1953) Veen (1980)

a

Only nonsymbiotically grown plants are included. If necessary, values were recalculated from data presented in the literature, assuming 40% C in the dry matter and a respiratory quotient of 1.0.

The proportion of carbohydrates respired in the roots increases when growth occurs at a low nutrient supply (Van der Werf et al., 1992a; Table 1). This is largely explained by the slower growth of roots at a limiting supply of nutrients; in addition, specific costs for maintenance or ion uptake might increase (Van der Werf et al., 1994). B.

Root Respiration as a Fraction of the Photosynthates Produced Daily

Eight percent to 52% of all carbohydrates produced per day in photosynthesis are respired in the roots during the same period (Table 2). This percentage increases with decreasing growth rate of the plants, be it due to an inherently low growth potential of the species (Poorter et al., 1990; Atkin et al., 1996) or to growth being restricted by a suboptimal nutrient supply (Boot et al., 1992; Van der Werf et al., 1992a). The percentage tends to decrease with increase in age. This decrease is probably not associated with an increased

efficiency of respiration, since the contribution of the nonphosphorylating, alternative path in root respiration of Glycine max Merr increases with increasing age of the plants (Millar et al., 1998). Therefore, this decrease may be due to a decrease in the demand for respiratory energy (e.g., in Carex acutiformis, where the energy required for root growth and ion uptake decreases drastically with increasing age; Van der Werf et al., 1988). The fraction of carbohydrates used in root respiration, including the respiration of microsymbionts, if present, is affected by both abiotic and biotic environmental factors (Table 2). In the presence of an N2 -fixing symbiont (Rhizobium), carbohydrate utilization by root respiration is greater than that of nonnodulated roots, supplied with nitrate as the source of nitrogen. This is explained by the greater energy requirement for N assimilation during N2 fixation in comparison with that for NO 3 assimilation (see Section VII.B). Also in the presence of a symbiotic mycorrhizal fungus (Glomus mosseae) the fraction of carbohydrates used

Respiratory Patterns

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Table 2 Utilization of Carbohydrates for Daily Root Respiration as a Percentage of Those Produced Daily in Photosynthesisa Species

Utilization for root respiration %

Hordeum distichum Lolium multiflorum Lupinus albus Lycopersicon esculentum Nardus stricta Pisum sativum

18 23 12 14 14–19 24 25 31 15 8 9 15 28 13–15 17 17 14 14–20 23 9–18 21 12–18

P. sativum P. sativum

16–20 25 21–24

Zea mays Monocots with a high potential growth rate Monocots with a low potential growth rate

18? 18? 52 16 38

Allium porrum Cucumis sativus Daucus carota Deschampsia flexuosa Festuca ovina F. pratensis Galinsoga parviflora Glycine max

Helianthus annuus Holcus lanatus

a

Special remarks Nonmycorrhizal Mycorrhizal High light þ long days Low light þ short days Decreases with age High light Low light Grown in soil High potential growth rate Nonsymbiotic With Rhizobium With Rhizobium þ Glomus

Reference Snellgrove et al. (1982) Challa (1976) Steingro¨ver (1981) Poorter (1991) Atkinson and Farrar (1983) Johansson (1991) Poorter et al. (1990) Harris et al. (1985)

Szaniawski (1981) Poorter (1991)

High light Low light High at low light Nonnodulated with Rhizobium Increases with age; grown in soil Nonnodulated; NO 3 fed; decreases with age With Rhizobium; decreases with age Nonnodulated; grown in soil decreases with age 56 days old High nutrient supply Low nutrient supply High nutrient supply Low nutrient supply

Farrar (1981) Hansen and Jensen (1979) Pate et al. (1979) Whipps (1987) Atkinson and Farrar (1983) De Visser (1985)

Duarte et al. (1988) Whipps (1987) Massimino et al. (1980) Van der Werf et al. (1992a) Van der Werf et al. (1992a)

When the plants were grown in soil, part of the respiration may be due to respiration of microorganisms.

in root respiration of Allium porrum is larger than that of nonsymbiotic plants (Table 2). It is well documented that during growth at a low light intensity, the rate of root respiration is relatively low (Lambers and Posthumus, 1980; Kuiper and Smid, 1985). Less information is available on the effects of light intensity during growth on the contribution of root respiration to the plant’s total carbon budget. Long-term exposure to a combination of reduced light intensity and short days has very little effect on the fraction of carbohydrates that are used in root respiration of Cucumis sativus (Table 2; Challa, 1976). Poorter (1991) found that
30% of all the

photosynthates produced per day are respired in the roots of Deschampsia flexuosa and Holcus lanatus, whether the grasses were grown at high or at low irradiance. The wide variation in the fraction of carbohydrates translocated to the roots and subsequently respired (Table 1) is partly associated with variation in the growth rate of the roots (Fig. 1). Slow-growing plants consume a far greater proportion of the daily produced photosynthates in root respiration than fast-growing ones. This is true for a comparison of species which vary in their potential growth rate (Poorter et al., 1990; Atkin et al., 1996), as well as for a comparison of

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Figure 1 Carbohydrate consumption in root respiration as a proportion of those produced in photosynthesis for 24 herbaceous species, grown in a nutrient solution with an optimum supply of nitrate. (Based on data from Poorter et al., 1990.)

plants of the same species which vary in growth rate, due to variation in the nutrient supply (Van der Werf et al., 1992a). Compared with fast-growing species, roots of inherently slow-growing species have relatively large costs for nitrate uptake, due to a major nitrate efflux component (Scheurwater et al., 1998, 1999). A similar explanation may apply for plants that grow slowly owing to a limiting nitrate supply, but further experiments are required to confirm this (Lambers et al., 1998a).

III.

VARIATION IN ROOT RESPIRATION

It is well documented that both the rate of root respiration and the respiratory quotient may vary with species, environmental conditions, and ontogeny. In this section we will analyze this variation and its consequences for the roots’ costs of functioning. A.

Variation in Rate of Respiration

It has been known for a long time that the rate of respiration of the primary root varies with distance from the tip. Machlis (1944) found a gradual decline of both the rate of oxygen uptake and that of carbon dioxide release (expressed on a root volume basis) for the primary root of Hordeum vulgare seedlings. The rates are two to three times higher in the root tip

than at a distance 90 mm from the tip. Similarly, the rate of oxygen uptake (expressed on a fresh mass basis) of the 10- to 15-mm segment of Allium cepa roots is 40% less than that of the apical 0–5 mm (Berry and Brock, 1946). A partial explanation for the decreasing respiration rate (on a volume or fresh mass basis) with increasing distance from the tip is offered when the data are expressed on a dry-mass basis and per cell (Goddard and Bonner, 1960). This shows that the higher respiration rate per unit fresh mass in the tip is due to the lower water content of the smaller, meristemic cells in this region. Expressing the data on a dry-mass basis leaves very little variation in the respiration rate. In Allium cepa roots, the respiration per cell and per gram nitrogen is very low in the smaller cells closest to the tip (Wanner, 1950; Fig. 2). It is about three times higher in the 2- to 5-mm zone than closer to or farther away from the tip. In Allium cepa, this is the zone where cell elongation and synthesis of protein and nucleic acids (both DNA and RNA) are most pronounced (Yemm, 1965). Presumably, the high respiration rate in this zone is due to the high requirement for respiratory energy for biosynthetic and uptake processes, as this is a major factor regulating the rate of respiration (see Section IV.A.2). Thus, variation in respiration along the primary root axis has a distinct physiological basis and is clearly understood when the data are expressed on the proper basis. However, it should be borne in mind that the data discussed here pertain to the primary (growing) root. No data are available on respiratory patterns in lateral roots, which may be of particular interest because of their restricted growth period. Consequently, there is still very little information on the partitioning of respiratory activity over the entire root system. Rates of root respiration vary widely among species (Table 3). Although fast-growing plants use less carbon in root respiration (expressed as a fraction of the carbohydrates fixed in photosynthesis on a daily basis) than slow-growing ones, their rates of root respiration are higher, especially when expressed on a dry-mass basis (Poorter et al., 1991; Van der Werf et al., 1992a; Atkin et al., 1996). The variation in the rate of root respiration (Table 3, Fig. 2) is largely due to differences in the demand for respiratory energy, e.g., for ion uptake and growth, rather than differences in respiratory efficiency. However, the specific energy requirements for ion uptake, growth or maintenance may differ as well (see Section V).

Respiratory Patterns

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Figure 2 Rate of O2 consumption by primary roots of Allium cepa and the number of cells/mm3 of root as a function of the distance from the tip. Data are expressed per gram of nitrogen (circles) and per cell (squares). (Based on data from Wanner, 1950.)

B.

Variation in the Respiratory Quotient

The respiratory quotient (RQ) of root respiration varies with the potential growth rate of a species and  depends on the source of N (NHþ 4 , NO3 , or N2 Table 4). In the absence of biosynthetic processes, the RQ of root respiration is expected to be 1.0, if sucrose is the only substrate for respiration. For roots of young seedlings, measured in the absence of a N source, values close to 1.0 have indeed been found

(Table 4). The RQ may be > 1, if more oxidized substrates, e.g., organic acids, are an additional substrate. Organic acids (malate) may be produced concomitant with the reduction of nitrate in leaves, followed by their transport and decarboxylation in the roots (Ben Zioni et al., 1971). If nitrate reduction proceeds in the roots, the RQ is also expected to be > 1, since per molecule of nitrate reduced to ammonium an additional two molecules of CO2 are produced. During synthesis of biomass, both carboxylating and decar-

Table 3 Activity of the Alternative Path, Assessed Using the Oxygen Isotope Discriminating Techniquea Species

Alternative path activity (%)

Control rate (nmol O2 g1 FM s1 )

Glycine max G. max G. max G. max G. max Nicotiana tabacum Poa alpina P. annua P. annua P. compressa P. pratensis P. trivialis Vicia radiata V. radiata

5 34 54 37 43 30 22 30 48 11 13 49 11 18

4.1 3.8 1.6 1.7 5.7 2.0 3.1 4.0 1.5 3.4 4.5 4.0 1.5 1.3

a

Special remarks 4 days old 7 days old 17 days old 14 C 28 C

low light

19 C 28 C

Reference Miller et al., 1998 Miller et al., 1998 Miller et al., 1998 Gonza`lez-Meler et al., 1999 Gonza`lez-Meler et al., 1999 Lennon et al., 1997 Millenaar et al., 2000b Millenaar et al., 2000b Millenaar et al., 2000a Millenaar et al., 2000b Millenaar et al., 2000b Millenaar et al., 2000b Gonza`lez-Meler et al., 1999 Gonza`lez-Meler et al., 1999

Rates are expressed as a percentage of the control rate, expressed in nmol O2 (g DM)1 s1 . All plants were grown in nutrient solution.

526

Lambers et al.

Table 4 Respiratory Quotient (RQ) of Root Respiration of a Number of Herbaceous and Wood Speciesa Species

RQ

Special remarks

Reference

Allium cepa

1.0 1.3 1.0 0.85 0.82 1.2 0.95 1.64 1.53 1.3 1.03 1.22 1.09 0.94 0.97 0.90 0.95 1.70 1.4 1.6 1.0 1.13 0.84 0.95 1.42 1.15 1.0 1.5 1.1 2.0 1.0 1.0 0.75

Root tips Basal parts

Berry (1949)

Avicennia marina A. marina Dactylis glomerata Festuca ovina Galinsoga parviflora Helianthus annuus Holcus lanatus Hordeum distichum H. vulgare H. vulgare H. vulgare

Lupinus albus Oryza sativa Pisum sativum

Triticum aestivum T. aestivum

Zea mays Z. mays

Fine roots Cable roots NO3 fed NO3 fed NO3 fed NO3 fed NO3 fed NO3 fed NH4 fed NO3 fed Root tips Basal parts N-deprived NHþ 4 added NO 3 added NO 3 fed N2 fixing NHþ 4 fed NO 3 fed NH 4 fed NO 3 fed N2 fixing 0–4 mm Basal parts N deprived NHþ 4 added NO 3 added Basal parts Fresh tips Starved tips

Burchett et al. (1984) Curran et al. (1986) Scheurwater et al. (1998) I. Scheurwater, unpublished I. Scheurwater, unpublished I. Scheurwater, unpublished Williams and Farrar (1990) Bloom et al. (1992) Machlis (1944) Willis and Yemm (1955)

Lambers et al. (1980) Brambilla et al. (1986) De Visser (1985)

Karlsson and Eliasson (1955) Barneix et al. (1984)

Greenway and West (1973) Saglio and Pradet (1980)

a All plants were grown in nutrient solution, with nitrate as the N-source, unless stated otherwise. The barley plants used by Willis and Yemm (1955) were pregrown without a nitrogen source and then provided with no N, ammonia, or nitrate at the start of the respiration measurements. The pea plants used by De Visser (1985) were grown with a limiting supply of combined N, so that their growth rate matched that of the symbiotically grown plants.

boxylating reactions occur, which also affect the RQ. For example, synthesis of oxidized compounds such as organic acids decreases the RQ, whereas the production of reduced compounds such as lipids leads to higher RQ values. Comparing the molecular formula of the biochemical compounds of the biomass with that of sucrose, RQ values > 1 are expected. The RQ will be < 1, if compounds that are more reduced than glucose, e.g., lipids and protein, are an additional substrate. This may happen upon starvation of excised root tips (Table 4; Saglio and Pradet, 1980; Brouquisse et al., 1991).

Like other slow-growing species from nutrientpoor habitats, the slow-growing Festuca ovina L. has a low RQ compared with those of the fastergrowing Dactylis glomerata L. and Holcus lanatus (Table 4; Scheurwater et al., 1998). Taking into account the respiratory energy costs of biomass synthesis from glucose, ammonia, and minerals, Penning de Vries et al. (1974) calculated a value for RQ of 1.25. Since respiration associated with transport processes and maintenance proceeds with a RQ of 1.0, lower values are expected for total root respiration if NHþ 4 is the source of N. Fast-growing

Respiratory Patterns

plants require a greater proportion of root respiration for growth (up to 45%), whereas slow-growing ones use proportionally more for transport and maintenance (up to 82%; Poorter et al., 1991). However, this would lead to RQ values, with ammonium being the N source, as different as 1.11 and 1.05 for fast- and slow-growing species, respectively. Faster-growing species also have higher specific rates of nitrate reduction in their roots than slowgrowing species, which offers an additional explanation (Scheurwater, 1999). If the fast-growing species were to make greater use of the malate shuttle—i.e., decarboxylate relatively more malate and exchanging the resultant HCO 3 for nitrate and take up relatively less Kþ (Ben Zioni et al., 1971)—then this might also partly account for their higher RQ values. However, on the basis of an analysis of their cation, anion, and organic N contents, H. Poorter (personal communication) concluded that fast- and slow-growing species do not differ in the proportion of NO 3 that is reduced in the root. Using several approaches to assess nitrate reductase activity in roots and shoots, Scheurwater (1999) confirmed such a similar pattern in inherently fast- and slow-growing grass species. An additional factor that accounts for lower RQ values in slow-growing species is their relatively inefficient nitrate uptake system, when plants are grown with free access to nitrate (see Section V.B). Since this additional respiration proceeds with an RQ of 1.0, it has the net effect of lowering the overall RQ value. We conclude that the relatively high RQ of fast-growing species reflects a rapid rate of nitrate reduction in these roots compared with those of slow-growing species as well as a more efficient nitrate uptake system. C.

Variation in Capacity and Activity of the Alternative Path

A substantial number of studies have investigated the level of alternative pathway respiration in roots, using respiratory inhibitors such as salicylhydroxamic acid (SHAM; a specific inhibitor of the alternative oxidase) and KCN (a specific inhibitor of the cytochrome pathway). These studies showed that there is a wide variation in the KCN-resistant component of respiration that is sensitive to SHAM (Table 3). The use of these inhibitors was based on the assumption that the cytochrome pathway is always fully saturated whenever the alternative pathway contributes to respiration (see Section VI.A). However, recent studies have demonstrated that the alternative

527

pathway actually shares electrons that are donated by reduced ubiquinone with the cytochrome pathway. As a result, the cytochrome pathway does not have to be fully saturated for the alternative pathway to become engaged (Hoefnagel et al., 1995; Ribas-Carbo et al., 1995; Wagner and Krab, 1995; Day et al., 1996). Previous estimates of alternative pathway respiration using the inhibitor SHAM may therefore have resulted in substantial underestimates of alternative pathway activity. To accurately estimate alternative pathway respiration in intact roots, the oxygen–isotope fractionation technique (Guy et al., 1989, 1992; Henry et al. 1999) needs to be used. However, only a few studies have used this technique to estimate alternative pathway activity in roots (Millar et al., 1998; Millenaar et al., 2000, 2001). Millar et al. (1998) showed a gradual decline in activity of the alternative path with increasing age of the roots of Glycine max, while Millenaar et al. (2001) found that alternative pathway respiration represents a greater proportion of total root respiration in inherently fast-growing species compared with their slow-growing counterparts. The activity of the alternative pathway remains constant in roots exposed to long nights and short days, following transfer from high light/short nights (Millenaar et al. 2000). Variation in the activity of the nonphosphorylating alternative path alters the roots’ costs of functioning to a significant extent. When only the cytochrome path contributes to root respiration, the ATP yield per O2 taken up is three times greater than when respiration is entirely due to the activity of the alternative path (Table 5). In vivo, the ADP:O ratio is likely to vary between 3 and 1 (Lambers, 1995). It is therefore important that further work using the 18 O isotope fractionation technique be conducted to determine the extent to which alternative pathway activity varies in roots.

IV.

REGULATION OF ROOT RESPIRATION

A.

Short-Term Effects

The rate of root respiration responds both to the demand for respiratory energy and the carbohydrate supply (Lambers et al., 1998b). These are rapid effects that can be explained by the response of specific enzymes and/or the electron transport systems. To understand the rapid responses upon changes in the environment, the regulation of root respiration is discussed in some detail below.

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Lambers et al. Table 5 In vivo ADP:O Ratios in Root Tips of Zea mays, as Determined with the Saturation Transfer 13 -P-NMR Technique and O2 Uptake Measurementsa Exogenous substrate Glucose Glucose None Glucose Glucose Glucose Succinate

O2 concentration 100 0 100 100 100 100 100

Inhibitor

Rate of O2 uptake

Rate of ATP production

ADP:O ratio

22.3 0 14.9 13.7 3.7 21.1 16.1

142.6 < 20 93.0 26.2 < 20 136.7 63

3.19 – 3.03 0.96 – 3.24 1.96

None None None KCN KCN þ SHAM SHAM None

Rates of ATP production and O2 consumption are expressed as nmol g1 (FM) s1 . Exogenous glucose and succinate were supplied at 50 mol m3 . The concentration of KCN was 0.5 mol m3 and that of SHAM 2 mol m3 , which is sufficiently high to fully block the alternative path in maize root tips (H. Lambers, unpublished). Only means values are presented, so that the ADP:O ratio cannot be calculated from the primary data given in the table. Source: Roberts et al., 1984. a

1. Regulation by Carbohydrate Supply

2.

Challa (1976) found a diurnal fluctuation both in the level of soluble sugars (Fig. 3A) and in CO2 production (Fig. 3B) for roots of Cucumis sativus, grown under conditions of a low light intensity and a short day. No such fluctuation in root respiration of C. sativus (Challa, 1976) or of four grass species (Scheurwater et al., 1998) is found when the plants are grown at a high light intensity and a long day. Plants grown under such conditions have a high level of carbohydrates in their roots throughout the entire day. Respiration of Zea mays root tips, depleted of sugars, is stimulated by 0.2 mol m3 exogenous glucose (Saglio and Pradet, 1980; Brouquisse et al., 1991). Bryce and ap Rees (1985a) also found such a stimulation of respiration for the apical 40 mm roots of Pisum sativum by exogenous sucrose (25–100 mol m3 ; Fig. 4), even without depleting them of substrate. The effect of exogenous sucrose on pea root respiration increases with increasing time of exposure. This points to adjustment of the respiratory apparatus, similarly to what has been found for roots of intact plants (see Section IV.B). Exogenous sugars do not invariably stimulate the rate of root respiration (Farrar, 1981). This is unlikely due to poor absorption of the added sugars. Rather, it reflects the adjustment of the respiratory capacity to the root’s carbohydrate level (see Section IV.B). Root respiration is regulated by adenylates and/or by the level of respiratory substrate. Adenylate control of electron transport and/or of glycolysis often coincides with regulation by the availability of substrate.

Glycolysis in roots is controlled by ‘‘energy demand’’ (Lambers, 1985). This is largely due to the allosteric regulation of two key enzymes, phosphofructokinase and pyruvate kinase, by adenylates. This regulation explains the ‘‘Pasteur effect’’, i.e., the stimulation of glycolysis under low-oxygen conditions, when oxidative phosphorylation is curtailed and the level of ATP drops, whereas that of ADP increases. Adenylates do not only exert control over glycolysis, but also over mitochondrial electron transport. Saturation of the cytochrome path may be due to a limiting capacity of the electron transport chain per se. Alternatively, it may be due to a restriction of the flux of electrons through the cytochrome path by oxidative phosphorylation. In the absence of ADP, the protonmotive force across the inner mitochondrial membrane increases, and so restricts the flow of electrons to O2 . This can be analyzed in vivo by application of an uncoupler (e.g., DNP, CCCP, FCCP, or preferably S13; Lambers, 1995) in the presence of an inhibitor which fully blocks the alternative path (e.g., SHAM). If addition of an uncoupler stimulates respiration in the presence of SHAM, then the cytochrome path was restricted by adenylates when the alternative path was blocked. However, these results cannot show if the cytochrome path was similarly controlled when the alternative path is not inhibited, because inhibition of the alternative path may have shifted electron flow toward the cytochrome path. If SHAM does not inhibit respiration, and an uncoupler does not stimulate oxygen uptake, mitochondrial electron flow is not

Regulation of Glycolysis and Electron Transport

Respiratory Patterns

529

Figure 4 Respiration of excised apical 40-mm root segments of Pisum sativum as dependent on the concentration and time after addition of exogenous sucrose. The dots refer to the original measurements; the plane was calculated using a graphics program (Surf, version 3.00, copyright Golden Software, Inc., 1987). (Based on data from Bryce and ap Rees, 1985a.)

the control of glycolysis. Table 6 shows that adenylates control the rate of oxygen uptake in Zea mays L. roots via an effect through limitation of the flux of electrons through the respiratory chain. In contrast, adenylates

Figure 3 Diurnal course of the soluble sugar concentration (A), and the rate of CO2 production (B) in the roots of Cucumis sativus. The data are expressed on the basis of starch- and sugar-free dry mass. (Based on data from Challa, 1976.)

restricted by adenylates, but is limited by the supply of respiratory substrate. In intact roots, mitochondria often operate in a state where ADP is present in a concentration which restricts the flow of electrons through the cytochrome path to a greater or lesser extent (Millar et al., 1998; see Section IV.A.1). If the addition of an uncoupler, e.g., DNP or FCCP, stimulates the rate of respiration in the absence of an inhibitor of the alternative path, respiration must have been limited by adenylates. However, this does not imply a limitation of oxidative phosphorylation by ADP. Rather, it may be the substrate supply to the mitochondria that is restricted by adenylates, e.g., via

Table 6 Control of Respiration of Intact Roots by Adenylates, via Their Effect on the Cytochrome Path (A, Zea mays), or via Their Effect on the Substrate Supply to the Mitochondria (B, Phaseolus vulgaris) Conditionsa A. None þKCN þSHAM þCCCP þSHAM þ CCCP B. None þKCN þSHAM þCCCP þSHAM þ CCCP

Oxygen consumptionb (%) 100 47 70 141 91 100 69 95 122 92

Applied concentrations: CCCP, 2 mmol m3 ; SHAM, 10 mol m3 ; KCN, 0.5 mol 3 . b Rates are expressed as percentages of the basal rate [64 and 89 nmol (g DM)1 s1 for Z. mays and P. vulgaris, respectively]. Source: Day and Lambers, 1983. a

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control respiration in roots of Phaseolus vulgaris via the substrate supply to the mitochondria, presumably via their effect on glycolysis (Pasteur effect; Day and Lambers, 1983). The relief of ADP limitation of the cytochrome path not only enhances the flux of electrons through this path but may also decrease the electron flow through the alternative path. Respiration is not invariably controlled by adenylates. Respiration of Zea mays root tips, depleted of sugars, cannot be stimulated by uncoupler (Saglio and Pradet, 1980; Williams and Farrar, 1992). Uncoupler only has an effect in the presence of a high endogenous level of sugars and when glucose is exogenously supplied. Thus, adenylate control is more important at a high level of endogenous respiratory substrate than at low levels. 3. Partitioning of Electrons Between the Two Internal NADH Dehydrogenases Internal NADH, i.e., NADH originating in the tricarboxylic acid cycle, can be accepted by the electron transport pathway via two internal NADH dehydrogenases, both located in the inner mitochondrial membrane. Electron transport from internal NADH to ubiquinone via the rotenone-sensitive dehydrogenase is coupled to proton extrusion, and hence to oxidative phosphorylation. Electron transport via the rotenoneresistant internal dehydrogenase is not coupled to proton extrusion. The Km of the internal rotenone-sensitive dehydrogenase is an order of magnitude lower than that of the resistant one (Møller and Palmer, 1982; Agius et al., 1998). This lower affinity together with the lack of any association with proton extrusion of the rotenoneresistant dehydrogenase suggests that it is operative when the NADH/NAD ratio is high, or when the availability of ADP is low. Thus it appears that the rotenone-insensitive path can only become engaged in the presence of a high level of NADH in the mitochondria. However, so far there is no information on the operation of the rotenone-insensitive path in intact roots. In the absence of any suitable inhibitor of the rotenone-insensitive bypass, such evidence will be hard to obtain. 4. Partitioning of Electrons between the Cytochrome Path and the Alternative Path The existence of two mitochondrial respiratory pathways, both transporting electrons from NADH, produced inside the mitochondria, to oxygen, raises the question how the diversion of electrons between these

Lambers et al.

paths is regulated. This is particularly relevant since transport of electrons via the cytochrome path is coupled to proton extrusion and hence oxidative phosphorylation. In contrast, transport of electrons to O2 via the alternative path is not coupled to the generation of a proton-motive force, at least not beyond the branching point with the cytochrome path, i.e., ubiquinone. Bahr and Bonner (1973a,b) initially concluded that simple competition for electrons between the alternative pathway and the cytochrome pathway cannot explain the experimental data obtained with isolated mitochondria. This manner of partitioning of electrons between the two electron transport pathways could be explained by the differential response of the cytochrome path and the alternative path to their common substrate, i.e., reduced ubiquinone. In mitochondria isolated from cotyledons of Glycine max, the activity of the cytochrome path increases linearly with the fraction of ubiquinone that is in its reduced state. In contrast, the alternative path shows no appreciable activity until a substantial (30–40%) fraction of the ubiquinone is in its reduced state, after which the activity increases very rapidly (Dry et al., 1989). Until recently, the widely held view was that the alternative path invariably acts as an ‘‘overflow’’ and never shares electrons with the cytochrome path. However, it has now been established that the activity of the cytochrome path can increase upon inhibition of the alternative path and that the electron transport pathways share electrons from reduced ubiquinone (see Section VI.A). B.

Long-Term Effects

The respiratory apparatus can be modified by the internal and/or by the external environment of the plant. These are relatively slow responses (several hours), compared with the fast responses discussed in Section IV.A. They are likely to significant in the plant’s response to environmental factors such as light and nutrients. After the removal of all but one seminal root of Hordeum distichum seedlings, the soluble sugar concentration and respiration of the remaining seminal root increase (Farrar and Jones, 1986). After pruning of the shoot to one leaf blade, both the soluble sugar concentration and the respiration of the seminal roots decrease. The effects on respiration reflect the coarse control of the respiratory capacity upon pruning or sucrose feeding (Bingham and Farrar, 1988; Williams and Farrar, 1990).

Respiratory Patterns

531

Changes induced by pruning are due to long-term responses of the roots’ respiratory metabolism to the carbohydrate supply (Fig. 4). The protein pattern of the roots of pruned plants is affected within 24 h (Williams et al., 1992). Mitochondria isolated from such roots show changes in respiratory properties and activities of cytochrome oxidase (McDonnel and Farrar, 1992). Glucose feeding to leaves enhances the activity of several glycolytic enzymes in these leaves, due to regulation of gene expression by carbohydrate levels (Krapp and Stitt, 1994). C.

Diurnal Fluctuations in Root Respiration

When plants are grown at a constant temperature, a relatively high light intensity, and a long photoperiod, the rate of root respiration tends to be rather constant throughout the day (Challa, 1976; Veen, 1977; Fig. 3). However, this is not invariably so, and diurnal patterns in CO2 production by roots have been recorded (Neales and Davies, 1966; Osman, 1971). At a high photon input, diurnal patterns in CO2 production are unlikely to be due to fluctuations in the level of carbohydrates (Huck et al., 1962; Farrar, 1981). Rather, they tend to correlate with the rate of ion uptake (Huck et al., 1962; Hansen, 1980; Casadesus et al., 1995). Root respiration is not a simple function of the carbohydrate supply from the shoot. When both CO2 production and O2 consumption are measured, the latter tends to show less diurnal variation (Fig. 5). Possibly, the variation in CO2 production is associated with that in nitrate reduction in the roots. Additionally, it may be associated with variation in the excretion of bicarbonate, which is released upon decarboxylation of malate that is produced in the leaves, coupled to nitrate reduction, and subsequently transported via the phloem to the roots (Ben Zioni et al., 1971). Like root growth, the rate of ion uptake, though correlated with the carbohydrate supply from the shoots, does not simply depend on carbohydrates because these are the source of energy for uptake (Bowling, 1981). Bowling (1981) concluded that ion uptake in Helianthus annuus is controlled by an ‘‘ion uptake controller,’’ imported from the shoots. Both amino acids (Imsande and Touraine, 1994) and glutathione (Lappartient et al., 1999), which are imported via the phloem, are such controlling factors. Simultaneous changes in the rates of ion uptake and root respiration therefore probably reflect the regulation of respiration by adenylates (‘‘energy demand’’). Most likely, root growth, though correlated with the

Figure 5 Diurnal variation in the rate of O2 consumption (nmol O2 g1 ðDMÞ s1 , filled symbols) and CO2 production (nmol CO2 g1 ðDMÞ s1 , open symbols) by roots of Holcus lanatus growing in nutrient solution with 2 mM nitrate. The light period started at 5 a.m. and finished at 7 p.m. Because of greater variation in CO2 production than in O2 consumption, the respiratory quotient (RQ) varies diurnally. (Based on data from Scheurwater et al., 1998.)

supply of carbohydrates from the shoot, is also regulated, rather than actually limited by this supply (McDonnel and Farrar, 1992). We conclude that the diurnal variation in root respiration in plants grown under constant-temperature conditions is a reflection of variation in the energy demand for growth and ion uptake. However, if respiration is controlled by the demand for respiratory energy, the question remains how the variation in this demand originates. This question cannot yet be answered but is of obvious interest to those attempting to model the growth of roots as dependent on carbohydrate supply and ion uptake.

D.

Effects of Plant Hormones

Little work has been done on the effects of plant hormones on root respiration. Some more data are available for other tissues and for isolated mitochondria. However, in the latter case the concentrations required may indicate that the effects which have been reported are not physiologically relevant. Only a brief survey on effects of plant growth substances on respiration is provided here. Markhart (1982) found an increase in CO2 production by Glycine max roots upon exposure to ABA.

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During 6 h of exposure to 44 mmol m3 cis-trans or 6 0 mmol m3 trans-trans ABA, root respiration gradually rises to 130% of the original value. In the absence of more information on which respiratory pathway is stimulated by ABA and on other aspects of carbohydrate transport and metabolism, a satisfactory explanation for this effect of ABA on root respiration cannot yet be given. Respiration of slices of storage tissue of Beta vulgaris roots (Palmer, 1966) increases upon aging. This increase is prevented in the presence of 50– 1000 mmol m3 kinetin. Since the rate of respiration decreases parallel to that of phosphate absorption, the effect of kinetin on respiration may result from a decreased demand for metabolic energy for phosphate uptake. Benzyladenine (108 mol m3 ), a synthetic cytokinin applied to the roots of Plantago major L., delays the decrease in root respiration found upon transfer from a solution with an optimum nutrient supply to a solution where nutrients are limiting (Kuiper and Staal, 1987). In these roots, cytokinins probably increase the roots’ demand for metabolic energy for ion uptake and thus account for the higher rate of respiration in roots exposed to benzyladenine. A number of cytokinins inhibit the oxidation of NADH by submitochondrial particles, but not by intact mitochondria (Miller, 1980, 1982), suggesting that only the oxidation of NADH originating in the tricarboxylic acid cycle, and not that of external NADH, is inhibited. The cytokinins are effective in the range of 50–500 mmol m3 , which is rather high compared with other known physiological effects of these hormones, e.g., on root growth and aerenchyma formation of Zea mays roots, where kinetin is already effective at 10 mmol m3 (Konings and De Wolf, 1984). Preincubation of isolated mitochondria in cytokinin solutions for several minutes does not lower the effective concentration, so it remains obscure if these effects have any physiological relevance. Benzylaminopurine and several adenine analogs, with or without cytokinin activity, inhibit the alternative path (Dizengremel, 1982; Miller, 1982). Titrations with benzyladenine mimic those obtained using SHAM, but the effective concentration is high: between 100 and 1000 mmol m3 . Exposure of Solanum tuberosum tubers to ethylene (10 ppm) causes an upsurge of their respiration rate (Reid and Pratt, 1972). The rate rapidly rises to 5–10 times the level of control tubers within 30 h and then slowly falls. Treatment of potato tubers (Solomos and Laties, 1976; Rychter et al., 1978) with ethylene enhances the capacity of the cyanide-resistant path in

Lambers et al.

mitochondria isolated from these tissues. The extent of the increase depends on the duration of the exposure and on the concentration of ethylene. The physiological meaning of such high concentrations of ethylene on the alternative path remains obscure; it is presumably linked to an increased demand for metabolic energy, e.g., associated with the synthesis of specific proteins (Christofferson and Laties, 1982). Respiration of the storage tissue of beet (Beta vulgaris L.) roots decreases in the presence of 1 mol m3 indolylacetic acid or other auxins. The rate of phosphate absorption also decreases in the presence of these compounds, though not in parallel (Palmer, 1966). A partial explanation for the decreased respiration may be the decreased demand for metabolic energy. However, since phosphate absorption and root respiration did not decrease in the same manner for all auxins, this is unlikely to be the full explanation. Neither respiration nor phosphate absorption by the storage tissue of Beta vulgaris roots are significantly affected by gibberellin (Palmer, 1966). The above compilation of effects of plant growth substances on respiration does not lead to a complete model on cause and effect. Some of the effects described above may lack physiological relevance. Others can possibly be explained by a modified requirement for metabolic energy or by changes in allocation of carbohydrates to the roots. The effects of environmental conditions on root respiration (see Section VII) might be mediated by hormones, but no clear-cut correlations are available. E.

Conclusions

The current view is that root respiration is controlled by a delicate balance between the roots’ demand for ATP (adenylate control) and the availability of respiratory substrate. Adenylates exert their effect both on glycolysis and on the activity of the cytochrome path. Whenever the availability of substrate is large compared with the demand for ATP, e.g., at low temperatures or upon a sudden increase in carbon flow to the roots, firstly, the alternative path may become increasingly engaged. In the long term, the higher availability of respiratory substrate enhances the capacity of various components of the respiratory apparatus, due to gene transcription followed by de novo protein synthesis. It should be kept in mind that many of the elements leading to the present model on the regulation of root respiration need further investigation. For example, the change in oxidation reduction of the disulfide

Respiratory Patterns

bonds of the alternative oxidase in intact roots so far has only been studied in roots of Glycine max (Millar et al., 1998) and a range of Poa species (Millenaar et al., 2001). The in vivo activity of the alternative oxidase can change without a change in the disulfide bonds of the alternative oxidase or in the regulation by pyruvate or other organic acids (Millenaar et al., 1998, 2001). Therefore, although our understanding of the regulation of root respiration has increased dramatically in the last decade, far more work needs to be done before a solid model can be constructed.

V.

GROWTH AND MAINTENANCE OF ROOTS

The first methods to quantify the various energyrequiring processes in roots discerned only two components of root respiration—i.e., respiration for growth, which included that for ion uptake, and respiration for the maintenance of root biomass. Subsequent methods included ion uptake as a third major process requiring respiratory energy (Veen, 1980). Most investigators have not accounted for the engagement of the alternative path. Since variation in the requirement of respiratory energy might be due to variation in the engagement of the alternative path, and since the contribution of this pathway to total root respiration may vary with age (Millar et al., 1998), the energy requirement of roots has been expressed in terms of ATP rather than O2 (Lambers and Van der Werf, 1988). However, most of these data have been obtained using inhibitors to assess alternative path activity, which makes the conversion from oxygen to ATP invalid. A.

Methodology

The rate of root respiration depends on three major energy-requiring processes, i.e., maintenance of root biomass, root growth, and ion uptake. This can be summarized in the following overall equation (Lambers et al., 1998b): r ¼ rm þ 1=Ygrowth RGR þ 1=Utransport TR where r is the rate of root respiration (
mol O2 or CO2 g1 day1 ); rm is the rate of root respiration to produce ATP for the maintenance of biomass; 1=Ygrowth (mmol O2 or CO2 g1 ) is the root respiration to produce ATP for the synthesis of cell material; RGR is the relative growth rate of the roots (mg g1 day1 ); 1=Utransport (mol O2 or CO2 mol1 ) is the rate of

533

respiration required to support TR, the transport rate (
mol g1 day1 ). In roots TR equals the net ion uptake rate (NIR) and the rate of xylem loading. The respiratory energy requirement for maintenance of root biomass is assumed to be linearly related to the fresh or dry mass of the root biomass to be maintained. Similarly, it is assumed that the respiratory energy requirement for anion uptake is proportional to the amount of anions taken up, whereas that for root growth is assumed to be proportional to the relative growth rate of the roots. Thus, the specific costs for all three processes are assumed to be constant during the experimental period. Based on these assumptions, the rate of respiration per gram of roots can be related to the relative growth rate of the roots and the rate of anion uptake by the roots. Superimposed is the maintenance respiration. If there is no tight correlation between growth and ion uptake, the costs of the three processes can be determined using a multiple regression analysis (Van der Werf et al., 1988). The analysis is graphically presented in a three-dimensional graph for the data of two Carex species (Fig. 6A). The dotted plane gives the rate of root respiration (y-axis) as a function of both the relative growth rate of the roots (z-axis) and the net rate of anion uptake by the roots (x-axis). Since the specific costs for maintenance are assumed to be constant, it appears as the intercept of the plane with the y-axis. The plane intersects both the y–z plane and the x–z plane. The slope of the plane with the x-axis and the z-axis give 1=Ygrowth , and 1=Utransport , respectively. Since the processes of root growth and ion uptake are often tightly correlated, it may be necessary to perturb the growth of a plant by pruning roots or shoots to discriminate between the three processes (Bouma, 1995). Alternatively, a linear regression can be carried out and the rate of root respiration can be plotted against the roots’ relative growth rate. In such a plot, the intercept with the y-axis gives the rate of maintenance respiration, whereas the slope includes costs for both root growth and for ion uptake. Figure 6B shows an example for roots of Quercus suber (Mata et al., 1996). B.

Variation Among Species and Among Environmental Conditions

Very few multiple regression analyses of the kind depicted in Fig. 6A have been carried out, because of tight correlations between growth and ion uptake, forcing authors to take refuge in an analysis as given in Fig. 6B. However, we can be quite sure that the specific

534

Lambers et al.

costs for one or more of the three major energy-requiring processes vary both among species (Poorter et al., 1991) and among growth conditions (Van der Werf et al., 1994). This conclusion is based on the observation that the rate of root respiration tends to vary considerably less than that of growth and ion uptake, both among species with different growth potential (Poorter et al., 1991, Atkin et al., 1996; Scheurwater et al., 1998) and among plants with different N supply (Granato et al., 1989; Van der Werf et al., 1992a). The construction costs per unit biomass may vary with the chemical composition of the plant. The oxygen requirement for the synthesis of roots of fast-growing species is higher than that for slow-growing ones, owing to their higher protein content (Poorter et al., 1991), and hence does not account for the relatively high rates of respiration of species with a low growth potential. The specific costs for ion uptake are considerably greater for plants with a low growth potential than for fast-growing ones (Scheurwater et al., 1998; Fig. 7), owing to the relatively greater importance of nitrate efflux (Scheurwater et al., 1999; Mata et al., 2000). This explains the relatively high rate of respiration of inherently slow-growing plants grown with free access to

Figure 6 (A) Rate of O2 consumption (
mol ðg FMÞ1 day1 , in roots of Carex acutiformis and C. diandra as related to both the relative growth rate (RGR) of the roots and their net rate of anion uptake (NIR). (B) Rate of O2 consumption (
mol ðg FMÞ1 day1 ) in roots of Quercus suber as related to the relative growth rate of the roots. The plane in (A) and line in (B) give the predicted mean rate of O2 consumption. The symbols are experimentally determined points (and SE, n ¼ 6 and 8?? in (A) and (B), respectively). The intercept of the plane in (A) and the line in (B) with the y-axis gives m. The slope of the projection of the line on the y–z plane gives 1=Y GR ; when projected on the x–y plane, the slope gives 1=U I . In (B) the slope gives costs for growth including ion uptake. (A Based on data from Van der Werf et al. (1988); B based on data from Mata et al., 1998.)

Figure 7 Respiratory energy budgets for roots of 24 herbaceous species, grown in a nutrient solution with nitrate. The budgets were constructed assuming that the specific costs for maintenance are the same for all species, i.e. similar to the ones for Carex diandra (Fig. 6). Costs for growth were calculated from the roots’ biochemical composition and the remaining part of the experimentally determined rate of O2 consumption was then apportioned to ion uptake. (Based on data from Poorter et al., 1991.)

Respiratory Patterns

nutrients. There is also some indirect evidence that the specific costs for ion uptake increase with a decline in growth rate owing to greater limitation by the nitrogen supply (Van der Werf et al., 1994; Lambers et al., 1998a). Such variation in specific costs with variation in the rate of nitrate uptake might explain why the rate of root respiration does not necessarily increase in proportion with the increase in nitrate uptake (Granato et al., 1989). Specific maintenance costs may also vary, but when grown with free access to all nutrients, the total maintenance costs appear to be small compared with those for growth and ion uptake (Fig. 7). Moreover, current evidence suggests that the specific costs for maintenance respiration of the roots of slow-growing species are very similar to those of inherently fast-growing species and thus cannot explain the relatively high rates of root respiration of the slow-growing species (Scheurwater et al., 1998). One possibility is that the specific maintenance costs increase with decreasing N supply (Van der Werf et al., 1994); they may also become substantial in older plants, in which growth and the rate of ion uptake has slowed down (Van der Werf et al., 1988). Protein turnover is probably a major process contributing to maintenance respiration (De Visser et al., 1992). So far, the only roots for which protein half-life values have been published are those of the fast-growing Dactylis glomerata and of the slow-growing Festuca ovina, both grown at an optimum nutrient supply (Van der Werf et al., 1992b; Scheurwater et al., 2000). These half-life values vary between 5 and 10 days. Assuming biosynthetic costs of 15 mol ATP/ mol peptide bond (Van der Werf et al., 1994), only 15– 30 nmol ATP g1 s1 is needed for the maintenance of the protein pool. These costs account for less than half of the total maintenance respiration, depending on the degradation constant used (Van der Werf et al., 1992a,b). The energy costs associated with maintaining ion gradients across root membranes may represent a substantial portion of the remaining energy requirements of maintenance. Bouma and De Visser (1993) calculated that it may account for most of the roots’ maintenance requirements. Table 7 summarizes the information on specific respiratory energy costs for growth, ion uptake, and maintenance of root biomass, obtained with the methods illustrated in Fig. 6A, a closely related method (Veen, 1980), or a method which separates only two components of respiration. The latter method yields values for maintenance respiration and values for growth, including ion uptake. It should be kept in

535 Table 7A Specific Respiratory Energy Costs for the Maintenance of Root Biomass, for Root Growth, and for Ion Uptakea

Growth, mmol O2 (g DM)1 Maintenance, nmol O2 (g DM)1 s1 Anion uptake, mol O2 (mol ions)1

Carex

Solanum

Zea

6.3

10.9

9.9

5.7

4.0

12.5

1.0

1.2

0.53

a

The values were obtained using a multiple regression analysis (Van der Werf et al., 1988: average values for Carex acutiformis and C. diandra (sedges); Bouma et al., 1996; Solanum tuberosum; Veen, 1980: Zea mays).

Table 7B Specific Respiratory Energy Costs for the Maintenance of Root Biomass and for Root Growth Including Costs for Ion Uptakeb Dactylis Festuca Quercus Triticum Growth þ ion uptake, mmol O2 (g DM)1 Maintenance, nmol (g DM)1 s1

11

19

12

18

26

21

6

22

b The values were obtained using a linear regression analysis (Scheurwater et al., 1998: Dactylis glomerata and Festuca ovina; Mata et al., 1996; Quercus suber; R. Van den Boogaard, unpublished: Triticum aestivum).

mind that the present method, like any other used so far in this area of research, is based on a number of assumptions. Reasonable as these assumptions may appear, it should be stressed that no experimental data are available to support that the specific costs for maintenance and anion uptake do not vary with age. If they do, they will bias the results in a manner that is not quite predictable.

VI.

CAPACITY OF THE ALTERNATIVE PATH

A.

Assessment of Alternative Path Activity

The capacity of the alternative path has been defined as the fraction of respiration that proceeds in the presence of an inhibitor which fully blocks the cytochrome path

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Lambers et al.

and which is sensitive to an inhibitor of the alternative path. Only if the cytochrome path is fully saturated can both the capacity and the activity of the alternative path be assessed using the specific inhibitors of the alternative and cytochrome pathways. However, the cytochrome path is probably never saturated in vivo, since the control coefficient of the cytochrome path to overall flux is quite low (Van den Bergen et al., 1994; Millar et al., 1995). As a result, noninvasive measurements of alternative pathway respiration, using the 18 O isotope fractionation method, should be used (Guy et al., 1989, 1992; Henry et al., 1999). That SHAM additions can result in underestimates of actual alternative pathway activity (see Section IV.A.1) means that previous measurements of alternative and cytochrome pathway activity must be treated with great caution. The previous studies do, however, demonstrate that the degree of SHAM inhibition and KCN resistance do vary between species and treatments. This suggests that differences in alternative and cytochrome pathway activity do occur. Given the low number of noninvasive studies that assess the level of alternative pathway respiration, we must at present rely on the SHAM and KCN results to get some insight into what variations in alternative and cytochrome pathway respiration occur. The following sections therefore present the results of those inhibitor studies. The reader is advised to treat these studies as an indicator of what levels of alternative pathway respiration occur in response to environmental conditions, rather than the changes that do occur.

B.

compatible solute, accumulates. The amount of glucose required for sorbitol synthesis in the roots during the first 12 h (6:6
mol glucose=g fresh mass) matches the amount saved in root respiration by a temporary reduction in (SHAM-sensitive) root respiration (62
mol glucose/g dry mass; Fig. 8). When osmotic adjustment is completed, the SHAM sensitivity returns to the level before the water potential decreased. The two examples cited above have been used to illustrate that the activity of the alternative path in roots appears to decrease when the availability of carbohydrates for respiration decreases. On the other hand, it supposedly increases when the demand for

Physiological Significance of the Alternative Path

In the short term, inhibition of respiration by SHAM decreases with decreasing carbohydrate concentration in the roots (Fig. 3). However, when plants are exposed to a low light intensity for an extended period, SHAM inhibition of root respiration is the same as in plants grown at a higher light intensity (see Section VII.A.7). Upon lowering the water potential in the root environment of Plantago coronopus, the demand for carbohydrates for osmotic adjustment increases, and sorbitol, a * There is at least one exception where the capacity of the alternative path cannot be determined in this manner—that is, when the capacity of the alternative path exceeds the rate proceeding in the presence of an inhibitor of the cytochrome path. In practice it means that there must be some inhibition of respiration by an inhibitor of the cytochrome path to assess the capacity of the alternative path.

Figure 8 Root respiration and sorbitol accumulation in the roots of Plantago coronopus. (A) SHAM-resistant root respiration; (B) SHAM-sensitive respiration; (C) sorbitol accumulation. Plants were pregrown in a nonsaline nutrient solution and transferred at time zero to a nutrient solution containing 50 mol m3 NaCl (closed symbols). Control plants (open symbols) were kept in a nonsaline solution throughout. (Based on data from Lambers et al., 1981.)

Respiratory Patterns

carbohydrates for other processes decreases, so more is left for respiration. Considering that the interpretation of the results was based on inhibitor studies, no firm conclusion is possible. In fact, in roots of Poa annua the alternative pathway activity (assessed with the 18 O isotope fractionation technique) was constant for 4 days after the transfer from high light and short nights to low light and long night (Fig. 9). During this period both the activity of the cytochrome pathway and rate of total respiration decreased, coinciding with a decline in the soluble sugar concentration (Millenaar et al., 2000). In addition to changes in the carbohydrate availability, changes in the requirement for respiratory energy may also affect the SHAM sensitivity of root respiration. De Visser et al. (1986) found a transient increase in SHAM sensitivity in Pisum sativum roots upon addition of nitrate to 2-week-old nitrogendeprived plants. In older pea plants, whose cytochrome path was no longer controlled by adenylates, an increased SHAM sensitivity was found upon addition of nitrate. Variation in SHAM sensitivity of respiration is transient and tends to be followed by further adjustments in the roots’ metabolism. The transient changes in SHAM sensitivity suggest that the nonpho-

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sphorylating path may play a role in coping with relatively rapid changes in the environment. As postulated by Purvis and Shewfelt (1993), a possible general function of the alternative pathway is to stabilize the reduction state of the ubiquinone pool, thereby preventing the extra production of oxygen free radicals. The in vivo reduction state of the ubiquinone pool can be stabilized by the alternative pathway (Millenaar et al., 1998). Transgenic tobacco plants that have a lower AOX expression show an increased oxygen free-radical production (Maxwell et al., 1999). The function of the alternative pathway seems to be the prevention of the formation of extra oxygen free radicals. However, there is no full proof, since there is no published report in which oxygen free-radical production, AOX activity, superoxide dismutase (SOD) activity, and the reduction state of the ubiquinone pool have been measured. VII.

Any factor that affects the respiratory energy requirement or the balance between carbohydrate supply and carbohydrate demand in the roots is bound to affect root respiration. Both the rate of respiration and the partitioning of electrons between the cytochrome path and the alternative path may be affected by such environmental conditions. In the following section both biotic and abiotic environmental factors, known to affect root respiration, are discussed. As mentioned earlier, SHAM additions frequently result in underestimates of actual alternative pathway activity (Section VI.A). The reader is therefore advised to treat the following studies as an indicator of what changes in alternative pathway respiration may occur in response to environmental conditions. A.

Figure 9 The activity (nmol O2 g1 FM1 ) in Poa annua roots of the alternative oxidase (circles) and cytochrome pathway (squares) with sucrose (open symbols) or without sort-term sucrose addition (closed symbols). Measurements were made with the 18 O isotope-fractionation technique and at different times after the transfer to low-light conditions. Error bars represent standard error (n ¼ 2 to 5). (Based on data from Millenaar et al., 2000a.)

EFFECTS OF ENVIRONMENTAL CONDITIONS

Abiotic Factors

Most environmental effects on root respiration can be understood on the basis of the regulation of respiration as discussed in Section IV.A. In the following sections we analyze some pertinent effects of nutrient supply, adverse soil conditions, including salinity and drought, temperature, and light conditions. 1.

Nutrient Supply

The well-documented rapid effect of ions on root respiration, coined ‘‘salt respiration’’ (Lundegardh, 1946, 1955), is probably partly due to the increased

538

demand for respiratory energy for ion transport (De Visser et al., 1986). Part of it might be due also to a replacement of osmotically active sugars by inorganic ions, leaving a large amount of sugars to be respired. A sudden increase of the uptake and assimilation of nitrogen can have a significant effect on root respiration. For example, exposure of nitrogen-starved Triticum aestivum seedlings (pregrown on CaSO4 ) to nitrate results in a transient increase in root respiration (Barneix et al., 1984). This may be due to the increased demand for ion transport and nitrogen assimilation. Addition of ammonium to the CaSO4-grown roots resulted in an even larger increase (200%) in total respiration (Barneix et al., 1984). Exposure to nitrate can result in higher rates of oxygen consumption relative to plants exposed to ammonium (Lambers et al., 1980; De Visser and Lambers, 1983), possibly reflecting the greater demands for metabolic energy associated with nitrate uptake (Pate, 1983). However, in other cases, a decrease in oxygen consumption is reported (Blacquie`re et al., 1987; Bloom et al., 1992; Atkin and Cummins, 1994, 1995). Bloom et al. (1992) found that lower rates of respiration in nitrate-grown Hordeum vulgare plants depend on the ability of the roots to reduce nitrate: exposure of ammonium-grown plants to nitrate only results in a decreased oxygen consumption when the root has the ability to induce nitrate reductase. No effect of nitrate treatment was observed in plants deficient in nitrate reductase (Bloom et al., 1992). A rise in the RQ is also associated with the induction of nitrate reductase activity in Hordeum vulgare (Willis and Yemm, 1955; Jessop and Fowler, 1977; Bloom et al., 1992). The reduction in oxygen consumption and the rise in RQ of nitratetreated roots (relative to ammonium) may be due to competition for reductant by nitrate reductase (Reed and Hageman, 1977; Sawhney et al., 1978; Oaks and Hirel, 1985; Bloom et al., 1992). Under steady-state conditions, when all plants are growing at the same rate, the respiration of Pisum sativum roots is the same with nitrate and with ammonium as the growth-limiting source of N (De Visser and Lambers, 1983). Somewhat higher rates of root respiration occur in Plantago lanceolata and P. major when grown with an optimum supply of ammonium than when grown with nitrate (Blacquie`re et al., 1987). When plants are grown at a low supply of nutrients, their rate of root respiration is lower than that of plants well supplied with mineral nutrients (Kuiper, 1983; Van der Werf et al., 1992a). Under phosphate deprivation, there is no effect on the rate of root

Lambers et al.

respiration of Phaseolus vulgaris (Rychter and Mikulska, 1990), which is distinctly different from the situation under limitation of all nutrients. It appears that the capacity of the cytochrome path is greatly reduced, as demonstrated by the much lower rate of respiration in the presence of an uncoupler and SHAM under phosphate deprivation. The decline in activity of the cytochrome path may be compensated by an increased activity of the alternative path, as suggested by a relatively strong effect of SHAM in vivo (Rychter and Mikulska, 1990). Further studies on isolated mitochondria revealed that the activity of cytochrome oxidase was reduced in the roots of phosphatedeficient bean plants (Rychter et al., 1992). 2.

pH

Root respiration rates can be affected by soil pH. For example, Yan et al. (1992) reported that root respiration rates of Zea mays and Vicia faba increase as nutrient solution pH values decrease. Net Hþ release by Hþ -ATPase activity is a basic necessity for continued root growth, and limits root growth at very low, critical pH values (Van Beusichem, 1982; Schubert et al., 1990). One way of coping with excess Hþ uptake is to increase active Hþ pumping by plasma membrane ATPases: such a response will increase the demand for ATP. Yan et al. (1992) suggested that the increased root respiration in response to noncritical low pH values is the result of increased Hþ ATPase activity (Yan et al., 1992). Increased respiration rates can therefore allow plants to maintain root growth at noncritical low pH values, by increasing the supply of ATP for Hþ pumping by plasma membrane ATPases. At very low, critical pH values (< 3:5 and 4.1 for Zea mays and Vicia faba, respectively), root growth, net Hþ release, and respiration rates decline (relative to rates at pH 7.0; Yan et al., 1992). Limitations in Hþ pumping do not appear to be responsible for the limited net Hþ release and root growth below the critical pH values. Rather, the increased entry of Hþ into the roots appears to be responsible (Yan et al., 1992). Such increased uptake of Hþ would tend to disturb cytosolic pH and ultimately root growth (Gerendas et al., 1990). The decrease in root respiration at very low pH values might therefore be the result of decreased demand for growth respiration and/or the direct effects of low cytosolic pH on the respiratory pathways. 3.

Aluminum

High concentrations of soluble aluminum also affect root respiration rates (Tan and Keltjens, 1990a,b;

Respiratory Patterns

Collier et al., 1993; De Lima and Copeland, 1994) in soils of low pH: the solubility of aluminum increases with decreasing pH (Foy et al., 1978; Fageria et al., 1988). The effects of aluminum on root respiration depend on whether whole root systems or root tips are considered. In intact whole roots, Collier et al. (1993) found an increase in respiration in both aluminum-tolerant and -sensitive cultivars of Triticum aestivum, during growth in the presence of a wide range of aluminum concentrations (0–900 mmol m3 ). The increase is entirely due to an enhanced level of SHAM-resistant respiration. The pattern of the changes in respiration is similar for the tolerant and the sensitive cultivar, in that the peak in respiration occurs at a lower aluminum concentration for the sensitive cultivar (100 vs. 350 mmol m3 ). At higher aluminum concentrations, respiration rates gradually decline again to the control rates. This coincides with effects on growth, which is not affected until much higher aluminum concentrations, in the tolerant cultivar. A similar stimulation of root respiration has been observed for a sensitive (Tan and Keltjens, 1990a) and a tolerant (Tan and Keltjens, 1990b) cultivar of Sorghum bicolor. De Lima and Copeland (1994) found an inhibition of the respiration of excised root tips of a sensitive cultivar of Triticum aestivum, even at 75 mmol m3 aluminum. Initially (12 h after exposure), only SHAM-resistant respiration is inhibited, whereas later SHAM-sensitive respiration is also reduced. Mitochondria isolated from roots of exposed plants have a diminished oxidative capacity. The data on whole root systems and those on root tips suggest that the latter are inhibited more readily by aluminum. The increase in respiration of the intact roots suggests that root functioning in the presence of aluminum imposes a demand for additional respiratory energy. These increased costs have little to do with the mechanism explaining tolerance, i.e., excretion of chelating organic acids, since such excretion does not occur to any major extent in the sensitive cultivar (Delhaize et al., 1993). 4.

Heavy Metals

Root respiration appears to be inhibited by lead. For example, Koeppe (1977) found that respiration of Zea mays root tips decreases by up to 40% within 3 h of exposure to 20 mol m3 lead. The degree of inhibition increased with the duration of exposure. Concomitant with the decrease in respiration of the root tips was a decrease in the energy charge of the treated tissue

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(Koeppe, 1977). The response of whole root systems to lead is not clear (see Section VII.A.3 for a comparison of root tips and whole root systems). It is also not clear to what extent respiration of roots is affected by lead in natural soils. Exposure of mitochondria isolated from etiolated Zea mays seedlings (using succinate as a substrate) to lead also results in a 80% decrease in respiration (Koeppe and Millar, 1970). The addition of inorganic phosphate reverses the lead-induced succinate inhibition. Koeppe (1981) suggested that lead decreased oxidative phosphorylation. Cadmium and zinc also appear to inhibit seedling mitochondrial respiration in a manner similar to that of lead (Bittell et al., 1974). 5.

Salinity and Drought

The rate of root respiration of the salt-tolerant Plantago coronopus declines when the plants are transferred from a nonsaline nutrient solution to one containing 50 mol m3 NaCl (Fig. 8). Prolonged exposure to 50 mol m3 NaCl, a concentration sufficiently low not to affect growth, has no effect on the rate of root respiration or the engagement of the alternative path (Blacquie`re and Lambers, 1981). This similarity in growth and respiratory pattern under saline and nonsaline conditions suggests that for roots of P. coronopus the respiratory costs to cope with this salinity level are negligible. Similar results were obtained for other salt-tolerant species, e.g., Plantago maritima (Lambers, 1979). Root respiration of the salt-tolerant gray mangrove Avicennia marina (Forsk.) is stimulated by moderate concentrations of NaCl, which stimulate growth, and inhibited by higher concentrations, which are inhibitory for growth (Burchett et al., 1984); that is, root respiration follows the pattern of growth. Different patterns are found for species that do not tolerate high levels of salinity. Root respiration of two Hordeum vulgare cultivars increased upon exposure to 10 mol m3 NaCl or KCl (Bloom and Epstein, 1984). Exposure to a solution from which ions can be accumulated may lead to the replacement of sugars by inorganic ions and thus cause increased engagement of the alternative path. Alternatively, the increased respiration might reflect the increased costs for ion transport. Williams et al. (1991) exposed half of the roots of Hordeum distichum to a nonpermeating solute (mannitol or sorbitol at 0:4 MPa). This leads to a rapid but transient increase in (11 C- or 14 C-labeled) carbon import and O2 consumption of the exposed root half, without any effect on the other part of the root system. The increased import of carbon may be the cause for

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the rise in respiration, but it cannot be excluded that the causal relationship is actually the other way around. The rise in respiration may be due to a rise in the engagement of the alternative path or reflect the energy demand of osmotic adjustment. The respiration of those roots of the salt-sensitive Opuntia ficus-indica which are exposed to 30 or 100 mol NaCl m3 increases, compared to that of roots of the same plants growing without NaCl. Contrary to the results with H. distichum, this is not a transient effect; it is associated with a reduction in growth of the exposed roots (Gersani et al., 1993). The rate of root respiration (expressed per g DM) of Lycopersicon esculentum and L. pennellii exposed to 100 mol m3 NaCl for 10 days decreases by 10% and 15%, respectively, in comparison with plants grown in a nonsaline root medium (Taleisnik, 1987). At this salinity level, growth of both species is reduced, which may offer an explanation for the reduced respiration rate. Exposure to a dry soil leads to a gradual decline in root respiration of Triticum aestivum, predominantly of the SHAM sensitivity (Nicolas et al., 1985). The decline in respiration correlates with the accumulation of organic solutes. The authors interpreted the data in the same manner as those on the short-term exposure of Plantago coronopus to NaCl (Fig. 8), i.e. accumulation of osmotic solutes reduces the availability of sugars. This subsequently leads to less grist for the mill of the alternative path. The difference in response when comparing different species is likely to be associated with the extent to which growth is affected. Plants whose growth is unaffected by salinity show a transient decline in root respiration or no response at all. In contrast, root respiration either is enhanced when growth is inhibited by salinity or declines parallel to the inhibition of growth. The respiratory costs of functioning in a saline environment for adapted species that accumulate NaCl are likely to be relatively small. For salt-excluding glycophytes this conclusion may be quite different. Experimentation, in the sense as illustrated in Fig. 7, may provide further insight. 6. Temperature Root respiration increases as a function of temperature, with the degree of increase being dependent on the temperature coefficient (Q10 ) of respiration. Q10 values range between 1.1 and 2.9. Differences in Q10 values occur in some plants experiencing contrasting growth temperatures (e.g., Gifford, 1995; Fitter et al., 1998). Moreover, although the Q10 of root respiration

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is constant over a broad range of temperatures in many studies (e.g., Tjoelker et al., 1999b), several others have reported that the Q10 varies with measurement temperature (e.g., Higgins and Spomer, 1976; Crawford and Palin, 1981; Berry and Raison, 1981; Palta and Nobel, 1989). Clearly, substantial variability in the temperature sensitivity of root respiration often occurs. The rate of root respiration at any given measuring temperature may also depend on the degree to which the roots have acclimated to the growth temperature. Acclimation of respiration to the growth temperature results in homeostasis of respiration, such that warmacclimated and cold-acclimated plants display similar rates of respiration when measured at their respective growth temperatures (Ko¨rner and Larcher, 1988; Atkin et al., 2000). Acclimation of root respiration to temperature occurs in Plantago lanceolata (Smakman and Hofstra, 1982) and Zostera marina (Zimmerman et al., 1989). However, no acclimation occurs in roots of Picea glauca (Weger and Guy, 1991), Picea engelmannii, or Abies lasiocarpa (Sowell and Spomer, 1986). Similarly, while acclimation to changes in growth temperature results in near-perfect homeostasis in Citrus volkameriana in wet soils, no acclimation occurs in roots of the same species growing in dry soils (Bryla et al., 1997). In species in which no thermal acclimation takes place, root respiration rates will depend entirely on ambient temperature, with low temperatures severely restricting root metabolism (Weger and Guy, 1991). In addition to the acclimation potential of total root respiration, acclimation may also occur within partitioning of electrons between the cytochrome and alternative pathways. For example, transfer of Plantago lanceolata roots from 21 C to 13 C initially resulted in a transient decline in SHAM-resistant respiration and an increase in SHAM-sensitive respiration (after 2 h of exposure; Smakman and Hofstra, 1982). Extended exposure ð> 2 hÞ to 13 C resulted in recovery in SHAM resistance to its original level (Smakman and Hofstra, 1982). The response of SHAM-sensitive respiration to extended 13oC exposure was more complex, first declining to a very low level, and then returning to its original 21 C rate within 24 h. Transfer from 13oC to 21oC causes similar acclimation in SHAM-resistant/-sensitive respiration (Smakman and Hofstra, 1982). Presumably, some adjustment to temperature at the level of energy-requiring processes or of the mitochondria occurs in roots that exhibit acclimation of electron transport chain partitioning. Such mitochondrial

Respiratory Patterns

adjustment occurs in callus-forming potato tuber disks: when grown at 28 C, the total respiratory capacity and the cyanide resistance of isolated mitochondria are greater than when grown at 8 C (HemrikaWagner et al., 1982). In contrast to the data on Plantago lanceolata, Weger and Guy (1991) did not find any acclimation of root mitochondrial electron transport to the growth temperature for Picea glauca. At any one temperature, SHAM-sensitive and SHAM-resistant respiration rates were independent of the growth temperature (4, 11, or 18 C; Weger and Guy, 1991). Thermal acclimation of root respiration, therefore, appears to depend on both species and environment. The probable mechanisms that underlie differences in the degree of acclimation (and Q10 values) are discussed in Atkin et al. (2000). The concentration of AOX protein often increases after transfer to lower temperatures (Vanlerberghe and McIntosh, 1992; Gonza´lez-Meler et al., 1999). Also KCN-insensitive, SHAM-sensitive respiration increases as found frequently (Vanlerberghe and McIntosh, 1992, and references therein). However, in a chilling-sensitive maize cultivar the activity of the alternative pathway (18 O isotope fractionation) was higher during the recovery period than in a less chilling-sensitive cultivar (Ribas-Carbo et al., 2000). Some plant species that are characteristic of cold environments exhibit inherently higher root respiration rates relative to species characteristic of warmer environments. For example, Higgins and Spomer (1976) found that root respiration rates were inherently higher in the alpine Geum rossii than in the subalpine Geum trifolium L. Similarly, alpine populations of Achillea millefolium had higher respiration rates than populations from more subalpine habitats (Higgins and Spomer, 1976). Soil temperature decreases with increasing elevation or latitude (Higgins and Spomer, 1976). The differences between the alpine and subalpine plants were maintained even when all plants were grown under identical conditions (cold and warm soils). Similarly, Sowell and Spomer (1986) reported that high-elevation populations of Picea engelmannii and Abies lasiocarpa had inherently higher root respiration rates than populations collected from warmer, lower elevations, regardless of the growth treatment or measuring temperature. In contrast, Keller (1967, as cited by Sowell and Spomer, 1986) found no consistent trend in root respiration rates of high- and low-elevation populations of those two species. Although high-altitude populations of Picea abies had inherently higher

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respiration rates than low-altitude populations, the opposite was observed in Larix decidua populations. Similarly, specific rates of root respiration were not higher in alpine Poa species than lowland Poa species, when both groups of species were grown under common, constant environment conditions (Atkin et al., 1996). Thus, the relationship between elevation (and thus soil temperature) and respiration appears to be species dependent.

7.

Light Conditions

As illustrated in Fig. 3, the rate of root respiration and the SHAM sensitivity are low when the carbohydrate concentration in the roots is low. However, as long as the nights are not too long, the rate of O2 consumption tends to be constant throughout the entire day, also when plants are grown at a relatively low light intensity (see Section IV.C). During growth at a low light intensity, the rate of root respiration of Plantago major is 30–50% lower than that of plants grown at a high light intensity (Kuiper and Smid, 1985). Similar results have been obtained for many other species (Lambers and Posthumus, 1980; Poorter, 1991; Millenaar et al., 2000). This lower respiration rate is likely to be associated with the low metabolic activity of roots, when plants grow at a low light intensity. Four days after transfer of Poa annua plants from an environment of high light intensity and short nights to one of a low light intensity and long nights, the sugar concentration and total respiration decreased to 10% and 60%, respectively. During this low-light period, the concentration and reduction state of the alternative oxidase protein did not change as compared with that before the transfer (Fig. 10). Also, the activity of the alternative oxidase did not change (Fig. 9), despite the large decrease in sugar concentration (Millenaar et al., 2000).

Figure 10 Immunoblot of alternative oxidase in whole-root extracts of Poa annua at different times after the transfer to low light conditions (0 ¼ control, and day 1, 2, and 3 after the transfer). (Based on data from Millenaar et al., 2000a.)

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8. Partial Pressures of O2 and CO2 in the Rhizosphere The partial pressures of both O2 and CO2 in the soil may differ vastly from those in normal air, especially upon flooding. Under such conditions the partial pressure of O2 drops to very low levels, and it is well documented that plants that occur naturally in flooded soils have a well-developed aerenchyma, allowing diffusion of O2 to the roots (Jackson and Armstrong, 1999). Such an aerenchyma avoids inhibition of respiration due to lack of O2 , which is inevitable for plants that are not adapted to wet soils (Perata and Alpi, 1993). CO2 partial pressures increase upon flooding of the soil. Nobel and Palta (1989) found values of 2400 and 4170
L CO2 L1 (0.2% and 0.4%, respectively) in flooded soils supporting the growth of desert succulents, as opposed to 540 and 1080
L L1 in the same soils when well drained. Under flooded conditions, the O2 concentration in these soils is virtually the same as that of well-drained soils. Considerably lower O2 and higher CO2 concentrations have been found by other authors, depending both on soil type and the metabolic activity of the plant cover. For example, Good and Patrick (1987) found O2 concentrations as low as 1.2% and CO2 concentrations of 56% and 38% in silt loam, supporting the growth of Fraxinus pennsylvanica and Quercus nigra, respectively. In the air spaces of these roots, CO2 concentrations of 10.4 (Fraxinus) and 15% (Quercus) were found. Compared with the wealth of papers on the inhibition of leaf respiration by an elevated CO2 concentration (700 vs. 350
L CO2 L1 in normal air; e.g., Drake et al., 1999), little research has been done on effects of CO2 on root respiration. Nobel and Palta (1989) found a reversible inhibition of root respiration by 5000
L CO2 L1 of 35% and 46% for the cacti Opuntia ficusindica and Ferocactus acanthodes, respectively. For both species, root respiration is fully inhibited by 20,000
L CO2 L1 (2%), which is an irreversible effect if lasting for 4 h, leading to death of cortical cells. Very similar effects were found for another desert succulent: Agave deserti (Palta and Nobel, 1989). Death of the cortical cells is unlikely to be due to inhibition of respiration, since exposure to a root atmosphere without O2 for a similar period is fully reversible for the same plants. The effects of CO2 on root respiration of desert succulents are possibly indirect, and due to inhibition of energy-requiring processes. However, the effects may also in part be direct, since Palet et al. (1991) and Gonza´lez-Meler et al. (1996) showed that the

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activity of the cytochrome path is inhibited by CO2 , apparently at the level of cytochrome oxidase. Qi et al. (1994) and Burton (1997) also found inhibition of root respiration by soil CO2 levels in a range normally found in soil for C3 plants (Pseudotsuga menziessii and Acer saccharum, sugar maple), whereas no such inhibition was found for a range of other species studied by other authors (e.g., Bouma et al., 1997a,b; Scheurwater et al., 1998). Because respiration is only affected by CO2 , and not by bicarbonate (Palet et al., 1992), the pH of the root environment might affect experimental results. The information in the literature is too scanty to conclude that the effects observed for desert succulents are valid for other plants also. However, long-term exposure to CO2 concentrations in the root gas phase as high as 10.4% and 15.0% allows growth of Fraxinus pennsylvanica and Quercus nigra, respectively (Good and Patrick, 1987). Also, growth of Avena sativa (Stolwijk and Thimann, 1957) and Nicotiana tabacum (Williamson and Splinter, 1968) is relatively little affected by exposure to 6.5% and 18.5% CO2 , respectively. Roots of other species (Pisum sativum, Phaseolus vulgaris, and Vicia faba; Stolwijk and Thimann, 1957) are more sensitive; e.g., cell division of Vicia faba is inhibited as soon as the CO2 concentrations exceeds 6% (Williamson 1968), and the growth of Triticum aestivum L. is less when the roots are exposed to aerobic conditions with 10% CO2 compared with an aerobic control (Trought and Drew, 1980). However, it appears that most species are considerably less sensitive to a high CO2 concentration in the root environment than are desert succulents. In the absence of further information, it would seem that root respiration of plants other than desert succulents is not particularly sensitive to CO2 . What, then, is so special about the respiration of desert succulents? Do the investigated desert succculents, all of which are CAM plants, perhaps have a large capacity for net CO2 fixation via PEP carboxylase in their roots? Hew (1976) provided evidence for CO2 fixation by aerial roots of tropical orchids, showing CAM metabolism in their thick leaves. CO2 assimilation is only apparent during the day, suggesting that it may not be mediated by PEP carboxylase and lead to malic acid production. To our knowledge, there is no information available on the significance of net dark CO2 fixation in nonaerial roots of CAM plants other than that which normally occurs in all roots to provide the major substrate for the mitochondria (Bryce and ap Rees, 1985b). If net dark CO2 fixation via PEP carboxylase does occur, it might lead to a rapid accumulation of malic acid

Respiratory Patterns

beyond what can be stored in vacuoles or exported via the xylem. This might lead to acidification of the cytosol and be the cause of the death of cortical cells (Nobel and Palta, 1989). The lack of such presumed capacity for net CO2 fixation in non-CAM plants might account for their relatively low sensitivity to the CO2 concentration in the rhizosphere; however, so far this is mere speculation. B.

Biotic Factors

1.

Effects of Symbiotic Organisms

Carbon costs of N assimilation of legumes have been extensively studied by Pate and coworkers (e.g., Pate et al., 1979). The costs of N2 fixation are invariably higher than those of nitrate assimilation (Table 2). Ryle and coworkers made a more detailed analysis of the respiratory costs of N2 fixation. When the oxygen concentration surrounding nodulated roots is lowered to 3%, which is sufficiently low to completely stop the respiration of the nodules but high enough for that of the roots to proceed, the production of carbon dioxide declines to
30% of the control roots (Ryle et al., 1984). Using this approach, the respiration of roots has been separated from that of nodules. For Glycine max, completely dependent on the symbiont Rhizobium for its nitrogen supply, the specific respiration rate of the nodules is almost five times higher than that of the roots (Ryle et al., 1984). The average respiratory costs of N assimilation in these nodulated roots is 13.2 mg CO2 (mg NÞ1 , of which 80% is associated with the activity of nitrogenase and ammonium assimilation and the rest with growth and maintenance of nodules. Assuming a respiratory quotient of 1.4 (De Visser, 1985) and an ADP:O ratio of 3 (which is a likely value, since the alternative path does not contribute to respiration in infected cells of nodules; Kearns et al., 1992), the cost can be expressed in the same units as the uptake of ions (Table 7). Since nitrate is the major anion taken up, the main part of the respiratory energy for anion uptake is for the absorption of nitrate. In addition, the values presented in Table 7 may include some costs for synthesis of amino acids to be translocated to the shoot and for transport of nitrogenous compounds to the shoot (Van der Werf et al., 1988). A valid comparison with the values for anion uptake and the costs for symbiotic fixation of nitrogen and associated costs is thus possible. The cost for the symbiotic system is 18 mol ATP (mol NÞ1 . A comparison with the data on the cost of a nonsymbiotic system using nitrate shows that the assimilation of atmo-

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spheric nitrogen in symbiosis with Rhizobium is a costly process. This explains why the respiration of nodulated roots of Trifolium repens is largely associated with the process of nitrogen fixation (Ryle et al., 1985a). Expressed in the units used in Table 2: in T. repens, N2 fixation requires 23% of the photosynthates produced daily (Ryle et al., 1985b). The legume–Rhizobium symbiosis has been studied more intensively than other systems, e.g., where cyanobacteria or actinomycetes are the N2 -fixing symbionts. Some work has been done on the Alnus rubra-Frankia system (Tjepkema and Winship, 1980; Winship and Tjepkema, 1982) and the Cycas circinalis-Nostoc system (Tredici et al., 1988). The carbon costs of another symbiotic system, that between higher plants and mycorrhizal fungi, has been given less attention. Mycorrhizal roots of Vicia faba (Pang and Paul, 1980), Allium porrum (Snellgrove et al., 1982), Glycine max (Harris et al., 1985), Plantago major (Baas et al., 1989), and Citrus volkameriana (Peng et al., 1993) have a higher rates of respiration than nonmycorrhizal ones. However, the rates of root respiration of mycorrhizal and nonmycorrhizal Trifolium subterraneum plants are the same (Silsbury et al., 1983). The costs of the mycorrhizal symbiosis have been estimated in different ways, e.g., by comparing the translocation of 14 C assimilates to the mycorrhizal and the nonmycorrhizal half of a split-root system (Koch and Johnson, 1984; Douds et al., 1988), by measuring the flow of 14 C-labeled assimilates into soil and external hyphae (Jakobsen and Rosendahl, 1990), and by comparing the rate of root respiration of nonmycorrhizal with that of mycorrhizal plants growing at the same rate (Snellgrove et al., 1982, Baas et al., 1989). The estimates vary between 4% and 20% of the carbon fixed in photosynthesis. However, symbiotic plants tend to have a higher rate of photosynthesis per plant, partly owing to their greater leaf area. Therefore, as long as phosphate is limiting, mycorrhizal plants usually grow faster, despite the large carbon sink of the symbiotic system. Baas et al. (1989) further investigated the increase in root respiration in Plantago major ssp. pleiosperma infected with the vesicular-arbuscular mycorrhizal fungus Glomus fasciculatum. In comparison with nonmycorrhizal plants growing at the same rate, the estimated rate of ATP production in root respiration is increased by 80% (Fig. 11). Since mycorrhizal P. major plants absorb more ions from the substrate than the nonmycorrhizal ones, Baas and coworkers analyzed whether the increased respiration could be explained by the

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increased energy demand for ion uptake. They concluded that only a minor part (15%) of the increased rate of ATP production is associated with an increased rate of ion uptake by the mycorrhizal roots. The major part (83%) is explained by the respiratory metabolism of the fungus and/or other effects of the fungus on the roots’ metabolism. Construction costs of fibrous roots are also higher for mycorrhizal than for nonmycorrhizal roots owing to their higher fatty acid concentration (Peng et al., 1993). 2. Effects of Parasitic Organisms Root respiration of both Brassica campestris and Lycopersicon esculentum is
50% higher in plants infected by the angiosperm parasite Orobanche aegyptica or O. cernua than in control plants (Singh and Singh, 1971). The respiration is increased most near the point of infection. Tap roots of Beta vulgaris plants, infected with beta virus 4, also respire more than uninfested plants, except when stored at a rather cool temperature of 2 C (Lo¨hr and Mu¨ller, 1952). Infection of Ipomoea batatas root tissue slices by the parasitic fungus Ceratocystis fimbriata ex. Ipomoea batatas (14137 C.F. Andrus) induces a fourfold increase in the rate of respiration in 40 h (Uritani and Asahi, 1980). The pattern of the increase in respiration is biphasic. The first phase, reaching a maximum after 20 h, occurs also in wounded tissue. It is probably associated with the normal wound response and presumably due to an increased energy demand for the synthesis of cell walls and other compounds. The second phase is more specifically associated with the infection and may be caused by the increased energy demand for the production of various metabolites (e.g., phytoalexins) whose synthesis is induced upon infection. In leaves, an increase in respiration upon infection with powdery mildew appears to be associated with an increase in activity of both the cytochrome and the alternative path (Farrar, 1992). Upon attack by pathogens, energy-requiring processes tend to increase. Infection of roots of a susceptible variety of Lycopersicon esculentum by the nematode Meloidogyne incognita, race 2, Kofoid and White, root respiration first increases but returns to the level of uninfested plants 8 days after inoculation (Zacheo and Molinari, 1987). A resistant variety responds in exactly the opposite way: at first there is no effect on root respiration, but after 8 days the rate exceeds that of control plants. The initial increase in the susceptible plants is associated with an increased

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SHAM sensitivity, whereas the much slower response in the resistant plants is not. Further studies using the oxygen isotope–fractionation technique are required before any conclusion can be reached on the contribution of the alternative path in the observed changes in respiration.

VIII.

CONCLUDING REMARKS

In the last decade our understanding of the regulation of root respiration and of the carbon costs of the various processes in roots of higher plants has increased considerably. Some specific areas need further development. In view of recent developments on the biochemical regulation of the alternative oxidase in isolated mitochondria, the control of the activity of the alternative path in intact roots deserves further attention. Our understanding of the specific respiratory cost that is associated with ion uptake in fast- and slow-growing species has increased substantially. However, we still have no information on the biochemical basis of the likely environmentally induced variation in the specific costs of ion transport. A full understanding of the survival value of the nonphosphorylating, alternative pathway, which may affect the carbon costs of the roots’ functioning to a major extent, is still lacking. Further development is to be expected from investigations of genetically modified plants which lack alternative path capacity, due to a transformation using antisense DNA (Vanlerberghe et al., 1994). The fraction of carbon produced in photosynthesis that is subsequently utilized in the growth and respiration of roots and their symbionts is certainly of major quantitative importance. Recent developments that are described in the present chapter have uncovered some of the carbon costs of ‘‘the hidden half.’’ This information may prove useful in future modeling of plant growth and has already helped us to understand optimum patterns of investment in relation to environmental conditions (Van der Werf et al., 1993). Only too often such models incorporate intricate details of the production of carbon in photosynthesis, while only including ballpark estimates of the consumption of carbon for growth and respiration in roots. Since information compiled in this chapter demonstrates that there is no fixed ratio between the utilization of carbon for root growth and that for root respiration, more detailed information may have to be included in models referred to above. Such information is now widely available for a range of species.

Respiratory Patterns

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Lambers et al. Scheurwater I, Cornelissen C, Dictus F, Welschen R, Lambers H. 1998. Why do fast- and slow-growing grass species differ so little in their rate of root respiration, considering the large differences in rate of growth and ion uptake? Plant Cell Environ 21:995–1005. Scheurwater I, Clarkson DT, Purves J, Van Rijt G, Saker L, Welschen R, Lambers H. 1999. Relatively large nitrate efflux can account for the high specific respiratory costs for nitrate transport in slow-growing grass species. Plant Soil 215:123–134. Scheurwater I, Du¨nnebacke M, Eising R, Lambers H. 2000. Respiratory costs and rate of protein turnover in the roots of a fast- and a slow-growing grass species. J Exp Bot 51:1089–1097. Schubert S, Schubert E, Mengel K. 1990. Effect of low pH of the root medium on proton release, growth, and nutrient uptake of field beans (Vicia faba). Plant Soil 124:239–244. Silsbury JH, Smith SE, Oliver AJ. 1983. A comparison of growth efficiency and specific rate of dark respiration of uninfected and vesicular-arbuscular mycorrhizal plants of Trifolium subterraneum L. New Phytol 93:555–566. Singh JN, Singh JN. 1971. Studies on the physiology of hostparasite relationships in Orobanche. I. Respiratory metabolism of host and parasite. Physiol Plant 24:380–386. Smakman H, Hofstra R. 1982. Energy metabolism of Plantago lanceolata as affected by change in root temperature. Physiol Plant 56:33–37. Snellgrove RC, Splittstoesser WE, Stribley DP, Tinker PB. 1982. The distribution of carbon and the demand of the fungal symbiont in leek plants with vesiculararbuscular mycorrhizas. New Phytol 92:75–87. Solomos T, Laties GG. 1976. Induction by ethylene of cyanide-resistant respiration. Biochem Biophys Res Commun 70:663–671. Sowell JB, Spomer GG. 1986. Ecotypic variation in root respiration rate among elevational populations of Abies lasiocarpa and Picea engelmannii. Oecologia 68:375–379. Steingro¨ver E. 1981. The relationship between cyanide-resistant root respiration and the storage of sugars in the taproot in Daucus carota L. J Exp Bot 32:911–919. Stolwijk JAJ, Thimann KV. 1957. On the uptake of carbon dioxide and bicarbonate by roots and its influence on growth. Plant Physiol 32:513–520. Szaniawski RK. 1981. Shoot:root functional equilibria. Thermodynamic stability of the plant system. In: Brouwer R, Gasparikova O, Kolek J, Loughman BG, eds. Structure and Function of Plant Roots. The Hague; Martinus Nijhoff/Dr W. Junk, pp 357–360. Szaniawski RK, Kielkiewicz M. 1982. Maintenance and growth respiration in shoots and roots of sunflower plants grown at different root temperatures. Physiol Plant 54:500–504.

Respiratory Patterns Taleisnik EL. 1987. Salinity effects on growth and carbon balance in Lycopersicon esculentum and L. pennellii. Physiol Plant 71:213–218. Tan K, Keltjens WG. 1990a. Interaction between aluminium and phosphorus in sorghum plants. I. Studies with the aluminium sensitive genotype TAM428. Plant Soil 124:25–23. Tan K, Keltjens WG. 1990b. Interaction between aluminium and phosphorus in sorghum plants. II. Studies with the aluminium tolerant genotype SC0 283. Plant Soil 124:25–32. Theologis A, Laties GG. 1978. Relative contribution of cytochrome-mediated and cyanide-resistant electron transport in fresh and aged potato slices. Plant Physiol 62:232–237. Tjepkema JD, Winship LJ. 1980. Energy requirement for nitrogen fixation in actinorhyzal and legume root nodules. Science 209:279–281. Tredici MR, Margheri MC, Giovannetti L, De Philippis R, Vincenzini M. 1988. Heterotrophic metabolism and diazotrophic growth of Nostoc sp. from Cycas circinalis. Plant Soil 110:199–207. Trought MC, Drew MC. 1980. The development of waterlogging damage in young wheat plants in anaerobic solution cultures. J Exp Bot 31:1573–1585. Umbach AL, Siedow JN. 1993. Covalent and noncovalent dimers of the cyanide-resistant alternative oxidase protein in higher plant mitochondria and their relationship to enzyme activity. Plant Physiol 103:845–854. Umbach AL, Wiskich JT, Siedow JN. 1994. Regulation of alternative oxidase kinetics by pyruvate and intermolecular disulfide bond redox status in soybean mitochondria. FEBS Lett 348:181–184. Uritani I, Asahi T. 1980. Respiration and related metabolic activity in wounded and infected tissues. In: Davies DD, ed. The Biochemistry of Plants, Vol 2. Metabolism and Respiration. London; Academic Press, pp 463–485. Van Beusichem ML. 1982. Nutrient absorption by pea plants during dinitrogen fixation. 2. Effects of ambient acidity and temperature. Neth J Agric Sci 30:85–97. Van den Bergen CWM, Wagner AM, Krab K, Moore AL. 1994. The relationship between electron flux and the redox poise of the quinone pool in plant mitochondria; interplay between quinol-oxidizing and quinone-reducing pathways. FEBS Lett 226:1071–1078. Van der Werf A, Kooijman A, Welschen R, Lambers H. 1988. Respiratory costs for the maintenance of biomass, for growth and for ion uptake in roots of Carex diandra and Carex acutiformis. Physiol Plant 72:483–491. Van der Werf A, Raaimakers D, Poot P, Lambers H. 1991. Evidence for a significant contribution of peroxidasemediated O2 -uptake to root respiration of Brachypodium pinnatum. Planta 183:347–352.

551 Van der Werf A, Welschen R, Lambers H. 1992a. Respiratory losses increase with decreasing inherent growth rate of a species and with decreasing nitrate supply: a search for explanations for these observations. In: Lambers H, Van der Plas LHW, eds. Molecular, Biochemical and Physiological Aspects of Plant Respiration. The Hague; SPB Academic Publishing, pp 421–432. Van der Werf A, Van den Berg G, Ravenstein HJL, Lambers H, Eising R. 1992b. Protein turnover: a significant component of maintenance respiration in roots? In: Lambers H, Van der Plas LHW, eds. Molecular, Biochemical and Physiological Aspects of Plant Respiration. The Hague; SPB Academic Publishing, pp 483–492. Van der Werf AK, Schieving F, Lambers H. 1993. Evidence for optimal partitioning of biomass and nitrogen at a range of nitrogen availabilities for a fast- and slowgrowing species. Funct Ecol 7:63–74. Van der Werf A, Poorter H, Lambers H. 1994. Respiration as dependent on a species’s inherent growth rate and on the nitrogen supply to the plant. In: Roy J, Garnier E, eds. A Whole-Plant Perspective of Carbon-Nitrogen Interactions. The Hague; SPB Academic Publishing, pp 61–77. Vanlerberghe GC, Vanlerberghe AE, McIntosh L. 1994. Molecular genetic alteration of plant respiration. Silencing and overexpression of alternative oxidase in transgenic tobacco. Plant Physiol 106:1503–1510. Veen BW. 1977. The uptake of potassium, nitrate, water, and oxygen by a maize root system in relation to its size. J Exp Bot 28:1389–1398. Veen BW. 1980. Energy costs of ion transport. In: Rains DW, Valentine RC, Hollaender A, eds. Genetic Engineering of Osmoregulation. Impact on Plant Productivity for Food, Chemicals and Energy. New York; Plenum, pp 187–195. Wagner AM, Krab K. 1995. The alternative respiration pathway in plants: role and regulation. Physiol Plant 95:318–325. Wang SY, Steffens GL, Faust M. 1983. Occurrence of alternative respiratory pathway in freshly excised apple root tissue. J Am Soc Hort Sci 108:1059–1064. Wanner H. 1950. Histologische und physiologische Gradienten in der Wurzelspitze. Ber Schweiz Bot Gesellsch 60:404–412. Wanner H, Schmucki S. 1950. Hemmung der Wurzelatmung durch Ausscheidungen des Wurzelsystems. Ber Schweiz Bot Gesellsch 60:413–425. Weger HG, Guy RD. 1991. Cytochrome and alternative pathway respiration in white spruce (Picea glauca) roots. Effects of growth and measurement temperature. Physiol Plant 83:675–681. Whipps JM. 1987. Carbon loss from the roots of tomato and pea seedlings grown in soil. Plant Soil 103:95–100.

552 Williams JHH, Farrar JF. 1990. Control of barley root respiration. Physiol Plant 79:259–266. Williams JHH, Farrar JF. 1992. Substrate supply and respiratory control. In: Lambers H, Van der Plas LHW, eds. Plant Respiration. Molecular, Biochemical and Physiological Aspects. The Hague; SPB Academic Publishing, pp 471–475. Williams JHH, Minchin PEH, Farrar JF. 1991. Carbon partitioning in split root systems of barley: the effect of osmotica. New Phytol 42:453–460. Williams JHH, Winters AL, Farrar JF. 1992. Sucrose: a novel plant growth regulator. In: Lambers H, Van der Plas LHW, eds. Plant Respiration. Molecular, Biochemical and Physiological Aspects. The Hague; SPB Academic Publishing, pp 463–469. Williamson RE. 1968. Influence of gas mixtures on cell division and root elongation of broad bean, Vicia faba L. Agronomy J 60:317–321. Williamson RE, Splinter WE. 1968. Effects of gaseous composition of root environment upon root development and growth of Nicotiana tabacum L. Agronomy J 60:365–368.

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33 Root pH Regulation Jo´ska Gerenda´s University of Kiel, Kiel, Germany

R. George Ratcliffe University of Oxford, Oxford, England

I.

pH VALUES AND THEIR MEASUREMENT

A.

The pH values in and around roots are frequently measured, and the expected values are well known. Thus soils vary in pH from < 3 to > 9 (Marschner, 1995); the root apoplast (Grignon and Sentenac, 1991; Yu et al., 2000) and the root vacuoles (Guern et al., 1991) are typically mildly acidic; and the pH of the root cytoplasm is usually in the range 7.2–7.6 (Guern et al., 1991). Our knowledge of these pH values, their regulation, and the physiological significance of the changes observed under normal and adverse conditions depends on the availability of techniques for measuring pH in and around functioning roots. Microelectrodes, fluorescence probes, and nuclear magnetic resonance (NMR) spectroscopy are the principal techniques, and this review of root pH regulation begins with a brief survey of the present status of these methods in root research. All three approaches yield values that ultimately depend on a calibration procedure, and although this inevitably becomes less certain for intracellular measurements, there is reasonable agreement between the pH values obtained with different techniques when direct comparison is possible (Gibbon and Kropf, 1994).

Microelectrodes

Microelectrodes allow fast, real-time monitoring of the pH in the cytoplasm (pHcyt ) and vacuole (pHvac ) of impaled cells (Felle, 1993), as well as the pH of the apoplast (Felle, 1998; Yu et al., 2000) and the rhizosphere (Zieschang et al., 1993). For intracellular measurements, the pH microelectrode has the advantage that it provides simultaneous measurements of the functionally related membrane potential, and by using a triple-barreled electrode it is even possible to make simultaneous measurements of pH and other ions, e.g., pH and potassium (Walker et al., 1998) or pH and nitrate (Miller and Smith, 1996). The precision of the microelectrode measurements is also useful in characterizing cellular heterogeneity, e.g., providing good evidence for the existence of vacuolar populations with different pH values in barley (Hordeum vulgare) root epidermal cells (Miller et al., 2000; A.J. Miller, personal communication). Microelectrodes are also frequently used for measuring pH gradients in the rhizosphere, either by means of electrode arrays (Fischer et al., 1989), by making multiple measurements with stationary electrodes (Newman et al., 1987; Miller et al., 1991) or by

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using ion-selective vibrating electrodes (Kochian et al., 1992). Vibrating electrodes have considerable advantages over their stationary counterparts because of better sensitivity and temporal resolution (see Chapters 20 and 29 by Porterfield in this volume). As well as providing pH maps around the root surface, spatially resolved pH measurements in the rhizosphere can also be used to quantify the Hþ fluxes in and out of roots (Newman et al., 1987; Miller et al., 1991; Kochian et al., 1992; Shabala et al., 1997). A further option is to combine microelectrode measurements with colorimetric methods in which the roots are grown in an agar medium containing a pH indicator such as bromocresol purple (Marschner and Ro¨mheld, 1983; Gollany and Schumacher, 1993). Videodensitometric analysis of the pH dye provides an accurate method for quantifying the pH gradients that develop around roots (Jaillard et al., 1996), and there is good agreement between the H+ fluxes calculated from colorimetric and potentiometric data (Plassard et al., 1999). B.

Fluorescence Probes

Fluorescence probes provide an increasingly powerful alternative to microelectrodes for the measurement of intracellular (Gilroy, 1997; Roos, 2000) and apoplastic (Kosegarten et al., 1999; Yu et al., 2000) pH values in roots. The method depends on introducing a fluorescent probe with a suitable pH dependence into the region or compartment of interest, and then deducing the pH from the intensity ratio of the fluorescence observed at two different excitation frequencies. The dye can be introduced by microinjection into specific cells, e.g., into root hairs, or by incubating the roots with the dye or a more lipophilic precursor. The latter approach is preferable since it is noninvasive and easy to implement, but it can be difficult to control and this may lead to uncertainty about the location of the dye. For example, the commonly used pH probe 2 0 ; 7 0 -bis-(2-carboxyethyl)-5-(and 6-)carboxy fluoroscein (BCECF) is often loaded by incubating roots with the acetomethyl ester (BCECF-AM). This procedure can lead to cytoplasmic and vacuolar pools of BCECF (Brauer et al., 1996) and it may be difficult to distinguish between them. For example, changes in the BCECF fluorescence ratio in maize (Zea mays) root hairs have been interpreted by different authors in terms of changes in both cytosolic pH (Kosegarten et al., 1997; Wilson et al., 1998) and vacuolar pH (Brauer et al., 1997a). This disconcerting disparity emphasizes the fact that there is a kinetic dimension

to BCECF pH measurements and that the localization of the dye is likely to be a function of both time and loading conditions. Short loading times (e.g., Plieth et al., 1999) or microinjection with dextran linked BCECF (e.g., Scott and Allen, 1999) minimize the sequestration of the dye into the vacuole. Calibration of fluorescence probes can also be problematic on occasion, since procedures for in situ calibration may be difficult to implement (Bibikova et al., 1998), but in general it is reasonable to assume that changes in pH can be accurately determined from changes in fluorescence (Fricker et al., 1997). An important feature of this approach to pH is that spatially resolved fluorescence measurements can be obtained with confocal laser scanning microscopy (LSM), allowing the construction of pH maps within cells and tissues (Gibbon and Kropf, 1994; Taylor et al., 1996). Unfortunately, optical sectioning by confocal LSM is restricted to the first two or three cell layers of a root, excluding many potentially interesting applications (e.g., Kawai et al., 1998), but much can still be achieved, particularly in Arabidopsis, and the method is a powerful alternative to the use of microelectrodes for probing cell-specific pH events (Bibikova et al., 1998; Scott and Allen, 1999). The power of the fluorescence technique has recently been increased still further through the development of pH-dependent green fluorescence protein (GFP) probes that can be targeted to particular subcellular locations (Kneen et al., 1998; Llopis et al., 1998). This approach is currently being extended to pH measurements in roots, since it clearly has the potential to probe the pH in a variety of previously inaccessible subcellular compartments in situ, including the endoplasmic reticulum, the mitochondria, and the plastids. In fact, pH-independent GFP probes are already having a major impact on research in plant cell biology (Fricker and Oparka, 1999), and it seems likely that the pH-dependent variants will be equally important. C.

NMR Spectroscopy

Like the fluorescence method, NMR measurements of pH-depend on the detection of tissue signals with pHdependent properties (Roberts, 1987; Ratcliffe, 1994). In its most widely applied form, 31 P NMR is used to detect the cytoplasmic and vacuolar pools of inorganic phosphate, and the position of the corresponding signals in the NMR spectrum, i.e., the chemical shift of each signal, is used to deduce pHcyt and pHvac on the basis of a suitable pH curve. The effective pKa of the

Root pH Regulation

inorganic phosphate at 6.8 is rather too high for the accurate determination of pHvac in many cases, but the method provides a good method for measuring pHcyt . In fact, NMR has been used extensively for measurements of both pHcyt and pHvac in plant tissues, e.g., in studies of the pH response of roots to anoxia (Ratcliffe, 1997), and although frequently applied to excised tissues, it can also be successfully adapted to measurements on the roots of intact seedlings (Roberts and Testa, 1988). NMR measurements of pH can also be made from the pH-dependent signals observed in the 13 C NMR spectra of 13 C-labeled root tissues, e.g., malate signals (Chang and Roberts, 1989), and the lower pKa values of the organic acids make them more suitable probes for the pH of the acidic vacuole. Other NMR methods are used occasionally for intracellular pH measurements in roots, e.g., a vacuolar pH of 3.7–3.8 was measured in the roots of Norway spruce (Picea abies), on the basis of the pH dependence of the fine structure of the proton coupled 14 N ammonium signal (Aarnes et al., 1995). There are several interesting possibilities that have yet to be exploited. For example, difluoroalanine and trifluoroalanine are readily taken up by plant tissues, and the resulting 19 F NMR signals have pH-dependent chemical shifts that could provide better time resolution for the measurement of pHcyt , and a more accurate determination of pHvac , than the conventional approach using 31 P NMR (Y. Shachar-Hill, personal communication). In fact, the temporal resolution of NMR measurements of intracellular pH is generally poor in comparison with that which can be achieved with fluorescence and microelectrodes; e.g., a pHcyt determination in < 5 min would only be possible in the most favorable cases with 31 P NMR, but this disadvantage is compensated by the metabolic information in the NMR spectrum (Ratcliffe, 1994). A more significant problem is the lack of spatial discrimination at the cellular level. The NMR signal is obtained from the whole sample, so the resulting pH values are averages that may mask cellular variation in pHcyt or pHvac . NMR spectra sometimes provide evidence for heterogeneity in the values, e.g., through the line broadening of the vacuolar malate signals in maize root tip spectra (Chang and Roberts, 1989), but such observations have been made infrequently and they give no indication of the spatial location of the different fractions. In principle, some form of spatially resolved spectroscopy based on the principles of NMR imaging (Ratcliffe, 1994; Chudek and Hunter, 1997) might solve this problem, but there have been no

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reports of such an approach being applied to roots or indeed other plant tissues so far.

II.

FACTORS INFLUENCING pH

The pH values recorded in and around roots are not necessarily constant, and changes in pH are frequently observed. Some of the factors that influence pH are likely to occur during normal growth and development, while others only arise if the plant is affected by adverse conditions. It is convenient to use this distinction as a basis for classifying the changes that are commonly observed. A.

pH Changes During Normal Plant Growth

In leaves, fluctuations in the aerial environment cause several well characterized pH changes, including the acidification of the thylakoids and the alkalinization of the chloroplast stroma in the light (Heldt et al., 1973; Kramer et al., 1999) and the diurnal fluctuation of pHvac in the leaves of CAM plants (Stidham et al., 1983). In contrast, most roots find themselves in a more stable environment and pH fluctuations due to diurnal changes in the immediate environment of a root are less likely. Exceptions to this generalization are likely to include the autotrophic roots of the leafless species of the genus Campylocentrum, as well as roots in rapidly fluctuating aquatic environments. For example, the aquatic resurrection plant Chamaegigas intrepidus grows in rock pools on granitic outcrops in Namibia, and it experiences a diurnal fluctuation in pH that ranges from < pH 6 in the morning to > pH 10 in the late afternoon. However, the plant is well adapted to its harsh environment, and NMR measurements have shown that the pHcyt is largely unaffected by the variation in the external pH (Schiller et al., 1998). A more important influence on the pH values in and around roots is growth itself (Raven, 1986; Raven and Wollenweber, 1992). The outcome depends on the mineral nutrients available to the plant, since the net proton balance is largely determined by the cation/ anion uptake ratio and the nature of the nitrogen source. Growth on nitrate is more likely to be proton consuming than growth on ammonium (Raven and Smith, 1976; Raven, 1986), and this tends to produce an alkalinization of the growth medium when plants are grown hydroponically on nitrate, and an acidification on ammonium (Kirkby and Mengel, 1967;

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Marschner, 1995). A similar effect is observed in the rhizosphere (Marschner and Ro¨mheld, 1983), but it is superimposed on a spatial variation along the root axis that tends to ensure that the pH around at the root tip is relatively alkaline (Fischer et al., 1989; Miller et al., 1991; Taylor and Bloom, 1998; Plassard et al., 1999). However, although rather large pH differences, typically 1–2 pH units, can develop in the growth medium or the rhizosphere as a result of differences in the nitrogen supply, only small differences are observed between the intracellular pH values for the roots of nitrate and ammonium grown plants (Gerenda´s et al., 1990). The growth-related pH changes that occur in the rhizosphere can have a profound effect on nutrient availability (Marschner, 1995; Chapter 36 by Neumann and Ro¨mheld in this volume). Thus, the acidification of the rhizosphere and root apoplast that accompanies growth on ammonium, or to a lesser extent nitrogen fixation, favors the uptake of a range of nutrients, including both macronutrients, such as phosphate, and minor nutrients, such as boron, iron, and manganese. Moreover, a shortage of certain nutrients can trigger specific pH changes, as in the acidification of the rhizosphere that occurs when organic acids are released in response to a shortage of phosphorus (Hoffland, 1992), and the acidification of the rhizosphere (Ro¨mheld et al., 1984) and the apoplast (Toulon et al., 1992) that occurs in response to iron deficiency. The magnitudes of the resulting pH changes are likely to be very dependent on the conditions experienced by the plant, and it is more difficult, for example, to reduce the apoplastic and rhizosphere pH values in high-carbonate soils (Hauter and Mengel, 1988). In fact, the optimum rhizosphere pH for growth is a complicated function of the soil type and the nutrient requirements of the plant, and the increased availability of certain nutrients at acidic pH values may also lead to toxic levels for others. Thus, iron and manganese toxicity can be a significant problem in acid soils (Gupta and Gupta, 1998), whereas on other soil types it may be advantageous to use an ammonium fertilizer to increase the availability of the same nutrients (Cummings and Xie, 1995; Malhi et al., 2000). The availability of aluminum also increases at acidic pH values, and aluminum toxicity is the most important constraint on plant growth in acidic soils (Horst, 1995; Marschner, 1995; Chapter 46 by Matsumoto in this volume). In this regard, growth-related alkalinization of the rhizosphere can also be observed, and recent evidence suggests that this may be important in

conferring resistance to aluminum (Degenhardt et al., 1998; Kollmeier et al., 2000). The normal growth and development of a plant also require localized or cell-specific pH changes within the roots. The acid growth theory of cell elongation lays particular emphasis on the acidification of the apoplast (Pritchard, 1994), and confocal LSM provides a convenient method for observing the pH changes in this compartment. For example, a fall in apoplastic pH from 4.9 to 4.5 was observed in gravitropically stimulated maize roots, and it was argued that this was sufficient to account for the cell growth required to allow the roots to bend (Taylor et al., 1996). A similar approach, using confocal ratio imaging of dextran conjugated dyes to measure pH values in the roots of Arabidopsis thaliana, has revealed an acidification of the apoplast at the root hair initiation site that coincided with a localized alkalinization of the cytoplasm (Bibikova et al., 1998). Other examples of localized pH changes associated with developmental events in roots include the acidification of specific cortical cells that precedes cell death and aerenchyma formation in rice (Oryza sativa) roots (Kawai et al., 1998), and the alkalinization of the root hair cells of alfalfa (Medicago sativa) in response to Rhizobium meliloti Nod factors (Felle et al., 1996). B.

pH Changes Under Adverse Conditions

In the laboratory, apoplastic pH can be altered by incubating roots in buffered solutions (e.g., Winch and Pritchard, 1999), while the intracellular pH values can be manipulated by incubating the roots with permeable weak acids and bases (e.g., Walker et al, 1998; Gerenda´s and Ratcliffe, 2000). Weak acids tend to accumulate preferentially in the more alkaline compartments of roots, and weak bases in the acidic compartments. However, since the accumulation of either type of compound tends to reduce the pH differences between the compartments, the intracellular partitioning of these compounds is usually rather dependent on the loading conditions and the duration of the experiment. Manipulations of this kind emphasize the finite capacity of the pH regulatory mechanisms, and although primarily of interest in a laboratory context, they can also be relevant in the field. For example, the use of ammonia-based fertilizers can lead to both high soil pH values and a high ammonium content (Liu et al., 1995), and this combination has the potential to perturb root pH values. The intracellular pH values in roots also respond to several agronomically important abiotic stresses,

Root pH Regulation

including flooding, drought, salt stress, and unfavorable soil pH values. The effect of oxygen deprivation on flooding intolerant plants is particularly important, since it causes an acidification of the cytoplasm (Roberts et al., 1984a) that leads to cell death if unchecked (Roberts et al., 1984b). For example, in maize root tips (Saint-Ges et al., 1991; Roberts et al., 1992; Fox et al., 1995) and alfalfa root hairs (Felle, 1996), the imposition of anoxia causes an immediate decrease of 0.5–0.6 pH units in the cytoplasmic pH. This fall in pH, which occurs in the first 15–20 min of oxygen deprivation in the case of maize root tips, is usually followed by a period of stabilization or partial recovery, before the onset of a further acidification that eventually proves lethal (Roberts et al., 1984b). The response of the pH to anoxia is influenced by the external pH experienced by the roots (Fox et al., 1995; Xia and Roberts, 1996), with lower external pH values promoting cytoplasmic acidosis. Moreover, the fall in pH is reduced if the roots are first acclimatized to a low oxygen level by a hypoxic pretreatment (Xia and Roberts, 1994). This latter observation is particularly relevant to field conditions where the onset of anaerobiosis is likely to be gradual (Drew, 1997). The effects of other abiotic stresses on root pH values are generally less marked than the effect of oxygen deprivation. Hyperosmotic shock caused only small increases in pHcyt and pHvac in excised maize root tips, with exposure to an osmotic potential of 1:35 MPa resulting in an alkalinization of the cytoplasm of just 0.1 pH units after 3 h (Spickett et al., 1992). The effect of NaCl on pHcyt is similar to that of nonionic osmotica at comparable osmotic potentials, but the influx of Na into the vacuole can lead to a substantial increase in pHvac (Fan et al., 1989; Spickett et al., 1993; Katsuhara et al., 1997). For example, a vacuolar alkalinization of 0.6 pH units was observed when maize root tips were exposed to NaCl concentrations > 200 mM, but the effect was considerably smaller (0.3 pH units) in the root tips of the halophyte Spartina anglica (Spickett et al., 1993). Intracellular pH values are also sensitive to the external pH, but observations on plant cell suspensions (Fox and Ratcliffe, 1990; Gout et al., 1992) suggest that this factor is only likely to be important at extreme pH values. Thus, varying the external pH between 8 and 9.25 had no effect on pHcyt and pHvac in maize root tips (Gerenda´s and Ratcliffe, 2000), and increasing the external pH from 6 to 10 caused an increase of only 0.1 pH unit in the pHcyt of Chamaegigas intrepidus roots (Schiller et al., 1998). Similarly, modest decreases in the external pH from around neutral to 4.5 or 4 have

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little or no effect on pHcyt in maize root tissues (Gerenda´s et al., 1990; Kasai and Bayer, 1995), although there is good evidence from observations on cell suspensions that the passive influx of protons at or below pH 4.5 can overwhelm the capacity of the plasma membrane H+-ATPase leading to acidification of the cytoplasm (Gout et al., 1992). The observed decrease in the net proton release at low external pH values correlates with poor root growth in Vicia faba and maize (Schubert et al., 1990; Yan et al., 1992), but it is unclear whether this can be attributed to the expected acidification of the cytoplasm.

III.

pH REGULATION

The inherent pH dependence of many cellular events, which arises from the prevalence of ionizable groups among the metabolites and macromolecules that make up the cell, indicates that pH regulation must be an essential feature of all living systems. The need for pH regulation is further emphasized by the fact that even normal cell growth can lead to the net production or consumption of protons (Raven, 1986). So, while it is clear from the preceding section that there are many situations in which intracellular pH values might change, these changes actually occur against a general background of pH homeostasis. This is achieved by maintaining an interactive balance between the proton-consuming and proton-generating processes within the cell. Moreover, if these processes are to act as pH regulatory mechanisms, their activity has to be modulated, either directly or indirectly, by the pH changes that are occurring. There are two general solutions to the problem of pH regulation—transporting the protons elsewhere (biophysical pH regulation), or arranging for them to be consumed by other metabolic processes (biochemical pH regulation)—and these mechanisms have to be deployed in such a way as to allow functionally important pH changes to occur at specific locations at specific times. A.

Contribution of Membrane Transport to pH Regulation

Membrane transport processes contribute to pH regulation by providing pH-dependent mechanisms for the movement of ions and metabolites between compartments (Fig. 1). While attention focuses on the protontransporting ATPases in the plasma membrane and the tonoplast, their contribution to pH regulation generally depends on charge compensating movements of

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Figure 1 Transport processes across the plasma membrane and tonoplast that can contribute to the biophysical regulation of the apoplastic, cytoplasmic, and vacuolar pH values in a root. Proton pumping from the cytoplasm is accompanied by the movement of strong ions through a variety of channels and carriers. The net change in ion balance, together with any change in the weak acid content of the compartment of interest, determines the overall effect on the pH.

other ions, so the biophysical regulation of pH actually depends on the integrated response of both primary and secondary membrane transport. However, the potential importance of the Hþ -ATPases in pH regulation is reinforced, because many of the secondary fluxes are themselves strongly influenced by the gradients established by the electrogenic pumps. The likely involvement of multiple membrane transport processes in pH regulation is further emphasized by a rigorous analysis of the factors that determine pH. Such an analysis shows that the pH of a solution in equilibrium with a gas phase containing CO2 depends on the partial pressure of the CO2 (pCO2 ), the concentration of the weak acids in the solution ð½A1;tot ; ½A2;tot  . . . ½Ai;tot  . . .), and the strong ion difference ([SID]) of the solution, i.e., the net charge carried by the ions that are fully dissociated under the prevailing conditions (Stewart, 1983). This analysis

leads to an equation for ½Hþ  that takes the form of a fourth order polynomial ½Hþ 4 þ a½Hþ 3 þ b½Hþ 2 þ c½Hþ  þ d ¼ 0 where a, b, c, and d are determined by the dissociation constants for the ionic equilibria involving water, the weak acids and CO2 , and by the values of [SID], [Ai;tot  and pCO2 (Gerenda´s and Schurr, 1999). This equation, which can be solved numerically if sufficient analytical information is available, can be used to calculate the pH of the solutions in intracellular and extracellular compartments, e.g., in the xylem and phloem (Gerenda´s and Schurr, 1999). The equation also has implications for pH regulation since it indicates that changes in pH can only occur through changes in the independent variables [SID], [Ai;tot , and pCO2 . Thus biophysical pH regulation may actually be the result of a multiplicity of membrane transport processes leading

Root pH Regulation

to a net change in [SID] and/or [Ai ; tot] (Fig. 1). Moreover, proton transport alone is insufficient to cause a change in pH, and while the operation of a proton cotransport system, such as a Naþ =Hþ antiport (Fig. 1), can change pH, proton pumps can only be effective in tandem with other transport processes. These and other considerations indicate that the plasma membrane Hþ -ATPase has the potential to play a key role in biophysical pH regulation in the apoplast, including the xylem vessels (Clarkson and Hanson, 1986), and in the cytoplasm, provided its action can be coupled to the movement of other ions. Thus, the increase in ATPase activity that occurs as pHcyt falls below its normal level (Luo et al., 1999; Morsomme and Boutry, 2000) has to be linked to enhanced cation uptake and/or enhanced anion efflux to avoid the inactivation of the ATPase by the hyperpolarization of the membrane (Blatt et al., 1990). Similarly, the reduced activity of the proton pump as pHcyt increases has to be coupled to the net influx of negative charge to avoid the depolarization of the membrane. Examples of these pH- and/or voltagedependent compensating ion movements under conditions that activate the plasma membrane Hþ -ATPase include the activation of chloride efflux through a pHdependent anion channel in Chara corallina (Johannes et al., 1998) and the activation of potassium influx through an inwardly rectifying potassium channel (KIRC) in barley roots (Amtmann et al., 1999). Interestingly, it has been found that pHcyt declines in parallel with cytoplasmic potassium activity in potassium-starved barley roots (Walker et al., 1996, 1998), and while this may merely reflect changes in the composition of the cytoplasmic solution, there is also the possibility that pH regulation itself is impaired when potassium is in short supply. The role of the plasma membrane Hþ -ATPase in opposing the passive influx of protons across the plasma membrane has been clearly demonstrated in an NMR investigation of cytoplasmic pH regulation in a sycamore (Acer pseudoplatanus) cell suspension (Gout et al., 1992). Cytoplasmic pH regulation failed abruptly when the external pH was reduced to < 4:5, and at this critical pH it was observed that the oxygen consumption rate had risen to its maximum possible value and that the ATP level had fallen to < 50% of its normal steady-state concentration. It was concluded that the plasma membrane Hþ -ATPase was no longer able to compensate for the influx of protons at the low external pH because of a shortage of ATP. This conclusion was confirmed by performing experiments on phosphate-starved cells and observing that pHcyt only

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recovered to its normal value of 7.5 at external pH values of 4.5 and 6.0 after the resupply of inorganic phosphate had allowed the ATP level to recover to its normal value (Gout et al., 1992). The importance of the plasma membrane Hþ -ATPase in establishing and maintaining the normal steady-state value of pHcyt is further demonstrated by the changes that occur during the adaptation of maize roots to low external pH (Yan et al., 1998). Adaptation increased the proton pumping activity of the Hþ -ATPase, and it was concluded that this effect was a significant factor in the survival of the roots at low pH values. Biophysical pH regulation is also important in controlling the pH in the cytoplasm and apoplast of roots in many other situations. For example, the assimilation of ammonium releases protons (Raven and Smith, 1976; Gerenda´s and Ratcliffe, 2000), and since there are only limited possibilities for restoring the proton balance metabolically (Raven, 1986), it has been found that the regulation of pHcyt during growth is dominated by proton transport (Raven and Smith, 1976; Allen and Raven, 1987). This has the effect of increasing the likelihood of adverse effects at acid pH values, and so one response to growth on ammonium is to increase the activity of the plasma membrane Hþ ATPase (Yamashita et al., 1995; Schubert and Yan, 1997). In contrast, in nitrate-grown plants, both the theoretical analysis (Raven and Smith, 1976) and the experimental evidence (Allen and Raven, 1987) suggest that the contribution of proton transport to pH regulation is supplemented by biochemical mechanisms, resulting in tighter pH regulation in nitrate-grown roots (Gerenda´s et al., 1990). Anaerobic conditions present a severe test for the pH regulatory mechanisms of most roots and the role of membrane transport in opposing the acidification of the cytoplasm that occurs under anaerobiosis is unclear (Ratcliffe, 1999). Substrate limitation of the ATPases was advanced as a possible cause of the acidification of the cytoplasm in anoxic maize root tips (Saint-Ges et al., 1991), but subsequent work on similar tissues suggested that pH regulation was unaffected by changes in the ATP level within the normal physiological range (Xia et al., 1995; Ratcliffe, 1999). In fact, the significance of these experiments really depends on whether the plasma membrane Hþ -ATPase can function under anoxia, and there is conflicting evidence on this point. Thus, microelectrode data indicated a depolarization of alfalfa root hairs under anoxia, suggesting that the proton pump was deactivated (Felle, 1996), whereas measurements of external pH showed that proton release from maize root tips was stimulated

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by fusicoccin under anoxia, suggesting that the plasma membrane Hþ -ATPase could indeed operate under these conditions (Xia and Roberts, 1996). While several other observations on the maize root tips were consistent with stimulation of the plasma membrane Hþ -ATPase by reduced pHcyt under anoxia, it was nonetheless concluded (1) that proton pumping made little contribution to cytoplasmic pH regulation in anoxic maize root tips at an external pH of around 6, and (2) that proton pumping did not provide an explanation for the improved cytoplasmic pH regulation observed in acclimated tissues (Xia and Roberts, 1996). The contribution of the proton pumps in the tonoplast to pH regulation under anoxia is also of interest, both because of the potential significance of the vacuole as a source or sink for transportable ions and metabolites (Fig. 1), and because the tonoplast Hþ -pyrophosphatase has been shown to be induced by anoxia in rice seedlings (Carystinos et al., 1995). Fluorescence and NMR measurements on maize root hairs led to the conclusion that pHvac is maintained by the tonoplast Hþ -ATPase under aerobic conditions and by the tonoplast Hþ -pyrophosphatase under anoxia (Brauer et al., 1997b). Lactate efflux is another membrane transport process with the potential to contribute to cytoplasmic pH regulation in anoxic roots. Hypoxic pretreatment of maize roots increases the efflux of lactate, a fermentation end product, from anoxic root tips (Xia and Saglio, 1992), and it also improves cytoplasmic pH regulation under anoxia (Xia and Roberts, 1994). While these observations suggest that a lactate transport process, such as Hþ =lactate cotransport or lactate efflux through an anion channel, could contribute to intracellular pH regulation in anaerobic plant tissues, subsequent work has shown that the greater lactate efflux from the acclimated maize root tips was accompanied by a smaller net efflux of protons (Xia and Roberts, 1996). This indicates that lactate efflux did not make a major contribution to the observed changes in the external pH and this is difficult to reconcile with a significant role for the process in intracellular pH regulation. B.

Contribution of Metabolism to pH Regulation

Metabolism contributes to pH regulation by providing pH-dependent pathways that allow a shift toward proton consumption as pH falls, and toward proton production as pH increases (Fig. 2; Davies, 1986; Raven,

1986). This biochemical mechanism of pH regulation depends on the existence of enzymes with pH-dependent activities that catalyze proton-consuming or proton-producing reactions. In fact, the systemic nature of metabolism means that the relationship between the fluxes through these pathways and the activities of the enzymes with nonzero elasticity coefficients for Hþ is generally unpredictable (Fell, 1997); thus, empirical measurements are essential to demonstrate that a particular flux is indeed responding to a change in intracellular pH in a way that contributes to pH regulation. Moreover, in analyzing the contribution that such fluxes might make to pH regulation, it is necessary to consider the impact of the step of interest within the context of metabolism as a whole, and this entails a consideration of the pathways that supply the substrate, as well as the pathways that allow the regeneration of any coenzymes that might be involved in the reaction (Figs. 3, 4). The most commonly encountered example of biochemical pH regulation is based on the pH-dependent balance between the synthesis and degradation of malate (Fig. 3; Davies, 1986; Sakhano, 1998). The synthesis of malate from carbon skeletons derived ultimately from glucose generates protons þ C6 H12 O6 þ 2CO2 ! 2C4 H4 O2 5 þ 4H

and the step catalyzed by PEP carboxylase has an alkaline pH optimum; while the oxidative decarboxylation of malate to pyruvate, and the subsequent regeneration of NADþ using oxygen as a terminal electron acceptor, consumes protons þ  1 C4 H4 O2 5 þ H þ 2O2 ! C3 H3 O3 þ H2 O þ CO2

and the step catalyzed by malic enzyme has an acidic pH optimum. Thus malate metabolism can respond to pH changes in a way that compensates for those changes, and one well-known consequence of this effect is the higher organic acid content of the roots and other tissues of nitrate grown plants (Kirkby and Mengel, 1967; Raven, 1986). As discussed in the preceding section, this biochemical mechanism supplements the biophysical regulation of pH in nitrate grown plants (Raven and Smith, 1976; Allen and Raven, 1987) and thus confers greater flexibility in their response to pH perturbations. It is clear from this example that biochemical mechanisms can be important in maintaining pH homeostasis, and indeed in a recent paper it was argued that the importance of the PEP carboxylase/ malic enzyme biochemical pH stat for pH regulation

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Figure 2 Metabolic processes that can contribute to the biochemical regulation of pHcyt in a root. The diagram provides balanced equations for the aerobic and anaerobic oxidation of carbohydrate, the synthesis and degradation of malate, the reduction of nitrate, and the subsequent assimilation of ammonium into glutamate. These pathways contribute to pH regulation through the consumption and production of protons, but their net effect depends on the pathways available for the recycling of reducing power (Figs. 3 and 4).

could provide an explanation for the existence of the alternative oxidase, since a role for this enzyme can be envisaged in the proton consuming degradation of malate (Sakhano, 1998). However, it should be emphasized that the contribution of biochemical pH stats to pH regulation is quite variable, and it can only be established through experiment. For example, the ammonium-induced alkalinization of pHcyt in maize root tips stimulated carboxylate synthesis, but the contribution of this proton-generating process to pH regulation during and after the ammonium treatment was quantitatively insignificant (Gerenda´s and Ratcliffe, 2000). Similarly, an investigation of pH regulation in sycamore cells showed that increasing the external pH caused an increase in pHcyt , and this was associated with an increase in the levels of malate and citrate (Gout et al., 1992). However, the onset of carboxylate synthesis was too slow to account for the stabilization

of pHcyt , indicating that the biochemical pH stat made a negligible contribution to pH regulation in the immediate aftermath of the abrupt change in the external pH. Similarly, on switching the external pH back to its original value, pHcyt returned to its normal value before any detectable fall in the malate and citrate levels. Thus, while the stimulation of carboxylate synthesis by elevated pHcyt would undoubtedly have contributed to pH homeostasis in the cytoplasm during the continued exposure of the cells to alkaline pH values, it did not play a critical role in stabilizing the pH during the periods in which the cells were responding to the alteration in the pH of the external medium (Gout et al., 1992). The biochemical mechanisms of pH regulation appear to be particularly important in roots during anaerobiosis (Figs. 2–4; Ratcliffe, 1999). Considerable interest attaches to the link between

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Figure 3 Proton consumption and production during the aerobic and anaerobic oxidation of carbohydrate, and the synthesis and degradation of malate. (A) The proton balance before allowing for changes in oxidation state; (B) the proton balance under aerobic conditions, assuming that electrons are supplied by the complete oxidation of glucose according to the equation C6 H12 O6 þ 3O2 ! 6CO2 þ 12Hþ , and that electrons are consumed by the reduction of oxygen according to the equation O2 þ 4Hþ þ 4e ! 2H2 O; and (C) the proton balance under anaerobic conditions, assuming that electrons are supplied by þ  the conversion of carbohydrate to pyruvate according to the equation C6 H12 O6 ! 2C3 H3 O 3 þ6H þ 4e , and that electrons  þ  are consumed by the conversion of pyruvate to ethanol according to the equation C3 H3 O3 þ 3H þ 2e ! C2 H6 O þ CO2 : The proton balance of the steps involved in the synthesis and degradation of malate is different under aerobic (B) and anaerobic (C) conditions, although the net effect is the same.

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Figure 4 Proton consumption and production during the conversion of nitrate to GABA. (A) The proton balance before allowing for the provision of reducing power and a source of carbon skeletons; (B) the proton balance under aerobic conditions, assuming that electrons are supplied by the complete oxidation of glucose according to the equation C6 H12 O6 þ 3O2 ! 6CO2 þ 12Hþ ; and (C) the proton balance under anaerobic conditions, assuming that electrons are supplied þ  by the conversion of carbohydrate to pyruvate according to the equation C6 H12 O6 ! 2C3 H3 O 3 þ 6H þ4e . A comparison of (B) and (C) shows that nitrate assimilation consumes protons under aerobic conditions and releases them under anaerobic conditions. The overall proton balance for the incorporation of ammonium into glutamate is further influenced by the pathway used to replenish the 2-oxoglutarate pool. (Gerenda´s and Ratcliffe, 2000).

the commonly observed switch from lactate to ethanol production that occurs shortly after the onset of anaerobiosis, and the acidification of the cytoplasm. According to the biochemical pH stat model (Davies et al., 1974), lactate dehydrogenase, which has an alkaline pH optimum, is inhibited by the acidification, while at the same time pyruvate decarboxylase, which has an acidic pH optimum, is activated. The net result is that lactate production is expected to be transient, and the resulting switch from a protongenerating fermentation pathway to a proton-neutral pathway stabilizes pHcyt . NMR methods have been used extensively to probe the validity of this model in roots (Ratcliffe, 1997, 1999; Roberts and Xia, 1996), providing key evidence, e.g., for the role of pH as an in vivo regulator of pyruvate decarboxylase (Roberts et al., 1984a; Fox et al., 1995). While it has become clear from this work that the biochemical pH stat model does not provide a complete understanding of pH regulation under anoxia, there is little doubt that the pH-dependent switch from lactate

to ethanol production can be an important factor (Ratcliffe, 1999). There are several other proton-consuming pathways that can be activated by a fall in pHcyt and which could therefore be important in pH regulation under anoxia. First, NMR analyses of maize root tips have shown that the decarboxylation of malate (Fig. 3) makes a significant contribution to pH regulation during the anoxic response (Roberts et al., 1992; Edwards et al., 1998). These analyses show that it is the combined activation of pyruvate decarboxylase and malic enzyme that is responsible for the stabilization and partial recovery of pHcyt that occurs following the onset of oxygen deprivation (Roberts et al., 1992). Secondly, there is good evidence that pH is a regulator of glutamate decarboxylase in vivo, with a fall in pH leading to an increase in the proton consuming synthesis of -aminobutyrate (GABA) from glutamate (Carroll et al., 1994; Crawford et al., 1994; Ford et al., 1996). However, the synthesis of GABA was insignificant during the initial stabilization of pHcyt in

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maize root tips (Roberts et al., 1992), and it appears that GABA synthesis, which is a commonly observed response to oxygen deprivation, only contributes to pH homeostasis in the subsequent metabolism of the anaerobic root tissue. Finally, it is also possible that there is a contribution to pH homeostasis from nitrate reduction under anoxia (Fig. 4). Nitrate reductase is reversibly activated by a decrease in pH (Kaiser and Brendle-Behnisch, 1995), and nitrate reduction is known to be stimulated in anoxic barley roots (Botrel et al., 1996; Botrel and Kaiser, 1997). Moreover, nitrate prolongs the survival of anoxic maize root tips (Roberts et al., 1985), and it is known to be assimilated during the anaerobic germination of rice seeds (Reggiani et al., 1993) and in anoxic rice coleoptiles (Fan et al., 1997). The impact of anaerobic nitrate reduction on pH regulation in root tissues is unclear, and the hypothesis that the activation of nitrate reductase under anoxia can contribute to the stabilization of the cytoplasmic pH has yet to be conclusively tested. It is important to put the potential contribution of particular metabolic steps or pathways to pH regulation into their proper metabolic context before concluding that a biochemical pH regulatory mechanism is involved. For example, ammonium uptake caused a reduced cytosolic alkalinization of rice root hairs when ammonium assimilation was inhibited with methionine sulfoxime, and it was argued that this could be explained in terms of the reduced impact of the proton-consuming assimilation of ammonium on pH homeostasis (Kosegarten et al., 1997). In fact, the assimilation of ammonium is only likely to consume protons under conditions where there is inadequate mobilization of reducing power and carbon skeletons for the operation of the glutamine synthetase/glutamate synthase pathway (Gerenda´s and Ratcliffe, 2000). This suggests that it is perhaps the unusually rapid assimilation of ammonium by rice root hairs that is responsible for the observed pH effects during ammonium uptake rather than ammonium assimilation itself.

IV.

PHYSIOLOGICAL CONSEQUENCES OF pH CHANGES

Although the pH regulatory mechanisms available to plants are powerful, they have a finite capacity and there are many situations in which the pH may actually deviate from the notional normal value. Thus the limitation of the plasma membrane Hþ -ATPase in oppos-

ing the acidification of the cytoplasm at low external pH values has been clearly demonstrated (Gout et al., 1992), and a biochemical mechanism such as the decarboxylation of malate can only contribute to pH regulation until the available malate is exhausted. Moreover, it is also possible to envisage cell- and tissue-specific changes in the activity of particular mechanisms that will allow the establishment of different pH values in the same compartment under different circumstances. The net result is that changes in pH can occur, and these changes often have consequences for cell function. These changes occur against a background of generally tight pH regulation, and they can be considered to have signaling properties akin to those of a second messenger (Kurkdjian and Guern, 1989; Guern et al., 1991, 1992). As a result, there is considerable experimental interest in manipulating pH values in order to establish the functional significance of such changes. While these signaling functions have been explored extensively in leaves, where there is good evidence for the involvement of both xylem pH (Hartung et al., 1998; Wilkinson, 1999) and guard cell pH (Grabov and Blatt, 1998; Blatt and Grabov, 1999) in controlling transpiration, the investigation of similar pH-mediated processes in roots is rather less advanced. One area of interest is the extent to which changes in pHcyt cause changes in metabolic flux. Biochemical pH regulation depends on a significant pH-dependent metabolic response, and indeed there is good evidence for the pH-dependent activation of several pathways in roots, including the fermentation pathway to ethanol (Roberts et al., 1984a; Fox et al., 1995) and the conversion of glutamate to GABA (Ford et al., 1996). However, whether changes in pH are solely responsible for the observed alterations in metabolism in these and other cases is generally unclear, because full quantitative analyses of the in vivo pH sensitivity of the relevant enzymes and their control coefficients for the fluxes of interest have not been reported. The interaction that has been uncovered between the calcium and proton signaling pathways in guard cells (Grabov and Blatt, 1998) provides a good illustration of the complexity that can underpin an apparently simple pH response, and it may well be that a similar picture will emerge in roots in due course. Changes in pHcyt are often associated with changes in cytosolic calcium; e.g., the cytosolic calcium level in Arabidopsis roots increases in response to a low external pH (Plieth et al., 1999), and the demonstration that a calcium-dependent phosphorylation step regulates the plasma membrane Hþ ATPase from maize roots (De Nisi et al., 1999) empha-

Root pH Regulation

sizes the potential for cross-talk between calcium and pH signals. Moreover, as demonstrated in guard cells, pHcyt can influence the activity of ion channels, and the resulting changes in membrane potential and intracellular composition can be important in mediating the ultimate cellular response to an incoming signal (Zimmermann et al., 1999). Changes in apoplastic and/or cytoplasmic pH are also implicated in a wide range of developmental processes in roots, including root growth (Taylor et al., 1996; Winch and Pritchard, 1999), root hair development (Bibikova et al., 1998; Chapter 5 by Ridge and Katsumi in this volume), gravitropism (Scott and Allen, 1999; Chapter 30 by Pilet in this volume), and cell death (Kawai et al., 1998). For example, evidence has been obtained that suggests that changes in pHcyt in the columella cells serve as a second messenger in the perception of gravistimulation by Arabidopsis roots (Scott and Allen, 1999). The use of BCECF-dextran as a fluorescent pH probe showed: (1) that pHcyt in the cells on the lower side of the second tier of columella cells increased by 0.4 pH unit within the first minute of gravistimulation; (2) that a similar alkalinization occurred over a slower time scale in the cells on the upper side of the second tier of columella cells; and (3) that pHcyt in the third tier of columella cells decreased by 0.4 pH unit over the first 6 min of gravistimulation. Moreover, the gravitropic response was disrupted when the roots were incubated with a range of reagents that altered the normal pHcyt values. These reagents, which included benzoic acid, procaine, methylamine, and bafilomycin A1, had no effect on root growth, and their effect on the gravitropic response indicated that the changes in pHcyt in the gravity-sensing columella cells must play a key role in the signal cascade following gravistimulation (Scott and Allen, 1999). This paper provides a good illustration of the way in which manipulating intracellular pH values can shed light on the functional importance of the pH changes that occur in response to a physiological perturbation.

V.

PROSPECTS FOR FUTURE RESEARCH

Roots have a considerable armory at their disposal for coping with pH stresses. However, the interplay between the biochemical and biophysical approaches to pH regulation, coupled with the ubiquity of the

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proton and the need to permit pH changes in particular circumstances, can make it difficult to discern and quantify the contribution of specific mechanisms to the overall process of pH control. Further progress in this endeavor is likely to be increasingly dependent on (1) the investigation of mutants with interesting pH-related phenotypes, and (2) the manipulation of genes with putative roles in pH regulation or pH sensing. In fact, there have been relatively few applications of these approaches to the investigation of intracellular pH regulation in roots, and further studies would be welcome. The use of both mutants and transgenics for this purpose is contingent on finding conditions in which the plant displays a pH-related phenotype. Thus, in principle, a mutant such as the det3 mutant of Arabidopsis, which has a defective tonoplast Hþ ATPase (Schumacher et al., 1999), could shed light on the contribution of the tonoplast proton pump to cytoplasmic pH regulation. However, given the multiplicity of the pH regulatory mechanisms that can operate in the cytoplasm, it is always possible that the lesion in one mechanism may be compensated by the activities of the others, and the extent to which this occurs can only be determined empirically. For example, an analysis of the pH response to anoxia in maize lines differing in the activity of alcohol dehydrogenase showed that cytoplasmic pH regulation was only affected at exceptionally low activities (Roberts et al., 1989), indicating that the hypoxic induction of the enzyme is not a critical feature of the biochemical pH stat model of pH regulation under anoxia. Similarly, transgenic plants should provide numerous opportunities for probing the quantitative contribution of specific enzymes and transporters to pH regulation, and there has been considerable interest in altering the activities of the enzymes involved in fermentation (Rivoal and Hanson, 1994; Bucher et al., 1994; Tadege et al., 1998; Dennis et al., 2000; Quimio et al., 2000; Sweetlove et al., 2000). However these plants have been little used in studies of pH regulation, with most investigations focusing on the metabolic consequences of the manipulation. In conclusion, the tools exist for the dissection of the pH regulatory pathways in roots at the molecular level, and when combined with the powerful array of techniques that are available for observing pH, it should be possible to gain further insights, in unprecedented detail, into the significance of pH and its regulation in root physiology.

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Gerenda´s and Ratcliffe Marschner H, Ro¨mheld V. 1983. In vivo measurement of root induced pH changes at the soil-root interface: effect of plant species and nitrogen source. Z Pflanzenphysiol 111:241–251. Miller AJ, Smith SJ. 1996. Nitrate transport and compartmentation in cereal root cells. J Exp Bot 47:843–854. Miller AL, Smith GN, Raven JA, Gow NAR. 1991. Ion currents and the nitrogen status of roots of Hordeum vulgare and non-nodulated Trifolium repens. Plant Cell Environ 14:559–567. Miller AJ, Cookson SJ, Smith SJ, Wells DM. 2000. Vacuolar heterogeneity revealed by ion-selective microelectrode measurements. J Exp Bot 51(suppl):17. Morsomme P, Boutry M. 2000. The plant plasma membrane Hþ -ATPase: structure, function and regulation. Biochim Biophys Acta 1465:1–16. Newman IA, Kochian LV, Grusak MA, Lucas WJ. 1987. Fluxes of Hþ and K+ in corn roots: characterization and stoichiometries using ion-selective microelectrodes. Plant Physiol 84:1177–1184. Plassard C, Meslem M, Souche G, Jaillard B. 1999. Localization and quantification of net fluxes of Hþ along maize roots by combined use of pH indicator dye videodensitometry and Hþ -selective microelectrodes. Plant Soil 211:29–39. Plieth C, Sattelmacher B, Hansen UP, Knight MR. 1999. Low pH mediated elevations in cytosolic calcium are inhibited by aluminium: a potential mechanism for aluminium toxicity. Plant J 18:643–650. Pritchard J. 1994. The control of cell expansion in roots. New Phytol 127:3–26. Quimio CA, Torrizo LB, Setter TL, Ellis M, Grover A, Abrigo EM, Oliva NP, Ella ES, Carpena AL, Ito O, Peacock WJ, Dennis E, Datta SK. 2000. Enhancement of submergence tolerance in transgenic rice overproducing pyruvate decarboxylase. J Plant Physiol 156:516– 521. Ratcliffe RG. 1994. In vivo NMR studies of higher plants and algae. Adv Bot Res 20:43–123. Ratcliffe RG. 1997. In vivo NMR studies of the metabolic response of plant tissues to anoxia. Ann Bot 79(suppl A):39–48. Ratcliffe RG. 1999. Intracellular pH regulation in plants under anoxia. In: Egginton S, Taylor EW, Raven JA, eds Regulation of Acid–Base Status in Animals and Plants. Cambridge, U.K.: Cambridge University Press, pp 193–213. Raven JA. 1986. Biochemical disposal of excess Hþ in growing plants? New Phytol 104:175–206. Raven JA, Smith FA. 1976. Nitrogen assimilation and transport in vascular land plants in relation to intracellular pH regulation. New Phytol 76:415–431. Raven JA, Wollenweber B. 1992. Temporal and spatial aspects of acid-base regulation. Curr Top Plant Biochem Physiol 11:270–294.

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34 Nutrient Absorption by Plant Roots: Regulation of Uptake to Match Plant Demand Anthony D. M. Glass University of British Columbia, Vancouver, British Columbia, Canada

I.

INTRODUCTION

each nutrient, and standard deviations were larger than the mean values (Wolt, 1994). In addition, variations in pH may exert potent indirect effects on the availabilities of the essential nutrients, as well as impacting directly upon root growth and the concentrations of potentially toxic ions such as aluminum or manganese. In natural soils the local variation may be even more extensive; Jackson and Caldwell (1993) measured conþ centrations of NO 3 , inorganic phosphate (Pi), K , and þ NH4 in soils taken from a native sagebrush steppe. In a 10  12 m area the authors analyzed 362 soil samples þ and reported that concentrations of NO 3 and NH4 þ varied across 3 orders of magnitude. Pi and K concentrations varied by only 1 order of magnitude. In addition to such sources of variability, there is considerable seasonal variation in soil composition as a result of microbial and plant activities (Murphy et al., 2000). Major soil disturbances, such as those associated with forest fires or large-scale clear cuts of forest lands, and the large-scale erosion that commonly follows such disturbances, may further increase the variability of an already heterogeneous soil system (Vitousek et al., 1979). In addition to soil heterogeneity with respect to inorganic nutrients, plant demand for these nutrients also varies according to daily and seasonal growth patterns. In the context of such heterogeneity, plant roots must respond to local, regional, and seasonal

Most of what we know about ion uptake by plant roots is derived from studies using hydroponic methods of plant culture. Such methods generally provide high concentrations of the required nutrients in wellmixed, aerated, and buffered solutions maintained at moderate temperatures. If the plants suffer any deficiency, it is usually that of light, because most growth cabinets provide light at
10–15% that of full sunlight. In the real world, plants experience very different conditions. Soils are notoriously heterogeneous, in terms of both their chemistry and physics. Extreme soil types, the so-called problem soils, are those that limit plant growth by deficiencies or excesses of various elements. In global terms, such problem soils may constitute up to 3 billion hectares (Dudal, 1976). For example, Serpentine soils contain excessive levels of magnesium (Mg2þ ), combined with relatively low levels of calcium (Ca2þ ), as well as toxic levels of nonessential ions such as cobalt and nickel. But even when we set aside such extreme soils, available data for agricultural soils indicate that the concentrations of nitrogen, phosphorus, and potassium, elements that typically limit crop productivity, may vary across orders of magnitude. For example, soil solution Kþ , þ 2 nitrate (NO 3 ), ammonium (NH4 ) and sulfate (SO4 ) concentrations from 35 different U.S., Australian, and N.Z. soils ranged across 2–4 orders of magnitude for 571

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changes by means of adaptations that can optimize nutrient capture. Some of these adaptations operate at the physiological level and can be observed on a time scale of hours in a laboratory setting. Others are longer-term strategies that occur on a time scale of days or weeks, and may include adaptations associated with changes of root morphology, growth rates, and symbiotic associations with mycorrhizae. The almost universal occurrence of these different kinds of adaptation among higher plants argues for their importance in dealing with soil nutrient heterogeneity. However, while the same basic adaptive mechanisms are found among most plants, there are nevertheless differences that allow plants to occupy various ecological niches. These adaptations have obviously developed over evolutionary time. For example, tree species such as Picea glauca L. (white spruce) may grow on soils in which NO 3 is undetectable (Stark and Hart, 1997) and where available N is principally in the form of NHþ 4 or amino acids. In a controlled laboratory setting, roots of these plants  took up NHþ 4 up to 20 times faster than NO3 (Kronzucker et al., 1997). From the ecological perspective, a major focus of interest has been to consider how plants adapt to inadequacies of inorganic nutrients. However, physiological studies make it evident (see below) that plants rapidly reduce inorganic ion uptake when oversupplied. This may limit the development of osmotic problems or toxic excesses associated with specific ions. For example, excessive Pi or Cl accumulation produces symptoms of toxicity in many plant species (Rossiter, 1952; Green et al., 1973; Reisenauer et al., 1973). In the present chapter we shall explore the processes involved in acquiring inorganic ions by plant roots, as well as the mechanisms that enable plants to adapt to the soil heterogeneities outlined above. Through the operation of these homeostatic mechanisms, plants are able to optimize nutrient uptake.

II.

ROOT MORPHOLOGY IN RELATION TO SOIL PROPERTIES

Within weeks of seed germination, the tiny embryonic root is transformed into an elaborate and extensive root system that infiltrates a large volume of soil. Figure 1 reveals the extent of vertical and horizontal root growth of a maize plant at various ages from 2 to 14 weeks (Hall et al., 1953). Also shown are the percentages of 32 P-labeled phosphorus absorbed from the different soil layers at 2, 7, and 14 weeks. With increas-

Figure 1 Lateral and horizontal growth of the root system of a maize plant and the percent 32 P absorbed from the soil at various distances from the stem at 2, 7, and 14 weeks after planting. (From Hall et al., 1953.)

ing age, there was a spectacular increase in root biomass, and 32 Pi absorption occurred at increasingly greater distances from the stem axis. The extent of root growth in crop species is exemplified by data for a 1-month-old winter rye plant whose primary and secondary lateral roots had grown to a combined length of 620 km with a surface area of 237 m2 (Dittmer, 1937). Root hairs of this plant numbered 14:5  109 with a surface area of 400 m2 . Such an enormous root extension serves not only to absorb nutrients, but also to access water and to anchor the plant. However, with respect to acquiring inorganic nutrients, root proliferation and extension are crucial because of physicochemical constraints on the movement of inorganic ions in soils. Cations, such as Kþ , or polyvalent ions, such as Pi, which are strongly bound to soil particles, are considered to move through soil largely by diffusion (Barber, 1995). Using 86 Rbþ as a radiotracer for Kþ , Barber showed that zones of 86 Rbþ ðKþ Þ depletion developed in the soil around roots, because Kþ diffusion was too slow to replace

Nutrient Absorption

the Kþ removed by root absorption. Similar depletion zones were demonstrated for soil Pi (Bhat and Nye, 1974). Diffusion-limited movement across zones of ion depletion probably also applies to NHþ 4 . By conand Cl , are trast, monovalent anions, such as NO 3 poorly bound to soil particles and are thought to move to the root by bulk flow of the transpiration-driven flow of soil solution up to the root surface (Barber, 1995). When transpiration rates are high and large quantities of water and dissolved solutes reach the root, a greater proportion of the required inorganic nutrients are supplied by bulk flow, even when their concentration in the soil solution is low. As soils dry out, and transpiration declines, diffusion of nutrients becomes more important. But when water-filled cavities shrink substantially, and the pathway for diffusion along soil surfaces becomes more tortuous, rates of diffusion also decline. Where different plant species grow in close proximity, as in pastures and natural ecosystems, underground competition for nutrients is probably intense and depletion zones may overlap among adjacent roots. Thus, in addition to inherent regional differences in soil nutrient availability, biological activity may further increase soil heterogeneity. Thus, continuous root growth into unexplored soil, or greater local exploration by means of root hairs or mycorrhizal associations, is crucial for plant survival. In highly intensive agricultural systems, such as those which supply concentrated nutrient solutions to individual plants (e.g., hydroponic greenhouse crops or fertigated fruit trees), the imperative to develop extensive root systems is lessened. High-yielding cultivars of crop species, selected for such intensive methods of cultivation, have probably also been selected (inadvertently) for smaller root systems and greater aboveground biomass (see Discussion in Evans, 1980). Where the harvested material is aboveground, this reallocation of biomass is ideal. However, under natural conditions, especially in nutrient-poor soils, a well-developed root system is essential.

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primary root, labeling the region behind the root apex, corresponding to the zone of root hair proliferation, as the principal zone of nutrient absorption. It is certainly true that in various tree species, e.g., Picea alba (white spruce) and Pinus contorta (lodgepole pine), the influx of 13 NO 3 into young unsuberized roots may be as high as five times higher than into old roots (Kronzucker et al., 1995; Min et al., 1998). Also interesting is the observation that some high-affinity nitrogen transporter genes are most active in the root hair zone in tomato (Lauter et al., 1996). Nevertheless, considering that the total surface area of older roots is much larger than that of the young actively absorbing root apices, the contribution of older roots is not to be dismissed. Kramer and Bullock (1966), e.g., reported that > 99% of the roots of Pinus taeda L. and Liriodendrion tulipifera L. were suberized, and Kramer (1969) concluded that the major part of the water and solutes absorbed by these plants was through such roots. Indeed, in laboratory experiments the potential for ion absorption and ion translocation to the shoot has been shown to extend to all regions of the seminal roots of wheat (Rovira and Bowen, 1970) and pine seedlings (Bowen, 1970) and as far back as 50 cm from the root tip in leek (Clarkson, 1974). The notable exception was the capacity for Ca2þ translocation, which declined rapidly with distance from the root tip. Notwithstanding these observations, in soils that develop depletion zones around older roots, ion uptake may be limited not by the root’s capacity for uptake per se, but by the rate of ion movement across the depletion zone or by the root’s capacity to grow into unexplored regions of soil. In this regard, the formation of mycorrhizal associations greatly extends the zone of nutrient capture and is of paramount importance to plants in the wild. It is not only the age of the root that determines its activity, but also its position and structure (see also Chapter 9 by Waisel and Eshel in this volume). B.

III.

LOCALIZATION OF ION UPTAKE IN THE ROOT

A.

Longitudinal Axis

Because of the depletion effects discussed above, it is commonly believed that only the younger portions of the extending root system are active in absorbing inorganic ions, as they enter unexplored regions of soil. Many elementary textbooks present diagrams of the

Radial Axis

With respect to the root’s radial axis, it has long been considered that the epidermis and its root hairs, together with the cortical cells, provide a large surface area for ion absorption. According to this assumption, soil solution permeates through the cell wall intercellular space continuum (the apoplasm) as far as the endodermis. All cells adjacent to this continuum absorb and transfer ions through the cytoplasmic continuum (the symplasm) across the endodermis and into

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Glass

xylem vessels for onward delivery to the shoot. In wellwatered soils or in hydroponic growth facilities with high levels of nutrients, this is probably the case. However, when ion concentrations in the soil solution are low, or as they decline owing to plant absorption, creating zones of depletion around the root, it is unlikely that any but the epidermal cells are active in ion uptake. IV.

ADAPTIVE CHANGES IN ROOT MORPHOLOGY

A.

Root:Shoot Ratio

Changes in root morphology occur in response to numerous edaphic factors, including availability of water, nutrients, and physical impediments (Miller, 1938). An extensive literature dealing with effects of soil inorganic nutrients on the direction, number, and extent of root growth dates back as far as the classical studies of Nobbe (1862) and Tucker and Von Seelhorst (1898). Probably the best-known response to deprivation of inorganic nutrients is the increased root-toshoot ratio (Harris, 1914). This response allows for a more efficient allocation of available plant resources toward soil exploration. However, nutrient deprivation

usually results in a smaller absolute root biomass than is the case under nutrient sufficient conditions. The use of soil-grown and solution-cultured roots has confirmed that, in virtually every plant species investigated, increased root:shoot ratios result from deprivations of Pi (Asher and Loneragan, 1967; Whiteaker et al., 1976; Lefebvre and Glass, 1982), Kþ (Asher and Ozanne, 1967; Siddiqi and Glass, 1983), and NO 3 (Bradshaw et al., 1964). In the study by Asher and Loneragan (1967), growth of eight pasture species was compared at external Pi levels from 0.04 to 25
M. On average, root:shoot ratios declined from 0:82  0:3 to 0:23  0:05 while root and shoot dry weights increased 3:2  2:37-fold and 13  10:8-fold, respectively. The large standard deviations, as in the study by Bradshaw et al. (1964), indicate the extent of differences among species. Changes of shoot and root biomasses and root:shoot ratios for Hypochoeris glabra L. (Asher and Loneragan, 1967) are presented in Fig. 2A. Absolute root and shoot weights increased with increasing Pi, while root:shoot ratios declined. The figure also shows that changes in root:shoot ratios were limited to the most extreme conditions, where Pi was below 1
M. Above this concentration root:shoot ratios were unchanged. This response was typical of all species examined in this study, although

Figure 2 (A) Root (~) and shoot (*) dry weights and root : shoot ratios (*) (100) for smooth flatweed (Hypochoeris glabra L.) in response to various concentrations of external Pi. (From Asher and Loneragan, 1967.) (B) Root (~) and shoot (*) dry weights and root : shoot ratios (*) (1000) for smooth flatweed (Hypochoeris glabra L.) in response to various concentrations of external Kþ . (From Asher and Ozanne, 1967.)

Nutrient Absorption

lupins (Lupinus digitatus Forsk) showed only small changes of biomass and root:shoot ratios in response to increasing Pi. Barley seedlings grown from seed in solutions containing either 0 or 15
M Pi showed no differences in root:shoot ratios until day 14 (Lefebvre and Glass, 1982). At this age the root:shoot ratios were 0.94 and 0.47, respectively, for P and þP plants. Presumably the large reserves of seed P buffered the seedlings for a considerable period of time. Similar results (Fig. 2B) were obtained when available Kþ was varied for 14 pasture species (Asher and Ozanne, 1967). Likewise, increasing NO 3 availability decreased the root:shoot ratios in several pasture species, and again there were significant differences in the extent of changes among the species (Bradshaw et al., 1964). In particular, Agrostis stolonifera and Lolium perenne showed similar yield responses but very different root:shoot responses. The authors concluded that ‘‘overall differences in shoot:root ratios between species is [sic] likewise difficult to interpret, since it bears little apparent relationship to overall yield, responses to nitrogen or ecological distribution.’’ Clearly, the observed morphological changes are only one part of an arsenal of adaptive changes invoked by inadequate nutrition. Others include physiological changes such as increased capacity for nutrient uptake (see below) and the efficiency of nutrient utilization. Differences within species in root:shoot ratios, as well as among species have been documented for Pi and K+ (Gerloff, 1976; Siddiqi and Glass, 1983; Whiteaker et al., 1976). There was also an age-dependent (from 2 to 6 weeks) decline in the root:shoot ratio in response to Kþ levels in barley varieties (Siddiqi and Glass, 1983). The mechanisms underlying changes of root:shoot ratios are unclear. We have observed that nutrient-deprived plants translocate a much smaller proportion of absorbed nutrients to the shoot than nutrient-rich plants. For example, in Kþ -starved barley, Kþ was not translocated to leaves for about 6 h even when roots were supplied with an external solution containing 10 mM KCl (Glass, 1978). Likewise, during the first 10 min of exposure to 13 NHþ 4 , Nstarved rice plants translocated only
5% of the incoming N to the shoot, compared to
30% in Nsufficient plants (Kronzucker et al.,1998). These observations indicate that regulatory mechanisms controlling translocation rates result in the retention of a greater proportion of the limiting nutrient in the roots of nutrient-deprived plants. This would provide the resources necessary to produce a higher root:shoot ratio than would be possible with higher rates of trans-

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location of the limited resource to the shoot. Whether these effects are the cause or result of the increased root:shoot ratio is unknown. However, the rapidity with which changes in the proportions of Kþ or N translocation to shoots take effect suggests that they may be part of the causal mechanism. In summary, nutrient deprivation generally reduces the absolute growth of both roots and shoots, while increasing the root:shoot ratio and blocking translocation from root to shoot. B.

Changes in Root Morphology

Under conditions of nutrient deprivation, there are also significant changes in the dimensions of roots and the extent of lateral root development. Using hydroponic methods, Hackett (1969) demonstrated that K or Pi deficiency was without effect on seminal roots, but reduced the numbers, surface areas, dry weights, and volumes of nodal roots and primary and secondary laterals. Likewise, using soil-grown wheat seedlings, Bowen et al. (1974) demonstrated that P deficiency had little effect on the formation of adventitious roots or first-order laterals, but reduced the second-order laterals by 50%. Their most severe treatment (zero P addition) reduced the elongation rates of all axes except the longest and first- and second-order laterals. Other studies have shown that growth of the main root axis is increased and lateral root formation retarded by deficiencies of Kþ (Jensen, 1982) or N (Drew, 1975; Chapter 9 by Waisel and Eshel in this volume). Root diameters of P-deficient barley plants declined to roughly one-third of those of P-supplied plants (Lefebvre and Glass, 1982). Roots of P plants also exhibited a prolific development of elongated root hairs, virtually absent from the þP roots. Taken together, under natural conditions such developmental changes serve to increase the likelihood that roots may reach a source of the limiting nutrient. The inhibition of lateral root growth in regions of nutrient-impoverished soil was demonstrated as early as 1862 by Nobbe, who allowed roots of corn plants to grow through infertile soil contained in glass cylinders. Various fertilizers (ammonium sulfate, calcium nitrate, and potassium phosphate) were added to specific layers of the soil. Branching of primary and secondary roots was small as roots grew through infertile zones but was prolific in the fertilized regions. This observation has been confirmed repeatedly (cf. Drew and Goss, 1974). Figure 3 and Table 1 provide details of this phenomenon in barley roots, including numbers and dry

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In summary, plants respond to nutrient impoverishment through a number of morphological adaptations that improve their capacity to acquire limiting inorganic nutrients. Such responses are relatively slow compared to the physiological changes that can be initiated within hours of perturbing the inorganic nutrient supply. The next section is devoted to these physiological adaptations.

Figure 3 Lateral root development in barley roots supplied with (H) or without (L) inorganic nutrients in the zones shown. (From Drew and Goss, 1974.)

weights of laterals in fertilized and unfertilized zones. Given the soil heterogeneity described above, the capacity to proliferate in nutrient-rich regions of the soil optimizes the roots’ use of their resources to capture nutrients where they are available. Table 1 Numbers of Laterals and Their Dry Weights Resulting from Provision (H) or Absence of Provision (L) of Nutrient Supply to Specific Regions of Barley Rootsa Nutrient treatment (H or L) H H H L H L

Number of laterals 1st Order: 3.2 2nd Order: 3.9 1st Order: 4.9 2nd Order: 3.6 1st Order: 4.6 2nd Order: 2.2 1st Order: 1.5 2nd Order: 0 1st Order: 7.8 2nd Order: 5.4 1st Order: 2.3 2nd Order: 0

V.

PHYSIOLOGICAL ADAPTATIONS

A.

Homeostats for Ion Accumulation

Roots of hydroponically grown wheat plants that were deprived of a particular nutrient (e.g., NO 3, Ca2+, Kþ , Na+, or Pi) increased their subsequent uptake of that nutrient by up to fourfold (Table 2) when it was resupplied (Brezeale, 1906). Hoagland and Broyer (1936) rediscovered this phenomenon, using roots of barley plants, and recognized that there are upper limits for nutrient accumulation, possibly dictated by osmotic effects or by the ‘‘saltsaturation of protoplasmic constituents.’’ The literature of these bygone days indicates that absorbed ions were thought to bind to protoplasmic substances and that when these substances became saturated, further binding (and hence further uptake) was reduced. Clearly, no direct evidence for such mechanisms was forthcoming from these studies. The increased capaTable 2 Effects of Removal of One Nutrient (-NO 3, -Ca2þ , -Kþ or -PO3 4 ) at a Time Upon Uptake Rate (% of control in parenthesis) for That Nutrient When Measured 18 h Later

Dry weight of laterals (mg)

Parts per million of nutrients removed from complete nutrient solution per 100 g water transpired by seedlings

19.6

Treatment

NO 3

Ca2þ

Kþ

PO3 4

13.6

Complete -NO 3

0.61 (100%)

1.38 (100%)

1.45 (100%)

19.3

6.21 (100%) 20.76 (334%)

2.0

-Ca2þ

61.1 4.5

-Kþ -PO3 4

a

These data correspond to those of Fig. 3. Source: Drew and Goss, 1974.

Source: Brezeale, 1906.

1.36 (223%) 5.6 (407%) 2.54 (175%)

Nutrient Absorption

city for ion uptake which develops following nutrient deprivation, and the opposite effect, the downregulation of uptake as that element accumulates in the plant, allow for rapid restoration of what Cram (1976) referred to as the set points, or ‘‘required’’ internal levels of particular ions. These set points are presumably determined by the physicochemical requirements for individual ion functions. For example, cytoplasmic Kþ is held at concentration values of
100–150 mM (Memon et al., 1985; Walker et al., 1996). It has been proposed that the fidelity of protein synthesis from mRNA has a strict requirement for this range of Kþ concentration (Leigh and Wyn Jones, 1984). Likewise, cytosolic Pi is maintained at
5 mM (Lee et al., 1990) because of its participation in intermediary metabolism and energy transduction (ADP/ATP conversions). Vacuolar ion concentrations are less strictly regulated and may drop substantially in the process of maintaining cytosolic concentrations, when uptake from the external medium is inadequate. Simultaneous measurements of Pi concentrations in maize root vacuoles and cytoplasm (Fig. 4), during several days growth without Pi, revealed that cytoplasmic Pi remained constant at the expense of the larger vacuolar pool (Lee et al., 1990). Apparently, there are upper limits for vacuolar ion accumulation which may be related to osmotic limits or in some cases perhaps the potential for toxic effects of excess accumulation (Hoagland and Broyer, 1936).

Figure 4 Cytoplasmic and vacuolar Pi concentrations (
mol cm3 root volume) in roots of maize plants during P deprivation. During the period of P deprivation, root samples were analyzed by NMR to determine cytoplasmic and vacuolar Pi concentrations. With increasing duration of P deprivation, vacuolar Pi declined but cytoplasmic Pi concentrations remained constant until vacuolar Pi was exhausted. Different symbols represent data from separate experiments. (From Lee et al., 1990.)

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B.

Multiple Transport Systems

Kinetic studies of the ion transport systems of roots, beginning with the extensive studies of Epstein and coworkers (Epstein, 1976), established that the absorption of most inorganic ions could be modeled as the result of the operation of two types of plasma membrane carriers. These carriers, considered to function at low and high external ion concentrations, respectively, have become known as the high-affinity transport systems (HATS) and low-affinity transport systems (LATS). In some cases, where the influx of ions such þ as NHþ 4 or K has been examined in detail, and using sufficiently short exposures to measure ion influx rather than net uptake (influx minus efflux), the LATS failed to saturate but responded in a linear fashion to increasing external ion concentration (Kochian and Lucas, 1982; Siddiqi et al., 1990; Wang et al., 1993). This may indicate a very low affinity of the LATS for the ion in question. In the case of NO 3 uptake, kinetic analyses suggest that three types of carriers participate (Glass and Siddiqi, 1995). Two are high-affinity transporters that operate at low external NO 3 concentration, saturating . One of these, a high-capacity and at < 100
M NO 3 high-affinity transporter (iHATS), is induced within hours of exposure to NO 3 , while the other is a lowcapacity, high-affinity carrier (cHATS) expressed constitutively (without prior exposure to NO 3 ). The third transport system is a low-affinity transporter (LATS) which becomes evident at higher NO 3 concentrations (
0:5 mM) and is also expressed without prior exposure to NO 3 in barley roots (Siddiqi et al., 1990). This transporter, like other LATS, also fails to saturate at elevated external NO 3 concentrations. Recent studies, using molecular biology methods, have suggested that both low-affinity Kþ and NO 3 transporters (encoded by the akt1 and Nrt1 genes, respectively) may be important components of the uptake of these ions even in the high-affinity range of concentration (Hirsch et al., 1998; Wang et al., 1998; Liu et al., 1999). These observations may require that we reevaluate traditional concepts of high- and lowaffinity transport. However, it is important to await further confirmation of these studies and consider the evidence carefully before we throw the baby out with the bath water. In the case of the LATS for Kþ influx, it was observed that a mutant with a disrupted akt1 gene absorbed less 86 Rb (a tracer for Kþ ) than the wild-type plants at low external ion concentration. However, the plants were grown with high levels of 86 Rb fluxes at low Kþ and NHþ 4 prior to measuring

578

Glass

external concentration. Pretreatment with high Kþ is known to suppress expression of the HATS for Kþ influx (see the section dealing with kinetic constants, below), and NHþ 4 is strongly inhibitory to HATS for Kþ influx. Thus, the conditions used by Hirsch et al. (1998) must have virtually suppressed all HATS for Kþ influx. Under conditions in which the HATS is expressed, as shown in the kinetic analysis of 86 Rbþ influx in corn roots by Kochian and Lucas (1982), the relative contribution of the LATS to HATS influx was extremely small. Likewise, the evidence for a major role of the Nrt1 gene product in HATS for NO 3 influx is problematic. The plants used for the studies by Wang et al. (1998) and Liu et al. (1999) were grown on high N prior to measuring uptake. These conditions suppress high-affinity NO 3 uptake (Glass and Siddiqi, 1995). The study by Liu et al. (1999) also included expressing the Nrt1 gene product in Xenopus oocytes, which resulted in the expression of both high- and low-affinity transport. While the results are thought provoking, they must be interpreted with caution because the gene was clearly being expressed in a foreign host in a novel membrane system. Can we be certain the same applies in planta? In summary, in both the above examples, the experimental conditions were biased in a manner that suppressed the HATS activity. There is no reason to presume that LATS may not contribute to HATS activity; the question is, how significant these contributions are at low external ion concentration, under conditions where the HATS transporters make a large contribution to influx? In the section dealing with gene cloning it will be seen that for each ion there may exist several genes encoding functional transporters for both low and high-affinity transport. Only time will tell if each of these transporters can contribute to both low- and high-affinity transport. C.

Active and Passive Transporters

Ion fluxes via the HATS have been demonstrated to be the result of thermodynamically active fluxes against their respective electrochemical gradients, and evidence suggests that the proton gradient across the plasma membrane serves as the source of free energy for these active fluxes. In the case of NHþ 4 , it has frequently been claimed that the electrical gradient across the plasma membrane,
’ is the driving force for influx (e.g., Frommer et al., 1994; Sohlenkamp et al., 2000). However, at low external NHþ 4 concentration
’ was shown to be inadequate to drive NHþ 4 influx into Lemna and rice roots (Ullrich et al., 1984; Wang et

al., 1994). Ion uptake via the LATS is thought to be passive for cations but active for anions such as NO 3 or Pi. These conclusions arise from the use of the Nernst equation, using measured electrical potentials across the plasma membrane, together with estimates of external and cytoplasmic ion concentrations (see Ullrich et al., 1984; Wang et al., 1994). In the earlier literature, effects of metabolic inhibitors and temperature coefficients for ion uptake were commonly used in attempting to identify active and passive transport systems. However, such data can only provide very general indications of energy dependence, because of indirect effects of metabolic perturbations. In particular, electrical potentials, which contribute to passive uptake, are themselves subject to effects of inhibitors and low temperatures. Thus, definitive statements concerning active versus passive transport depend upon thermodynamic evaluations, such as the Nernst evaluation.

D.

Influx and Efflux

When the rate of root absorption of a particular ion is measured by depletion of that ion in solution, the experimenter actually determines net uptake—the difference between influx and efflux. Radiotracers, by contrast, allow us to distinguish between influx and efflux. Such studies have revealed that the influx of ions via high-affinity transport systems is rapidly downregulated or upregulated, respectively (over periods of hours), in response to increases or decreases of the external ion concentration (Hodges and Vaadia, 1964; Pitman et al., 1971; Cram, 1973; Glass, 1978; Wang et al., 1993; Kronzucker et al., 1998; Lappartient et al., 1999). Efflux of ions also rises as internal ion concentrations rise in response to increasing external ion concentration (Cram, 1983; Memon et al., 1985; Wang et al., 1993). By contrast, ion efflux falls as internal ion concentration declines, when the ion concentrations of external solutions are reduced. As a consequence, of these effects, net uptake (influx– efflux) via HATS declines as external ion concentration increases. However, the decline of influx is generally much larger than the increase of efflux (Kronzucker et al., 1999), and under these conditions ion influx via LATS assumes a greater importance. Transport of ions through their respective LATS also declines as tissue ion concentrations increase, but generally more slowly than is the case for the HATS (Clement et al.,1978; Glass and Dunlop, 1978; Kochian and Lucas, 1982).

Nutrient Absorption

E.

Mechanisms Responsible for Regulating the Influx of Ions

It has been assumed that the flux changes, associated with perturbations of external ion concentrations, result from internal events. Such perturbations of supply increase or decrease cellular ion concentration or concentrations of metabolites (e.g., amino acids in the cases of N or S absorption). By contrast, plants might respond to external perturbations by monitoring the external ion concentration by means of plasma membrane sensors. However, when Kþ or Pi was provided to only one part of the root system, while the other parts were deprived of these nutrients, rates of Kþ and Pi uptake increased in the fed root. This occurred despite the constant external ion concentration experienced by the fed root (Borkert and Barber, 1983; Drew and Saker, 1984). Such findings are inconsistent with the hypothesis of external sensing of ion concentrations. 1.

Kinetic Constants, Vmax , and Km for Ion Influx

Using influx isotherms, several studies have demonstrated that the Vmax for influx via the HATS increases as ion-starved roots deplete internal reserves. This has  been demonstrated for Pi, SO2 4 and Cl (Lee, 1982), þ  K (Glass, 1978), NO3 (Siddiqi et al., 1990), and NHþ 4 (Wang et al., 1993). In some studies, the Km values for influx also responded to deprivation, decreasing their values and thereby increasing the affinities of the carriers for their ions. This was particularly pronounced for Kþ (Glass, 1978) and NHþ 4 (Wang et al., 1993), and for Pi in sterile excised barley roots (Cartwright, 1972). Altered values of Km were not evident in other studies, including those that examined the influx of Pi (Lee, (Lee, 1982), or NHþ 1982), SO2 4 4 (Rawat et al., 1999). It is unclear why different experimenters should obtain apparently opposite results. When different plant species were employed, genuine interspecies differences may exist. However, Cartwright (1972) and Lee (1982) were both studying Pi influx in barley roots. Perhaps the differences may depend upon the extent of deprivation required to induce the observed changes. In summary, it can be stated that in response to perturbations of ion supply, changes of Vmax values are essentially universal, while changes of Km have been consistently reported only for Kþ . The changes of Vmax can be explained by changes in the number of ion transporters. This might result from increased expression of the genes encoding transpor-

579

ters or from changes in their turnover. Changes of Km are more likely the result of direct (noncovalent) effects of ions or other regulatory molecules upon the transporter proteins, or of (covalent) posttranscriptional effects such as protein phosphorylation. 2.

Cloning of Genes That Encode Ion Transporters

Prior to the recent developments in plant molecular biology, it was possible only to speculate about the mechanisms outlined above, but beginning with the cloning of a low-affinity Kþ transporter (Anderson et al., 1992), genes encoding many ion transporters have been characterized (Chrispeels et al., 1999). This is still a rapidly developing field, but we are beginning to obtain some insights into how these genes are regulated and, in particular, how they respond to perturbations of inorganic nutrient supply. Nevertheless, the large number of genes that have been cloned for each ion, often multiple members of the same gene family, raises more questions than answers. For example, at the time of writing (August 2000), the NRT2 gene family, considered to encode high-affinity NO 3 transporters, numbers seven members in Arabidopsis (Gene Bank Accessions). However, since over 90% of the Arabidopsis genome has now been sequenced, it is unlikely that a large number of new members will be discovered. Do all members of this family encode functional transporters? Are they all expressed in the same cells or are they localized in different tissues with different functions? Are they developmentally regulated? Only time will tell! The techniques used to clone genes encoding ion transporters are quite simple in principle and have involved three types of approach: 1. Transformation of microbial mutants defective in the transport of a particular ion, by means of plant cDNA. Ideally, the mutant is ‘‘rescued,’’ becoming capable of growth on low concentrations of that ion, when before transformation they were incapable of growth on that medium. This method has been successfully employed to isolate genes encoding Kþ and NHþ 4 transporters (Anderson et al., 1992; Ninnemann et al., 1994). 2. Selection of survivors of toxic analogs of a particular ion, e.g., using chlorate (ClO 3 ) as a toxic analog of NO 3 (Tsay et al., 1993). This methodology relies upon the presumption that mutants capable of surviv ing ClO 3 treatment are impaired in NO3 uptake. However nitrate reductase (NR) mutants are also obtained by this method.

580

3. Use of conserved sequences of genes encoding transporters that have already been cloned from other organisms to develop primers that are then used to isolate their homologous plant genes. This approach was used to isolate the AtKUP genes, encoding Kþ transporters of Arabidopsis thaliana (Kim et al., 1998) and high-affinity NO 3 transporters from barley and Arabidopsis (Trueman et al., 1996; Zhuo et al., 1999). At the time of writing this chapter, genes encoding 2+ , Mg2þ , transporters for Kþ , Ca2þ , Na+, NHþ 4 , Fe  2  NO3 , Cl , SO4 , and Pi have been isolated from higher plants (Hirschi et al., 1996; Lurin et al., 1996; Briat and Lobreaux, 1997; Chrispeels et al., 1999; Apse et al., 1999; Shaul et al., 1999). In every case in which the regulation of gene expression has been examined, it was found that ion deprivation and/or subsequent provision of the limiting nutrient, resulted in a rapid increase/decrease, respectively, of the mRNA encoding for that particular transporter. In addition, posttranscriptional regulation was also suggested in the cases of and NO uptake (Rawat et al., 1999; NHþ 3 4 Sohlenkamp et al., 2000; Vidmar et al., 2000). Space does not permit an encyclopedic coverage of this literature; moreover, it is proceeding at a rapid pace as the Arabidopsis genome is almost completely sequenced. Rather, one example of each methodology—namely, a high-affinity NHþ transporter, 4 AtAMT1.1, from Arabidopsis for the first methodology; a low-affinity NO 3 transporter (originally called CHL1, now renamed AtNRT1.1) for the second methodology; and a high-affinity Kþ transporter family (AtKUP) as representative of the third methodology—will be described. These examples exemplify the approaches that are possible.

Glass

grown plants to 100
M NH4 NO3 caused both influx and AtAMT1 mRNA to increase by > 10-fold within 24 h. Likewise, when NH4 NO3 was resupplied to Nstarved plants, 13 NHþ 4 influx and AtAMT1 mRNA declined to the level of N-rich plants within 12 h (Fig. 5a, left panel, and Fig. 5b). However, unlike nonmetabolized ions, such as Kþ or Ca2þ , NHþ 4 is rapidly converted to glutamine and other amino acids, following absorption by the roots, due to the activity of the enzyme glutamine synthetase. Thus, either NHþ 4 or

3. Three Case Studies in Molecular Biology a. AtAMT1 AtAMT1 was cloned from Arabidopsis by method 1 above, making use of the yeast (Saccharomyces) Mep1Mep2 double mutant, which is defective in NHþ 4 transport and grows very slowly at low external concentration (Ninemann et al., 1994). NHþ 4 AtAMT1.1 is one of three members of this gene family in Arabidopsis, which in tomato has been shown to be preferentially expressed in root hairs (Lauter et al., 1996). AtAMT1.1 mRNA levels and 13 NHþ 4 influx in roots of Arabidopsis plants grown on 1mM NH4 NO3 were < 10% of the values found in N-deprived plants (Rawat et al., 1999). Transfer of the 1mM NH4 NO3 -

Figure 5 (a) Northern blots of AtAMT1 expression in Ndeprived roots of Arabidopsis thaliana, 0, 3, 6, and 9 h after þ resupply of NHþ 4 (lanes 1–4, respectively) and effects of NH4 in the presence of MSX, Gln plus MSX, NHþ plus Gln plus 4 MSX, and Gln alone (lanes 5–8, respectively). (b) 13 NHþ 4 influx (*), and AtAMT1 mRNA levels (*), as percentages of their maximum values, at intervals following resupply of NHþ 4 to N-deprived roots of Arabidopsis thaliana, in the presence (þMSX) or absence (MSX) of methionine sulfoximine. (From Rawat et al., 1999.)

Nutrient Absorption

amino acids might be responsible for the changes of AtAMT1 mRNA levels and NHþ 4 influx. By treating plants with methionine sulfoximine (MSX), an inhibitor of glutamine synthetase, NHþ 4 assimilation can be blocked and the effects of NHþ 4 can be distinguished from effects of glutamine. When N-starved roots of Arabidopsis were resupplied with NH4 NO3 in the presence of MSX, root NHþ 4 levels increased 27-fold and glutamine levels remained low (Rawat et al., 1999). Under these conditions, AtAMT1 mRNA levels remained at 88% of those of N-starved plants. By contrast, in roots supplied with NH4 NO3 in the absence of MSX, glutamine concentration increased ninefold, and AtAMT1 mRNA levels declined to
20% of the level of the N-starved plants (Fig. 5a, right panel, and Fig. 5b). These observations strongly support the hypothesis that AtAMT1 expression is regulated through effects of glutamine rather than of NHþ 4 . Nevertheless, this same study provided evidence that accumulated NHþ 4 may also exert negacarrier through posttranslative effects on the NHþ 4 tional regulation, in addition to the documented effects of glutamine at the level of AtAMT1.1 mRNA. Since the cloning of the first higher plant NHþ 4 transporter by Ninnemann et al. (1994), the same research team has isolated two more members of the AtAMT1 family from roots of Arabidopsis (Gazzarrini et al., 1999). When these genes were expressed in yeast, their transporters showed Km values for NHþ 4 ranging from an estimated 0:5mM (AtAMT1.1) to 30 and 36
M, respectively, for AtAMT1.2 and AtAMT1.3. These findings, as well as similar studies of other transporter gene families, reveal an apparent redundancy in representation among particular families, whereby even in the highaffinity category for a particular ion there may be several functional genes. In roots of Arabidopsis the three genes of the AtAMT1 family responded differentially to N deprivation, and all three showed increased expression during daylight hours, which was correlated with increased NHþ 4 influx during this time of day. b.

AtNRT1.1

The compound chlorate (ClO 3 ) behaves as a toxic ana log of NO 3 by virtue of its absorption by the NO3  transporter, and by its reduction to chlorite (ClO2 ) through the action of NR. For this reason, ClO 3 has been employed as an effective herbicide. This toxicity provides a means of isolating two categories of mutants—those with defective transport systems for NO 3 ; or those with defective NR—by their capacity to survive exposure to ClO 3 . Using this method

581

Arabidopsis mutants defective in NR and in NO 3 transport have been isolated (Doddema et al., 1978). The transport mutant was named B1 and found to be affected only in the low-affinity transport system for NO 3 uptake, which was reduced, but not eliminated, by this mutation. Mutants generated by the random insertion of Agrobacterium T-DNA into the plant genome have an advantage over other kinds of mutants because the bacterial DNA provides a convenient tag, enabling the gene in which it is inserted to be identified. By exposing T-DNA insertional mutants of Arabidopsis to ClO 3 it was possible to isolate individual plants that were immune to the toxic effects of this compound. Among these were mutants in NO 3 transport (Tsay et al., 1993). Figure 6 (see color insert) shows wild-type plants and T-DNA insertional mutants after treatment with ClO 3 . The gene encoding this transport activity corresponded to the B1 mutant isolated by Doddema et al., in 1978, and was named CHL1 initially; for systematic reasons it is now called AtNRT1.1. The gene was cloned and sequenced, and its physiological function was evaluated by expressing the gene in Xenopus oocytes. In this foreign host, the gene’s expression conferred properties that are typical of plant nitrate transporters. These included the accumulation of NO 3 by the oocytes and membrane depolarization following exposure to NO 3 . Surprisingly, when the detailed kinetics of 13 NO 3 influx were measured using these mutants, low-affinity transport appeared no different from that in wild-type plants when plants were grown on KNO3 as sole source of N (Touraine and Glass, 1997). However, in NH4 NO3 -grown plants, low-affinity NO 3 influx was substantially reduced, but not eliminated, in the mutant plants compared to wild-type plants. The reason that low-affinity transport was not completely eliminated in the mutant plants became apparent when a second NRT1 gene, named AtNRT1.2, was cloned from Arabidopsis (Huang et al., 1999). The expression patterns of AtNRT1.1 and AtNRT1.2 have been examined by means of in situ hybridization experiments, which revealed that both genes are expressed primarily in the epidermis close to the root tip, but in the cortex or endodermis, farther back from the root tip. These results indicated that expression patterns vary as a result of developmental changes as the root matures. AtNRT1.1 expression in roots of Arabidopsis was found to be induced by exposure to NO 3 (Tsay et al., 1993), while its homolg AtNRT1.2 appears to be constitutive. Thus, in its response to induction by

582

Glass

NO 3 , the AtNRT1.2 more closely resembles the physiological behavior of the (constitutive) low-affinity transporter in barley roots. c.

AtKUP

The third method of cloning genes encoding ion transporters relies upon already published sequences of genes encoding ion transporters from other organisms. This method presumes that despite the vast potential for changes in gene sequence that might have occurred through evolution, base sequences that encode critical regions of the corresponding protein will be conserved. By searching for base sequences that are homologous to bacterial Kþ transporters among partially sequenced genes of Arabidopsis in the Genbank database, a new family of putative Kþ transporters was identified (Kim et al., 1998). At least four members of this family, named AtKUP1, AtKUP2, AtKUP3, and AtKUP 4, were identified in Arabidopsis. The authors provided strong evidence that the plant genes encode legitimate Kþ transporters by expressing the genes in an E. coli mutant which possesses null mutations in all three of its major Kþ transporter systems (Trk, Kdp, and Kup). Figure 7 shows Petri plates inoculated with the triple mutant transformed with the Arabidopsis AtKUP1 cDNA. The left and right halves of the plate contained 134 and 2 mM Kþ , respectively, and the upper quartiles were inoculated with bacteria transformed with the plant cDNA. The lower quartiles of the plate

Figure 7 Growth of an E. coli mutant defective in its three major Kþ transporters that has been transformed with an Arabidopsis cDNA corresponding to the AtKUP1 gene (Kim et al., 1998). At low Kþ the mutant is unable to grow (bottom right quartile) in the absence of the plant cDNA. At high Kþ the mutant grows with or without the plant cDNA (upper and lower left quartiles, respectively).

were inoculated with bacteria lacking the plant cDNA (labeled vector on the accompanying Petri plate map) as controls. Clearly, at very high external Kþ the triple mutant can grow effectively with or without the plant cDNA. However, at low external Kþ , growth was only possible when the bacteria expressed the plant cDNA. Further evidence for a role in HATS Kþ transport was given by the demonstration that transgenic Arabidopsis cell cultures, overexpressing AtKUP1 displayed increased Rbþ uptake. The Km of this transport was 22
M, indicating that it is a highaffinity Kþ transporter. When expression levels of the 4 AtKUP genes was compared in roots of plants grown at low and high external Kþ , it was observed that low levels of Kþ resulted in elevated levels of expression of AtKUP3 (Fig. 8), while the opposite was true for AtKUP2 . The AtKUP3 gene may therefore be responsible for the observed increase of Kþ influx associated with Kþ deprivation. Perhaps the AtKUP2 gene encodes a Kþ transporter responsible for delivering Kþ to the stele. Recall that Kþ deprivation reduced the transport of Kþ to the shoots. 4.

Cycling of Nutrients Within Xylem and Phloem

The concept of regulating ion influx according to demand, via effects of internal (cytoplasmic or vacuolar) feedback upon transcript abundance, applies equally to single-celled organisms as to multicellular organisms. Where the site of absorption in the roots may be far away from the site of utilization, at the tops of large trees, there is a need for additional levels of communication and integration over and above those that operate in single-celled organisms. By use of tracers and split-root experiments, it has been demon-

Figure 8 Expression levels of the AtKUP2 and AtKUP3 genes in roots of Arabidopsis plants grown at low and high Kþ , respectively. (From Kim et al., 1998.)

Nutrient Absorption

strated that ions or their metabolic products not only proceed from root to shoot via the xylem, but may return to the roots via the phloem, and even recycle back to the shoot (Cooper and Clarkson, 1989; Marschner et al., 1997). It has been proposed that this cycling/recycling provides a means whereby root:shoot signaling of whole-plant nutrient status can be achieved, leading to a proper integration of root activity in response to whole plant demand. This proposal and some potential pathways of feedback control are illustrated in Fig. 9. In the experiments discussed above involving the localized provision of nutrients to a limited region of the root (Borket and Barber 1983; Drew and Saker, 1984), increased nutrient uptake by the fed root provided evidence of feedback from the rest of the plant to the fed root. However, this does not preclude also a localized control. In experiments involving the provision of a particular nutrient to nutrient-deprived plants, it was demonstrated that initially there is no transfer of that nutrient to the shoot for several hours. For example, it was noted that following the provision of Kþ (Glass, 1978) or NHþ 4 (Kronzucker -deprived plants, et al., 1998) to roots of Kþ - or NHþ 4 transfer of these elements to the shoot was limited until several hours had elapsed. Yet, during this time there was strong downregulation of influx, indicating the potential for autonomous control of root activity by information residing in the root itself. It may be hypothesized that the root nutrient status is ultimately the determinant of transcriptional events and ion fluxes in the root. Recycling of nutrients within the xylem and phloem may influence this nutrient status through the exchange of ions between the stele and the root

583

cortical regions via the symplasm. This would indicate the importance of long-distance feedback effects, particularly those originating from the shoot operating ultimately at the level of transcript abundance in the root, as described above. Such a mechanism could be one means of responding to both local and long-distance signals. F.

Summary and Conclusions

It is evident that plants must adapt to the great heterogeneity of the soil environment by means of root systems that are morphologically and physiologically plastic. The recent literature has revealed that there are many more genes encoding ion transporters than might have been anticipated from earlier kinetic studies of ion transport. It may be that this apparent redundancy will be explained as the result of differential expression of these genes in different tissues or organs. In addition, there is evidence that some of the genes may encode transporters that are capable of functioning as both high- and low-affinity transporters (Wang et al., 1998; Liu et al., 1999; Hirsch et al., 1998). It should be emphasized that most of the evidence advanced in support of these claims was either based on plant gene expression in a foreign host (e.g., in yeast or Xenopus oocytes) or suppressed highaffinity transporters. Notwithstanding these shortcomings, such studies have forced us to reevaluate some of our long-held beliefs and have raised challenging questions. It is almost a century since Brezeale (1906) noted the adaptive response of plant roots to ion deprivation. Considerable progress in understanding how plants respond to the heterogeneity of nutrient supply has been achieved since that date. The next decade will require even more intensive efforts, using an integrated physiological and molecular methodology to resolve the many exciting and challenging questions that have emerged from molecular studies of the last decade. REFERENCES

Figure 9 A diagram showing the pathway of inorganic nutrient transport between roots and shoots, and possible pathways of feedback regulation. Based on an hypothesis by Cooper and Clarkson (1989). Complete lines represent ion fluxes; dotted lines represent feedback effects on the genes that encode transporters.

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584 Asher CJ, Ozanne PG. 1967. Growth and potassium content of plants in solution cultures maintained at constant potassium concentrations. Soil Sci 103:155–161. Asher CH, Loneragan JF. 1967. Response of plants to phosphate concentration in solution culture. I. Growth and phosphorus content. Soil Sci 103:225–233. Barber SA. 1995. Soil Nutrient Bioavailability. New York: John Wiley & Sons. Bhat KKS, Nye PH. 1974. Diffusion of phosphate to plant roots in soil. II. Uptake along the roots at different times and the effect of different levels of phosphorus. Plant Soil 41:365–382. Borket CM, Barber SA. 1983. Effect of supplying P to a portion of the soybean root system on root growth and P uptake kinetics. J Plant Nutr 6:895–910. Bowen GD. 1970. Effects of soil temperature on root growth and on phosphate uptake along Pinus radiata roots. Aust J Soil Res 8:31–42. Bowen GD, Cartwright B, Mooner JR. 1974. Wheat root configuration under phosphate stress. In :Bieleski RL, Ferguson AR, Creswell, eds. Mechanisms of Regulation of Plant Growth. Wellington, N.Z.: Royal Society N.Z. publications. Bradshaw AD, Chadwick MJ, Jowett D, Snaydon RW. 1964. Experimental investigation into the mineral nutrition of several grass species. IV. Nitrogen level. J Ecol 52:665–676. Brezeale JF. 1906. The relation of sodium to potassium in soil and solution cultures. J Am Chem Soc 28:1013–1025. Briat JF, Lobreaux S. 1997. Iron transport and storage in plants. Trends in Plant Sci 25:187–193. Cartwright B. 1972. The effect of phosphate deficiency on the kinetics of phosphate absorption by sterile excised barley roots and some factors affecting the ion uptake efficiency of roots. Soil Sci Plant Anal 4:313–322. Chrispeels MJ, Crawford NM, Schroeder JI. 1999. Proteins for transport of water and mineral nutrients across the membranes of plant cells. Plant Cell 11:661–675. Clarkson DT. 1974. Ion Transport and Cell Structure in Plants. New York; McGraw-Hill. Clement CR, Hopper MJ, Jones LHP. 1978. The uptake of nitrate by Lolium perenne from flowing nutrient solution. J Exp Bot 29:453–464. Cooper HD, Clarkson DT. 1989. Cycling of amino-nitrogen and other nutrients between shoots and roots in cereals—a possible mechanism integrating shoot and root in the regulation of nutrient uptake. J Exp Bot 40:753–762. Cram WJ. 1973. Internal factors regulating nitrate and chloride influx across the plasmalemma. J Exp Bot 24:328– 341. Cram WJ. 1976. Negative feedback regulation of transport in cells. The maintenance of turgor, volume and nutrient supply. In: Luttge U, Pitman MG, ed. Encyclopedia of Plant Physiology, New Series IIA. Berlin: SpringerVerlag, pp 284–316.

Glass Cram WJ. 1983. Characteristics of sulfate transport across plasmalemma and tonoplast of carrot root cells. Plant Physiol 72:204–211. Doddema H, Hofstra JJ, Feenstra WJ. 1978. Uptake of nitrate by mutants of Arabidopsis thaliana, disturbed in uptake or reduction of nitrate. I. Effect of nitrogen source during growth on uptake of nitrate and chlorate. Physiol Plant 43:343–350. Drew MC. 1975. Comparison of the effects of a localized supply of phosphate, nitrate, ammonium and potassium on the growth of the seminal root system, and the shoot in barley. New Phytol 75:479–490. Drew MC, Goss MJ. 1974. Environmental stress and the growth of barley root systems: the effect of nutrient ion concentration and mechanical impedance. In: Bieleski RL, Ferguson AR, Creswell, eds. Mechanisms of Regulation of Plant Growth. Wellington, N.Z.: Royal Society Publications. Drew MC, Saker LR. 1984. Uptake and long-distance transport of phosphate, potassium and chloride in relation to internal ion concentrations in barley: evidence of non-allosteric regulation. Planta 160:500–507. Dittmer HJ. 1937. A quantitative study of the roots and root hairs of a winter rye plant. Am J Bot 24:417–420. Dudal R. 1976. Inventory of the major soils of the world with special reference to mineral stress hazards. In: Wright MJ, ed. Plant Adaptation to Mineral Stress in Problem Soils. Ithaca, NY: Cornell University Agricultural Experiment Station. Epstein E. 1976. Kinetics of ion transport and the carrier concept. In: Luttge U, Pitman MG, ed. Encyclopedia of Plant Physiology, New Series 2B. Berlin: SpringerVerlag, pp 70–94. Evans LT. 1980. The natural history of crop yield. Am Sci 68:388–397. Frommer WB, Kwart M, Hirner B, Fischer WN, Hummel S, Ninnemann O. 1994. Transporters for nitrogenous compounds in plants. Plant Mol Biol 26:1651–1670. Gazzarrini S, Lejay T, Gojon A, Ninnemann O, Frommer WB, Von Wiren N. 1999. Three functional transporters for constitutive, diurnally regulated, and starvation-induced uptake of ammonium into Arabidopsis roots. Plant Cell 11:937–947. Gerlof GC. 1976. Plant Efficiencies in the use of nitrogen, phosphorus and potassium. In: Wright MJ, ed. Plant Adaptation to Mineral Stress in Problem Soils. Ithaca, NY: Cornell University Agricultural Experiment Station. Glass ADM. 1978. The regulation of Kþ influx into intact roots of barley (Hordeum vulgare (L.) cv. Conquest) by internal Kþ . Can J Bot 56:1759–1764. Glass ADM, Dunlop J. 1978. The influence of potassium content on the kinetics of potassium influx into excised ryegrass and barley roots. Planta 141:117–119. Glass ADM, Siddiqi MY. 1995. Nitrogen Absorption by Plant Roots In: Srivastava HS and Singh RP, eds.

Nutrient Absorption Nitrogen Nutrition in Higher Plants. New Delhi: Associated, pp. 21–56. Green DG, Ferguson WS, Warder F. 1973. Accumulation of toxic levels of phosphorus in phosphorus-deficient barley. Can J Plant Sci 53:241–246. Hackett C. 1969. A study of the root system of barley. II. Relationships between root dimensions and nutrient uptake. New Phytol 68:1023–1030. Hall NS, Chandler WF, Van Bavel CHM, Reid PH, Anderson JH. 1953. A tracer technique to measure growth and activity of a plant root system. NC Agr Exp Sta Tech Bull 101. Harris FS. 1914. The effect of soil moisture, plant food and age on the ratio of tops to roots in plants. J Am Soc Agron 6:65–75. Hirsch RE, Lewis BD, Spalding EP, Sussman MR. 1998. A role for the Akt1 potassium channel in plant nutrition. Science 280:918–921. Hirschi KD, Zhen RG, Cunningham KW, Rea PA, Fink GR. 1996. CAX1, an Hþ =Ca2þ antiporter from Arabidopsis. Proc Natl Acad Sci USA 93:8782–8786. Hoagland DR, Broyer TC. 1936. General nature of the process of salt accumulation by roots with description of experimental methods. Plant Physiol 11:471–507. Hodges TK, Vaadia Y. 1964. Uptake and transport of radiochloride and tritiated water by various zones of onion roots of different chloride status. Plant Physiol 39:104– 108. Huang N-C, Chiang C-S, Crawford NM, Tsay Y-F. (1996) CHL1 encodes a component of the low-affinity nitrate uptake system in Arabidopsis and shows cell type-specific expression in roots. Plant Cell 8:2183–2191. Huang N-C, Liu KH, Lo HJ, Tsay YF. 1999. Cloning and functional characterization of an Arabidopsis nitrate transporter gene that encodes a constitutive component of low-affinity uptake. Plant Cell. 11:1381–1392. Jackson RB, Caldwell MM. 1993. The scale of nutrient heterogeneity around individual plants and its quantification with geostatics. Ecology 74:612–614. Jensen P. 1982. Effects of interrupted Kþ supply on growth and uptake of Kþ , Ca2þ , Mg2þ , and Naþ in spring wheat. Physiol Plant 56:259–265. Kim EJ, Kwak JM, Uozumi N, Schroeder JI. 1998. ATKUP1—an Arabidopsis gene encoding high-affinity potassium transport activity. Plant Cell 10:51–62. Kochian LV, Lucas WJ. 1982. Potassium transport in corn roots. I. Resolution of kinetics into a saturable and linear component. Plant Physiol 70:1723–1731. Kramer PJ. 1969. Plant and Soil Water Relationships: A Modern Synthesis. New York: McGraw-Hill. Kramer PJ, Bullock ??.1966. Seasonal variation in the proportion of suberized and unsuberized roots of trees in relation to the absorption of water. Am J Bot 53:200– 204. Kronzucker HJ, Siddiqi MY, Glass ADM. 1995. Kinetics of NO 3 influx in spruce. Plant Physiol 109:319–326.

585 Kronzucker HJ, Siddiqi MY, Glass ADM. 1997. Root discrimination against soil nitrate and the ecology of forest succession. Nature 385:59–61. Kronzucker HJ, Schjoering JK, Erner Y, Kirk GJD, Siddiqi MY, Glass ADM. 1998. Dynamic interactions between root NHþ 4 influx and long-distance N translocation in rice: insights into feedback processes. Plant Cell Physiol 39:1287–1293. Kronzucker HJ, Glass ADM, Siddiqi MY. 1999. Inhibition of nitrate uptake by ammonium in barley: analysis of component fluxes. Plant Physiol 120:283–291. Lappartient AG, Vidmar JJ, Leustek T, Glass ADM, Touraine B. 1999. Inter-organ signaling in plants: regulation of ATP sulfurylase and sulfate transporter genes expression in roots mediated by phloem-translocated compound. Plant J 18:89–95. Lauter F-R, Ninnemann O, Bucher M, Riesmeier J, Frommer WB. 1996. Preferential expression of an ammonium transporter and of two putative nitrate transporters in root hairs of tomato. Proc Natl Acad Sci USA 93:8139–8144. Lee RB. 1982. Selectivity and kinetics of ion uptake by barley plants following nutrient deficiency. Ann Bot 50:429– 449. Lee RB, Ratcliffe RG, Southon TE. 1990. Phosphorus-31 NMR measurements of the cytoplasmic and vacuolar inorganic phosphate content of mature maize roots. Relationships with phosphorus status and phosphate fluxes. J Exp Bot 41:1063–1078. Lefebvre DD, Glass ADM. 1982. Regulation of phosphate influx in barley roots: Effects of phosphate deprivation and reduction of influx with provision of orthophosphate. Physiol Plant 54:199–206. Leigh RA, Wyn Jones RG. 1984. A hypothesis relating critical potassium concentration for growth to the distribution and function of this ion in the plant cell. New Phytol 97:1–13. Liu KH, Huang CY, Tsay YF. 1999. CHL1 is a dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake. Plant Cell 11:865– 874. Lurin C, Geelen D, Barbier-Brygoo H, Guern J, Maurel C. 1996. Cloning and functional expression of a plant voltage-dependent chloride channel. Plant Cell 8:701– 711. Marschner H, Kirkby EA, Engels C. 1997. Importance of cycling and recycling of mineral nutrients within plants for growth and development. Bot Acta 110:65–273. Memon AR, Saccomani M, Glass, ADM. 1985. Efficiency of potassium utilization by barley varieties: the role of subcellular compartmentation. J Exp Bot 36:1860– 1876. Miller EC. 1938. Plant Physiology: With Reference to the Green Plant. New York; McGraw-Hill. Min X, Siddiqi MY, Guy RD, Glass ADM, Kronzucker HJ. 1998. Induction of nitrate uptake and nitrate reductase

586 activity in trembling aspen and lodgepole pine. Plant Cell Environ 21:1039–1046. Murphy DV, MacDonald AJ, Stockdale EA, Goulding KWT, Fortune S, Gaunt JL, Poulton PR, Wakefield JA, Webster CP, Wilmer WS. 2000. Soluble organic nitrogen in agricultural soils. Biol Fertil Soils 30:374– 387. Ninnemann O, Jauniaux J-C, Frommer B. 1994. Identification of a high affinity NHþ 4 transporter from plants. EMBO J 13:3464–3471. Nissen P. 1996. Uptake mechanisms. In: Waisel Y, Eshel A, Kafkafi U, eds. Plant Roots: The Hidden Half. New York: Marcel Dekker. Nobbe F. 1862. U¨ber die feinere Vera¨stelung der Pflanzenwurzeln. Landw Versuchs-Stat 4:212–224. Pitman MG, Mowat JL, Nair H. 1971. Interactions of processes for accumulation of salt and sugar in barley plants. Aust J Biol Sci 24:619–631. Rawat SR, Silim SN, Kronzucker HJ, Siddiqi MY, Glass ADM. 1999. AtAMT1 gene expression and NHþ 4 uptake in roots of Arabidopsis thaliana: evidence for regulation by root glutamine levels. Plant J 19:143– 152. Reisenauer HM, Walsh LM, Hoeft RG. 1973. Testing soils for sulfur, boron, molybdenum and chlorine. In: Walsh LM, Beaton JD, eds. Soil Testing and Plant Analysis. Madison, WI: Soil Science Society of America, pp 173–200. Rossiter RC. 1952. Phosphorus toxicity in subterranean clover and oats grown on Mulchea sand and the modifying effect of lime and nitrate-nitrogen. Aust J Agric Res 3:227–243. Rovira AD, Bowen GD. 1970. Translocation and loss of phosphate along roots of wheat seedlings. Planta 93:18–25. Shaul O, Hilgemann DW, de-Almeida-Engler J, Van Montagu M, Inze D, Galili G. 1999. Cloning and characterization of a novel Mg2þ =Hþ exchanger. EMBO J 18:3973–3980. Siddiqi MY, Glass ADM. 1983. Studies of growth and mineral nutrition of barley varieties. I. Effect of potassium supply on K uptake and growth. Can J Bot 61:671–678 Siddiqi MY, Glass ADM, Ruth TJ, Rufty TW. 1990. Studies of the uptake of nitrate in barley. I. Kinetics of 13 NO 3 influx. Plant Physiol 93:1426–1432. Sohlenkamp C, Shelden M, Howitt S, Udvardi M. 2000. Characterization of Arabidopsis AtAMT2, a novel ammonium transporter in plants. FEBS Lett 467:273–278. Stark JM, Hart SC. 1997. High rates of nitrification and nitrate turnover in undisturbed coniferous forests. Nature 385:61–64.

Glass Touraine B, Glass ADM. 1997. Nitrate and chlorate fluxes in the chl1-5 mutant of Arabidopsis thaliana. Does the CHL1-5 gene encode a low-affinity nitrate transporter. Plant Physiol 114:137–144. Trueman LJ, Richardson A, Forde BG. 1996. Molecular cloning of higher plant homologues of the high-affinity nitrate transporters of Chlamydomonas reinhardtii and Aspergillus nidulans. Gene 175:223–231. Tsay Y-F, Schroeder JI, Feldmann KA, Crawford NM. 1993. A herbicide sensitivity gene CHL1 gene encodes a nitrate-inducible nitrate transporter. Cell 72:705–713. Tucker M, Von Seelhorst C. 1898. Der Einfluss, welchen der Wassergehalt und der Reichtum des Bodens auf die Ausbildung der Wurzeln und der oberirdischen Organeder Haferpflanze ausu¨ben. J Landw 46: 52–63. Ullrich WR, Larsson C-M, Larsson M, Lesch S, Novacky A. 1984. Ammonium uptake in Lemna gibba G1, related membrane potential change and inhibition of anion uptake. Physiol Plant 61:369–376. Vidmar JJ, Zhuo D, Siddiqi MY, Schjoerring JK, Touraine B, Glass ADM. 2000. Regulation of HvNRT2 expression and high-affinity nitrate influx in roots of Hordeum vulgare by ammonium and amino acids. Plant Physiol 123:307–318. Vitousek PM, Gosz JR, Grier CC, Melillo JM, Reiners WA, Todd RL.1979. Nitrate losses from disturbed ecosystems. Science 204:469–474. Walker DJ, Leigh RA, Miller AJ. 1996. Potassium homeostasis in vacuolated (?) plant cells. Proc Natl Acad Sci USA 93:10510–10514. Wang MY, Siddiqi MY, Ruth TJ, Glass ADM. 1993. Ammonium uptake by rice roots. II. Kinetics of 13 NHþ 4 influx across the plasmalemma. Plant Physiol 103:1259–1267. Wang M, Glass ADM, Shaff JE, Kochian LV. 1994. Ammonium uptake by rice roots. III. Electrophysiology. Plant Physiol 104:899–906. Wang R, Liu D, Crawford NM. 1998. The Arabidopsis CHL1 protein plays a major role in high-affinity nitrate uptake. Proc Natl Acad Sci USA 95:15134– 15239. Whiteaker G, Gerloff GC, Gabelman WH, Lindgren D. 1976. Intraspecific differences in growth of beans at stress levels of phosphorus. J Am Soc Hort Sci 101:472–475. Wolt JD. 1994. Soil Solution Chemistry: Applications to Environmental Science and Agriculture. New York; Wiley. Zhou D, Okamoto M, Vidmar JJ, Glass ADM. 1999. Regulation of a putative high-affinity nitrate transporter (Nrt2;1At) in roots of Arabidopsis thaliana. Plant J 17:563–568.

35 Dynamics of Nutrient Movement at the Soil–Root Interface Albrecht O. Jungk Georg-August University of Go¨ttingen, Go¨ttingen, Germany

I.

INTRODUCTION

Soil is a multiphasic system containing nutrients in various degrees of solubility. The nutrients bound to the solid soil phase are virtually immobile. Only a small fraction is dissolved in the liquid phase. Because roots absorb nutrients in a dissolved state only, the soil solution is the immediate source of plant nutrients. Nevertheless, the major part of nutrients taken up originates from the solid soil material. As a result of nutrient uptake, the concentration of the soil solution near roots usually decreases and a gradient is created which causes the nutrient to diffuse from the bulk of the soil toward the root surface. Furthermore, the depletion of the solution disturbs the equilibrium between the nutrients in solution and those bound to the solid soil phase. As a result, nutrients from the solid phase are released, and the concentration of the solution is replenished. The sequence: root growth—nutrient uptake—depletion of solution—transport from soil—desorption from the solid phase (Fig. 1) is a fundamental feature in supplying plants with mineral nutrients. However, plants have evolved other strategies to mobilize nutrients from the soil matrix. One of them is the modification of the chemical composition of the rhizosphere by exuding substances which enhance the release of nutrients from the soil solid phase (see Chapter 36 by Neumann and Ro¨mheld in this volume). Another widespread strategy is the association of roots with micro-organisms, particularly with fungi, that support roots in acquiring mineral nutrients by enlarging the absorbing surface area (see Chapters 47 by Vance, 48 by

Supplying plants with inorganic nutrients is one of the major functions of roots. The ability of the roots to fulfil this function depends on two complex phenomena: nutrient availability in soil and nutrient acquisition by plants. The term nutrient availability summarizes the soil properties affecting nutrient supply to the plants, and it comprises two aspects: a chemical one and a positional one. The chemical aspect depends on the chemical bonds between the element and other ions or the soil matrix, and the concentration of the element in the soil. The positional aspect is related to both the distribution of the element in the rooting volume and its mobility in the soil. Mobility determines the rate of ion transport, and thus the amount of and the distance from which the ion can move through the soil toward the surface of a root. The term nutrient acquisition encompasses the plant properties that take part in nutrient supply to the plants. This phenomenon includes the physiological processes that are responsible for nutrient entry into the plant (see also Chapters 34 by Glass and 37 by Silberbush in this volume). Nutrient acquisition from a given soil may vary according to genus, species, or even variety. In many soils nutrient availability is inadequate for crop growth unless fertilized. Because of their importance for plant production, both phenomena have been studied intensively (for overview see Barber, 1995; Marschner, 1995; Tinker and Nye, 2000). 587

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amounts of nutrients that actually enter the plant. This chapter provides an insight into the processes involved in the transfer of nutrients from soil into plants and the factors affecting them (see Table 1). II.

ACCESS OF PLANT ROOTS TO THE NUTRIENTS IN SOIL

Contact between the root surface and soil nutrients, which is a prerequisite for uptake, is brought about in two ways: growth of roots to the sites where nutrients are located, and transport of nutrients from the bulk soil to the root surface. Figure 1 The soil–root system, schematic representation of the basic elements: a root embedded in the soil solid phase with sorbed ions and the pore space with liquid (and air). Circles symbolize ions. The processes related to nutrient dynamics are: (1) uptake, (2) transport to root in the liquid phase, (3) desorption from the solid into the liquid phase, and (4) root exudation that may enhance the release of nutrients from the solid phase. (Modified from Claassen and Steingrobe, 1999.)

Kapulnik and Okon, 49 by Sieber, and 50 by Kottke in this volume). Soil and plant properties, i.e., nutrient availability in soil and acquisition by plants, interact at the soil–root interface and thus determine rates of uptake and

A.

Root Interception

The term root interception was introduced by Barber et al. (1963) to describe the amount of nutrients that do not have to move to the root to be available for uptake. The quantity of nutrients intercepted by a growing root equals the amount present in a volume of soil identical to the root volume. The volume of the roots is usually < 1% of the soil volume, even in the densely rooted upper soil layer of agricultural fields. Therefore, root interception provides the plants with < 1% of the soil nutrients. For this reason, only a small part of the nutrient requirement is usually supplied by root interception. There are only a few exceptions to that rule, mainly the amount of calcium

Table 1 Processes Involved in Nutrient Transfer from Soil to Plant and Factors Affecting Them Processes

Factors

Root development

Root length Root distribution Root morphology (architecture, diameter, hairs) Concentration at root surface Kinetics of uptake Transpiration Concentration of soil solution Concentration gradient Diffusion coefficient Depletion of soil solution Root exudates (Hþ =OH ions, reducing agents, carboxylates, chelates) Chemical soil composition (pH, humus, minerals) Physical soil properties (texture, density, hardiness) Soil biology Mycorrhizal infection Bacteria

Nutrient uptake Transport from soil to root mass flow, diffusion

Mobilization by roots desorption, dissolution hydrolysis of org. compounds Mobilization by associated organisms

Nutrient Movement at Soil–Root Interface

589

B.

ð1Þ

Ft ¼ Fm þ Fd

intercepted by roots in a high-calcium soil, which may even exceed plant demand (Table 2). Root growth could therefore seem unimportant in providing access to soil nutrients. However, nutrients of low mobility, such as P and micronutrients, move only short distances through the soil within the life span of a root segment. Therefore, the root system will have to be placed and distributed throughout the rooting zone in a way that will allow adequate amounts of the soil nutrients to reach the root surface by their own movement. For this reason root length density, which is the length of root per unit of soil volume, is of major importance for total uptake, if the mobility of the nutrient is low (Barber, 1995; Claassen, 1990), but also for subsoil depletion of a relatively mobile ion like nitrate (Kage, 1997).

1.

Mass Flow

Mass flow is the convective transport of nutrients dissolved in the solution from the bulk of the soil toward the root. The flux of nutrients established in this way, Fm (mol m2 s1 ), depends on the flux of water,  (m3 m2 s1 ), and the concentration of the soil solution CL (mol m3 ). Fm ¼ CL

ð2Þ

The amount of a nutrient supplied to the plant by mass flow can be estimated by multiplying the volume of water transpired by the mean concentration of the nutrient in the bulk soil solution (Barber, 1962). This figure is often named apparent mass flow, because it does not allow for concentration gradients around roots and other influences. Recent results suggest that this method may markedly overestimate real ion supply by mass flow. Water flux to the root, caused by water uptake, is related to plant growth and is not equally distributed within the rooting zone. Nutrient concentration of the soil solution, on the other hand, is determined by the soil properties and may vary widely within the rooting zone. Therefore, influx of a nutrient into roots is usually different from the rate of supply by mass flow. If mass flow is lower than influx, the soil around the absorbing root will be depleted of the nutrient. In this way a concentration gradient is created from bulk soil toward the root. This is generally the case with phosphate, and potassium as seen from Table 2. Nitrate is often assumed to move largely by mass flow to roots. However, in crop rotations of wheat, barley and sugar beet only 15–33% of the total N uptake were supplied by mass flow (Strebel and

Nutrient Transport from Soil to Plant Roots

The bulk of nutrients has to move over certain distances through the soil in order to reach the root surface (Bray, 1954). Barber (1962) suggested that nutrient transport from soil to root proceeds by mass flow and diffusion, and proposed a method to estimate the proportion of these two mechanisms. This led the way to a quantitative description of the movement of nutrient in the rhizosphere. The driving forces for both transport mechanisms are mediated by the plant. When roots absorb water and nutrients, they create gradients in the soil water potential and the nutrient concentration of the ambient solution. As a result, water and nutrients move along these gradients by simultaneous mass flow and diffusion. The total flux, Ft , is the sum of mass flow, Fm , and diffusive flux, Fd :

Table 2 Significance of Root Interception, Mass Flow, and Diffusion in Supplying a Maize Crop with Nutrients (kg ha1 )

Nutrient Nitrogen Phosphorus Potassium Calcium Magnesium Sulfur Source: Barber, 1995.

Approximate amounts supplied by

Amount needed for 9500 kg yield of grain ha1

Root interception

Mass flow

Diffusion

190 40 195 40 45 22

2 1 4 60 15 1

150 2 35 150 100 65

38 37 156 0 0 0

590

Duynisveld, 1989). Similar values were obtained for fava bean, maize, and oats (Wiesler and Horst, 1994; Kage, 1997). It must therefore be concluded that diffusion is superior to mass flow also in supplying nitrate to the plants. If mass flow is higher than influx, the nutrient accumulates at the soil–root interface. This was often observed with calcium because of its high concentration in the soil solution (Table 2). Magnesium was accumulated or depleted at the root, depending on the Mg concentration of the soil solution (Seggewiss and Jungk, 1988). If anions are present, such as sulfate or carbonate, which form Ca salts of low solubility, it may even lead to precipitation of solid salts (Fig. 2). At tree roots, where the process of accumulation may continue for years, Barber (1995; 99) found macroscopic sheaths of calcium carbonate. Dinkelaker et al. (1989) observed crystals of Ca citrate in the region of cluster roots of lupin resulting from root-released citrate. It is

Jungk

assumed that solid salts at the root surface may damage root tissues and impede water uptake. If highly soluble salts, such as NaCl, accumulate around roots, the water potential of the soil solution may be lowered to detrimental levels (Schleiff, 1986). This process creates severe problems in irrigated crop production under arid conditions. However, salt-adapted species like mangroves (Waisel et al., 1986), and other species growing in saline habitats (Wu and Seliskar, 1998), apparently do not suffer. 2.

Diffusion

Diffusion is the random movement of ions or molecules caused by thermal agitation (Brownian motion). The driving force of net diffusion is a concentration gradient. The diffusive flux, Fd , (mol m2 s1 ) in a homogeneous medium under planar conditions is described by equation (3), which is known as Fick’s first law (Jost, 1952): Fd ¼ D

dC dx

ð3Þ

dC=dx is the concentration gradient (mol m3 m1 ), and D the diffusion coefficient (m2 s1 ), a proportionality factor which relates the diffusive flux to the concentration gradient. Hence, D is the mobility parameter of the diffusing solute. The minus sign indicates that net diffusion is directed toward the lower concentration. The concentration gradient, which develops when ion influx is greater than the rate of supply by mass flow, determines the diffusive flux from soil to root. If, however, a nutrient accumulates at the root as the result of mass flow being higher than influx, net diffusion would tend to be in the opposite direction, opposite to the water flux. As a result of its ion and water uptake, the root can be viewed as a cylindrical sink located in a homogeneous medium (cf. Chapter 37 by Silberbush in this volume). The radial change of concentration due to diffusion around that sink can be estimated by some form of the ‘‘continuity’’ equation (Eq. 4) as used by Barber (1995; 92):   dC 1 d dC ¼ rD ð4Þ dt r dr dr

Figure 2 Gypsum crystals developed on roots of Rhododendron indicum L. grown in a peat substrate, irrigated with water containing Ca2þ and SO2 4 concentrations of 3.9 and 2.1 mol m3 , respectively.

C is the concentration, t is time, and r the radial distance from the center of the root. To calculate diffusive fluxes and ion distributions around roots in soil, diffusion coefficients must be known. These were measured in homogeneous media and are known for nutrient ions dissolved in water. The soil, however, is a nonho-

Nutrient Movement at Soil–Root Interface

591

mogeneous porous system. Ions diffuse essentially in the pore spaces filled with water, and may interact with those bound at the solid soil phase. Diffusion in soil is therefore much slower than in water, and varies with soil conditions and the ion regarded (cf. Tinker and Nye, 2000). Studying the applicability of diffusion theory to ion diffusion in soil, Nye (1966b) suggested equation (5) to make allowance for the impact of soil properties on diffusion. De ¼ DL f

dCL dC

ð5Þ

where De is the effective diffusion coefficient in soil (m2 s1 ), DL the diffusion coefficient in water (m2 s1 ), and  the volumetric water content of the soil (m3 m3 ) which determines the fraction of the cross-sectional area available for diffusion. The impedance factor, f , allows for the restrictions of the mobility of ions in soil compared to water. A major constituent is the distance of diffusion which is, because of the tortuous pathway of diffusion in the water-filled soil pores, longer than the straight line distance. f decreases linearly with the soil water content  (Barraclough and Tinker, 1981; So and Nye, 1989; Bhadoria et al., 1990). dCL =dC is the reciprocal of the soil buffer power for the ion concerned, where CL (mol m3 ) is the concentration of the ion in soil solution, and C (mol m3 ) is the total diffusible amount of this ion. The latter is the sum of the ion in the soil solution plus that sorbed at a unit volume of solid soil material, which is in equlibrium with the ion in solution. The determination of C is difficult because desorption depends on the depletion of the surrounding solution, and may not be instantaneous (Staunton and Nye, 1989). Owing to the various factors impeding ion diffusion in soil, De is usually several orders of magnitude lower than DL , as seen from Table 3, and may vary widely depending on soil conditions (Kaselowsky et al., 1991; Jungk and Claassen, 1997). De is the general parameter for the mobility of a nutrient in soil. It determines the distance out of

Table 3 Diffusion Coefficients of Nutrient Ions in Water, DL , and Order of Magnitude in Soil, De (m2 s1 ) Ion NO 3 Kþ H2 PO 4

DL Water (25 C)

De Soil

1:9  109 2:0  109 0:9  109

1010 –1011 1011 –1012 1012 –1015

which a unit of root can draw the nutrient. The average distance of diffusion,
x, increases with time proportional to the square root of De . It can be estimated by equation (6) (Syring and Claassen, 1995). pffiffiffiffiffiffiffiffiffiffi ð6Þ
x ¼ De t
x is the distance at which the decrease of concentration is 20% of the maximum decrease at the root surface. Nitrate depletion calculated with this equation agreed well with measured data (Claassen and Steingrobe, 1999). It may be used to estimate interroot competition. Equation (6) applies to planar conditions, although the values for cylindrical conditions were found to be similar (Syring and Claassen, 1995). According to equation (6), nitrate ions with a diffusion coefficient of Dl ¼ 1:9  109 m2 s1 would move in water 28 mm per day, but in a soil with De ¼ 5  1011 m2 s1 only 5 mm. The corresponding values for potassium diffusion in the soil (De ¼ 5  1012 m2 s1 ) would be 1.4 mm and for phosphate (De ¼ 1  1013 m2 s1 ) only 0.2 mm per day. As a result, with the root length densities usually found in arable soils, position limits phosphate availability to a fraction of the plough layer (Hendriks et al., 1981), but almost all the nitrate from about a 1-m soil layer is available to cereals (Wehrmann and Scharpf, 1986). Mass flow and diffusion occur concomitantly and are interdependent. It is impossible to calculate exactly what proportion of the total solute absorbed by a plant has arrived by mass flow and how much reached the roots by diffusion (Tinker and Nye, 2000). Nevertheless, Barber’s (1962) idea that transport of nutrients from soil to plant roots is mediated by mass flow and diffusion, has provided a physically defined basis for understanding the interactions between root and soil in the transfer of nutrients.

III.

INTERACTIONS BETWEEN ROOTS AND SOIL

Acquisition of nutrients and water by roots creates gradients of water potential and ion concentration and disturb the ionic equilibria between the solid and the liquid soil phases in the vicinity of roots. This triggers two types of processes in the soil: (1) transport of water and nutrients through the soil toward the root, and (2) exchange of ions between the solid and the liquid soil phases. The rate and the degree of such processes at the interface between root and soil mirror the dynamics of nutrient movement, and they are most

592

Jungk

important constituents of the quantitative aspects of nutrient acquisition by plants. A.

Methods for Studying Nutrient Distribution Around Roots

Perception of the system, to some degree, depends on the methods of measurement. Therefore, a few remarks are made on methods. A simple method for obtaining information on the rhizosphere soil is to take roots out of the soil and shake them gently. The soil adhering to roots is regarded as the rhizosphere soil that can be analyzed separately from the bulk soil (Lorenz et al., 1994). However, the separation of the two samples is poorly defined. To make ion distribution around roots visible, Walker and Barber (1962) introduced the autoradiographic method. For this purpose soil is uniformly mixed with a radioisotope. Roots are caused to grow along a glass plate. An x-ray film, mounted on a plastic foil, is then pressed to the surface. After development, the film provides a visible impression of the nutrient distribution around the root (Fig. 3). To obtain quantitative information, such autoradiographs of soil labelled with 32 P, 33 P or 86 Rb have been scanned by a micro-densitometer (Bhat and

Nye, 1973; Claassen et al., 1981a). The data reflect the change of the isotopically exchangeable P or the total Rb concentration. In order to relate isotopically exchangeable P to the soil solution P concentration, a desorption study must also be carried out. It should relate the amount of phosphate sorbed to the soil matrix to the equilibrium concentration in the soil solution. The spatial resolution of autoradiographs depends on the radiation energy of the nuclide. The use of 33 P, a -emitter of relatively weak energy, represents P concentration profiles rather precisely, because the radiation penetrates only about 0.3 mm of soil. Profiles obtained by 32 P or 86 Rb, nuclides of higher  energy, may deviate markedly from concentration profiles because of their ‘‘crossfire.’’ A disadvantage of roots growing along a glass plate is that the arrangement of root hairs and the geometry of nutrient flow toward roots growing along a glass plate is different from the natural situation. To determine concentration gradients under radial diffusive flow without these disturbances, Kraus et al. (1987) made autoradiographs from cross sections of roots growing in soil followed by densitometric measurements. The method of growing roots along a glass plate was also applied by Marschner and Ro¨mheld (1983)

Figure 3 Autoradiographs of maize roots grown along a glass plate in an agar layer labeled with 32 P-phosphate 4, 6, and 9 days after germination (left to right), respectively. Bar indicates 1 cm. (From Hendriks, 1980.)

Nutrient Movement at Soil–Root Interface

for other nondestructive measurements and observations. After removal of the glass, the soil and root surfaces are covered with a layer of agar containing indicators to visualize chemical changes in the rhizosphere. With this procedure not only pH changes around individual roots and along their axes have been shown in vivo, but also the dynamics of a variety of other elements including enzyme activities (Dinkelaker et al. 1993). Similarly, Reidenbach and Horst (1997) have measured rates of nitrate uptake in different zones of maize roots. To measure nutrient concentrations in soil with conventional methods, relatively large samples are needed. These are difficult to obtain from roots under natural conditions in the spatial resolution necessary to determine ion gradients. To overcome this problem, Kuchenbuch and Jungk (1982) have grown plants in small containers in which roots were separated from soil by a fine-meshed tense screen which can be penetrated only by soil solution and root hairs, not by roots. Seedlings of small-seeded plants produce rapidly a mat of roots, which can be regarded as a planar soil– root interface. The soil is then frozen and cut by a microtome parallel to the screen into layers tenths of a mm in thickness. The samples thus obtained have a known distance from the root, and are large enough to be analyzed for various chemical constituents, enzymes and microorganisms. The results presented in Figs. 13, 15, and 17 were obtained with this procedure. Other authors have applied and modified the method for various purposes. For example, phosphate depletion of the rhizosphere was studied by Hinsinger and Gilkes (1996, 1997), of nonexchangeable potassium by Kuchenbuch and Jungk (1984), the root-induced destruction of soil minerals by Hinsinger et al. (1993), and nonexchangeable ammonium by Scherer and Ahrens (1996). To control pH and nutrient supply, a more sophisticated version was developed by Gahoonia and Nielsen (1991, 1996) and used to evaluate the phosphate uptake efficiency of plants. Zoysa et al. (1997) adapted the approach to rhizosphere processes in tree crops. The method of thin-slicing soil blocks was also applied to measure concentration gradients in soil for the determination of the effective diffusion coefficient (Kaselowsky et al., 1990). Microanalytical methods are becoming available for the determination of ion fluxes into individual cells (Kochian et al., 1992). To measure ion concentrations on a microliter scale, Fritz et al. (1994) applied x-ray microanalysis in a transmission electron microscope, and obtained information on the ion distribution in the soil solution around single roots grown under nat-

593

ural conditions. However, to apply methods like this, techniques of microsampling have still to be developed. Noninvasive imaging methods have begun to be applied to measure dynamic processes around roots in a resolution near 0.1 mm (cf. Gregory and Hinsinger, 1999). B.

Pattern of Soil Depletion Around Roots

To demonstrate the change of nutrient concentration around single roots as a result of nutrient uptake, an autoradiograph is shown in Fig. 3. Before plant growth started, the x-ray film was uniformly darkened by the tracer, indicating homogeneous distribution in the medium. As seen from the brightened areas, the concentration was rapidly decreased soon after germination while the nutrient accumulated in the roots. The depleted zone extended radially with time and included other parts of the medium with the progress of root growth. After 9 days the depleted zone had expanded over the total volume of the rooting medium. In this case, agar was used as a substrate to avoid phosphate adsorption to the solid phase. In soil, phosphate mobility is strongly impeded because phosphate is adsorbed to the soil matrix. The depleted zone is therefore much smaller than that shown here. It indicates that the volume of soil out of which a root can draw a nutrient depends on the mobility of the nutrient, which in turn is largely a function of its binding to the soil matrix. Some anions, such as nitrate and chloride, are hardly bound to soil, and thus mobile in soil about as much as shown in Fig. 3 for phosphate in agar. Quantitative information on soil depletion around root segments of a defined age was obtained by scanning the density of autoradiographs (Claassen et al., 1981a,b). The concentration of 86 Rb, as a tracer for potassium, decreased drastically within the first day, with only small changes occurring later (Fig. 4). The depletion profile extended with time in a radial direction, indicating that, after a short initial phase, root supply depends mainly on transport from further distant soil. The depletion extended
5 mm into the soil perpendicular to the root. The isotopically exchangeable phosphate is the source of soil solution replenishment (Morel and Plenchette, 1994; Morel and Torrent, 1997). A depletion profile of the isotopically exchangeable phosphate (Fig. 5) was obtained by scanning an autoradiograph of a 3-d maize root segment. The depletion zone extended to only 1.5 mm. The difference between P and K/Rb (Fig. 4) is due to their diffusion coefficients (Table 3). The isotopically exchangeable phosphate at

594

Figure 4 Development of a rubidium depletion profile around a maize root segment in a sandy soil, 1, 2, and 4 days old, respectively. (From Claassen et al., 1981b.)

Figure 5 Depletion of two phosphate fractions in the rhizosphere of a maize root 3 days old in a sandy soil. Isotopically exchangeable P was obtained by scanning a 33 P autoradiograph, and soil solution P concentration by the buffer curve shown in Fig. 6. (From Hendriks et al., 1981.)

Jungk

the root surface decreased to about half the initial concentration (Fig. 5). This decrease would amount to as much as 400 kg P ha1 if the total volume of a 30-cm plough layer (3000 m3 ha1 ) were depleted to that level. This would be more than 10 times the P demand of an arable crop. The quantity of P depleted at the root surface may be assumed as that available. However, the volume of soil depleted to this level extended to a radius of hardly 1 mm around the root. Taking this radius and a root length density of 3 cm root per cm3 of soil, only 10% of the plough layer volume would contribute to the phosphate supply of these plants. Hence, a quantity of 40 kg P ha1 could be regarded spatially available in the plough layer (Hendriks et al., 1981). The soil solution P concentration around the root, which is important for both P influx and P desorption, cannot be measured directly. It was derived from the desorption curve shown in Fig. 6. As seen in Fig. 5, the soil solution phosphate concentration was depleted from 25 mmol m3 in the bulk soil to a minimum of 1 mmol m3 at the root surface, only 4% of the initial value. Thus, a steep concentration gradient was created. Only small fractions of the total soil reserves are usually present in solution. Assuming a soil volume of 3000 m3 , with 20% of it filled with water, and a

Figure 6 A phosphate buffer curve: relationship between P concentration of soil solution and isotopically exchangeable phosphate of a sandy soil, used to obtain soil solution P from isotopically exchangeable P in Fig. 5. (From Hendriks et al., 1981.)

Nutrient Movement at Soil–Root Interface

concentration of 25 mmol m3 P (cf. Fig. 5) gives a P quantity of only 0.465 kg ha1 present in the soil solution of the plough layer. For comparison: the P demand of a well-yielding arable crop is
40 kg ha1 . Results of this type demonstrate that the soil solution must be many times replenished from the soil matrix. To achieve that efficiently, roots have to reduce the ion concentration in the soil solution to a very low level. Maize and soybean reduced the phosphate concentration in nutrient solution to < 0:1 mmol m3 (Jungk et al., 1990). Potassium concentration was decreased to 0.4 mmol m3 by wheat and sugar beet (Meyer and Jungk, 1993). This means that plant roots are able to create almost the maximum possible concentration gradient, and thus cause the highest possible diffusive flux from soil to root. This is the mechanism that enables plants to acquire substantial proportions of the nutrients sorbed to the soil matrix. The shape of depletion profiles varies widely among nutrients, depending on the effective diffusion coefficient. Gradients of nitrate, potassium, and phosphate (Barber 1995) from modal De values are shown in Fig. 7. The slopes of the curves indicate that nitrate, because of its high diffusion coefficient (Table 3), moves in the soil over much greater distances than phosphate or potassium. As a result of its superior mobility, nitrate concentration at the root surface remains at a higher level for longer periods of time than that of phosphate and potassium. Thus, at root length densities often found in soil, the whole rooted soil layer is entirely depleted of nitrate (Wehrmann and Scharpf, 1986; Kuhlmann et al., 1989; Strebel and

595

Duynisveld, 1989; Wiesler and Horst, 1993, 1994; Kage, 1997). Uptake is then restricted, unless replenished by nitrification or fertilization. On the contrary, phosphate, which remains undepleted between neighboring roots, continues to be accessible if the roots continue to grow. If the rhizoplane, the surface of root axes, would be the only sink for P uptake, the slope of the curves should become increasingly steep as the root surface is approached (Fig. 7). This is in contrast with the data presented in Figs. 4 and 5, showing that a soil layer of
0:5 mm was almost uniformly depleted. The thickness of this layer equals approximately the average length of the root hairs, exclaiming the contribution of root hairs to soil exploitation (Lewis and Quirk, 1967; Bhat and Nye, 1973; Fo¨hse et al., 1991; Gahoonia et al., 1997; Gahoonia and Nielsen, 1998). C.

Soil Properties Affecting Nutrient Supply to Plants

Nutrient mobility determines transport from soil to root. If concentrations are too low to make mass flow sufficient, diffusion becomes the major mode of ion transport. In such a case, the parameters of ion diffusion determine the rate of supply. 1.

Soil Solution Concentration

Depending on the fluxes toward and into the root, the soil solution concentration may drastically change around root. Analytical measurements of ion concentrations at the root surface are still difficult under natural conditions. Therefore, indirect methods have been used. Under steady-state conditions the influx into roots equals the flux toward the root, which in turn depends on the concentration gradient around the root. Based on this assumption, Barraclough (1986) suggested equation (7), using the steady-state model of Baldwin et al. (1973) that describes ion diffusion to a randomly dispersed root system in soil.
CL ¼ CL  CLr0 ¼  

Figure 7 Comparison among calculated concentration gra þ dients of NO 3 , H2 PO4 , and K ions caused by plant roots in soil when supplied mainly by diffusion. (From Barber, 1995.)

In 4DL f

1 1 1 1n 1  r20 Lv r20 Lv



ð7Þ

where CL is the concentration of the bulk soil solution; CLr0 the concentration at the root surface; In is net inflow per unit root; r0 is root radius, and Lv is root length density. If influx is known, which can be

596

Jungk

calculated from uptake and from root length, the concentration difference between bulk soil and root surface can be estimated. With this method, Barraclough (1986) found that winter wheat needed soil solution concentrations of 165
M nitrate, 14
M phosphate, and 56
M potassium to drive the observed influxes. If the roots fully deplete the soil solution, which may often happen with nitrate, the gradient equals the concentration of the soil solution. However, if the influx, In , and the bulk soil solution, CL , are known, equation (7) can also be used to calculate the concentration at the root surface. Wiesler and Horst (1994) have used this equation to evaluate the efficiency in which maize cultivars utilize soil nitrate by quantifying the factors affecting CLr0 . Seeling and Claassen (1990) applied the same method to experiments with several levels of potassium to determine the kinetic parameters Imax and Km for potassium influx of maize growing in soil. 2. Soil Buffer Power Nutrients move from the soil to the roots essentially in solution, which is in equilibrium with ions bound to the solid soil phase. The mobile and the immobile fractions interact by exchanging nutrients between them, a process termed buffering. The buffer power of a nutrient, b, is defined by b¼

dC dCL

ð8Þ

where C is the total diffusible quantity of an ion in the soil, i.e., the sum of ions dissolved in the soil solution plus those sorbed at the solid phase in equilibrium with CL , the concentration of this ion in the soil solution. The fractions present in the solid or the liquid phase depend on the nutrient, the chemical nature of the soil matrix, and the degree of saturation with the nutrient. To allow for that, the buffering behavior of a soil is usually described by a buffer curve (cf. Fig. 6). When plant roots absorb phosphate from a well-supplied soil, the conditions change. With the decrease of the P solution concentration, the value of b increases. For the example shown in Fig. 6, b increased from 50 to as much as 5000 when the isotopically exchangeable P decreased by 50% of its initial level (cf. Fig. 5). With progressing depletion, the P solution concentration tends to be stabilized, but possibly below the level necessary for adequate supply. The process of buffering does not influence nutrient uptake per se. However, according to equation (5), the

effective diffusion coefficient, De , is inversely proportional to b. Large differences of De exist among nutrients (Table 3). They result mainly from differences in the buffer power. For example, b for nitrate ranges between 0.1 and 0.5, which equals  because soil nitrate is almost fully dissolved in the soil solution. In contrast, b for potassium ranges between 10 and 100, and that for phosphate between 50 and 10,000. For this reason the buffer power often dominates the diffusive transport from soil to root. The buffering properties of soils depend on the chemical and mineralogical nature of the soil material. They are important for a variety of chemical processes in soil, not only the nutrient supply of plants (Lindsay, 1979; Barrow, 1983, 1985). The buffer power for phosphate depends mainly on the number and the properties of ferric iron and aluminum oxy/hydroxy groups at the surface of soil colloids (Barrow, 1985; Geelhoed et al., 1997). In calcareous soils P is also buffered by sorption and precipitation by calcium carbonate (Castro and Torrent, 1998). The kinetics varies among soils, and depends on the aggregation of the soil (Hansen et al., 1999). The exchangeability of phosphate from these surfaces is highly relevant for the bioavailability of this nutrient, and may be affected by root exudates (Morel and Hinsinger, 1999; Geelhoed et al., 1999). The buffer power for potassium is largely determined by the mineralogical composition of the soil, particularly its clay fraction. Therefore, b for potassium varies with the composition of micacious minerals (Sparks, 1987). In soils of similar origin, the value of b generally increases with their clay content. Accordingly, the shape of potassium depletion profiles near the roots may be markedly affected. As seen in Fig. 8, plants utilized larger proportions of potassium from the soil of high De , which was due to a low value of b. Estimation of a generally applicable value of b is still difficult because adsorption and desorption to and from the soil material may not be instantaneous (Staunton and Nye, 1989). Large differences in the value of b for phosphate were found when determined by adsorption or desorption (Bhadoria et al., 1991). This phenomenon is attributed to the reaction between soil and phosphate in which phosphate is initially adsorbed and subsequently diffuses beneath the adsorbing surface (Barrow, 1983, 1985). Cations, such as Kþ , Rbþ , Csþ , and NHþ 4 , may enter the interlayers of micaceous clay minerals (Sparks, 1987). The mobility and instantaneous exchangeability of these ions may be highly restricted (Scherer and Ahrens, 1996).

Nutrient Movement at Soil–Root Interface

597

near roots extended from 0.10 cm at  ¼ 0:14 to 0:20 cm at  ¼ 0:20. The favorable influence of soil moisture on nutrient supply of plants is at least partly the reason for the high fertility of loamy soils compared to sandy ones because they have a higher water storage capacity. The continuity of water films between the soil and the root surface is a basic requirement for nutrient uptake. It may gradually be interrupted during drying of the soil. Read et al. (1999) proposed that the mucilage, produced by the root cap, may play a role in maintaining soil–root contact (see Chapter 3 by Sievers et al. in this volume). 4.

Figure 8 Influence of soil texture on rubidium depletion around 3-day-old maize root segments. (From Claassen et al., 1981b.)

3.

Soil Water-Holding Capacity

The soil water-holding capacity, i.e., the quantity of water present at a certain water potential, is a function of the pore volume and the pore size distribution, which in turn depend on soil texture. Fine-textured (clay) soils have a higher water-holding capacity than coarse-textured (sandy) soils. The volumetric fraction of water in soil, , is an important factor of the effective diffusion coefficient, De (Eq. 5), because nutrients essentially diffuse in water-filled pores. In a silt-loam soil, with the increase of  from 0.20 to 0:37 m3 m3 , De increased almost linearly by a factor of 6 (Kuchenbuch et al., 1986). The impedance factor, f (Eq. 5), which mainly accounts for the tortuosity of the diffusive pathway, is also affected by soil water content. It increases linearly with the volumetric water content (Barraclough and Tinker, 1981; So and Nye, 1989). Therefore, in a given soil, De is proportional to the product of the water content, , and f . The situation may be more complex in aggregated soils because the access to exchange sites is restricted (Nye and Staunton, 1994). An increase of the soil water content, which results in the increase of De , enables roots to draw nutrients from a larger volume of soil. For example, Gahoonia et al. (1994) found that the depletion zone of NaHCO3 -extractable phosphate

Soil Bulk Density

The chemical and physical soil properties affect the function of roots (Glinski and Lipiec, 1990) and the dynamics of nutrient movement at the soil–root interface. Natural settling or pressure exerted by animal, human, or vehicle traffic may cause soil compaction and thus increase soil bulk density. The soil pore space, particularly the larger pores, which provide most of the oxygen supply to the roots, is reduced. There are strong interactions between soil water and soil aeration. If oxygen partial pressure in the soil decreases below a certain level, root growth and function will be impaired by anoxia (Goss et al., 1990; see also Chapter 42 by Armstrong and Drew in this volume). Under such conditions the mobility of nutrients, which can be chemically reduced to more soluble forms, increases; particularly, Fe2þ and Mn2þ ion concentrations may dramatically increase and reach detrimental levels. However, plants adapted to waterlogged soils, such as rice, are able to form aerenchyma, which conducts oxygen from shoot to roots, and release part of it into the rhizosphere (see Chapter 42 by Armstrong and Drew in this volume). Reduced metals may there be reoxidized and toxic levels eliminated (Trolldenier, 1988; Flessa and Fischer, 1992). Uptake of nutrients, especially phosphate, may be impaired by soil compaction (Boone and Veen, 1982; Goss et al., 1990). It is often assumed that compaction decreases the mobility of nutrients in soil. To understand the influence of soil compaction on nutrient movement and supply of plants, several factors have to be taken into account. Primarily, soil compaction decreases the fractions of larger pore and increases the fraction of the smaller ones. The water storage properties of the compacted soil are therefore changed. Both factors affect ion movement. Furthermore, soil compaction may also restrict root growth by increasing

598

the mechanical soil resistance (Ehlers et al., 1983; Chapter 45 by Masle in this volume). Some of the interactions between soil bulk density and phosphate behavior may be illustrated by Fig. 9. The parameters of phosphate diffusion are summarized in Table 4. Soil compaction has changed De slightly. At the same gravimetric water content, the volumetric water content, , was increased by soil compaction (simply because the same quantity of water was concentrated in a smaller volume of soil), and this improved phosphate diffusion, as seen by De . The buffer capacity was slightly increased, because more solid material is contained in a unit volume of compacted soil. This tended to decrease diffusion. Overall, the effective P diffusion coefficient, De , was increased rather than decreased. Phosphate movement from soil to root should therefore not be impaired by compaction of this soil. In comparison, other factors had stronger influence on P diffusion. The addition of water from external sources had markedly increased both  and f . The overriding influence, however, was caused by phosphate fertilizer application, which lowered the buffer power, b, and thus increased De by orders of magnitude. Soil bulk density may affect nutrient transfer also through its effect on root development. Because of the increase of soil strength, the root/shoot ratio (Fig. 9a) was decreased by soil compaction whereas in this experiment (Hoffman and Jungk, 1995), average root diameter was increased from 0.19 to 0.22 mm. As expected from the change of De , and root diameter,

Jungk

influx per unit root surface was increased in most cases (Fig. 9b). Finally, as the overall effect, phosphate supply of the plants, in terms of shoot P concentration, remained almost unchanged (Fig. 9c). Hence, the reduction of root length caused by mechanical soil resistance was apparently compensated for by an increase in influx per unit root length. Shoot growth was also decreased in this case because of soil compaction, but this decrease was not related to the phosphate content of the plants. 5.

Soil Temperature

Diffusive transport is the result of thermal motion, and thus depends on temperature. However, the rate of reaction of nutrients with the soil may also be affected (Barrow, 1979; Sparks, 1987). When the temperature was raised, Buhse (1992) found an increased bonding of phosphate in a fertilized soil at the expense of P in solution. Caused by the increase of the phosphate buffer power, b, the effective diffusion coefficient, De , decreased. Variation of temperature may cause increases and decreases of the bonding of inorganic ions with the soil matrix. Barrow (1992) concluded that the adsorption process is rapid but complex because temperature may separately affect the ions in solution, the charge on the sorbing surface, and the affinity of the ions for the surface. Moreover, adsorption is followed by subsequent slow diffusion of the ions beneath the surface, tending to enhance their retention with temperature. It is therefore difficult to

Figure 9 Root/shoot ratio (a), P influx per unit root length (b), and P concentration of shoot dry matter (c) as related to soil bulk density of sugar beet grown on a luvisol under the influence of two soil moisture and P levels (P-O: no P applied; P-300: 300 mg P [kg soil]1 ). (From Hoffmann and Jungk, 1995.)

Nutrient Movement at Soil–Root Interface

599

Table 4 Factors of Phosphate Diffusion in Soil as Affected by Soil Bulk Density, P Fertilizer Application, and Soil Water Content of a Luvisol from Loess Bulk density (g cm3 ) 1.30

1.50

1.65

P application (mg kg1 )

Water content (g g1 )

0 0 300 300 0 0 300 300 0 0 300 300

0.20 0.32 0.20 0.32 0.20 0.27 0.20 0.27 0.20 0.23 0.20 0.23

CL a (
mol L1 )

Ca (mmol L1 ) 0.29 0.29 8.56 9.02 0.31 0.31 9.34 9.33 0.31 0.31 10.12 10.38

0.1 0.1 108 115 0.1 0.1 112 109 0.1 0.1 115 111

b

 (cm cm3 )

f

De 1013 (m2 s1 )

2926 2906 79 78 3097 3048 83 86 3087 3074 88 93

0.26 0.42 0.26 0.42 0.30 0.41 0.30 0.41 0.33 0.38 0.33 0.38

0.25 0.51 0.25 0.51 0.27 0.40 0.27 0.40 0.28 0.34 0.28 0.34

0.21 0.66 7.32 24.44 0.23 0.48 8.69 16.97 0.27 0.32 9.35 10.74

3

a CL : per liter soil solution; C: per liter soil. Source: Hoffmann and Jungk, 1995.

separate the influence of temperature on these two different processes (Barrow, 1992). In any case, compared to its influence on uptake, the effect of temperature on the behavior of nutrients in soil seems to be small (Moorby and Nye, 1984; Engels and Marschner, 1990; Buhse, 1992; Garnett and Smethurst, 1999). D.

Plant Properties Affecting the Exploitation of Soil Nutrients

50% of the maximum rate of uptake. Saturation, on the other hand, prevents excessive uptake. Net ion influx, In , can often be formally described by equation (10), which was derived from the Michaelis-Menten equation (Nielsen, 1976). Although deviations were found, depending on species and nutritional status of the plant, and on the element regarded (see Chapter 34 by Glass in this volume), the equation can be used to quantify and compare kinetic uptake properties of roots with three parameters under various conditions.

The efficiency of root systems depends on genetic factors (Von Wire´n et al., 1997). Their quantitative influence on the nutrient supply of plants can be expressed by three parameters: (1) net inflow per unit root length, I; (2) the ratio of root length, RL, to shoot weight, SW; and (3) the duration of the uptake activity of each root segment, ta . The relationship between these factors may be described by equation (9) (Claassen and Jungk, 1984): X ¼I

RL t 100 SW a

ð9Þ

where X is the percentage of a nutrient in the shoot. 1.

Kinetics of Nutrient Absorption

Nutrient influx into plants often follows a saturation curve (Fig. 10). Generally, roots are highly efficient in absorbing nutrients in the low concentration range. For example, a P concentration of only 2.6 mmol m3 in the external solution was sufficient to obtain

Figure 10 Michaelis-Menten kinetics of phosphate inflow per cm root length of intact french bean plants, 22 days old, in relation to the P concentration of a complete nutrient solution determined by short-term depletion of the external solution. (From Jungk, 1974.)

600

In ¼

Jungk

Imax ðCL  CL min Þ Km þ CL  CL min

ð10Þ

where Imax ðpmol m2 s1 Þ is the maximum influx, Km ðmmol m3 ) is the Michaelis-Menten constant, i.e., the concentration (CL  CL min ) where In is 50% of Imax , and C min ðmmol m3 ) is the concentration where net influx is zero. Imax defines the capacity or potential of a root to absorb the nutrient when present in high concentration. Km describes the affinity of the root to the nutrient, which indicates the uptake efficiency in the range of low concentrations. CL min defines to what concentration a nutrient may ultimately be depleted at the root surface. The parameters of ion uptake kinetics may vary depending upon the type of nutrient, plant species and genotype, the age of the plant and the root segment, and the nutritional status of plants. Whenever the nutrient is readily available, its total flux into the root system is mainly determined by the demand of the shoot. Demand regulates the uptake by changing the root length per unit of shoot, and net influx per unit of root, In , the parameter of uptake efficiency. Nitrate influx was found to be close to Imax in order to match the demand of plants (Steingrobe and Schenk, 1991; Kirk and Solivas, 1997). Steingrobe and Schenk (1994, 1997) developed a simulation model for nitrate uptake based on the relationship between growth rate and Imax . Uptake properties of root systems varied among various species (Fig. 11) (Fo¨hse et al., 1988).

Inflow of phosphate in French bean and ryegrass was well below 1  1012 mol m1 s1 whereas spinach had five times that influx. Even varieties of the same species may differ markedly (Nielsen and Barber, 1978; Schenk and Barber, 1980; Ro¨mer and Fahning, 1998; Ro¨mer and Schenk, 1998; Egle et al., 1999; Hylander et al., 1999). The ability to deplete phosphate from the soil-root interface differs among species, as seen from Fig. 12. This may be ascribed to differences in Imax and/or CL min . Decrease of influx with the increase of plant or root age was reported (Mengel and Barber, 1974; Jungk and Barber, 1975; Bhat et al., 1979). This might be partly attributed to the demand of shoots per unit of root, and to aging of the roots. Reidenbach and Horst (1997) have shown that nitrate influx of maize was
300 mmol m2 s1 at the root tip, and declined to one-third of that at a distance of 30 cm from the root tip. Similar results were found for Na and K uptake (Eshel and Waisel, 1996). Uptake kinetics of roots may vary markedly with the supply of a plant with that nutrient. (Glass, 1977; de Willigen and Van Noordwijk, 1987; Buysse et al., 1996). To obtain plants with different phosphate status, Jungk et al. (1990) have grown maize and soybean in flowing nutrient solution culture with different phosphate concentrations, each kept constant within treatments. Subsequent short-term measurements of kinetic parameters have shown maximum inflow, Imax , fivefold higher in plants which had been raised in solution

Figure 11 Root/shoot ratio (r/s), phosphate inflow (I), shoot P concentration, and relative yield of seven plant species. (From Fo¨hse et al., 1988.)

Nutrient Movement at Soil–Root Interface

Figure 12 Phosphate depletion around 5-day-old maize and rape roots in a sandy soil labeled with 33 P-phosphate. Data obtained by scanning autoradiographs. (From Hendriks et al., 1981.)

of low compared with that of high phosphate concentration. Imax declined drastically with the increase of percentage of P in the plants (Fig. 13). In comparison, the Michaelis constant, Km , as determined by Hanes plot, did not change much. The minimum P concentration, CL min , decreased with the decrease of P supply from 0.3 to as low as 0.06 mmol m3 . When potassium was withdrawn, Drew et al. (1984) found a decrease of Km by a factor of 4 and no change of Imax in 1 day, but a large increase of Imax thereafter. These results indicate that plants adapt their root uptake kinetics to the demand of the shoot, mainly by adjusting Imax . It is well known that ions interact competitively or synergistically in the uptake process. (see also Marschner, 1995; Chapters 34 by Glass, 36 by Neumann and Ro¨mheld, and 37 by Silberbush in this volume). However, kinetics of uptake is important not only for the flux across the root surface, but also for the movement of nutrients in the ambient soil. The latter may also be influenced by interactions caused by plant demand and the behavior of the ions in soil. For example, magnesium absorption by Lolium perenne was increasingly impeded when potassium concentration at the root surface was > 50 mmol m3 .

601

Figure 13 Maximum phosphate inflow, Imax , measured in short-term solution depletion experiments, as affected by the phosphorus concentration of maize and soybean plants grown under steady-state conditions in flowing solution culture. (From Jungk et al., 1990.)

Because plants need more K than Mg, K influx is usually higher than that of Mg. The soil–root interface was therefore rapidly depleted of K but not of Mg. As soon as the K concentration decreased to < 20 mmol m3 , Mg influx increased abruptly (Seggewiss and Jungk, 1988). K depletion at the soil– root interface may therefore be necessary for adequate Mg supply of plants growing in soils with high K content. Presumably, there are more cases that the removal of one element affects the acquisition of others. For example, Hinsinger and Gilkes (1997) assumed that Ca depletion of the rhizosphere could be a supporting factor for the mobilization of phosphate from apatitic sources. 2.

Size of the Root System

Roots are often considered efficient when Imax is high. However, the benefit of a high Imax to the plants is small if uptake is limited by transport from the bulk soil, as commonly happens with the lesser mobile nutrients. For adequate total uptake under these circumstances, plants have to develop root systems with a large surface area and distributed in a way that ade-

602

Jungk

quate proportions of the soil nutrients can reach the root. The size of the root system is therefore a major factor in plant nutrition. Root systems as the sink for soil nutrients may be described in terms of length, surface area, or weight, and be related to the size of the shoot, to volume of soil, or to soil surface area. Total root size is expressed as root length per unit of area, e.g., km ha1 . Root length per volume of soil, RLv (m m3 ), is the parameter related to interroot competition for nutrients. It determines the average half-distance between roots, r1 , and thus the volume of soil out of which a root can draw nutrients. It can be estimated from equation (11) (Claassen, 1990), under the simplifying assumption that the roots are distributed regularly in soil. 1 r1 ¼ pffiffiffiffiffiffiffiffiffiffiffiffi RLv

shoot ratio (Steffens and Mengel, 1980; Trehan and Claassen, 1998; Sadana and Claassen, 1999). Root/ shoot ratio may also vary among cultivars (Horst et al., 1993; Gahoonia and Nielsen, 1996), and therefore is used as a selecting criterion in breeding for phosphate efficient cultivars (Ro¨mer and Schenk, 1998; Egle et al., 1999). Root/shoot ratio changes within the life cycle of a plant (Jungk and Barber, 1975; Mu¨ller, 1988), and plants have the ability to modify this trait according to nutritional requirements. As shown in Fig. 9a, Pdeficient sugar beet plants had developed much more root length per unit shoot weight than those amply supplied with P, presumably because shoot growth is more limited than root growth. Similar results were reported by Fist (1987) for tropical grain legumes (cowpea, pigeonpea, black gram, guar, and soybean) with phosphate, by Wiesler and Horst (1994) for maize with nitrate, and by Chen and Gabelman (1995) for tomato with potassium. If only part of a root system is supplied with nitrate or phosphate, the part that had received the nutrient increases root length and thus compensates uptake for the other (Drew and Saker, 1978a,b; Laine´ et al., 1998).

ð11Þ

Some data on root size and distribution of arable crops are summarized in Table 5. They indicate large differences among species and, generally, a gradual decrease of root density with soil depth. The root/shoot ratio determines the amount of roots that has to feed a unit of shoot. Root/shoot ratio also varies among species, and with plant age. As shown in Fig. 11, rape and spinach were efficient in phosphate acquisition mainly because of high inflow per unit root length, whereas wheat and ryegrass depended more on a high root/shoot ratio. Onion and French bean were the least successful among these species, apparently because they are low in both properties. Compared to many other plants, potassium uptake efficiency of wheat and other cereals is high because of their high root/

3.

Morphological Root Properties

Morphological root properties may affect the acquisition of nutrients by several factors in various ways. Root architecture determines the spatial configuration and distribution of roots in soil. It is fundamental for the access of plants to the soil nutrients, and affects the competition among roots (Berntson, 1996; Lynch and

Table 5 Root Distribution and Distance Between Roots and Three Arable Crops Root length density, RLV

Average half distance between roots, r1

Soil layer (cm)

Wheat

Maize (cm cm3 )

Spinach

Wheat

Maize (cm)

Spinach

0–30 30–60 60–90 90–120 120–150 150–180

8.2 1.7 1.0 0.7 0.27 0.03

3.8 1.5 0.4 0.1 0.01 —

2.3 0.06 — — — —

0.2 0.4 0.6 0.7 1.1 3.2

0.3 0.5 0.9 1.8 5.6 —

0.4 2.3 — — — —

36

Total root length (km m2 ) 17

7

0–180

Source: Adapted from Claassen and Steingrobe, 1999.

Nutrient Movement at Soil–Root Interface

603

Nielsen, 1996; Chapters 2 by Fitter and 22 by Page`s in this volume). Ge et al. (2000) concluded that, owing to phosphorus deficiency, plants may alter their root architecture. By changing their gravitropism, roots would concentrate in soil layers near the surface which usually contain more phosphate than deeper layers. The ratio between the surface area of a root system and its weight may be markedly affected by the radius of the roots. Radii of feeding roots range in diameter from < 50 to > 300
m with frequent values around 100
m. Root hairs have radii of
5
m and numbers between 100 and 1000/cm root. The hairs appear a few millimeters behind the root apex, and the hair zone may extend between several millimeters and a few centimeters toward the base (Tinker and Nye, 2000; Chapters 5 by Ridge and Katsumi and 37 by Silberbush in this volume). Root hairs are very efficient in increasing the root surface area, although their effect on uptake may not be proportional because of competition among them. In seven species, root hairs roughly doubled the total root surface, compared to naked roots (Fo¨hse et al., 1991). Root hairs extend into pores, and voids in the soil (Misra et al., 1988). The hairs develop perpendicularly to the root axis, and thus increase the soil volume out of which a less mobile nutrient can be drawn (Nye, 1966a). In case of densely haired roots, this volume can be estimated by the average length of hairs as can be concluded from Figs. 5 and 12. However, the influence of root hairs on the depletion profile of roots grown in the bulk soil may have been overestimated because of the technique used. The roots were growing along a glass plate and were forced to concentrate hairs on the plate. Moreover, the radiation of 33 P reached the x-ray film only from a distance of
0:3 mm. Root hairs are assumed to have an influx per unit area similar to that of the root axes (Claassen, 1990; Gahoonia and Nielsen, 1998; Tinker and Nye, 2000). Nevertheless, per unit area, and if they do not interfere with each other, they may exceed root axes in the ability to extract nutrients from soil. Nutrients diffuse from the bulk soil to the root surface in radial direction. Therefore the amount of soil contributing nutients to a unit of root surface depends on the curvature of the sink. The specific soil volume, Vs , out of which a nutrient diffuses to a sink, is negatively related to the root radius as seen from equation (12) (Claassen and Steingrobe, 1999). Vs ¼
r þ


r2 2r0

ð12Þ

where
r is the distance of diffusion and r0 is the root radius. When the sink has a smaller radius, the concentration gradient necessary to drive a certain diffusive flux is therefore attained at a lower concentration of the nutrient in the bulk soil. Feeding roots have radii often between 50 and 150
m whereas the radius of root hairs is
5
m and that of mycorrhizal hyphae is only
1:5
m. As shown in Table 6, the Vs of hyphae may exceed roots by a factor of 100. Thus, because of their morphological properties, root hairs and mycorrhizal hyphae are potentially more efficient than roots as a sink for diffusing nutrients. This is particularly important for nutrients of low mobility. Fo¨hse et al. (1991) have calculated that root hairs contributed up to 90% to the total phosphate absorption. Root hair formation depends on the plant genetics (Gahoonia et al., 1997) and on environmental factors (Marschner, 1995). The length and number of root hairs are apparently among the means to adapt the uptake the efficiency of a root system to the demand of the shoot for some of the nutrients. As seen from Fig. 14, rape plants were equipped with long root hairs if phosphate and nitrate supply was low, and it decreased to almost zero with the increase of supply of these nutrients. K, Ca, and Mg did not have such an effect (Fo¨hse and Jungk, 1983). Plants and fungi have developed symbiotic associations (Tinker, 1984; Chapter 50 by Kottke in this volume). They act, at least partly, in a similar way, but are more efficient than root hairs and provide for transport from the soil to the root over much larger distances than allowed by diffusion. Mycorrhizal fungi are known to improve phosphate supply to plants if available soil P is low (Tinker, 1984). Comparison of soil depletion profiles of mycorrhizal and nonmycorrhizal roots (Fig. 15) shows that the fungus collected phosphate out of a much larger soil volume than roots, and decreased the soil P to a lower level at the root

Table 6 Specific Soil Volume, Vs , per Unit Surface Area of Root Cylinder, Root Hair, and Mycorrhizal Hypha as a Function of Their Radius, r0 a

Root cylinder Root hair Hypha a

r0 ð102 cmÞ

Vs (cm3 cm2 )

1.5 0.05 0.015

1.5 40 133

Calculated with equation (12). The average distance of diffusion,
r, was assumed to be 0.2 cm.

604

Figure 14 Average root hair length in rape plants as affected by phosphate and nitrate concentrations in the nutrient solution. (From Fo¨hse and Jungk, 1983.)

surface. The high extracting efficiency of arbuscular mycorrhizae does not seem to be due to special dissolving abilities, because, after 32 P labeling of the soil, the specific activity of P in both mycorrhizal and nonmycorrhizal plants was equal to that in the soil solution (Tinker, 1975). The efficiency of the fungus may thus

Jungk

mainly be attributed to the small radius of the hyphae, and the large ectomycelium, which consisted in the case of Fig. 15 of a network of
1000 cm hyphae/ cm3 soil extending several centimeters away from the root surface into the soil (Viebrock, 1988). A characteristic of certain species of the Proteaceae are the so called ‘‘proteoid roots.’’ These are bottlebrushlike clusters of rootlets, which form along lateral roots in soils of low fertility (see Chapter 55 by Pate and Watt in this volume). Homologous clusters occur in other families under P deficiency. They increase the surface area of the root system, but furthermore, they can also excrete protons, carboxylic anions, and phenolics, which may cause the release of phosphate and other nutrients from the soil matrix (Dinkelaker et al., 1995; Neumann et al., 1999; Chapter 36 by Neumann and Ro¨mheld in this volume). Low availability of a nutrient means that the flux per cross-sectional area of soil, and thus the influx per unit absorbing area, is small. Root hairs and other systems that increase the absorbing root surface are regarded to be evolutionary adaptations to this situation. The large surface area provides for a relatively high total uptake of a nutrient although the influx per unit area of absorbing surface is relatively small. Plants of this type do not depend on high values of Imax to be efficient.

IV.

Figure 15 Influence of arbuscular mycorrhiza on soil phosphate depletion: Capsicum annuum L. was grown in an oxisol inoculated (þM) or not inoculated (M) with Glomus macrocarpum. Duration: 14 weeks; P extractant: 4M HCl. (From Viebrock, 1988.)

MODELING NUTRIENT DYNAMICS AT THE SOIL–ROOT INTERFACE

As shown above, the transfer of nutrients from soil into root results from both nutrient availability in soil and by the acquisition by plants. Because the two phenomena interact in various ways, it is difficult to measure the factors affecting them and to assess their significance for the overall process. To overcome this problem and to gain a more holistic understanding of the system, mathematical simulation has been attempted by various approaches and is increasingly used (cf. Nye and Marriott, 1969; Claassen and Barber, 1976; Rengel, 1993; Barber, 1995; Tinker and Nye, 2000; Chapter 37 by Silberbush in this volume). It is stressed here only to demonstrate the usefulness of modeling as a tool for better understanding the complexity, and the quantitative aspects of nutrient dynamics in the rhizosphere. Using equation (13) Claassen and Steingrobe (1999) simulated the change in concentration of the total diffusible nutrient in soil around a segment of root with

Nutrient Movement at Soil–Root Interface

605

time, making allowance for the nutrient flux under radial geometry and the influence of soil buffer power.   C 1 C rDe b L þ v0 r0 CL  A b L¼ ð13Þ r r t r where CL ¼ concentration of the nutrient in soil solution, multiplied with b; buffer power, gives the total concentration of diffusible nutrient in soil; t ¼ time; r ¼ radial distance from the root axis; De ¼ effective diffusion coefficient; v0 ¼ water flux across the soil– root interface; r0 ¼ root radius; and A is sink term that accounts for uptake by root hairs according to Michaelis-Menten kinetics using the quasi-steady state approach of Claassen (1990). To integrate equation (13) and run the model the following parameters are required: three soil parameters (initial soil solution concentration, CLi ; effective diffusion coefficient, De ; buffer power, b), and eight root parameters (maximum influx, Imax ; Michaelis constant, Km ; minimum concentration, Cmin ; water influx, v0 ; root radius, r0 ; half-distance between roots, r1 ; initial root length, L0 ; relative root growth rate, k), and for root hairs the three kinetic parameters (Imax , Km , Cmin ) and root hair radius, rh0 . The assumptions are: 1. 2. 3.

4.

A root segment can exploit a limited volume of soil, a cylinder of radius r1 . At the outer border of the cylinder the nutrient flux is zero. At the root surface transport by mass flow and diffusion is equal to influx, which in turn follows Michaelis-Menten kinetics. Roots distribute evenly in the soil; no allowance is made for changing distances between roots when roots grow.

The principle of the model is visualized by Fig. 16, where the distance of soil depletion and nutrient influx is projected to the same axis. Nutrient concentration around a single root changes in space and time. Depletion profiles depend on influx into roots and replenishment from soil. In turn, influx depends on the resultant soil solution concentration. Application of the model has shown that potassium depletion in the rhizosphere of rape seedlings in a luvisol was predicted in good agreement with experimental results (Fig. 17). Results like these have encouraged application of the model for evaluating the influence of factors that cannot be measured. Claassen (1990) and Claassen and Steingrobe (1999) used the approach to evaluate the sensitivity of soil and plant parameters under various constellations of other factors. This is

Figure 16 Schematic representation of the mathematical model relating soil solution concentration in the rhizosphere to nutrient uptake kinetics of roots and nutrient transport from soil.

demonstrated in Fig. 18 for the interactions among the parameters (1) root length density, RLv ; (2) initial soil solution potassium concentration, CLi ; and (3) soil K buffer power on the depletion pattern of the soil solution potassium concentration. In case of the low root

Figure 17 Potassium depletion by 4-day-old rape roots of a luvisol supplied with three K levels. Comparison of data measured with the thin slicing method of Kuchenbuch and Jungk (1982), and the curves obtained by model calculation. (From Claassen et al., 1986.)

606

Jungk

Figure 18 Influence of root length density on soil potassium depletion patterns under various conditions. Potassium concentration of soil solution around maize roots after 10 days uptake calculated for initial soil solution K concentrations, CLi ¼ 0:1 and 1:0
mol K cm3 , root length densities, RLV ¼ 1 and 10, and soil K buffer power, b ¼ 10 and 30. Note: Parallel vertical lines represent roots. (Adapted from Claassen and Steingrobe, 1999.)

density, RLv ¼ 1, and the high K buffer power, b ¼ 30 (Fig. 18a), the K depletion profiles that develop at the root surface do not extend to the average half-distance between roots. Therefore, in that part of the soil, the initially high K solution concentration, CLi , does not change; i.e., there is no (inter-) root competition for potassium. In conclusion: when no root competition occurs, the soil K buffer power does not much affect K flux to roots. In contrast (Fig. 18b), the high root density, RLv ¼ 10, causes a strong root competition for soil K, and the K concentration between roots is markedly reduced. The influence of the K buffer power is now drastically evident. Even at the high value of b, the initially high solution K concentration decreases markedly throughout the whole soil volume, and at the lower value of b to as low as 3% of the initial. The low CLi at the low buffer power is decreased almost to zero. Diffusion to a cylindrical sink is affected by the radius of the sink. Roots and root hairs differ in radius; they can therefore exploit different specific volumes of soil (see Table 6). To demonstrate the role of this factor for phosphate acquisition and its interaction with influx, depletion profiles of two plant species differing in Imax for P influx were calculated separately for single root cylinders and single root hairs as shown in Fig. 19. (More data of the same experiment are given in Fig. 11.) The P concentration at the surface of the root cylinder was reduced close to CL min , almost equally in both species, and thus P

uptake diminished. Around root hairs, however (influx per unit surface assumed equal to that of the root cylinder), because of the higher specific soil volume, depletion proceeds at a slower pace. Therefore, P concentration would remain on a higher

Figure 19 Soil depletion as affected by sink radius and uptake rate. Calculated depletion of soil solution P by root cylinders and single root hairs of two plant species differing in P uptake rate. Radius, r0 , of roots ¼ 100, and of root hairs ¼ 5
m; Imax of wheat ¼ 33, and of rape ¼ 98 mol 1010 m2 s1 . (From Claassen, 1990.)

Nutrient Movement at Soil–Root Interface

level, and therefore influx maintained on a higher rate over a longer period of time. The depletion profiles of root hairs extend over shorter distances than those of the root cylinders, and the concentration gradients around root hairs are steeper than those around the cylinders, which speeds up diffusion from soil. Root hairs can therefore absorb with a higher proportion of Imax than roots. Furthermore, the influence of differences in Imax between the two species are now evident. In conclusion: wheat, having the smaller Imax , will deplete the soil in a slower pace than rape, and sustain adequate influx over longer periods of time. Wheat should therefore succeed with a lower phosphate availability than rape, but wheat needs a higher root/shoot ratio than rape. The approach was also used to compare the measured phosphate uptake by a field crop with that predicted by the model in a soil with low P availability. As seen in Fig. 20, when P availability is adequate, the predicted uptake by wheat was in agreement with the experimental result, at least in the earlier stages of growth. However, without P application, i.e., under inadequate P availability, the measured P uptake was much higher than that predicted, and the difference increased with the progress of the season. This indicates that the model does not include all factors that the plants have used for the acquisition of scarcely available soil phosphate. Probably part of the phosphate was acquired by means of root exudates which are not accounted for in this model. The difference between measured and predicted uptake may be taken as an indication of the amount of nutrients mobilized by such exudates.

Figure 20 Phosphorus uptake of a winter wheat crop measured by field experiment on a luvisol from loess, compared to calculated P uptake. (From Jungk and Claassen, 1989.)

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V.

MOBILIZATION OF SOIL NUTRIENTS BY ROOT EXUDATES

Even though root systems expose large surfaces to the soil and make efficiently use of ion diffusion toward them, these root properties alone may not suffice to feed the plant. Plants have evolved other mechanisms, which increase the solubility of strongly bound soil nutrients in the rhizosphere, often in response to scarce supply. They consist of the release of acidifying, chelating, and reducing agents, and enzymes from roots. As a result, both chemical and biological properties of the rhizosphere may markedly differ from that of the bulk soil (Rengel, 1997). The physiological aspects of these processes are treated in Chapter 36 by Neumann and Ro¨mheld in this volume. Some remarks are made here on the dynamics of nutrient transport in the rhizosphere. The acquisition of some of the micronutrients often depends on root exudation, but that of phosphate and potassium may also be markedly increased (Hinsinger, 1998; Tinker and Nye, 2000), at least in case of low availability in soil. For example, in response to the differential uptake of cations and anions, the pH around roots may be markedly changed by net release of protons or bicarbonate ions (Grinsted et al., 1982). The process is strongly affected by the source of nitrogen. As shown in Fig. 21, the pH of the rhizosphere was decreased by more than 1 unit by ammonium N nutrition, whereas nitrate nutrition caused an increase of a half pH unit. As a result, phosphate was depleted from rhizosphere soil to a much lower level by roots  supplied with NHþ 4 compared to those with NO3 N nutrition. Acidification due to ammonium nutrition was found to be also the driving force for the mobilization of P from rock phosphate (Hinsinger and Gilkes, 1996) and phosphated calcite (Bertrand et al., 1999). However, the effect of a pH change may differ according to the chemical nature of soil P. In an acid oxisol a pH increase had mobilized P rather than a pH decrease (Gahoonia et al., 1992). By root-released protons, potassium (Kuchenbuch and Jungk, 1984) and ammonium (Scherer and Ahrens, 1996) may also be efficiently extracted from K sources of low solubility. Studying the K availability of the trioctahedral micaceous soil mineral phlogopite, Hinsinger et al. (1993) found the rhizosphere pH of rape decreased from 7.5 to 4 within 16 days. Within 4 d, the mineral was depleted from a large part of its interlayer potassium, and within 8 d even from part of the magnesium of the octahedral sheet. They showed clearly that phlogopite in the rhizosphere was thereby

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Jungk

Figure 21 Rhizosphere soil pH and phosphate depletion by ryegrass grown 10 days on a luvisol supplied with ammonium or with nitrate. (From Gahoonia et al., 1992.)

irreversibly transformed to vermiculite within a few days. The change of pH around roots may also affect the dynamics of other nutrients via dissolution or precipitation. In combination with reducing and complexing agents, the solubility of metals such as aluminum, iron, manganese, zinc, copper, and molydenum may drastically be changed (see Chapter 36 by Neumann and Ro¨mheld in this volume). However, an assessment of their influence on root acquisition is still difficult. Quantitative studies of zinc (Wilkinson et al., 1968) and manganese (Sadana and Claassen, 2000) indicate that the uptake of these elements can also be explained without root-induced mobilization. The anions of low-molecular-weight di- and tricaboxylic acids, such as citric, malic, succinic, and others, are often released by roots in relatively large quantities. Mainly the acids are released by roots of dicots, particularly by the cluster roots of lupins under phosphate deficiency (Dinkelaker et al., 1989; Hoffland et al., 1989; Gerke et al., 1994; Jones and Darrah, 1994, 1995; Zoysa et al., 1998; Kirk et al., 1999). Gramineae and other monocots seem to exude smaller amounts of them (Ro¨mheld, 1991; Neumann and Ro¨mheld, 1999). However, fumaric acid was recently found to be secreted in major quantities by barley roots when grown in sterile solution (Gahoonia et al., 2000). The role of these compounds in the rhizosphere has been studied intensively in the last decade (Jones, 1998). Gardner and Boundy

(1983), Fox et al. (1990), and Kamh et al. (1999) have shown that the roots of white lupine grown in phosphate-deficient soils are able to solubilize phosphate from sources that are unavailable to other species. Horst and Waschkies (1987) have descriptively demonstrated that soil phosphate was mobilized by lupin in such quantities that the P supply of wheat was markedly improved when both species were grown together. Citrate was found to be particularly effective in mobilizing sorbed phosphate (Gardner et al., 1983; Jones and Darrah, 1994; Gerke, 1995; Kirk et al., 1999; Geelhoed et al., 1999). The process includes at least two different mechanisms (Jones, 1998): (1) ligand exchange, whereby citrate directly replaces phosphate groups at exchange surfaces, e.g., AlðOHÞ3 or FeðOHÞ3 ; and (2) complexation of metal ions, which constitute the exchange matrix holding P, e.g., Ca2þ in rock phosphate or Fe3þ in FeOOH groups. Under such conditions citrate forms soluble Fe3þ complexes (Gardner et al. 1983) in the pH range of up to about pH 7 (Jones, 1998). The mobilization of phosphate by these processes depends on the sorbent and on the type of chemical bonding of phosphate (Barrow, 1985; Geelhoed et al., 1997). A source of phosphate that appears to be particularly well accessible in this way is the group of humate-Fe/Al-phosphate complexes (Gerke, 1993, 1995) that mainly occurs in podsolic soils.

Nutrient Movement at Soil–Root Interface

A quantitative assessment of phosphate mobilization by root exudation of carboxylates was repeatedly attempted. Nye (1983, 1984) developed a mathematical model to simulate the process which was later applied and modified by other authors (Jones and Darrah, 1995; Kirk, 1999; Gerke et al., 2000a,b). Darrah (1991a,b) measured diffusion coefficients of root exudates to provide parameters for the calculation. Nevertheless, the process is still not fully understood, particularly the form in which desorbed P diffuses from the location of release to the root. As the whole process, Gardner et al. (1983) suggested this sequence of events: complexation of iron by citrate and transport of a citrate-Fe(III)-P complex to the root, split of the complex by reduction of Fe(III) to Fe(II), and liberation of P for uptake. A modified model of the process was proposed by Kirk (1999). The dissolution of phosphate from apatitic sources that occurs in soils of high pH seems to require the combination of both proton and organic anion extrusion from roots (Imas et al., 1997; Neumann and Ro¨mheld, 1999). Using a soil of high CaCO3 content and low P availability, Dinkelaker et al. (1989) and Ro¨mheld and Marschner (1989) observed a strong release of protons and of citrate in the region of cluster roots of lupin, and a massive precipitation of Ca citrate. Therefore, acidification of the rhizosphere in combination with the elimination of Ca ions from the solution appears to be the mechanism of P mobilization from apatitic P by lupin. Not all plants seem to have this ability. Tyler (1992) proposed that the calcifuge plants lack the ability to solubilize the native phosphate of limestone soils. Approximately 50% of the total soil phosphate may be a constituent of soil organic matter. Part of it can be mobilized from, or immobilized into, this fraction by the microbial population of the rhizosphere (McLaughlin et al., 1987). Phosphate esters may be hydrolyzed by phosphatases produced by such microorganisms or by phosphatases released from plant roots. On the outside of roots, phosphatase activity was found in quantities markedly higher than in the bulk soil (Tarafdar and Jungk, 1987; Dinkelaker and Marschner, 1992). Under P-deficient conditions the secretion of phytase and acid phosphatase of 16 plant species increased in all cases (Li et al., 1997). In sugar beet the activity of exogenous root phosphatases increased by four to 20 times (Beissner and Ro¨mer, 1999a), and the kinetic parameters of acid phosphatase, Vmax and Km , indicated higher hydrolyzing efficiency (Beissner and Ro¨mer, 1999b). Nevertheless, the quantitative contribution of organic soil P to the P

609

supply of plants is not well understood. In aseptic nutrient solutions, roots of intact plants have readily hydrolyzed low molecular organic compounds such as sodium phytate, lecithine, and sodium glycerophosphate (Tarafdar and Claassen, 1988). The release rate of inorganic P even exceeded the P influx into roots. These compounds differ from those mainly occurring in soil, but it has been shown (Findenegg and Nelemans, 1993; Asmar et al., 1995; Seeling and Jungk, 1996) that at least part of the heterogeneous pool of organic soil P can be utilized as a P source of plants. Model calculations indicated that about onethird of the total P taken up by a field-grown crop may have been derived from the organic soil phosphorus (Jungk et al., 1993).

VI.

CONCLUSIONS

Research in the last decade has widened our perception of the transfer of nutrients from soil into plants, but many questions are still open. As a general remark, the dynamics of nutrient movement at the soil–root interface has been studied only with some of the nutrients so far, particularly with phosphate, nitrate, ammonium, and potassium, which have major importance in the fertilization of crops. Likewise, the number of plant species included in this field of research is small so far. Annual plants were studied more than perennials and woody plants. Moreover, to limit the complexity of the experiments, and the interpretation of their results, studies were often performed only at early developmental stages of the plants, and for short periods of time. The soil–root interface is the site of entry of mineral nutrients into the living system. The fluxes of nutrients across this interface depend on interactions between both soil and plant properties. When roots grow, the concentration of nutrients in the soil immediately surrounding the roots may dramatically change within a few days, because the rate of nutrient uptake is not matched by the rate of replenishment from further distant soil. The process is essential for the nutrient supply of plants, and may limit plant growth. Direct measurements of fluxes and concentrations at the soil–root interface are still hardly possible. However, in a number of cases, calculations with mechanistic models have shown good agreement between calculated and measured values of both soil depletion around roots and nutrient uptake, which is the result of such fluxes. It may therefore be concluded that we have a realistic perception of the transfer of nutrients

610

from soil into plants. Modeling has thus markedly contributed to the understanding of the dynamic interactions between root and soil in the transfer of nutrients from soil into plants, and the influence of factors that cannot be measured. However, in other cases the results of calculations deviate markedly from those of measurements. This indicates that not all of the mechanisms the plant uses for nutrient acquisition are fully understood. Unavoidably, the quantitative aspects of the transfer of nutrients from soil into plants is widely treated on the basis of simplifying assumptions, and the parameters are not always adequately defined or measurable. Among the soil parameters used for the quantitative description of the dynamics of nutrient mobility in soil around roots, the buffering properties are of major importance. The determination of an adequate parameter of the soil buffer power, b, for the strongly bound nutrients, such as phosphate and potassium, is still a problem, for several reasons. The buffering process is not instantaneous, and big differences occur if b is determined by either adsorption or desorption. The value also depends on the saturation with the respective nutrient, and may change markedly at the root surface while the roots withdraw nutrients from the adjacent soil. Because b is an important parameter of the effective diffusion coefficient in soil, the diffusive transport from the soil to the root depends on b. Therefore, as can be expected, calculated depletion profiles for nutrients of a low buffer power, such as nitrate (Claassen and Steingrobe, 1999) and potassium (cf. Fig. 17) were found in good agreement with those measured experimentally. Not much information on such data is available for other nutrients, but it is in line with this consideration that calculated phosphate uptake agreed with measured uptake only at an ample P supply (cf. Fig. 20), i.e., at a relatively low P buffer power. Therefore, research is still needed to clarify the influence of the change of b near the root surface, which occurs as the result of soil depletion by uptake. The determination of ion diffusion in aggregated soils also appears to be a process not sufficiently solved. The root properties relevant for the acquisition of nutrients present even more problems in regard to their quantitative description. One of them is the distribution of the roots in soil. The assumption usually made for model calculation is that roots are regularly distributed. It implies that the distances among roots are equal and that every segment of root has the same volume of soil to feed on. This assumption may be not far from reality in well-tilled arable soils of a med-

Jungk

ium texture although, in fact, roots are usually distributed at random. But soils of a high clay content, particularly when untilled, and below the plow layer, may form cracks. These cracks, and also large biopores, provide pathways for abundant root growth, which result in a clustered root distribution in soil. De Willigen and Van Noordwijk (1987) have described the problem in detail, but more research is required to assess its importance for nutrient acquisition. Another aspect is root turnover. Within the life span even of annual plants, part of the roots die while others continue to grow. The total root length a plant develops may therefore be several times higher than that present at any instant of time. This root turnover depends on the nutrient supply of the plant. It may thus be assumed to improve the spatial access to nutrients of low mobility, but evidence for the amount of root turnover and its role in nutrient acquisition is still scarce. The increase of the root/shoot ratio as observed under shortage of phosphate and nitrogen may be related to this phenomenon. Likewise, it is generally accepted that root hairs are important for the uptake of nutrients of a root system, and their function appears to be well understood. Nevertheless, the quantitative assessment of their contribution to the total uptake is still doubtful. One reason is the lack of verification for the assumption, that the uptake kinetics of root hairs is equal to that of the root cylinders; other reasons are the difficulty to determine the proportion of roots that develops hairs, and the longevity of the function of root hairs. A basic problem in quantitatively assessing the importance of root properties for nutrient acquisition is the ability of roots to adapt to their environment, because they may change in space and time. Plants develop different types of roots, such as seminal and nodal roots, with different growth patterns and uptake properties, adapted to different sites of the root environment (cf. Chapter 9 by Waisel and Eshel this volume). No report is available how to include these heterogeneities in a quantitative description of nutrient acquisition. Furthermore, in the course of the development of a plant, roots differ in the age of their different elements, position in soil, and distance from the shoot. It is unknown if root position affects nutrient uptake kinetics, but it is known that uptake kinetics changes with the age of a root segment. This may be unimportant during the period of exponential root growth, because the majority of roots are then only a few days old. The properties of the older roots may then be neglected in calculation, but this would cause serious errors when dealing with older plants.

Nutrient Movement at Soil–Root Interface

Adaptation of roots to plant demand and nutrient availability seems to be even more important for rootinduced mobilization of nutrients because the mechanisms applied are specific responses to the shortage of a nutrient. Model calculation that does not account for root exudates has underestimated phosphorus uptake (cf. Fig. 20). The difference between calculated and measured uptake by the Pdeficient plants may be attributed to root exudates, but direct measurements are lacking. Another phenomenon not well understood is the difference among species in the efficiency of potassium uptake (Steingrobe and Claassen, 2000). Differences in root/ shoot ratio and uptake kinetics do not fully account for these differences. There is some indication that plants liberate potassium from the interlayers of clay minerals by mechanisms of which some seem to be still unknown in nature. The adaptive mechanisms involved in the acquisition of nutrients may be assumed, at least partly, to be genetically controlled. Progress in elucidating their nature and their quantitative importance may thus be expected from genetic engineering. The possibility of identifying the controlling genes, production of plants with these genes switched either on or off, may lead the way to a new level of insight in this field.

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Nutrient Movement at Soil–Root Interface Gahoonia TS, Nielsen NE 1991. A method to study rhizosphere processes in thin soil layers of different proximity to roots. Plant Soil 135:143–146. Gahoonia TS, Nielsen NE. 1996. Variation in acquisition of soil phosphorus among wheat and barley genotypes. Plant Soil 178:223–230. Gahoonia TS, Nielsen NE. 1998. Direct evidence on participation of root hairs in phosphorus (32 P) uptake from soil. Plant Soil 198:147–152. Gahoonia TS, Claassen N, Jungk A. 1992. Mobilization of phosphate in different soils by ryegrass supplied with ammonium or nitrate. Plant Soil 140:241–248. Gahoonia TS, Raza S, Nielsen NE. 1994. Phosphorus depletion in the rhizosphere as influenced by soil moisture. Plant Soil 159:213–218. Gahoonia TS, Care D, Nielsen NE. 1997. Root hairs and phosphorus acquisition of wheat and barley cultivars. Plant Soil 191:181–188. Gahoonia TS, Asmar F, Giese H, Gissel-Nielsen G, Nielsen NE. 2000. Root-released organic acids and phosphorus uptake of two barley cultivars in laboratory and field experiments. Eur J Agron 12:281–289. Gardner WK, Boundy KA. 1983. The acquisition of phosphorus by Lupinus albus L. IV. The effect of interplanting wheat and white lupin on the growth and mineral composition of the two species. Plant Soil 70:391–402. Gardner WK, Barber DA, Parbery DC 1983. The acquisition of phosphorus by Lupinus albus L. III. The probable mechanism by which phosphorus movement in the soil–root interface is enhanced. Plant Soil 70:107–124. Garnett TP, Smethurst PJ. 1999. Ammonium and nitrate uptake by Eucalyptus nitens: effects of pH and temperature. Plant Soil 214:133–140. Ge Z, Rubio G, Lynch JP. 2000. The importance of root gravitropism for inter-root competition and phosphorus acquisition efficiency:results from a geomet ric simulation model. Plant Soil 218:159–171. Geelhoed JS, Findenegg GR, Van Riemsdijk WH. 1997. Availability to plants of phosphate adsorbed on goethite:experiment and simulation. Eur J Soil Sci 48:473–481. Geelhoed JS, Van Riemsdijk WH, Findenegg GR. 1999. Simulation of the effect of citrate exudation from roots on the plant availability of phosphate adsorbed on goethite. Eur J Soil Sci 50:379–390. Gerke J. 1993. Solubilization of Fe(III) from humic-Fe complexes, humic/Fe oxide mixtures and from poorly ordered Fe-oxide by organic acids—consequences for P adsorption. Z Pflanzenernahr Bodenk 156:253–257. Gerke J. 1995. Chemische Prozesse der Na¨hrstoffmobilisierung in der Rhizospha¨re und ihre Bedeutung fu¨r den U¨bergang vom Boden in die Pflanze. Go¨ttingen, Germany; Cuvillier Verlag. Gerke J, Ro¨mer W, Jungk A. 1994. The excretion of citric and malic acid by proteoid roots of Lupinus albus L.; effects on soil solution concentration of phosphate,

613 iron, and aluminum in the proteoid rhizosphere in samples of an oxisol and a luvisol. Z Pflanzenernahr Bodenkd 157:289–294. Gerke J, Beissner L, Ro¨mer W. 2000a. The quantitative effect of chemical phosphate mobilization by carboxylate anions on P uptake by a single root. I. The basic concept and determination of soil parameters. Z Pflanzenernahr Bodenkd 163:207–212. Gerke J, Beissner L, Ro¨mer W. 2000b. The quantitative effect of chemical phosphate mobilization by carboxylate anions on P uptake by a single root. II. The importance of soil and plant parameters for uptake of mobilized P. Z Pflanzenernahr Bodenkd 163:213– 219. Glass ADM. 1977. Regulation of K+ influx in barley roots evidence for direct control by internal K+. Aust J Plant Physiol 4:313–318. Glinski J, Lipiec J. 1990. Soil Physical Conditions and Plant Roots. Boca Raton, FL: CRC Press. Goss MJ, Barraclough PB, Powell BA. 1990. The extent to which physical factors in the rooting zone limit crop growth. Asp Appl Biol 22:173–181. Gregory PJ, Hinsinger P. 1999. New approaches to studying chemical and physical changes in the rhizosphere: an overwiew. Plant Soil 211:1–9. Grinsted M, Hedley M, White R, Nye P. 1982. Plant induced changes in the rhizosphere of rape seedlings. 1. pH changes and the increase in P concentration in the soil solution. New Phytol 91:19–29. Hansen HCB, Hansen PE, Magid J. 1999. Empirical modeling of the kinetics of phosphate sorption to macropore materials in aggregated subsoils. Eur J Soil Sci 50:317– 327. Hendriks L. 1980. Vera¨nderung der Phosphatkonzentration des Bodens in der Umgebung lebender Pflanzenwurzeln. PhD thesis, Universita¨t Hannover, Germany. Hendriks L, Claassen N, Jungk A. 1981. Phosphatverarmung des wurzelnahen Bodens und Phosphataufnahme von Mais und Raps. Z Pflanzenernahr Bodenkd 144:486– 499. Hinsinger P. 1998. How do plant roots acquire mineral nutrients? Chemical processes involved in the rhizosphere. Adv Agron 64:225–265. Hinsinger P, Jaillard B. 1993. Root-induced release of interlayer potassium and vermiculitization of phlogopite as related to potassium depletion in the rhizosphere of ryegrass. J Soil Sci 44:525–534. Hinsinger P, Gilkes RJ. 1996. Mobilization of phosphate from phosphate rock and alumina-sorbed phosphate by the roots of ryegrass and clover as related to rhizosphere pH. Eur J Soil Sci 47:533–544. Hinsinger P, Gilkes RJ. 1997. Dissolution of phosphate rock in the rhizosphere of five plant species grown in an acid, P-fixing mineral substrate. Geoderma 75:231– 249.

614 Hinsinger P, Elsass F, Jaillard B, Robert M. 1993. Rootinduced irreversible transformation of a trioctahedral mica in the rhizosphere of rape. J Soil Sci 44:535–545. Hoffland E, Findenegg G, Nelemans J. 1989. Solubilization of rock phosphate by rape. 2. Local root exudation of organic acids as a response to P starvation. Plant Soil 113:161–165. Hoffmann C, Jungk A. 1995. Growth and phosphorus supply of sugar beet as affected by soil compaction and water tension. Plant Soil 176:15–25. Horst W, Waschkies C. 1987. Phosphatversorgung von Sommerweizen (Triticum aestivum L.) in Mischkultur mit weiber Lupine (Lupinus albus L.). Z Pflanzenernahr Bodenkd 150:1–8. Horst WJ, Abdou M, Wiesler F. 1993. Genotypic differences in phosphorus efficiency of wheat. Plant Soil 155/ 156:293–296. Hylander LD, Ae N, Hatta T, Sugiyama, M. 1999. Exploitation of K near roots of cotton, maize, upland rice, and soybean grown in an Ultisol. Plant Soil 208:33–41. Imas P, Bar-Yosef B, Kafkafi U, Ganmore-Neumann R. 1997. Phosphate induced carboxylate and proton release by tomato roots. Plant Soil 191:35–39. Jones DL. 1998. Organic acids in the rhizosphere—a critical review. Plant Soil 205:25–44. Jones DL, Darrah PR. 1994. Role of root derived organic acids in the mobilization of nutrients from the rhizosphere. Plant Soil 166:247–257. Jones DL, Darrah PR. 1995. Influx and efflux of organic acids across the soil–root interface of Zea mays L. and its implications in rhizosphere C flow. Plant Soil 173:103–109. Jost W. 1952. Diffusion in Solids, Liquids, Gases. New York; Academic Press. Jungk A. 1974. Phosphate uptake characteristics of intact root systems in nutrient solution as affected by plant species, age and P supply. Proc Int Colloq Plant Anal Fertil Probl, pp 185–196. Jungk A, Barber SA. 1975. Plant age and the phosphorus uptake characteristics of trimmed and untrimmed corn root systems. Plant Soil 42:227–239. Jungk A, Claassen N. 1989. Availability in soil and aquisition by plants as the basis for phosphorus and potassium supply to plants. Z Pflanzenernahr Bodenkd 152:151–157. Jungk A, Claassen N. 1997. Ion diffusion in the soil-root system. Adv Agron 61:53–110. Jungk A, Asher CJ, Edwards DG, Meyer D. 1990. Influence of phosphate status on phosphate uptake kinetics of maize (Zea mays) and soybean (Glycine max). Plant Soil 124:175–182. Jungk A, Seeling B, Gerke J. 1993. Mobilization of different phosphate fractions in the rhizosphere. Plant Soil 155/ 156:91–94.

Jungk Kage H. 1997. Is low rooting density of faba beans a cause of high residual nitrate content of soil at harvest? Plant Soil 190:47–60. Kamh M, Horst WJ, Amer F, Mostafa H, Maier P. 1999. Mobilization of soil and fertilizer phosphate by cover crops. Plant Soil 211:19–27. Kaselowsky J, Bhadoria PBS, Claassen N, Jungk A. 1990. A method for determining phosphate diffusion coefficients by bulk diffusion in soil. Z Pflanzenernahr Bodenk 153:89–91. Kirk GJD. 1999. A model of phosphate solubilization by organic anion excretion from plant roots. Eur J Soil Sci. 50:369–378. Kirk GJD, Solivas JL. 1997. On the extent to which root properties and transport through the soil limit nitrogen uptake by lowland rice. Eur J Soil Sci 48:613–621. Kirk GJD, Santos EE, Findenegg GR. 1999. Phosphate solubilization of organic anion excretion from rize growing in aerobic soil. Plant Soil 211:11–18. Kochian LV, Shaff JE, Kutreiber WM, et al. 1992. Use of an extracellular, ion-selective, vibrating microelectrode system for quantification of K+, H+, and Ca2þ fluxes in maize roots and maize suspension cells. Planta 188:601–610. Kraus M, Fusseder M, Beck E. 1987. In situ determination of the phosphate-gradient around a root by radiography of frozen soil sections. Plant Soil 97:407–418. Kuchenbuch R, Jungk A. 1982. A method for determining concentration profiles at the soil–root interface by thin slicing rhizosphere soil. Plant Soil 68:391–394. Kuchenbuch R, Jungk A. 1984. Wirkung der Kaliumdu¨ngung auf die Kaliumverfu¨gbarkeit in der Rhizospha¨re von Raps. Z Pflanzenernahr Bodenk 147:435–448. Kuchenbuch R, Claassen N, Jungk A. 1986. Potassium availability in relation to soil moisture. 1. Effect of soil moisture on potassium diffusion, root growth and potassium uptake of onion plants. Plant Soil 95:221– 231. Kuhlmann H, Barraclough PB, Weir AH. 1989. Utilization of mineral nitrogen in the subsoil by winter wheat. Z Pflanzenernaehr Bodenk. 152:291–295. Laine´ P, Ourry A, Boucaud J, Salette J. 1998. Effects of a localized supply of nitrate on NO 3 uptake rate and growth of roots in Lolium multiflorum Lam. Plant Soil 202:61–67. Lewis DG, Quirk JP. 1967. Phosphate diffusion in soil and uptake by plants. III. 32 P autoradiography. Plant Soil 26:445–453. Li M, Osaki M, Rao IM, Tadano T. 1997. Secretion of phytase from the roots of several plant species under phosphorus-deficient conditions. Plant Soil 195:161–169. Lindsay W. 1979. Chemical Equilibria in Soils. New York; John Wiley. Lorenz SE, Hamon RE, McGrath SP. 1994. Differences between soil solutions obtained from rizosphere and

Nutrient Movement at Soil–Root Interface non-rhizosphere soils by water displacement and soil centrifugation. Eur J Soil Sci 45:431–438. Lynch J, Nielsen KL. 1996. Simulation of root system architecture. In: Waisel Y, Eshel A, Kafkafi U, eds. Plant Roots: The Hidden Half. 2nd ed. New York; Marcel Dekker, pp 247–257. Marschner H. 1995. Mineral Nutrition of Higher Plants. 2nd ed. London; Academic Press. Marschner H, Ro¨mheld V. 1983. In vivo measurement of root induced pH changes at the soil–root interface: effect of plant species and nitrogen source. Z Pflanzenernahr Bodenk 111:241–2251. McLaughlin MJ, Alston AM, Martin JK. 1987. Transformations and movement of P in the rhizosphere. Plant Soil 97:391–399. Mengel DB, Barber SA. 1974. Rate of nutrient uptake per unit of corn root under field conditions. Agron J 66:399–402. Meyer D, Jungk A. 1993. A new approach to quantify the utilization of non-exchangeable soil potassium by plants. Plant Soil 149:235–243. Misra RK, Alston AM, Dexter AR. 1988. Role of root hairs in phosphorus depletion from a macrostructured soil. Plant Soil 107:11–18. Moorby H, Nye PH. 1984. The effect of temperature variation over the root system on root extension and phosphate uptake by rape. Plant Soil 78:283–293. Morel C, Hinsinger P. 1999. Root-induced modifications of the exchange of phosphate ion between soil solution and soil solid phase. Plant Soil 211:103–110. Morel C, Plenchette C. 1994. Is the isotopically exchangeable phosphate of a loamy soil the plant-available P? Plant Soil 158:287–297. Morel C, Torrent J. 1997. Sensitivity of isotopically exchangeable phosphate in soil suspensions to the supporting solution. Soil Sci Soc Am J 61:1044–1052. Mu¨ller R. 1988. Bedeutung des Wurzelwachstums fu¨r die Phosphaterna¨hrung von Winterweizen, Wintergerste und Zuckerru¨ben. PhD thesis, University of Go¨ttingen, Germany. Neumann G, Ro¨mheld V. 1999. Root excretion of carboxylic acids and protons in phosporus-deficient plants. Plant Soil 211:121–130. Neumann G, Massonneau A, Martinoia, Ro¨mheld V. 1999. Physiological adaptations to phosphorus deficiency during proteoid root development in white lupin. Planta 208:373–382. Nielsen NE. 1976. A transport kinetic concept for ion uptake by plants. 3. Test of the concept by results from water culture and pot experiments. Plant Soil 45:659–677. Nielsen NE, Barber SA. 1978. Differences among genotypes of corn in the kinetics of phosphorus uptake. Agron J 70:695–698. Nye PH. 1966a. The effect of nutrient intensity and buffering power of a soil, and the absorbing power, size and

615 root-hairs of a root, on nutrient absorption by diffusion. Plant Soil 25:81–105. Nye PH. 1966b. The measurement and mechanism of ion diffusion in soil. 1. The relation between self-diffusion and bulk diffusion. J Soil Sci 17:16–23. Nye PH. 1983. The diffusion of two interacting solutes in soil. J Soil Sci 34:677–691. Nye PH. 1984. On estimating the uptake of nutrients solubilized near roots or other surfaces. J Soil Sci 35:439– 446. Nye PH, Marriott FHC. 1969. A theoretical study of the distribution of substances around roots resulting from simultaneous diffusion and mass flow. Plant Soil 30:459–472. Nye PH, Staunton S. 1994. The self-diffusion of strongly adsorbed anions in soil: a two-path model to simulate restricted access to exchange sites. Eur J Soil Sci 45:145–152. Read DB, Gregory PJ, Bell AE. 1999. Physical properties of axenic maize root mucilage. Plant Soil 211:87–91. Reidenbach G, Horst WJ. 1997. Nitrate-uptake capacity of different root zones of Zea mays (L.) in vitro and in situ. Plant Soil 196:295–300. Rengel Z. 1993. Mechanistic simulation models of nutrient uptake: a review. Plant Soil 152:161–173. Rengel Z. 1997. Root exudation and microflora populations in rhizosphere of crop genotypes differing in tolerance to micronutrient deficiency. Plant Soil 196:255–260. Ro¨mer W, Fahning J. 1998. Phosphataufnahme und-verwertung von drei Inzuchtlinien des Welschen Weidelgrases (Lolium multiflorum Lam.) und ihren Hybriden. Z Pflanzenernahr Bodenk 161:35–39. Ro¨mer W, Schenk H. 1998. Influence of genotype on phospate uptake and utilization efficiencies in spring barley. Eur J Agron 8:215–224. Ro¨mheld V. 1991. The role of phytosiderphores in acquisition of iron and other micronutrients in graminaceous species: an ecological approach. Plant Soil 130:127–134. Ro¨mheld V, Marschner H. 1989. Citric acid excretion and precipitation of calcium citrate in the rhizosphere of white lupin (Lupinus albus L.). Plant Cell Environ 12:285–292. Sadana US, Claassen N. 1999. Potassium efficiency and dynamics in the rhizosphere of wheat, maize, sugar beet evaluated by a mechanistic model. J Plant Nutr 22:939–950. Schenk MK, Barber SA. 1980. Potassium and phosphorus uptake of corn genotypes grown in the field as influenced by root characteristics. Plant Soil 54:65–76. Scherer HW, Ahrens G. 1996. Depletion of non-exchangeable NH4 -N in the soil–root interface in relation to clay mineral composition and plant species. Eur J Agron 5:1–7. Schleiff U. 1986. Water uptake by barley roots as affected by the osmotic and matric potential in the rhizosphere. Plant Soil 94:143–146.

616 Seeling B, Claassen N. 1990. A method for determining Michaelis-Menten kinetic parameters of nutrient uptake for plants growing in soil. Z Pflanzenernahr Bodenk 153:301–303. Seeling B, Jungk A. 1996. Utilization of organic phosphorus in calcium chloride extracts of soil by barley plants and hydrolysis by acid and alkaline phosphatases. Plant Soil 178:179–184. Seggewiss B, Jungk A. 1988. Einfluss der Kaliumdynamik im wurzelnahen Boden auf die Magnesiumaufnahme von Pflanzen. Z Pflanzenernahr Bodenk 151:91–96. So HB, Nye PH. 1989. The effect of bulk density, water content and soil type on the diffusion of chloride in soil. J Soil Sci 40:743–749. Sparks DL. 1987. Potassium dynamics in soils. Adv Soil Sci 6:1–63. Staunton S, Nye PH. 1989. The effect of non-instantaneous exchange on the self-diffusion of phosphate in soil. J Soil Sci 40:751–760. Steffens D, Mengel K. 1980. Das Aneignungsvermo¨gen von Lolium perenne im Vergleich zu Trifolium pratense fu¨r Zwischenschicht-Kalium der Tonminerale. Landwirtsch Forsch 36:120–127. Steingrobe B, Claassen N. 2000. Potassium dynamics in the rhizosphere and K efficiency of crops. J Plant Nutr Soil Sci 163:101–106. Steingrobe B, Schenk MK. 1991. Influence of nitrate concentration at the root surface on yield and nitrate uptake of kohlrabi (Brassica oleracea gongyloides L.) and spinach (Spinacea oleracea L.). Plant Soil 135:205–211. Steingrobe B, Schenk MK. 1994. A model relating the maximum nitrate inflow of lettuce (Lactuca sativa L.) to the growth of roots and shoots. Plant Soil 162:249–257. Steingrobe B, Schenk MK. 1997. Calculation of the total nitrate uptake of lettuce (Lactuca sativa L.) by use of a mathematical model to simulate nitrate inflow. Z Pflanzenernahr Bodenk 160:73–79. Strebel O, Duynisveld WHM. 1989. Nitrogen supply to cereals and sugar beet by mass flow and diffusion on a silty loam soil. Z Pflanzenernahr Bodenk 152:135–141. Syring KM, Claassen N. 1995. Estimation of the influx and the radius of the depletion zone developing around a root during nutrient uptake. Plant Soil 175:115–123. Tarafdar JC, Claassen N. 1988. Organic phosphorus compounds as a phosphorus source for higher plants through the activity of phosphatases produced by plant roots and microorganisms. Biol Fertil Soils 5:308–312. Tarafdar JC, Jungk A. 1987. Phosphatase activity in the rhizosphere and its relation to the depletion of soil organic phosphorus. Biol Fertil Soils 3:199–204. Tinker PB. 1975. The soil chemistry of phosphorus and mycorrhizal effects on plant growth. In: Sanders FE, Mosse B, Tinker PB, eds. Endomycorrhizas. London; Academic Press, pp 353–372.

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36 Root-Induced Changes in the Availability of Nutrients in the Rhizosphere Gu¨nter Neumann and Volker Ro¨mheld University of Hohenheim, Stuttgart, Germany

I.

INTRODUCTION

ties determining the delivery of nutrients from the solid phase to the soil solution; (2) the activity of soil microorganisms involved in the mineralisation of soil organic matter; and (3) the plant requirements and root uptake rates for a given nutrient. This implies the formation of gradients for nutrient availability in the rhizosphere: mobile nutrients, such as Ca2þ , Mg2þ , 2 NO 3 , and SO4 generally present in high concentrations in soil solutions can reach the root surface via mass flow driven by root water uptake and transpiration. The delivery rate may exceed the plant requirements, resulting in accumulation of Ca2þ and Mg2þ in the rhizosphere (Youssef and Chino, 1987) or even in precipitation of CaSO4 at the root surface (Jungk, 1991). In contrast, nutrients that exhibit low solubility in soil solutions, such as P, K, NHþ 4 , and most micronutrients (depending on type and amount of clay minerals in the soil), are rapidly depleted by root uptake in the rhizosphere soil (Kuchenbuch and Jungk, 1984; Ro¨mheld, 1998). Although the total soil content of these nutrients frequently exceeds the plant requirements by several orders of magnitude, the diffusion-based delivery from the soil solid phase is frequently much too slow to match the plant’s demand for proper growth. During a growth season, usually < 20% of the top soil is explored by roots. Theoretical considerations on plant nutrient requirements suggest that sparingly soluble nutrients (e.g., P and Fe) in the rhizosphere soil solution must be

The term rhizosphere was first introduced by Hiltner (1904) to describe the stimulation of microbial biomass and activity in the soil near the root surface. Research activities during the last decades provided increasing evidence that chemical changes at the soil–root interface not only influence plant microbial interactions but also have important effects on plant availability and the acquisition of soil mineral nutrients. Therefore, the term rhizosphere is now defined more generally as the volume of soil that is influenced by root activity (Hinsinger, 1998). The spatial extension of the rhizosphere is highly variable and can range from several millimeters in case of soluble nutrients (e.g., nitrate) and volatile compounds (Darrah, 1993), to < 1 mm for sparingly soluble nutrients such as phosphate (Hu¨bel and Beck, 1993). This depends on soil structure, and particle size, water content, and buffering capacity (Kuchenbuch and Jungk, 1984; Nye, 1986; Chapter 35 by Jungk in this volume), as well as on plant factors including root morphology, mycorrhizal colonization (Jacobsen et al., 1992), nutrient uptake, root exudation, and the physiological status of the plant (Fig. 1; Neumann and Ro¨mheld, 2000). Transport of nutrients in soils and root uptake is generally restricted to the soil solution phase. Thus, mineral nutrient availability in the rhizosphere largely depends on (1) soil-chemical and soil-physical proper617

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Figure 1 Gradients in the rhizosphere.

replaced 20–50 times per day (Marschner, 1995a). This is not simply explainable on base of diffusion processes and requires additional adaptations of higher plants for aquisition of nutrients with limited solubility (Fig. 2). These adaptations can be grouped into strategies toward (1) enhanced spatial acquisition of the available fraction of sparingly soluble nutrients by increasing the root surface involved in nutrient

Figure 2 Strategies for nutrient acquisition in the rhizosphere.

absorption (Anghinoni and Barber, 1980; Kothari et al., 1991), and (2) increasing nutrient solubility by root-induced chemical changes in the rhizosphere (Marschner, 1995b, 1998). The first group of adaptations comprises changes in root morphology and geometry, such as stimulation of root growth and enhanced formation of fine roots and root hairs, but also enhanced mycorrhizal colonization. Chemical strategies for nutrient mobilization include root-induced modifications of pH and redox potential in the rhizosphere, and release of nutrient mobilizing root exudates or of hydrolytic enzymes. Highaffinity uptake systems for mineral nutrients are expressed particularly under conditions of limited nutrient availability (Schachtman et al., 1998). This may support both the diffusion-mediated desorption of mineral nutrients from the soil solid phase by extensive depletion of the rhizosphere soil solution (Morel and Hinsinger, 1999) as well as the spatial exploitation of bulk soils with low nutrient concentrations in the soil solution (Fig. 2). Based on selected examples, this chapter will focus on the various aspects of nutrient acquisition in the rhizosphere. For a more detailed description of processes involved in transport and uptake of nutrients the reader is refered to the Chapters 35 by Jungk, 34 by Glass, and 37 by Silberbush in this volume.

Availability of Nutrients

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619

SPATIAL ACQUISITION OF NUTRIENTS

Adaptive changes in root growth and morphology increasing the surface area for nutrient absorption are most pronounced under conditions of P starvation but also in response to N, Fe, and Zn deficiency (Marschner, 1995b; Landsberg, 1996). Increases in root/shoot dry weight ratio, root branching, root elongation, and fine root production (Table 1) have been related to modifications of sink strength associated with enhanced shoot-to-root partitioning of photosynthates and N compounds (Marschner et al., 1996; Jeschke et al., 1996). A regulatory function of plant hormonal factors is suggested by changes in hormonal balances particularly of auxins, cytokinins, abscisic acid (ABA), and ethylene (Landsberg, 1996; Hare et al., 1997; Borch et al., 1999; Hartung and Jeschke, 1999) and by altered responsiveness to hormonal signals (Borch et al., 1999). Starvation in P, N, and Fe increases density and/or length of root hairs (Robinson and Rorison, 1987; Fo¨hse et al., 1991; Landsberg, 1996), but considerable differences exist between plant species (Robinson and Rorison, 1987) and cultivars (Fig. 3; Gahoonia et al., 1997). Radioactive 32 P tracer studies demonstrated that root hairs provided a significant proportion (63%) of the total P uptake in rye (Gahoonia and Nielsen, 1998). Surprisingly, root hair formation seems to be not affected by other diffusion-limited nutrients such as Zn2þ and Cu2þ , although evidence for uptake of most macro- and micronutrients (NO 3, þ 2þ  2þ 2þ H2 PO , K , Ca , Cl , Zn , Mn ) by root hairs is 4 now available (Gilroy and Jones, 2000). Again, hormonal factors such as ethylene (Lynch and Brown, 1997; Landsberg, 1996; Borch et al., 1999), auxins (Landsberg, 1996; Gilroy and Jones, 2000), cytokinins (Silverman et al., 1998), and abscisic acid (Hartung

Figure 3 Model for the P depletion in the rhizosphere as related to genotypic differences in root hair length.

and Jeschke, 1999) have been implicated in the regulation of root hair initiation and growth. Higher plants are able to exploit nutrients, particularly N and P, from rich patches in soils by localized proliferation and elongation of lateral roots (Fig. 4) as an adaptation to nonhomogeneous nutrient supply, which is rather a rule than an exception under field conditions (Drew, 1975; Leyser and Fitter, 1998; Hodge et al., 1999). Stimulation of lateral root growth has been related to a higher partitioning of photosynthates and auxins (Sattelmacher and Thoms, 1995) or to a local increase of intracellular sugar concentrations (Bingham et al., 1998) at the sites of high nutrient supply. However, recent findings suggest that NO 3 per se may act as an exogenous signal at the site of root proliferation, which rapidly activates the expression of a gene homolog to the MADS box transcription factor as a possible component of the signal

Table 1 Shoot and Root Growth of 12-Day-Old Maize Plants in Response to Increasing Duration of P Starvation Roots Days without P supply 1 2 4 6

Shoot biomass (g dry matter per pot) 2.10 2.34 1.93 1.65

Source: Anghinoni and Barber 1980.

Biomass (g dry matter per pot)

Total length (m per pot)

Diameter (mm)

0.27 0.31 0.40 0.43

46.4 57.7 75.7 90.8

2.27 2.23 1.99 1.84

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However, the mycorrhizal benefit depends very much on characteristics of root morphology, such as length and density of root hairs (see also Chapter 50 by Kottke in this volume).

III.

Figure 4 Responses of barley root growth to localized supply of phosphate and nitrate in different compartments along the root system. (Adapted from Drew et al., 1975.)

transduction chain (Zhang et al., 1999; Zhang and Forde, 2000). Although acquisition of nutrients with high mobility in soils, such as NO 3 ; doesn’t necessarily require the proliferation of lateral roots (Robinson, 1996), the mobile nitrate anion as a product of nitrification may act as a signal molecule to indicate the presence of patches with nutrients of lower soil mobility, such as NHþ 4 , organic N, and P (Forde and Zhang, 1998). Localized root proliferation in the upper soil layers with the greatest P availability has been related to a decreased root geotropic response under conditions of P limitation (Bonser et al., 1996), which may be regulated by enhanced production of ethylene (Lynch and Brown, 1997). For practical applications, fertilizer placement strategies can provide adequate nutrient supply, particularly during early stages of plant and especially root development in soils low in available nutrients (Bo¨hm, 1974). Deep placement of nitrogen fertilizers can promote deep rooting and thus tolerance to drought stress (Garwood and Williams, 1967). Ammonium fertilizers, locally applied in high concentrations, are slowly converted to NO 3 by nitrification (Malhi and Nyborg, 1988) and are exploited by localized root proliferation around the NHþ 4 depot. Particularly in horticultural practice, this may ensure adequate N supply and simultaneously counteract groundwater pollution by NO 3 leaching and excessive accumulation of NO 3 in the plant tissues (Titz and Sommer, 1988; Sommer, 1993). Localized application of P fertilizers in higher concentrations is a strategy to cope with a high capacity for P fixation, particularly in acid soils. In most plant species, mycorrhizal associations can also increase the spatial availability of nutrients such as P, N, Zn2þ , and Cu2þ , extending the nutrient absorptive surface by formation of mycorrhizal hyphae.

ROOT-INDUCED CHANGES IN RHIZOSPHERE pH

The pH in the rhizosphere may differ by up to 2–3 units from that in the bulk soil, with important consequences for the strongly pH-dependent solubility of mineral nutrients and toxic elements in the soil solution (Marschner, 1995a). pH changes in the rhizosphere depend on various soil and plant factors, such as soil buffering capacity, soil moisture level and aeration, CO2 production by plant roots and by microorganisms, microbial acid production, root exudation of carboxylates, plant genotype, and nutritional status of the plant. Quantitatively most important are, however, cation/anion uptake ratios and N assimilation. The driving force for nutrient uptake by root cells is Hþ extrusion, mediated by the activity of a plasma membrane-bound Hþ pumping ATPase, which creates an outward positive gradient in electropotential and pH between the cytosol (pH 7–7.5) and the apoplast (pH 5–6). This electrochemical potential gradient provides the energetization for anion uptake by proton–anion cotransport and for cation uptake via uniport or proton–cation countertransport. Excess uptake of anions over cations therefore leads to net removal of protons in the rhizosphere and to an increase in rhizosphere pH. In contrast, excessive uptake of cations is charge balanced by a net release of protons and consequently leads to rhizosphere acidification (Fig. 5). A.

N Form Effects

Since nitrogen is plant available both in cationic  (NHþ 4 ) and anionic (NO3 ) forms, which can comprise up to 80% of the total ion uptake, the cation/anion uptake ratio can be largely determined by the form of N supply (Marschner, 1995a). Preferential uptake of NO 3 , characteristic for plant growth in many well-aerated agricultural soils, promotes excess uptake of anions over cations and thus a rise in rhizosphere pH. Furthermore, assimilation of NO is associated with production OH 3  (3NO3 ! 2OH ), which is released into the rhizosphere during NO 3 reduction in the root tissue for intercellular pH stabilization (Fig. 5A). The prevailing localization of NO 3 reduction (roots or shoots)

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Figure 6 Effect of a root-induced increase in rhizosphere pH on availability of nutrients (P, Ca, Mg) and toxic elements (Al) to field-grown pearl millet in an acidic sandy soil in West Africa. (Adapted from Bagayoko et al., 2000.)

Figure 5 Model for extracellular pH changes and intracellular pH stabilization as affected by cation/anion uptake ratio and N assimilation.

depends on plant species, root zone temperature, and on the level of NO3- supply with corresponding effects on rhizosphere alkalinization (Goyon et al., 1994; Marschner, 1995a). On acid soils, an increase of the rhizosphere pH in response to NO 3 fertilization may enhance P availability, either by ligand exchange of with P adsorbed to Fe and Al oxides HCO 3 (Gahoonia et al., 1992; Jungk et al., 1993) or by indirect effects on microbial P mineralization (Bagayoko et al., 2000; Alvey et al., 2000). Rhizosphere alkalinization may also counteract Al toxicity in acid soils by increasing the availability of Ca2þ and Mg2þ but reducing the concentration of toxic Al species in the rhizosphere soil solution (Fig. 6; Marschner, 1998; Bagayoko et al., 2000). Also, increased availability of molybdate by reduced adsorption to sesquioxide surfaces in acidic mineral soils has been reported (Trobisch, 1966). Preferential uptake of NHþ 4 occurs under conditions of inhibited or delayed nitrification, particularly in

wetland soils, acid soils, and in soils of Arctic tundras (Chapin et al., 1993; Marschner, 1995b) or shortly after application of NHþ 4 fertilizers, organic fertilizers, and inhibitors of nitrification. Excess cation uptake as þ a consequence of preferential NHþ 4 uptake and H þ production during NH4 assimilation by the root tisþ sues (3NHþ 4 ! 4H ) leads to enhanced net extrusion þ of H and a decrease of pH in the rhizosphere (Fig. 5B; Marschner, 1995a). Particularly in neutral and alkaline soils, NHþ 4 -induced rhizosphere acidification can increase the availability of P out of acid soluble Ca-phosphates (Gahoonia et al., 1992; Logan et al., 2000), and of micronutrients such as Fe, Mn, Zn, Si, and B (Table 2; Reynolds et al., 1987; Thomson et al., 1993). It may increase the availability also of toxic elements such as Cd (Table 3; Wu et al., 1989). Enhanced resistance to plant deseases, such as take all (Gaeumannomyces graminis) and powdery mildew (Erysiphe graminis) by NHþ 4 supply in wheat, has been related to improved micronutrient availability as cofactors of enzymes involved in defense reactions, such as diaminoxidase (Cu), polyphenol oxidase (Cu), ascorbate oxidase (Cu), peroxidase (Mn), and lipoxigenase (Fe). Enhanced mechanical resistance of the cell walls due to higher incorporation of SiO2 has also been reported (Leusch and Buchenauer, 1988; Graham and Webb, 1991). In acid soils, however, rhizosphere acidification may even cause adverse effects on plant growth as a consequence of enhanced P adsorption

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Table 2 Effect of the N Form Applied to a Sandy Loam Soil (pH 6.8) on Rhizosphere pH and Nutrient Uptake in Bean (Phaseolus vulgaris L.) Plants Uptake (
g m1 root length) N form applied

Rhizosphere pH

P

K

Fe

Mn

Zn

Cu

CaðNO3 Þ2 ðNH4 Þ2 SO4 ðþnitrification inhibitor)

6.6 4.5

815 1818

2026 1756

68 184

23 37

11 21

2.7 3.7

Source: Thomson et al., 1993.

to Fe and Al oxides, solubilization of toxic Al species, or even acid-induced root injury (Marschner, 1995a). A cationic nutrient uptake pattern, associated with rhizosphere acidification and corresponding effects on nutrient availability in neutral and alkaline soils, is also characteristic for many leguminous plant species depending on symbiotic N2 fixation (Aguilar and Van Diest, 1981; Wallace, 1982). This can similarly occur during early seedling growth of leguminous plants, probably owing to preferential utilization of the nitrogen seed reserves (Neumann and Ro¨mheld, 1999). Although N form-induced modifications of the rhizosphere pH are a general response of higher plants, the quantitative expression exhibits considerable variation between plant species and even cultivars with important consequences for genotypic differences in nutrient acquisition (Ro¨mheld and Marschner, 1984; Ro¨mheld, 1986; Logan et al., 2000). This has been related to inherent differences in cation/anion uptake ratios (Bekele et al., 1983), the preferential sites of nitrate reduction, and plant adaptations to extremes in soil pH (Marschner, 1995a; Stro¨m et al., 1994). Nitrogen form-induced changes in rhizosphere pH may also affect root morphology (Bloom, 1997). Ammonium-induced stimulation of root elongation in tobacco and potato was mimicked by pH adjust-

ment of the external medium to pH 4.5, even when NO 3 was supplied as nitrogen source. In contrast, plants grown in nutrient solution with moderate NHþ 4 concentrations (2 mM) buffered to pH 7.0 resembled the root morphology of NO 3 -grown plants (Walch-Liu and Engels, 1998). This may be attributed to modifications in apoplastic pH and its impact on root elongation (cf. Hager et al., 1971; Winch and Pritchard, 1999). However, these effects may be superimposed by Al-induced inhibition of root growth in acid soils or by ammonia toxicity at higher NHþ 4 concentrations in combination with neutral or alkaline pH (Marschner, 1995b) B.

Effects of Nutrient Limitation

Irrespective of the nitrogen source, rhizosphere acidification seems to be an adaptive response in many nongraminaceous plant species, which can increase nutrient availability in neutral and alkaline soils particularly under conditions of P, Fe, and Zn deficiency (Ro¨mheld, 1987; Cakmak and Marschner, 1990; Hoffland et al., 1989; Neumann and Ro¨mheld, 1999). Inhibition of nitrate uptake as a response to nutritional stress (Cakmak and Marschner, 1990; Rufty et al., 1990; Gniazdowska et al., 1999) associated with high

Table 3 Effect of N Form on Rhizosphere pH and Cd Concentration in Lolium perenne N form applied NO 3 NH4 NO3 NHþ 4 Source: Wu et al., 1989.

Rhizosphere pH

Cd content in the shoot ðmg kg1 dry matter)

6.8 6.2 5.5

4.2 8.0 12.2

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inherent uptake rates for Ca2þ and Mg2þ (Moorby et al., 1988; Marschner, 1995a) may finally lead to excess uptake of cations, compensated by enhanced net extrusion of Hþ (Table 4; Landsberg, 1981; Marschner, 1995a). Protons are provided by increased biosynthesis of organic acids via nonphotosynthetic CO2 fixation by PEP carboxylase in the root tissue (Hoffland et al., 1992; Rabotti et al., 1995; Johnson et al., 1996; Neumann and Ro¨mheld, 1999). The remaining carboxylate anions may be either stored in the vacuoles of the root tissue, translocated to the shoot, or released into the rhizosphere with Hþ or Kþ as counterions (Neumann and Ro¨mheld, 2000). Carboxylate exudation charge-balanced by Hþ extrusion may additionally contribute to rhizosphere acidification (Dinkelaker et al., 1989, 1997; Hoffland et al., 1992; Neumann et al., 1999). Accordingly, enhanced root excretion of carboxylates and protons in P-deficient white lupin (Lupinus albus L.), as well as Fe deficiency-induced Hþ extrusion in roots of cucumber, was associated with increased activities of the root plasmalemma Hþ ATPase (Rabotti and Zocchi, 1994; Dell’Orto et al., 2000; Mu¨ller et al., 2001). In contrast to NHþ 4 -induced rhizosphere acidification, which generally spreads over the whole root system, Hþ extrusion in response to nutritional stress is frequently confined to distinct root zones (Fig. 7). In Lupinus albus and members of the Proteaceae adapted to extremely P-deficient soils in Western Australia, P deficiency-induced rhizosphere acidification is predominantly restricted to root clusters (proteoid roots)

Figure 7 Effect of N form and Fe nutritional status on rhizosphere acidification (detected by embedding the roots into agar with Bromocresol Purple) and net release of Hþ by roots of intact sunflower plants.

along first-order laterals. Moreover, it is restricted to distinct stages during cluster root development associated with intense excretion of carboxylate anions such as citrate and malate (Dinkelaker et al., 1997; Kamh et al., 1999; Neumann et al.; 1999, 2000; Chapter 55 by Pate and Watt in this volume). In oilseed rape and tomato, P deficiency-induced Hþ extrusion was confined to 1-cm subapical root zones

Table 4 Effect of the P-Nutritional Status on the Ratio of Cation/Anion Uptake, Uptake of NO 3 , and Net Extrusion of Protons by Roots of Different Plant Species Plant species P status Tomato þP P Chickpea þP P White lupin þP P Wheat þP P

Cation/anion uptake ratio

% change in NO3 uptake


pH (growth medium)

0.78 1.33

83:3

1:4

1.17 1.26

48:2

0:6

n.d. 1.38

55:6

1:1

0.39 0.29

not determined

þ1:6

Sources: Dinkelaker et al., 1989; Le Bot et al., 1990; Heuwinkel et al. 1993; Pilbeam et al., 1993; Neumann et al., 1999.

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(Hoffland et al., 1989; Neumann and Ro¨mheld, 1999), and the same acidification pattern is a characteristic response to Fe deficiency in many nongraminaceous plant species (Ro¨mheld and Marschner, 1984; Marschner et al., 1986). The highly localized release of Hþ can distinctly exceed the uniformly distributed þ NHþ 4 -induced H extrusion (Fig. 7). This may enable rhizosphere acidification even in well-buffered calcareous soils and thereby mediate the mobilization of acid-soluble nutrients such as P, Fe, Mn, and Zn (Marschner, 1995a). Additionally, spatial variations in nutrient uptake (Ro¨mheld, 1986; Bloom, 1997), metabolic activity (McCully, 1999; Neumann et al., 1999), Hþ extrusion related to root elongation (Hager et al., 1971; Ro¨mheld, 1986), and emergence of root hairs (Gilroy and Jones, 2000) may have an impact on the pattern of rhizosphere pH along the root system. C.

Impact on Nutrient Uptake

Apart from effects on nutrient availability in soils, processes involved in uptake of nutrients are also affected by alterations in rhizosphere pH. Generally, cation uptake declines with decreasing pH, whereas anion uptake is inhibited when the external pH increases. This may be attributed (1) to competition between Hþ and OH (HCO 3 ) with other cations or anions respectively, (2) to effects of external pH on the electrochemical potential gradient providing the driving force for nutrient uptake, and (3) to pH-induced alterations of root metabolism and function (Marschner, 1995b). These effects may, however, be superimposed by effects of pH on nutrient availability. For example, in hydroponic or sand culture, Mn2þ uptake is inhibited by low pH of the growth medium (Islam et al., 1980; Elamin and Wilcox, 1986), whereas NHþ 4 -induced rhizosphere acidification induces a stimulation of Mn2þ uptake in neutral and alkaline availability soils due to increased Mn2þ (Friedrichsen, 1967). As an example for pH effects on ion uptake by modification of the dissociation equilibrium, P uptake is stimulated at low external pH due to enhanced formation of monovalent H2 PO 4 , which is the preferential form for P uptake by higher plants (Marschner, 1995b). Effects of various phenolic compounds, present in the water-soluble fraction of the soil organic matter, on root growth, and on ion uptake are well documented. Depending on concentration and type of compounds, both stimulatory and inhibitory effects of the compounds have been reported (Maggioni et al., 1987;

Rao, 1990). It was recently demonstrated that humic substances are able to stimulate root extrusion of Hþ (Pinton et al., 1997) and that effects on ion uptake may be at least partially explained by interactions of phenolics such as humic substances with the root plasma membrane Hþ ATPase (Varanini et al., 1993; Pinton et al., 1999a).

IV.

REDOX POTENTIAL AND REDUCING PROCESSES IN THE RHIZOSPHERE

In aerated soils, the redox potentials range between þ500 and þ700 mV but decrease when the soil moisture level increases, reaching negative values in flooded soils. However, even in aerated soils and particularly in the rhizosphere, anaerobic or hypoxic microsites are quite abundant as a consequence of respiratory O2 consumption by rhizosphere microorganisms and plant roots but also due to root growth-induced soil compaction (Fischer et al., 1989). This has important consequences for the solubility of mineral nutrients, such as Mn2þ and Fe2þ , which can even accumulate to phytotoxic concentrations under reducing conditions in poorly aerated soils. Limited oxygen supply or flooding also increases the risk of losses of nitrogen (as N2 , N2 O, etc.) by denitrification or incomplete nitrification (Prade and Trolldenier, 1989). Moreover, þ waterlogging promotes the fixation of NHþ 4 and K by III clay minerals in response to Fe reduction (Chen et al., 1987; Schneider and Scherer, 1998), the production and emmission of methane (Kimura et al., 1991), and the accumulation of phytotoxic low-molecular-weight monocarboxylic acids (Harper and Lynch, 1982) and ethylene (Jackson, 1991). In alkaline soils and in soils rich in organic matter, Zn2þ and P availability may be reduced by adsorption to amorphous Fe hydroxides and carbonates, particularly under fluctuating water regimens (Kirk et al., 1990; Kirk and Bajita, 1995). Plants adapted to waterlogging (e.g., lowland rice) exhibit aerenchyma formation for transport of O2 from the aerial plant parts to the roots, where it is released and maintains high redox potentials in the rhizosphere (Armstrong, 1979; Chapter 42 by Armstrong and Drew in this volume). Aerenchyma formation is induced by increased ethylene concentrations not only under conditions of oxygen shortage but also in response to N and P deficiency (Lynch and Brown, 1997) and strongly depends on K and Si supply (Okuda and Takahashi, 1965; Trolldenier, 1977). In flooded soils, rhizosphere oxidation is an essential adaptation to prevent the accumulation of Fe2þ ,

Availability of Nutrients

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Mn2þ , H2 S, and monocarboxylic acids to phytotoxic levels. This is reflected by precipitation of FeIII hydroxide plaques at the root surface, which in turn can immobilize Mn2þ (Marschner, 1988) and also Zn2þ (Otte et al., 1989; Zhang et al., 1998). The Fe2+ oxidation process also strongly promotes rhizosphere acidification according to the reaction: 4Fe2þ þ O2 þ 10H2 O ! 4FeðOHÞ3 þ 8Hþ This effect is further enhanced by root uptake of NHþ 4 due to inhibition of nitrification and concomitant net extrusion of Hþ . The resulting decrease in rhizosphere pH (Fig. 8) can mediate the mobilization of Zn adsorbed to FeIII hydroxides (Kirk and Bajita, 1995), solubilization of acid-soluble soil P fractions (Saleque and Kirk, 1995), and also the release of fixed NHþ 4 (Schneider and Scherer, 1998). In aerated soils, root-induced reduction of Mn oxides is assumed to be an important mechanism for Mn acquisition as a result of combined effects of enzymatic reduction at the root surface, chemical reduction by excreted compounds such as phenolics and malate, and Mn reduction by Mn-reducing rhizosphere microorganisms (Godo and Reisenauer, 1980; Marschner, 1988; Chapter 43 by Hagemeyer and Breckle in this volume). A marked increase of the reductive capacity of the subapical root zones (Fig. 9C) seems to be a common response to Fe deficiency in dicotyledonous plant species and in nongraminaceous monocotyledons. This is associated with intense acidification of the root apo-

Figure 8 Root-induced Fe oxidation and pH changes in the rhizosphere of lowland rice. (From Begg et al., 1994.)

Figure 9 Model for Fe deficiency-induced changes in root physiology and rhizosphere chemistry associated with Fe acquisition in Strategy I plants. (From Neumann and Ro¨mheld, 2000.)

plast and the rhizosphere (Fig. 9A) and with enhanced release of Fe-chelating and Fe-reducing compounds (Fig. 9B) such as phenolics and carboxylates (Olsen et al., 1981; Ro¨mheld, 1987). Solubilization of Fe3þ is mediated by rhizosphere acidification, by complexation with chelating compounds, and by reduction to FeII (Fig. 9) and has been termed as ‘‘Strategy I’’ for Fe acquisition (Ro¨mheld, 1987). Recently, it was demonstrated that also the complexation of Fe3þ with soluble humic acid fractions and subsequent complex splitting by FeIII reduction may contribute to Fe acquisition of Strategy I plants (Pinton et al., 1998). Humic substances may even increase the root-induced responses to Fe deficiency (Pinton et al., 1999b). Reduced iron is subsequently taken up by the roots probably by a specific transporter for Fe2þ (Fig. 9D), identified by functional complementation of a yeast mutant defective in iron uptake (Eide et al., 1996). Distinct changes in root morphology, such as thickening of root tips, or formation of rhizodermal transfer cells, proliferation of root hairs and of lateral roots may increase the root surface area contributing to Fe deficiency-induced alterations in rhizosphere chemistry (Landsberg, 1982, 1996; Ro¨mheld and Kramer, 1983). Hormones (ethylene and indole acetic acid) have been implicated in the signaling of the coordinated Strategy I response to iron deficiency in nongraminaceous plants (Romera and Alcantara, 1994; Landsberg, 1996; Romera et al., 1999). However, recent studies with mutants of Arabidopsis and tomato suggest that ethylene-induced modifications of root morphology and the physiological responses to Fe deficiency, such as Hþ extrusion and enhanced reductive capacity at the root surface, may be regulated separately (Schmidt et al., 2000a,b).

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The reductive capacity is increased under Fe deficiency by enhanced or by additional expression of a plasmamembrane-bound reductase system (turbo reductase) with a low pH optimum (Bru¨ggemann et al., 1991; Holden et al., 1991), which is further activated by rhizosphere acidification (Fig. 9; Ro¨mheld and Marschner, 1983). Release of carboxylates and phenolics under Fe deficiency stress may be stimulated by a steeper electrochemical potential gradient. PEP carboxylase-mediated biosynthesis of organic acids in root tissues in response to Fe limitation does not only provide protons for rhizosphere acidification but also supplies the electrons for the Fe deficiency-induced plasma membrane-bound reductase system. Oxidation of citrate via cytosolic aconitase and cytosolic NADP-dependent isocitrate dehydrogenase (Bienfait, 1988, 1996) has been reported to be a major direct or indirect electron source for reductasemediated iron reduction. Other studies suggested an important role of NADH (Moog and Bru¨ggemann, 1994) and even of ascorbate (Askerlund and Larsson, 1991). Although no plant FeIII reductases have been isolated and sequenced so far, four candidate genes were identified in Arabidopsis by PCR, using primers based on conserved motivs of yeast FeIII reductases (Robinson et al., 1997). In transgenic tobacco plants, constitutive expression of two FeIII reductase genes from yeast increased Fe contents in leaves by 50% (Samuelsen et al., 1998). A close positive correlation exists between the quantitative expression of Strategy I responses and the resistance of plant species and cultivars to Fe deficiency (Fig. 10; Ro¨mheld, 1987). As a side effect, Fe deficiency-induced root responses in Strategy I plants can increase the avail-

Figure 10 Relationship between Fe efficiency under field conditions and reducing capacity at the root surface of soybean cultivars. (Adapted from Ro¨mheld, 1987.)

ability of Cu, Mn, and Mg (Moraghan, 1985; Welch et al., 1993) but also of toxic elements such as Cd (Rodecap et al., 1994; Cohen et al. 1998), and may even induce Mn toxicity in calcareous soils with low Fe availability but high Mn levels (Moraghan, 1979). Increased reductive capacity of the roots may also be an adaptive response to P (Dinkelaker et al., 1995) and Cu limitation (Welch et al., 1993).

V.

ORGANIC RHIZODEPOSITION

A large proportion of the photosynthetically fixed carbon (30–60% in annual plant species) is translocated to the root system. Out of this carbon fraction, a subtantial proportion (up to 40% in annual plants; up to 70% in forest trees) can be released into the root environment (Lynch and Whipps, 1990; Liljeroth et al., 1994). This has important consequences for the activity and composition of microbial populations, the availability of nutrients, and the solubility of toxic elements in the rhizosphere. Organic rhizodeposition comprises lysates of sloughed-off cells and dead tissues as well as root exudates, released from intact root cells either passively as diffusates or actively as root secretions or excretions. Quantity and composition of the released compounds are affected by multiple factors. Rhizodeposition can be stimulated in response to high light intensities (Rovira, 1959; Cakmak et al., 1998), temperature extremes (Rovira, 1959; Vancura, 1967), the mechanical impedance of the substrate (Boeuf-Tremblay et al., 1995; Groleau-Renaud et al., 1998), toxic elements and low pH in the soil solution (Ro¨mheld and Marschner, 1983; Kochian, 1995; Costa et al., 1997), limitation of nutrients (Marschner, 1998; Neumann and Ro¨mheld, 2000), and the presence of microorganisms (Meharg and Kilham, 1995). The net release of organic compounds is also strongly affected by the expression of absorption mechanisms in plant roots, which are able to retrieve up to 90% of amino acids and sugars released into the rhizosphere (Jones and Darrah, 1993; Darrah, 1996). These findings are in good agreement with the identification of transporters for distinct amino acids (Fischer et al., 1998), small peptides (Steiner et al., 1994), and sugars (Xia and Saglio, 1988) in plant roots. Retrieval mechanisms may thereby help to minimize passive losses of assimilates and particularly of organic nitrogen, which may comprise up to 20–30% of the total plant N assimilation during the growth period (Janzen, 1990). Even the preferential uptake of organic nitrogen has been reported for plant species adapted to ecosystems such

Availability of Nutrients

as Arctic tundras, where the rate of nitrogen mineralization is generally low (Chapin et al., 1993). A.

Sloughed-Off Cells and Tissues

Organic compounds released from sloughed-off root cells and tissues are the major carbon source for rhizosphere microorganisms, but may indirectly have an impact as microbial metabolites on the availability of mineral nutrients and on exclusion of toxic elements in the rhizosphere (Marschner, 1998; Schilling et al., 1998). However, recent studies suggest that even the liberation of sloughed-off root cells is a genetically controlled process, which exhibits genotypic differences between plant species, and can be modified by various environmental factors (Hawes et al., 2000). Production and release of the so-called root border cells is stimulated at the root cap in response to contact with free water and elevated CO2 concentrations. Embedded into a layer of mucilage polysaccharides, the cells are viable after detachment from the root cap for up to 1 week, and can be transported during root growth to more basal parts of the root (McCully, 1999; Hawes et al., 2000). Border cells are able to produce antibiotics and specifically attract root pathogens such as parasitic nematodes, fungal zoospores, and pathogenic bacteria, thereby counteracting infection of the apical root meristem. In response to infection with pathogenic bacteria and to toxic levels of aluminum, root border cells exhibit enhanced mucilage excretion, which seems to repel bacteria and alleviates toxic effects of Al on border cell viability (Hawes et al., 2000). It remains to be established whether border cells also promote nutrient aquisition by release of chelators such as carboxylates or by attraction of symbiotic or associative microorganisms. B.

High-Molecular-Weight Compounds

High-molecular-weight (HMW) compounds such as mucilage polysaccharides and ectoenzymes are released from roots via exocytosis (Battey and Blackbourn, 1993; Battey et al., 1999). Mucilage is released at the root cap as a gelatinous polyuronic acid polysaccharide, and is subsequently transferred to older root zones during maturation of growing cells. Secretion is mediated by Golgi vesicles of hypersecretory root cap cells. Subsequently the secretory cells degenerate or are sloughed off as root border cells (Battey and Blackbourn, 1993; Hawes et al., 2000). Mucilage has protective functions for the root meristem and improves the root soil contact by inclusion and aggre-

627

gation of soil particles (Marschner, 1995b; McCully, 1999). Mucilage with inclusions of soil particles and microorganisms is termed as mucigel (Bowen and Rovira, 1991). A putative function as lubricant, as pointed out in earlier studies, seems to be unlikely, since no water retention or swelling capacity can be demonstrated at water potentials lower than zero (McCully, 1999). Mucilage can to some extent promote P desorption from clay minerals (Matar et al., 1967; Grimal, 1994), probably mediated by the galacturonate component of the polysaccharide (Nagarajah et al., 1970; Grimal, 1994). A direct contribution of the cell wall to mobilization of sparingly soluble P forms was suggested by Ae et al. (1996) and Ae and Otani (1997). In dry soils, stimulation of mucilage secretion in response to increased soil-mechanical impedance can contribute to the maintainance of Zn2þ uptake by facilitating Zn2þ transport from embedded soil particles to the root surface (Nambiar, 1976). This process may be further promoted by root transfer of water from the subsoil and subsequent release into the dry top soil layer (hydraulic lift; Vetterlein and Marschner, 1993). Mucilage may also contribute to exclusion of toxic elements such as Al (Horst et al., 1982) and heavy metals (Cd, Pb) by complexation with galacturonates mainly in exchange with Ca2þ (Morel et al., 1986; Chapter 43 by Hagemeyer and Breckle in this volume). Secretory proteins, such as ectoenzymes (e.g., acid phosphatase, phytase, peroxidase, phenoloxidase), are synthesized by polysomes bound to the endoplasmic reticulum (ER) and enter the endomembrane system by vectorial segregation into the ER lumen. During the passage through the Golgi apparatus they are separated from proteins with vacuolar destination and subsequently are transported to the plasmalemma by transfer vesicles (Chrispeels, 1991; Chrispeels and Raikhel, 1992). Secretory processes involved in exocytosis strongly depend on intracellular and extracellular Ca2þ levels (Battey and Blackbourn, 1993; Battey et al., 1999). Under P-deficient conditions, enhanced secretion of phosphohydrolases by plant roots (e.g., acid phosphatases, nucleases, phytases) and also by rhizosphere microorganisms (acid and alkaline phosphatases, phytases) can enhance the availability of Pi in the rhizosphere by hydrolysis of soluble organic P esters (Fig. 11), which can comprise 20–80% of the total soil phosphorus (Tarafdar and Marschner, 1994; Li et al., 1997a,b; Hayes et al., 2000). However, considerable variation in the ability of plant species and even of cultivars for secretion of phosphohydrolases has been

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Figure 11 Model for root-induced mobilization of sparingly soluble P sources in the rhizosphere by release of Hþ , carboxylate anions, and root-secretory phosphohydrolases.

reported (Li et al., 1997b; Ro¨mer et al., 1995). Phosphorus deficiency-induced root secretion of acid phosphatases is probably regulated at the transcriptional level (Wasaki et al., 1997; Neumann et al., 2000), and may involve sensing of external P concentrations in the growth medium (Wasaki et al., 1999) and differential induction of isoenzymes (Gilbert et al., 1999). Recently, increased acquisition of Fe/Al phytates supplied to agar media was demonstrated by transgenic expression of a phytase gene from Aspergillus niger in Arabidopsis (Richardson et al., 2000). In many soils, however, enzymatic hydrolysis is limited by the low solubility of recalcitrant organic P forms such as Ca/Mg and Fe/Al phytates (Adams and Pate, 1992). Another limiting factor is the low solubility of the rootborne phosphohydrolases, which is restricted by immobilization at the cell wall and in the mucilage layer of apical root zones (Dinkelaker et al., 1997) or by adsorption and inactivation on clay minerals and organomineral associations (Rao et al., 1996). Root excretion of carboxylates such as oxalate and citrate may, on the other hand, enhance the solubility of organic P forms available for the hydrolysis by phosphohydrolases in the rhizosphere (Fig. 11; Beibner, 1997; Otani and Ae, 1999). Root-secretory acid phosphatases may also contribute to Pi retrieval by hydrolysis of organic P permanently lost into the

rhizosphere from sloughed-off and damaged root cells (Lefebvre et al., 1990). C.

Low-Molecular-Weight Compounds

A wide range of low-molecular-weight (LMW) compounds including sugars, amino acids, caboxylates, phytosiderophores, phenolics, vitamins, hormones, and others are released from plant roots (Grayston et al., 1996; Neumann and Ro¨mheld, 2000). Particularly, phenolics, carboxylates, and phytosiderophores, which exhibit reducing properties and/or the ability to form stable complexes with metal cations in the soil matrix, can directly affect the availability of nutrients and of toxic elements in the rhizosphere (Ro¨mheld, 1987; Marschner, 1988; Jones, 1998). A significant utilization of chelated metals by plant roots usually requires complex splitting by root-induced pH changes or reduction of metal species such as Fe, Mn, and Cu at the root surface or in the apoplast (Wallace, 1980; Welch et al., 1993). To a smaller extent, uptake of chelated metal species can take place in the root zones of emerging laterals via endodermal gaps and a yet unsealed endodermis (Marschner et al., 1987). However, a significant direct root uptake of chelated metals has so far been demonstrated only for graminaceaous plant species in case of metal complexes with phytosiderophores.

Availability of Nutrients

Metal complexation by carboxylates is, on the other hand, an important mechanism to exclude uptake of toxic elements such as Al by plant roots (Kochian, 1995; Rengel, 1996; Ma, 2000; Chapter 46 by Matsumoto in this volume). In contrast, direct interactions of sugars and amino acids with soil minerals are only poorly expressed owing to the lack of charges or slow reaction kinetics with metal ions (Jones et al., 1994) but may indirectly influence nutrient availability as products of microbial metabolization (Schilling et al., 1998). Root excretion of carboxylates, phytosiderophores, and phenolics is frequently enhanced by deficiencies of sparingly available nutrients such as P, Fe, or Zn or in response to Al toxicity (carboxylates, phenolics; Jones, 1998; Neumann and Ro¨mheld, 2000). Phosphorus deficiency-induced root excretion of carboxylates has been reported for a large number of mainly dicotyledonous plant species (Ohwaki and Hirata, 1992; Jones, 1998; Neumann and Ro¨mheld, 1999). Carboxylates can mediate the mobilization of sparingly soluble Ca phosphates in alkaline and calcareous soils and of Fe/Al phosphates or humic Fe/Al-P complexes in acidic soils by mechanisms of ligand exchange, dissolution, and occupation of P sorption sites (Fig. 11). In calcareous soils, a concomitant extrusion of Hþ can further contribute to dissolution of acid-soluble Ca phosphates. Among the various carboxylates identified in plant root exudates, citrate and oxalate are most efficient with respect to mobilization of sparingly soluble P forms. This can be attributed to high stability constants for complexation of Fe3þ , Al3þ , and Ca2þ (Jones, 1998). However, model experiments with a wide range of different soils revealed that significant Pi desorption usually requires high carboxylate concentrations ( 1 mM) in the equilibrium solution, corresponding to a carboxylate accumulation of > 10
mol g1 of rhizosphere soil (Amann and Amberger, 1988; Xing-Guo and Jordan, 1995; Gerke et al., 2000a). Comparable rhizosphere concentration levels have so far been reported only for a limited number of plant species such as Lupinus albus, Trifolium pratense, and members of the Proteaceae (Dinkelaker et al., 1989, 1997; Gerke et al., 1994, 2000b). Carboxylates in the rhizosphere soil solution are rapidly degraded by microorganisms with average half-life times of 2– 3 h and almost complete mineralization within 48–72 h (Jones and Darrah, 1994; Jones et al., 1996a; Schilling et al., 1998). Biodegradation of organic acids is highly expressed in soils with a high organic matter content but is strongly inhibited by carboxylate adsorption to

629

the soil matrix (Boudot, 1992; Jones and Darrah, 1994). Localized release of root exudates in cluster roots (Dinkelaker et al., 1989, 1995) or in apical root zones (Marschner et al., 1986; Hoffland et al., 1992) with a low density of microbial colonization (Scho¨nwitz and Ziegler, 1986; Ro¨mheld, 1991) and excretion peaks over a limited period of time (Ro¨mheld, 1991; Dinkelaker et al., 1997; Watt and Evans, 1999) may counteract microbial degradation and thereby increase the rhizosphere concentration level of exudate compounds involved in mobilization of nutrients. Root hairs are frequently most abundant in root zones with high secretory activity (e.g., subapical root zones, cluster roots) but their contribution to the exudation process remains to be established. Root growth rate is another important factor that may affect the accumulation of root exudates in the rhizosphere: Assuming a volume of
30
L cm1 root length for the rhizosphere soil solution at a distance of 1 mm from the root surface (Bar-Yosef, 1991) and a residence time of 5 h for the root apex in a given soil zone (Jones et al., 1996a), carboxylate exudation at a rate of 6 nmol h1 cm1 root length would be required to reach a concentration level of 1 mM in the rhizosphere soil solution. This is comparable to the exudation rates of citrate in cluster roots of P-deficient white lupin (Fig. 12; Keerthisinghe et al., 1998;

Figure 12 Effect of P supply on carboxylate exudation in apical root zones of different plant species grown in hydroponic culture.

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Kamh et al., 1999; Neumann et al., 1999), a plant species with a proven ability to mobilize sparingly available P sources in soils (Gardner et al., 1983; Dinkelaker et al., 1989). In mature cluster roots, the high density of root tips as well as the absence of growth activity (Neumann et al., 1999; Watt and Evans, 1999) will further contribute to increase the concentration level of root exudates effective in nutrient mobilization in the rhizosphere. In principle, root exudation of carboxylates and other LMW compounds can be mediated by diffusion along the steep concentration gradient of organic solutes between the cytosol (mM range) and the rhizosphere soil solution (
M range). At the cytosolic pH of 7.1–7.4 (Marschner, 1995b), carboxylic acids are usually present as carboxylate anions. Carboxylate exudation is further stimulated by an outward positive charge gradient, created by plasmalemma ATPasemediated extrusion of Hþ for energetization of ion uptake, and by a large cytosolic Kþ diffusion potential (Fig. 11). Theoretical calculations for root exudation of LMW compounds and their transport by diffusion revealed a rate of
0:3 nmol h1 cm1 root length or 120 nmol h1 g1 root fresh weight (Jones et al., 1994; Jones, 1998). This is in good agreement with experimental data in the range of 0.5–0:9 nmol h1 cm1 root length or 100–380 nmol h1 g1 root fresh weight reported for apical root zones of wheat, potato, tomato, and white lupin grown in hydroponic culture (Fig. 12; Neumann and Ro¨mheld, 1999). Exudation of LMW compounds can be enhanced to some extent by stress factors affecting membrane integrity, such as nutrient deficiency (K, P, Zn, B), microbial toxins, temperature extremes, salinity, drought, or oxidative stress (Svenningson et al., 1990; Meharg and Kilham, 1995; Sacchi et al., 2000). This has been attributed to increased leakiness of membranes (Ratnayake et al., 1978; Cakmak and Marschner, 1988) but also to impairment of retrieval mechanisms (Jones and Darrah, 1993; Sacchi et al., 2000). However, there is increasing evidence that the selective secretion of chelating compounds (e.g., citrate, oxalate, malate, or phytosiderophores) involved in nutrient mobilization or exclusion of toxic elements (Al3þ ) may be mediated by specific transport mechanisms, such as anion channels with a concomitant release of Hþ or Kþ to maintain charge balance (Ryan et al., 1995; Neumann et al., 1999; Sakaguchi et al., 1999). Phosphorus deficiency-induced root exudation of carboxylates is frequently preceeded by organic acid accumulation in the root tissue (Neumann and Ro¨mheld, 1999). This has been related to increased

Neumann and Ro¨mheld

biosynthesis of organic acids in roots by enhanced carbohydrate catabolism (Neumann et al., 2000) and nonphotosynthetic CO2 fixation via PEP carboxylase (PEPC) (Johnson et al., 1994, 1996). In many dicots with high inherent uptake rates for Ca2þ and Mg2þ , inhibition of NO 3 uptake in response to P limitation results in excess uptake of cations over anions, which is balanced by proton extrusion. The protons are provided by increased biosynthesis of organic acids in the root tissue. Moreover, compared with glycolytic PEP catabolization via pyruvate kinase, which depends on P supply as Pi and ADP, the PEPC reaction results in liberation of Pi , and may therefore reflect a more economic Pi utilization under conditions of P limitation (Plaxton, 1998). Improved acquisition of sparingly soluble Ca-P and Al-P has recently been reported after transgenic overexpression of citrate synthase in tobacco (Lo´pezBucio et al., 2000) and Arabidopsis plants (Koyama et al., 2000) or in carrot cells (Koyama et al., 1999) and has been attributed to an increased biosynthetic capacity for citric acid, associated with higher rates of citrate exudation. Increased accumulation of organic acids in the root tissue of P-deficient plants may be also related to enhanced shoot-to-root allocation of carboxylates (Hoffland et al., 1992) and to a reduction in turnover of organic acids (Neumann and Ro¨mheld, 1999). Accumulation of extraordinarily high amounts of citrate during cluster root development in white lupin (20–30
mol g1 root fresh weight) was associated with enhanced activity of enzymes involved in biosynthesis of organic acids (PEPC, MDH, and CS) (Johnson et al., 1994; Keerthisinghe et al., 1998) but decreased levels of soluble intracellular Pi, and a reduction of aconitase activity and root respiration (Table 5; Neumann et al., 1999). It was suggested that P-limited respiration causes a feedback inhibition of the dehydrogenases in the tricarboxylic acid (TCA) cycle, and thus a downregulation of citrate turnover, to counteract overreduction of the respiratory chain. Owing to the limited storage capacity in the vacuoles of cluster roots (Dinkelaker et al., 1995), citrate and protons may finally be released into the rhizosphere. This may prevent cytosolic overaccumulation, associated with cytoplasmic acidosis and interferences with Ca homeostasis by Ca2þ complexation with citrate (Neumann et al., 2000). Similar detoxification mechanisms with comparable rates of root exudation and intracellular accumulation of carboxylates have been discussed for accumulation and release of lactic acid in hypoxic maize root tips

Availability of Nutrients

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Table 5 Specific In Vitro Activities of PEP Carboxylase, Aconitase, and Root Respiration as Related to Citrate Accumulation in Roots of P-Sufficient and P-Deficient White Lupin nmol product min1 mg1 protein Root tissue þP nonproteoid roots P nonproteoid roots P mature proteoid roots

PEP carboxylase

Aconitase

0.12 0.21 0.34

0.019 0.014 0.014

Respiration Citrate concentration ð
mol O2 min1 g1 fwtÞ (
mol g1 fwt) 0.90 0.88 0.53

2.5 8.8 22.1

Source: Neumann et al., 1999.

(Xia and Roberts, 1994) and of citrate and malate in bicarbonate-tolerant rice genotypes (Yang et al., 1994; Hajiboland, 2000). In the latter case, high bicarbonate concentrations—a well-documented stress factor in many calcareous soils (Lee, 1998)—induced the accumulation of high carboxylate concentrations (malate and citrate at 30–70
mol g1 root fresh weight), particularly in the apical root zones of bicarbonate-sensitive varieties of rice, associated with a drastic inhibition of root growth. In contrast, root growth was not inhibited in bicarbonate-tolerant genotypes, which exhibited lower root concentrations and increased exudation of carboxylates, as well as higher shoot concentrations of Zn and Fe as major limiting nutrients in calcareous soils (Yang et al., 1993, 1994; Hajiboland, 2000). Similarly, higher exudation rates of carboxylates, reported in various calcicole plant species in comparison with calcifuge plants, have been related to improved acquisition of nutrients (P, Fe, Mn) in calcareous soils (Fig. 13; Stro¨m et al., 1994; Stro¨m, 1998). However, in alkaline soils, a major limiting factor for mobilization of micronutrients (and particularly of Fe3þ ) mediated by carboxylates such as citrate, is the low stability of Fe-citrate complexes at pH levels > 6:8 (Jones et al., 1996b). This is frequently associated with a high buffering capacity of calcareous soils, which depresses root-induced rhizosphere acidification and FeIII reduction involved in Fe acquisition by Strategy I plants (Chaney, 1984; Ro¨mheld, 1986). In graminaceous plant species, the release of considerable amounts of nonproteinaceous amino acids (mugineic acids; Fig. 14B)—so-called phytosiderohores (PS)—with a high capacity to form stable chelates with Fe3þ even at soil pH levels > 7 is an alternative strategy (termed Strategy II) to cope with low Fe availability in alkaline soils (Tagaki et al., 1984; Ro¨mheld, 1991), which is less dependent on the bulk soil pH. Phytosiderophores can also mediate the extraction of considerable amounts of Zn, Mn, Cu

(Treeby et al., 1989), and even Cd and Ni (Awad and Ro¨mheld, 2000) in calcareous soils (Fig. 14C), but there is only minimal competition by chelation with major soil cations such as Ca2þ , Mg2þ , and Al3þ (Ma and Nomoto, 1996). However, recent studies sugand particularly of P gest that application of SO2 4 fertilizers may counteract Fe-PS complexation, mainly by displacement of PS from the surface of Fe (hydr)oxides (Hiradate and Inoue, 1998). Phytosiderophoremediated Fe mobilization is not only restricted to Fe (hydr)oxides as a mineral Fe source: There is increasing evidence that graminaceous plants can aquire Fe by exchange chelation from complexes with native or partially degraded microbial siderophores such as rhizoferrin or dimerum acids (Fig. 14E; Yehuda et al.,

Figure 13 Proposed role of organic acid metabolism in adaptations of calcicole and calcifuge plant species and genotypes to high levels of soil bicarbonate and nutrient acquisition in calcareous soils.

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Neumann and Ro¨mheld

Figure 14 Model for root-induced mobilization of iron and other micronutrients (Zn, Mn, Cu) in the rhizosphere of graminaceous (Strategy II) plants, mediated by release of phytosiderophores (PS). (Adapted from Neumann and Ro¨mheld, 2000.)

1996; Ho¨rdt et al., 2000). Morever, Fe and Zn deficiency in groundnut and in guava grown in calcaerous soils is alleviated by intercropping with sorghum and maize. This effect has been at least partially attributed to a generally improved availability of Fe and micronutrients in response to PS exudation by the cereal crops (Khalil et al., 2000; Zuo et al., 2000). Tolerance of graminaceous plant species to Fe and Zn deficiency was found to be roughly related to the amount of PS released in response to Fe and Zn starvation (Hopkins et al., 1998; Marschner, 1998). However, considerable genotypic variation exists within each single plant species (Fig. 15; Kawai et al., 1988; Erenoglu et al., 1996), suggesting that PS exudation is not the only mechanism determining traits for Fe and Zn efficiency (Rengel and Ro¨mheld, 2000). It is however still a matter of debate whether PS exudation under micronutrient deficiency is a specific response. Intense PS exudation in Zn-deficient bread wheat may be an indirect effect due to an impaired Fe metabolism (Walter et al., 1994; Rengel and Graham, 1996). In contrast, Gries et al. (1998) suggested PS release in Hordelymus europaeus L. as a specific response to Cu deficiency. Biosynthesis of PS is probably regulated by the intracellular Fe level (Walter et al., 1995a). Accumulation of PS increases in the root tissue under limited Fe supply even before Fe chlorosis appears, and rapidly declines after resupply of iron

Figure 15 Relationship between Fe and Zn efficiency under field conditions and release of phytosiderophores in graminaceous plant species and cultivars.

Availability of Nutrients

(Tagaki, 1984). Phytosiderophores are derived from nicotianamine (NA) as a trimerization product of Sadenosyl methionine (Fig. 14A; Ma et al., 1995; Ma and Nomoto, 1996) with ubiquitous functions as an intracellular metal chelator in higher plants (Pich et al., 1994). Subsequent transamination and hydroxylation of NA leads to various PS (mugineic acids) with characteristic hydroxylation patterns in different graminaceous plant species (Ma et al., 1995). The ability to accumulate high amounts of PS in the rhizosphere soil solution (up to 1 mM in Fe-deficient barley) seems to be related to localized PS exudation concentrated in subapical root zones (1–2 cm). It is also attributed to a distinct diurnal rhythm of intense PS release over several hours after onset of the light period. This may counteract microbial degradation and diffusionmediated dilution into the bulk soil (Tagaki et al., 1984; Marschner et al., 1987; Ro¨mheld, 1991). In contrast, biosynthesis of PS in the root tissue proceeds during the whole day (Walter et al., 1995b), and initial steps of NA conversion to mugineic acids seem to be confined to large storage vesicles of the endoplasmic reticulum, particularly in epidermal root cells (Fig. 14A; Nishizawa and Mori, 1987; Nishizawa et al., 2000). However, subsequent PS exudation is probably mediated by an anion channel using the potassium gradient between the cytosol and the apolast (Fig. 14B; Sakaguchi et al., 1999; Neumann and Ro¨mheld, 2000). Several genes of enzymes involved in the PS biosynthetic pathway have been cloned including nicotianamine aminotransferase (NAAT), which is the key enzyme for PS biosynthesis (Nakanishi et al., 1993; Takahashi et al., 1997; Higuchi et al., 1999). Recently, it was possible to increase PS release in rice with a low inherent capacity for synthesis and secretion of PS by transgenic overexpression of NAAT genes from barley driven by the respective barley promoter or by the CaMV35S promoter. Finally, this resulted in a greater tolerance to Fe deficiency of the transformant lines grown in calcareous soils (Takahashi et al., 2000). Root uptake of FeIII -PS complexes is mediated by a specific transport system (Fig. 14D). The ys1 maize mutant, which is defective in FeIII -PS uptake, was recently used to clone the ys1 gene by transposon tagging. Heterologous expression of the ys1 protein restored growth of a yeast mutant defective in Fe uptake after application of FeIII -PS, suggesting that ys1 codes for the FeIII -PS transporter (Panaviene et al., 2000). Root uptake of PS complexes with Cu, Zn, and Co was found to be much lower than uptake of FeIII -PS but may still be sufficient to match the comparatively lower demand of plants for these micro-

633

nutrients (Gries et al., 1998). Based on Zn uptake studies with the ys1 maize mutant, the existence of two independent uptake systems in grasses was proposed by Von Wire´n et al. (1996), including direct uptake of the free Zn2þ ion after Zn-PS dissociation and uptake of Zn-PS complexes via the Fe-PS transporter (Fig. 14F). The further behavior of metal-PS complexes after entering the cytosol is still unknown. However, the reduction potential (102 mV for FeIII -PS) suggests that complex splitting is possible via reduction by common physiological available reductants such as NAD(P)H (–320 mV) or glutathione (–230 mV) (Ma and Nomoto, 1996).

VI.

NONINFECTING RHIZOSPHERE MICROORGANISMS

Root exudates and particularly sloughed-off cells and tissues act as a major carbon source for soil microorganisms (Haller and Stolp, 1985). Therefore, microbial activity and the population density of microorganisms are considerably higher at the rhizoplane and in the rhizosphere (by a factor of 5–50) than in the bulk soil (Lynch and Whipps, 1990). Also the composition of microbial communities in the rhizosphere is significantly influenced by rootborne carbohydrates (Kloepper et al., 1991; Marilley and Aragno, 1999). Both factors may differentially affect mineral nutrition of plants by positive or negative effects on (1) root growth and morphology, (2) physiologial and developmental processes, (3) availability of nutrients and nutrient dynamics in the rhizosphere, and (4) root uptake of nutrients (Marschner, 1998). For example, rapid microbial degradation limits the efficiency of rootborne chelators such as organic acids and phytosiderophores involved in mobilization of P, Fe and micronutrients (Von Wire´n et al., 1993; Jones and Darrah, 1994). On the other hand, positive effects on nutrient availability can be expected when quantitative and qualitative modifications of the rhizodeposition promote the microbial production of metal chelators (organic acids, siderophores). Up to 30% of cultivated soil microorganisms are able to mobilize sparingly soluble soil P sources (Mueller et al., 1997). Moreover, a modified composition of the sugar fraction in root exudates in response to P deficiency may increase the microbial production of P-mobilizing compounds in the rhizosphere (Schilling et al., 1998). Also, the mineralization of the various forms of organic soil P strongly depends on

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soil microbial activity but significant amounts of soil solution Pi can be sequestered into microbial biomass (Seeling and Zasoski, 1993). Therefore, it remains an open question to what extent microbial P mobilization in the rhizosphere also determines phosphorus availability for plants. Microbial production and subsequent degradation of siderophores may have an impact on plant availability of Fe by increased precipitation of amorphous Fe hydroxides at the rhizoplane as a source for rootinduced Fe mobilization (Marschner and Ro¨mheld, 1994). Iron may also be directly acquired from microbial siderophores and their partial degradation products by ligand exchange with phytosiderophores or reduction processes (Yehuda et al., 1996; Ho¨rdt et al., 2000). In contrast, direct uptake of microbial siderophores by plant roots is most likely of minor importance (Marschner and Ro¨mheld, 1994). The activity of noninfecting rhizosphere microorganisms is of particular significance for the manganese nutrition of plants, which depends on the oxidation state of Mn. Oxidation reactions are almost exclusively mediated by miroorganisms, whereas both microbial activity and root-induced changes in rhizosphere chemistry are involved in Mn reduction (Marschner, 1988). Since manganese is plant-available only in the reduced form (Mn2þ ), the balance of Mn-oxidizing bacteria (e.g., Arthrobacter spp.) to Mn reducers (e.g., fluorescent pseudomonads) strongly influences Mn availability in the rhizosphere (Marschner, 1988; Posta et al., 1994; Rengel, 1997). Root-induced changes in rhizosphere chemistry affecting the proportion of Mn oxidizers to Mn reducers can at least partially explain differences in Mn efficiency of wheat genotypes (Timonin, 1946; Rengel, 1997). Moreover, Mn deficiency, caused by a high proportion of Mnoxidizing microorganisms in the rhizosphere, may weaken the resistance of plants to fungal deseases, such as take-all (Gaeumannomyces graminis), and many soilborne pathogenes are effective Mn oxidizers (Rengel et al., 1994). However, in poorly aerated soils with low pH, a high microbial activity in response to a large supply of rootborne carbohydrates may lead to excessive mobilization of Mn in the rhizosphere and to Mn toxicity in plants (Marschner, 1988). Despite the high supply of carbohydrates by plant roots, rhizosphere microorganisms are often limited by the availability of nitrogen, which is also a major limiting nutrient for plant growth in most terrestrial ecosystems. Therefore, N competition between plants and microorganisms is believed to be intense (Hodge et al., 2000). For example, lower N concentrations of plants

Neumann and Ro¨mheld

grown under elevated CO2 concentrations have been at least partially attributed to increased root exudation of organic carbon associated with a higher sequestration of N by rhizosphere microorganisms (Diaz et al., 1993; Rouhier et al., 1994). Accordingly, elevated CO2 increases symbiotic nitrogen fixation in leguminous plants (Soussana and Hartwig, 1996). Increasing supply of fertilizer N usually increases the numbers of rhizosphere microorganisms. However, in poorly aerated soils under stress conditions which promote root exudation, high levels of N application may also increase the proportion of denitrifiers and therefore the potential of gaseous losses of N (Marschner, 1998). On the other hand, root exudation is also an important factor determining root colonization by associative diazotrophic (N2) fixing bacteria such as Azospirillum sp. or Enterobacter sp. These diazotrophic bacteria are abundant in the rhizosphere, at the rhizoplane, and even in the root cortical apoplast, particularly in graminaceous C4 plant species (Boddey and Do¨bereiner, 1988). Azospirillum strains preferably use malate as a carbon source (Alexander and Zuberer, 1989), which is a major carboxylate in root exudates (Neumann and Ro¨mheld, 1999). Specific attraction of Azospirrillum by mucilage polysaccharides has been reported by Mandimba et al. (1986). Associative diazotrophic bacteria may contribute to plant N acquisition by biological N2 fixation, particularly in tropical soils with low N levels and plants with high rates of root exudation (Marschner, 1995b). On the other hand, diazotrophic bacteria are also effective producers of phytohormones (Jagnow et al., 1991), which may have an impact on root growth and morphology (Martin et al., 1989). Phytohormone production, including auxins, cytokinins, and ethylene, has been similarly reported for many fungi and P-solubilizing bacteria and may be related to the presence of precursor compounds in root exudates and lysates of root cells (Arshad and Frankenberger, 1991; Glick, 1995). Holland (1997) even suggested that cytokinins may be exclusively produced by microbial symbionts of plants. It has recently been demonstrated that some bacterial strains, so-called mycorrhization helper bacteria (MHB), increase the ability of roots to establish ectomycorrhizal symbiosis (Garbaye, 1994), and there is increasing evidence that similar effects may be involved in the establishment of endomycorrhizae (Perotto and Bonfante, 1997). Even the presence of endosymbiotic bacteria has been reported in the cytoplasm of some endomycorrhizal fungi (Bonfante, 1996). Promoting effects have been related to the bacterial production

Availability of Nutrients

of unidentified compounds, which stimulate fungal growth, impact fungal gene expression, or mediate attachment of fungal hyphae to plant roots (Bonfante and Perotto, 2000).

VII.

MYCORRHIZAL ASSOCIATIONS

Mycorrhizae are the most widespread form of plant– microbial (fungal) associations, comprising about 80– 90% of all terrestrial plant species (Read, 1993). The formation of mycorrhizal associations probably coincided the colonization of land by higher plants (Taylor et al., 1995). Depending on structural interactions between plant roots and the fungal mycelium, at least two types of mycorrhizal associations can be distinguished. In endomycorrhizae, fungal hyphae penetrate root cortical cells and extraradical hyphae extend several cm into the surrounding soil. Among the various endomycorrhizal associations including ericoid, orchidaceous, and arbuscular (AM) mycorrhizae, AM formed by zygomycetous fungi of the order Glomales is most widely distributed. Arbuscular mycorrhizae are characterized by the intracellular formation of highly branched haustoria (arbuscules) in root cortical cells, which are the main sites of solute exchange with the host plant. Ectomycorrhizal (ECM) fungi are members of the Basidiomycetes, Ascomycetes, and Phycomycetes, and are mainly associated with roots of woody plants. Hyphae of ECM fungi are forming an interwoven mantle around the root surface, and a hyphal network (Hartig net) penetrates only the intercellular space of root cortical cells. Hyphal strands (rhizomorphs) extend well into the surrounding soil (Marschner, 1995b). Ectomycorrhizae are more abundant in N-limited ecosystems and boreal, temperate forests of the Northern hemisphere but can also occur simultaneously with AM. In contrast, AM associations are more characteristic for P-limited ecosystems in warmer climates, with drier soils, in pastureland, and in deciduous forests with a high turnover of organic matter (Read, 1991; Chapter 50 by Kottke in this volume) and are found in most crop plants (Fig. 16). Mycorrhizal associations can affect mineral nutrient acquisition of the host plant by (1) increasing the spatial availability of nutrients, (2) chemical mobilization of sparingly available nutrients, and (3) protective functions against root pathogens and abiotic stress factors such as metal toxicity, drought, salinity, and low temperatures (Marschner, 1995b; Leyval et al., 1995; Paradis et al., 1995). Mycorrhizal associations are

635

usually mutualistic, but negative effects on plant growth can be expected in stress situations. Apparently this occurs when the carbon costs (10– 20% of net photosynthesis) to establish and to maintain the mycorrhiza exceed the ability of the extraradical mycelium to compensate for the root’s function in uptake of mineral nutrients and water (Marschner, 1998). There is also evidence that aggressive colonization by certain AM fungi even in moderate- and high-P soils can reduce plant growth by producing carbon costs without beneficial effects for the plant nutritional status (Graham and Eissenstat, 1998; Graham and Abbott, 2000). According to the increased surface area for nutrient absorption by mycorrhizal associations, the contribution of mycorrhizal colonization to mineral nutrition of the host plant is particularly important for mineral nutrients with low mobility (e.g., P, 2þ 2þ NHþ 4 , Zn , Cu ) in nutrient-poor soils (Fig. 16) and in plants with coarse and poorly branched root systems (Table 6). There is also evidence for alterations of root morphology in response to AM colonization, such as promotion (Hetrick et al., 1988) or inhibition (Berta et al., 1995) of root elongation. Frequently, increased formation of laterals and of fine roots has been observed (Yano et al., 1996; Tisserant et al., 1996), which may be triggered by increased indole-3-butyric acid levels of infected roots (Kaldorf and Ludwig-Mu¨ller, 2000). Involvement of auxins is also indicated for the establishment of ECM associations (Barker et al., 1998; Tranvan et al., 2000). Also, increased cytokinin accumulation has been related to lateral root formation in response to AM infection (Barker et al., 1998). Stimulation of fine-root production may in turn increase the number of putative AM infection sites and thereby promote AM colonization (Kaldorf and Mu¨ller, 2000). However, the high contribution of AM to P acquisition has also been related to accumulation of polyphosphates in vacuoles with putative functions in P and energy storage, P transport to the host plant, and binding and transport of metal cations and basic amino acids (Smith and Gianninazzi-Pearson, 1988; Jennings, 1989). Hydrolysis of organic soil P by secretion of acid phosphatase (APase) and phytase from extraradical hyphae was reported for many ECM fungi (Fig. 16; Gourp and Pargney, 1991; Dinkelaker and Marschner, 1992; Colpaert et al., 1997) and also for some but not all AM fungi (Joner and Jakobsen, 1995). Mobilization of organic P was particularly expressed in presence of other acid phosphatase-producing fungi

Neumann and Ro¨mheld

636

Figure 16 Schematic presentation of the contribution of ectomycorrhizal (ECM) and arbuscular mycorrhizal (AM) associations to nutrient acquisition by plant roots. (Adapted from Marschner, 1995b.)

such as Aspergillus fumigatus (Tarafdar and Marschner, 1995). However, similar to root-induced secretion of phosphohydrolases, there is little evidence for hydrolysis of sparingly soluble organic P forms such as Fe/Al phytates, which dominate in many soils (Colpaert et al., 1997). According to the contribu-

tion of AM to P uptake and mobilization of at least some organic P fractions, downregulation of genes encoding for acid phosphatase and also for P transporters of the host plant has been recently reported for Medicago truncatula in response to AM colonization (Harrison, 1999).

Table 6 Relationship Between Root Hair Length and Mycorrhizal Benefit in Pasture Species Root hair Plant species Trifolium dubium Trifolium subterraneum Medicago polymorpha Ornithopaes compressus Lolium rigidum Source: Schweiger et al., 1995.

Length (
m)

Density (number mm1 root length)

Total length (mm mm1 root length)

Mycorrhizal benefit

127 215 255 477 1155

40 47 37 93 50

5.1 10.6 10.6 46.4 60.0

23.2 6.5 5.4 4.2 1.0

Availability of Nutrients

The mobilization of sparingly soluble P sources by release of large amounts of organic chelators, such as oxalate (Fig. 16), seems to be mainly restricted to ECM fungi (Marschner, 1998; Clark and Zeto, 2000) and may be not only involved in P acquisition (Lapeyrie et al., 1990) but also in weathering of mineral soils and rocks (Jongmans et al., 1997; Van Breemen et al., 2000). Acquisition of nitrogen from proteins and peptides by excretion of acid proteinases seems to be a unique feature of ECM (Fig. 16) and probably also of some ericoid mycorrhizal fungi (Marschner, 1998; Xiao and Berch, 1999). This may be an ecological advantage, particularly in N-limited forest ecosystems (Finlay et al., 1992). ECM fungi are also able to take up N from inorganic N sources with preference for NHþ 4 similar to the host plants (Plassard et al., 1991). Uptake of  NHþ 4 , NO3 , and amino acids has been also demonstrated for AM fungi (Fig. 16) but is probably not sufficient to sustain the plant’s N requirements (George et al., 1995; Hawkins and George, 1999). Information on acquisition of macronutrient cations such as K, Ca, and Mg by AM fungi is rather inconsistent, and positive effects have been mainly reported in acidic soils, generally low in these cationic bases (Clark and Zeto, 2000). In case of micronutrient acquisition, the role of AM is particularly evident in case of Zn and Cu (Fig. 16) and can account for up to 60% of the total uptake of nutrients (Kothari et al., 1991; Li et al., 1991). Also, enhanced acquisition of heavy metals and toxic elements, such as Cd, Ni, Pb, Co, and Cs, has been observed (Guo et al., 1996; Clark and Zeto, 2000). In contrast, ECM fungi have been reported to be effective in increasing heavy metal and Al tolerance of their host plants. This has been mainly attributed to a high retention capacity for heavy metals in the fungal mycelium and to exclusion by metal-chelating exudates such as organic acids and phenolics (Turnau et al., 1993; Martin et al., 1994; Denny and Ridge, 1995). However, alleviation of Al toxicity symptoms has been also observed in AM plants. Since root protection by hyphal mantles or production of chelating exudates seems not to be expressed in AM, increased metal tolerance may be rather an indirect effect resulting from enhanced growth and improved P and Si nutrition of mycorrhizal plants (Marschner, 1998; Clark and Zeto, 2000). In contrast to Zn and Cu, the reported effects of AM on Fe acquisition are controversial (Caris et al., 1998; Clark and Zeto, 2000). Uptake of Mn even seems to be markedly reduced by AM colonization.This

637

effect has been attributed to a marked decrease of Mn-reducing bacteria, such as fluorescent pseudomonads in the rhizosphere of AM plants by modifications of root exudation (Posta et al., 1994; Marschner et al., 1997). Suppression of Pseudomonas fluorescens strains has been also related to alleviatory effects on soil sickness by AM inoculation of grapevine (Vitis vinifera L.) seedlings (Waschkies et al., 1994). Protection of the host plant against soilborne pathogens seems to be also widely distributed in ECM fungi (Marschner, 1998).

VIII.

CURRENT PROBLEMS AND FUTURE ASPECTS OF RHIZOSPHERE RESEARCH

The importance of the soil–root interface for mineral nutrition of higher plants has long been recognized. During the last two decades, much progress has been made in the characterization of soil, plant, and microbial factors determining the availability of nutrients but also the levels of toxic elements in the rhizosphere. However, the complexicity of interacting rhizosphere processes, the variability within plant species and cultivars, and also the diversity at the ecosystem level require further investigation for a better understanding of the still widely distributed contradictions of experimental results. The role of rhizosphere processes in plant–microbial interactions, nutrient acquisition, and in plant adaptations to environmental stress or adverse soil–chemical conditions is not only of scientific interest. It also implies important practical aspects, associated with the need for production of healthy crop plants, for sustainable agricultural management systems, and in particular for low-input agricultural ecosystems. From the methodological point of view, there is an obvious lack of noninvasive techniques, which allow measurements of rhizosphere processes with a high spatial resolution. Further miniaturization of sampling and analytical techniques (e.g., use of specific microprobes for specific compounds, reporter bacteria, microsuction cups, capillary electrophoresis, chromatographical chip technologies), image analysis, and videodensitometry should fascilitate nondestructive measurements of rhizosphere processes at a high scale of resolution. However, the major limitation seems to be the sampling process itself. It has to be kept in mind that even rhizobox or root-window approaches (Smit et al., 2000) with minimal disturbance by the sampling procedure still represent artifi-

638

cial systems which do not necessarily reflect the natural growth conditions (see Chapter 18 by Polomski and Kuhn in this volume). New molecular methods, such as analysis of 16S rDNA clone libraries retrieved from environmental DNA, and in situ hybridization techniques provide powerful tools for studies on microbial diversity and microbial activity in the rhizosphere. These techniques can overcome restrictions of traditional methods linked with the extremely limited cultivability of soil bacteria (Assmus et al., 1997; Marilley and Aragno, 1999). Much more experience is required concerning effects of root exudates and rhizosphere products on mobility and plant availability of nutrients and toxic compounds (e.g., heavy metals and pesticides) in soils under different environmental conditions and at realistic rhizosphere concentration levels. In this context it is also important to consider the possibility of synergistic effects of simultaneous chemical reactions in the rhizosphere. Fertilization management, breeding technologies, or biotechnological approaches (see Chapter 17 by Bucher in this volume) may be employed for a directed manipulation of the soil–root interface toward improved efficiency of plants for nutrient acquisition, resistance to adverse soil–chemical conditions, or plant design for phytoremediation and phytomining strategies. However, this will require a detailed understanding of the physiological mechanisms involved in the regulation of root activity and of the related rhizosphere processes as well. Thus, future aspects of rhizosphere research will be based more and more on multidisciplinary approaches, integrating the fields of soil chemistry, soil microbiology, plant nutrition, plant physiology, and molecular biology.

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647 Tagaki S.1984. Mechanism of iron uptake regulation in roots and genetic differences. In: Agriculture, Soil Science and Plant Nutrition in the Northern Part of Japan. Tokyo; Japanese Society of Soil Science and Plant Nutrition, pp 190–195. Tagaki S, Nomoto K, Takemoto T. 1984. Physiological aspect of mugineic acid, a possible phytosiderophore of graminaceous plants. J Plant Nutr 7:469–477. Takahashi M, Yamaguchi H, Nakanishi H, Kanazawa K, Shioiri T, Nishizawa NK, Mori S. 1997. In: Ando T, ed. Purification, Characterization, and Sequencing of Nicotinamine Aminotransferase (NAAT-III) Expressed in Fe-deficient Barley Roots. Plant Nutrition for Sustainable Food Production and Environment. Dordrecht, Netherlands: Kluwer, pp 279–280. Takahashi M, Tanaks T, Nakanishi, H, Kawasaki S, Nishizawa NK, Mori S. 2000. Iron-deficiency-tolerant transgenic rice having enhanced secretion of mugineic acids in calcareous soils. In: Program and Abstracts of the Tenth International Symposium on Iron Nutrition and Interactions in Plants; Houston, TX, p 88. Tarafdar JC, Marschner H. 1994. Phosphatase activity in the rhizosphere and hyphosphere of VA mycorrhizal wheat supplied with inorganic and organic phosphorus. Soil Biol Biochem 26:387–395. Tarafdar JC, Marschner H. 1995. Dual inocculation with Aspergillus fumigatus and Glomus mossae enhances biomass production and nutrient uptake in wheat (Triticum aestivum L.) supplied with organic phosphorus as Na-phytate. Plant Soil 173:97–102. Taylor TN, Remy W, Hass H, Kerp H. 1995. Fossil arbuscular mycorrhizae from the Early Devonian. Mycologia 87:560–573. Timonin MI. 1946. Microflora of the rhizosphere in relation to the manganese deficiency disease of oats. Soil Sci Soc Am Proc 11:284–292. Tisserant B, Gianinazzi S, Gianinazzi-Pearson V. 1996. Relationships between lateral root order, arbuscular mycorrhiza development, and the physiological state of the symbiotic fungus in Platanus acerifolia. Can J Bot 74:1947–1955. Titz R, Sommer K. 1988. Ertragsstruktur sowie Nitratgehalte in Pflanzen und Bo¨den bei Freilandsalaten auf Ammonium-Basis gegenu¨ber konventioneller Du¨ngung. VDLUFA-Schriftenreihen 28:1321–1329. Thomson CJ, Marschner H, Ro¨mheld V. 1993. Effect of nitrogen fertilizer form on pH of the bulk soil and rhizosphere, and on the growth, phosphorus, and micronutrient uptake of bean. J Plant Nutr 16:493– 506. Tranvan H, Habricot Y, Jeanette E, Gay G, Sotta B. 2000. Dynamics of symbiotic establishment between IAAoverproducing mutant of the ectomycorrhizal fungus

648 Hebeloma cylindrosporum and Pinus pinaster. Tree Physiol 20:123–129. Treeby M, Marschner H, Ro¨mheld V. 1989. Mobilization of iron and other micronutrient cations from a calcareous soil by plant-borne, microbial, and synthetic metal chelators. Plant Soil 114:217–226. Trobisch S. 1966. Beitrag zur Auflka¨rung der pH- und Du¨ngungsabha¨ngigkeit der Mo-Aufnahme. Thaer Arch 10:1087–1099. Trolldenier G. 1977. Mineral nutrition and reduction processes in the rhizosphere of rice. Plant Soil 47:193–202. Turnau K, Kottke I, Oberwinkler F. 1993. Paxillus involutus–Pinus sylvestris mycorrhizae from heavily polluted forest. I. Elemental localization using electron energy loss spectroscopy and imaging. Bot Acta 106:213–219. Van Breemen N, Finlay R. Lundstro¨m U, Jongmans A, Giesler R, Olsson M. 2000. Mycorrhizal weathering: a true case of mineral plant nutrition? Biogeochemistry 49:53–67. Vancura V. 1967. Root exudates of plants. III. Effect of temperature and cold shock on the exudation of various compounds from seeds and seedlings of maize and cucumber. Plant Soil 27:319–328. Von Wire´n N, Morel JL, Guckert A, Ro¨mheld V, Marschner. 1993. Influence of soil microorganisms on iron acquisition in maize. Soil Biol Biochem 25:371–376. Von Wire´n N, Marschner H, Ro¨mheld V. 1996. Roots of iron-efficient maize also absorb phytosiderophore-chelated zinc. Plant Physiol 111:1119–1125. Varanini Z, Pinton R, DeBiasi MG, Astolfi S, Maggioni A. 1993. Low molecular weight humic substances stimulate Hþ -ATPase activity of plasma membrane vesicles isolated from oat (Avena sativa L.) roots. Plant Soil 153:61–69. Vetterlein D, Marschner H. 1993. Use of a microtensiometer technique to study hydraulic lift in sandy soil planted with pearl millet (Pennisetum americanum L. Leeke). Plant Soil 149:275–282. Walch-Liu P, Engels C. 1998. Pflanzenartenunterschiede im Wurzelwachstum bei verschiedener N-Erna¨hrung: NForm Effekt und/oder pH-Effekt? In: Merbach W, ed. 8. Borkheider Seminar zur O¨kophysiologie des Wurzelraumes. Pflanzenerna¨hrung, Wurzelleistung und Exsudation. Stuttgart, Germany: B.G. Teubner Verlagsgesellschaft, pp 73–79. Wallace A. 1980. Effect of chelating agents on uptake of trace metals when chelating agents are supplied to soil in contrast to when they are applied to solution culture. J Plant Nutr 1:171–175. Wallace A. 1982. Effect of nitrogen fertilizer and nodulation on lime-inducedchlorosisinsoybean.JPlantNutr5:171–175. Walter A, Ro¨mheld V, Marschner H, Mori S. 1994. Is the release of phytosiderophores in zinc-deficient wheat

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649 iron from microbial siderophores by graminaceous plants. Plant Physiol 111:1273–1280. Youssef RA, Chino M. 1987. Studies on the behaviour of nutrients in the rhizosphere. I. Establishment of a new rhizobox system to study nutrient status in the rhizosphere. J Plant Nutr 10:1185–1195. Zhang H, Forde BG. 2000. Regulation of Arabidopsis root development by nitrate availability. J Exp Bot 51:51– 59. Zhang X, Zhang F, Mao D. 1998. Effect of iron plaque outside roots on nutrient uptake by rice (Oryza sativa L.): zinc uptake by Fe-deficient rice. Plant Soil 201:33–39. Zhang H, Jennings A, Barlow PW, Forde BG. 1999. Dual pathway for regulation of root branching by nitrate. Proc Natl Acad Sci USA 96:6529–6534. Zuo Y, Zhang F, Li X, Cao Y. 2000. Sudies on the improvement in iron nutrition of peanut by intercropping with maize on a calcareous soil. Plant Soil 220:13–25.

37 Simulation of Ion Uptake from the Soil Moshe Silberbush Ben-Gurion University of the Negev, Sede-Boker, Israel

I.

INTRODUCTION

but the emphasis is on recent approaches to modeling of nutrient uptake from the soil, and recent development in modeling. Crop simulation models were divided into two classes: scientific approach—models that aspire to represent our understanding of the physiology of plants and their environmental interactions; and engineering approach—models that aspire to provide sound management advice and prediction to farmers and policy makers (Passioura, 1996). Gregory (1996) has classified the ‘‘scientific’’ models into three groups:

Ion uptake from the soil by plant roots entails several biological, chemical, and physical processes taking place concomitantly in a variable and changing environment. The processes themselves are not only complex and often nonlinear, but they also interact with and affect each other. Therefore, simulation of such these processes is complicated, and even today one fails to describe them in detail. Computer models are intended for one of two purposes. On one hand, they serve as research tools, summarizing the scientific understanding of the processes in question. On the other hand, they can serve as tools for decision makers, in agriculture (fertilization management, water consumption), environmental protection (contamination by fertilizers, decomposition of residues), and ecology (e.g., cycling of nutrient in an ecosystem, limiting growth factors). Models are tools for description of reality and prediction of outcomes of various changes. Thus, they may vary according to their purpose, treating in detail some parts of the soil–plant–atmosphere system, whereas simplifying other components the detailed description of which seems to be less crucial. This is usually done because the more complex a model is, the more room it has for errors and deviations from the actual behavior of the real case. Yet, a model is useless unless it is verified by actual data of the real system. In the following the basic structure of the different types of models will be presented (cf. Silberbush, 1996),

1. Models without roots: Models based on measured or estimated plant uptake efficiencies in which root growth and distribution is not explicit. Competitive ability is inferred in such models from shoot growth and by its demand. 2. Process-based models: Models based on measured or calculated root length and distribution. Those are based on variables such as time or temperature. Uptake by the whole root system is an integration of the uptake by all the individual roots. 3. Models that allow interaction between root and shoot growth and functioning, either by physiological mechanisms and/or by environmental impacts, both aerial and edaphic. Type 1 models are mainly those that were classified by Passioura (1996) as engineering models and empirical models by Silberbush (1996). Little if any knowledge may be gained from this approach regarding the 651

652

Silberbush

Table 1 List of Water and Ion Uptake Models Type of model Model/authors

Characteristics

Reference

1-Da

Hagin-Amberger Gardner Rao-Mathur LEACHM

N transformation and uptake Water uptake Cd transformation and uptake Chemical reactions and uptake

SNAPS TROIKA Helyar-Munns

Pesticide uptake Radial water flow to a single root Single root with root hairs, P transformation, and uptake Radial convection-diffusion flow and uptake by roots, extendible r1 b Radial convection-diffusion flow Baldwin’s model þ uptake by root hairs Extendible r1 (without competition) Constant r1 (root competition)

Hagin et al., 1976 Gardner, 1991 Rao and Mathur, 1994 Hutson and Wagenet, 1995b Jemison et al., 1994a,b Behrendt et al., 1995 Lambert and Penning de Vries, 1973 Helyar and Munns, 1975

Single root

Nye-Marriott Baldwin-Nye-Tinker Bhat-Nye-Baldwin Claassen-Barber Barber-Cushman (‘‘Nutrient Uptake’’) Itoh-Barber Bar Yosef-FishmanTalpaz De Willigen–Van Noordwijk Hoffland et al. COMP8

Cushman, 1980 Leadley-Reynolds-Chapin Bouldin 2-D

Reginato et al. NST 3.0 Silberbush-SorekYakirevich Yanai 2DSOIL

3-D

a b

Geelhoed et al. Geelhoed et al. Somma et al.

One-dimensional (vertical) model. The external boundary of the soil rhizocylinder.

As Barber-Cushman, with root hairs Zn uptake, different chemical forms, water depletion Constant and zero-sink for high and low concentrations, respectively r1 adjusted to root density As Baldwin-Nye-Tinker, variable buffer power, variable r1 , competition between plant species Barber-Cushman þ sink/source (root hairs or microorganisms) As Barber-Cushman, N components’ uptake Multi-ions without interaction, linear influx functions ‘‘Free boundary’’ (variable root radius) Uptake by root hairs Adjusted r1 , multi-ion uptake, interactions among ions, salinity Depletion and steady-state ion influx, root competition Modular set of models; includes transport, uptake, exchange between phases Exudation of protons and citrate Uptake by root hairs, zero sink Solute transport and uptake, and root growth, using finite-element technique

Nye and Marriott, 1969 Baldwin et al., 1973 Bhat et al., 1976 Claassen and Barber, 1976 Cushman, 1979; Barber and Cushman, 1981; Oates and Barber, 1987 Itoh and Barber, 1983 Bar-Yosef et al., 1980 De Willigen and Van Noordwijk, 1994 Hoffland et al., 1990 Smethurst and Comerford, 1993, Ibrikci et al., 1998 Cushman, 1980 Leadley et al., 1997 Bouldin, 1989 Reginato et al., 1990 Claassen, 1990. Silberbush et al., 1993 Yanai, 1994; Williams and Yanai, 1996 Timlin et al., 1996; Acock and Pachepsky, 1996 Geelhoed et al., 1999 Geelhoed et al., 1997a,b Somma et al., 1998

Simulation of Ion Uptake

effects or for the relative importance of the mechanisms involved. A typical example is the fertilization management model of Greenwood and Karpinets (1997), which is freely accessible to users via Internet (http://www.qpais.co.uk/moda-djg/potass.htm). Another example is the model of Mankin and Fynn (1996), who included the effects of plant demand for the nutrients, microclimate, and photosynthesis. The models discussed in this chapter are the scientific or mechanistic models, of types 2 and 3 on Gregory’s list. These models allow us to study and to analyze water and ion uptake by root systems from soils, with different degrees of interaction with the aerial parts of the plant. The mechanistic models may be also divided into one-, two-, and three-dimensional models as summarized in Table 1.

653

II.

ONE-DIMENSIONAL (VERTICAL) MODELS

One-dimensional (1-D) models of nutrient and water uptake take into account differences along the vertical dimension of the soil profile, assuming uniform, lateral distribution of all variables (root, nutrients, water). This type of model can be applied to a lawn of turf grass or to a field of alfalfa, where the landscape is homogeneous and plants are distributed over the whole area with a uniform, high density. These models are often used to simulate chemical, physical, and biological processes in a layered soil (Hagin et al., 1976; Gardner, 1991; Nye, 1992; Rao and Mathur, 1994; Hutson and Wagenet, 1995a,b; Kage, 1997; Wu et al., 1999). They emphasize changes in the soil with

Figure 1 Schematic description of the one-dimensional (vertical) uptake model—the distinct form: n, number of soil layers; i, index for the soil layers (between 1 and n); Ai, root uptake capacity per unit soil volume; Ci, solute concentration in the soil solution; Qi , the amount of solute in the soil layer; Fi, flux of solute between soil layers; Si, a sink/source term for the ith layer;
zi; layer thickness; Z, depth of the root zone bottom (distance from soil surface).

654

depth and with time, but oversimplify the characteristics of the root system (Richter, 1987). An example for 1-D models is illustrated in Fig. 1. One-dimensional models may be treated either as continuous or as discrete systems. In the first case, that applies to uniform soils in which the characteristics of the soil, the flow of soil solution, chemical reactions and source/sink relationships are continuous with depth. In a layered soil, with discrete changes along the root zone, the system may be treated as a set of distinct, vertically arranged cells, with a lateral cross section of a unity. Solute transport in the continuous case may be solved by a convection–dispersion equation (Hutson and Wagenet, 1995a):

@Cl @C @ @C

Dð ;qÞ l  qCl  Sðz;tÞ  Uðz;tÞ

þ s ¼ @z @t @t @z ð1Þ where Cl is the solute concentration in the soil solution and Cs (mass of the solute per volume unit of soil; see equation [8b] below) the soil content of ‘‘diffusible’’ (i.e., the fractions that are in a relatively fast equilibrium with the soil solution, mainly the exchangeable ions that may be readily transported by diffusion) forms, respectively. The other parameters are , the soil volumetric moisture content; D, the effective dispersion coefficient (affected both by soil moisture and the flow velocity); and q, the water flux density in the vertical direction. The term S may either represent a ‘‘sink,’’ and will then have a negative value in case of ion utilization by microorganisms, chemical precipitation, or adsorption by the soil matrix; or represent a ‘‘source’’ with a positive value in case the ions are released within the soil layer by decomposition of organic matter, by mineralization of insoluble, organic compounds. U is a sink which stands for uptake by the plant roots (both in terms of ion amount per unit of soil volume); z and t are depth and time indices, respectively. The example in Fig. 1 is of a discrete model. The convention in such models is to relate all calculations to a soil column with one unit area cross section, divided into n layers from the soil surface (z ¼ 0) down to the bottom at depth Z (positive direction downward). The layers are not necessarily equal in thickness; they are determined arbitrarily, based on the gradients and changes along the soil profile. Each layer i is characterized by its root abundance and its chemical and physical parameters: Depth (zi ), thickness ð
zi ), moisture content [ð zi ) (v/v), soil texture, and structure which determine water and ion retention

Silberbush

properties, and soluble ion quantities ðQi ) throughout the layer. The change in ion quantity Qi with a time step
t is described by equation (2):   Qiðtþ
tÞ  QiðtÞ ¼ FiðinÞ  FiðoutÞ þ ðSi  Ui Þ
zi
t ð2Þ where Fin and Fout are flow rate of the solute into and out of the layer, respectively. The sinks, S and U, are presented in terms of amount of the solute per unit of soil volume. Rate of uptake of the solute from each soil layer by the roots, Ui is defined as: Ui ¼ Ai ðCl ðiÞÞ ClðiÞ

ð3Þ

where A is root absorption activity (specific uptake capacity, expressed as root abundance, absorption surface area, mass or length per unit soil volume);  is root absorption power per root activity unit, assumed to be a function of the Cl (Nye and Tinker, 1977): max ðCl ðiÞ Þ ¼ ð4Þ Km þ ClðiÞ where max is maximal absorption power, and Km the Michaelis-Menten coefficient. Total uptake by the whole root system, Utotal , may be obtained from the sum of uptake from all the n layers that comprise the root zone, along the simulation period, which is divided into m time steps: Utotal ¼

m X n X

ðU
zÞi
tj

ð5Þ

j¼1 i¼1

The corresponding result in the continuous case will be obtained by the integration of equation (1) over depth and time. Some examples for 1-D models are presented in Table 1. The book edited by Loepert et al. (1995) contains various examples and applications of this type of models. This approach is widely used to simulate 1-D water and nutrient flow, and uptake by plants (Harrison et al., 1999; Wu et al., 1999; Lai and Katul, 2000). III.

RADIAL FLOW MODELS

The simplest form of this group is of the ‘‘single-roottype models’’ that describe radial flow of water and solutes toward a single root surrounded by a uniform soil. These are type 1-D models of a unique structure. Early such models like TROIKA dealt with radial flow only (Lambert and Penning de Vries, 1973). The Claassen-Barber model (Claassen and Barber, 1976) and its followers also accounted for uptake along a

Simulation of Ion Uptake

655

growing root, which made them 2-D models, but with radial and longitudinal dimensions. A schematic description of this configuration is presented in Fig. 2. The simulated system includes a cylindrical root with radius r0 , surrounded by an outer cylinder of soil, with radius r1 . The soil solution flows toward the root, carrying the solutes with it, and the solutes also diffuse in the soil, from all its sides in the radial direction, since no gradients occur in other directions. These models also consider root elongation. The various radial flow models differ in their boundary conditions of the radial flow, as will be discussed later.

A.

Radial Flow of Water and Solutes

@Cs þ vr Cl @r

where v0 is water radial velocity at the root surface (r ¼ r0 ), and b the soil buffer power for the solute, defined as: b¼

@Cs @Cl

ð6Þ

where vr is the radial water velocity, De the effective diffusion coefficient, and Cs as in equation (1). The

Figure 2 Schematic description of the radial-flux models. Radial flux of the solute (closed circles), Jr, to a unit of root length, by convection and diffusion of the soluble fraction in the soil solution Cl which is in equilibrium with the exchangeable fraction on the soil solids, Cx. The solute flows from the outer soil boundary, r1 , to r0 , the inner soil boundary at the root surface, where it equals net influx (In) to the root.

ð8aÞ

On the other hand, Cs (ion mass per unit soil volume), being the sum of the soluble and the adsorbed (Cx , ion mass per unit soil mass) fractions, equals: Cs ¼ Cl þ b Cx

Simulating water and solute flow in the radial direction is the most important improvement of this type of models over the 1-D (vertical) ones. Such flow brings about a change in the gradient of solute concentration in the soil solution (Cl ) with the radial distance r from the root center. Radial flow (flux), Jr , of the solute through the soil toward the root by means of convection (mass-flow) and diffusion is expressed as: Jr ¼ D e

differential form of equation (6) was formulated for radial flow by Nye and Marriott (1969):   @Cl 1 @ @C v r C r De l þ 0 0 l ¼ ð7Þ r @r @r @r b

ð8bÞ

where b is soil bulk density (mass of dry soil per soil volume unit). Introducing the slope of the adsorption isotherm as Kd ¼ @Cx =@Cl yields (Van Rees et al., 1990): b¼

@Cs ¼ þ b Kd @Cl

ð8cÞ

which allows the adjustment of the buffer power of the soil to the concentration of the adsorbed ion (Smethurst and Comerford, 1993; Hutson and Wagenet, 1995a) rather than using a constant value, as was done earlier by Claassen and Barber (1976) and Barber and Cushman (1981). In certain cases, the buffer power was used as a retardation factor of  the form R ¼ 1 þ b Kd (Jardine et al., 1988), which is the same as the buffer power if one takes R as dimensionless, equals to b . The use of the retardation factor enabled Silberbush et al. (1993) to account for concomitant flow of interacting solutes: ( ) mn 1  K ; if m ¼ 6 n mn s d  ð9Þ R ¼  1 1þ s Kdmn ; if m ¼ n where Rmn is the retardation factor for a combined flow of two solutes m and n; s is the specific density of the soil solids; is void fraction (v/v), and m and n are indices of the interacting ion species; Kdmn is the exchange factor for the two interacting ion species simultaneously. The effective diffusion coefficient, De , may be evaluated indirectly (Nye and Tinker, 1977; 77):  @C De ¼ Dl fl l ; ¼ Dl fl b ð10Þ @Cs where Dl is the diffusion coefficient of the solute in water (l), and fl is the soil impedance factor for its diffusion (So and Nye, 1989).

656

Silberbush

Equations (6) and (7) describe the radial flow of the solute between r1 and r0 (Fig. 2). At the root surface (r0 , the inner boundary), the flux from the soil Jr0 has to equal net influx In (influx–efflux) to the root. The uptake of ions and water by the root are the mechanisms that drive their flows through the soil toward the root in the first place. Figure 3 illustrates the depletion of orthophosphate-P concentration in the soil solution perpendicular to the root, simulated by the BarberCushman model (Oates and Barber, 1987).

B.

The Inner Boundary at the Soil–Root Interface

Uptake of the solute and of water by the root surface is the driving force for their flow toward the root. It defines the inner boundary for the processes that take place in the soil. In the simple case moisture content is assumed to be constant, and equally distributed in the soil. The model of Bar-Yosef et al. (1980) accounted also for moisture depletion gradients next to the root. Ion influx to the root is often assumed to obey in certain cases the Michaelis-Menten-type kinetics, namely, active uptake by the root as a function of ion concentration in the soil solution at the root surface. There are two ways to modify the original Michaelis-Menten equation in order to account for a concentration threshold:

In ¼

ð11aÞ

Imax ðCl  Cmin Þ Km þ ðCl  Cmin Þ

ð11bÞ

or: In ¼

where E stands for efflux, while Cmin is concentration threshold, at which In equals 0; Imax is maximal influx, and Km is the concentration (in equation [11b]: Cl Cmin ) at which In equals half Imax . Note that, unlike in equations (3) and (4) which describe ion disappearance from the soil, influx here is calculated in terms of mass of ion taken up per unit root surface area per unit of time (for example:
mol cm2 s1 ). Until the 1990s, the mechanistic models used constant values for influx parameters, although it was already known that adjustments are needed to Imax and Km according to the plant internal concentration of the nutrient (Siddiqi and Glass, 1982; Barber, 1984; 73). The effect of root aging (Barber, 1984; 215) was also not accounted for although its importance was illustrated by sensitivity analyses of models to their parameters (Barber and Silberbush, 1984; Yanai, 1994). Buysse et al. (1996) added a correction for Imax of nitrate influx based on the deficient concentration (deviation from sufficiency) in the root tissues. A different way to describe the inner boundary was proposed by De Willigen and Van Noordwijk (1994a,b). They divided nutrient uptake by roots from the soil into two cases: (1) when the restriction to uptake is due to the supply by the soil, and (2) when the limitation is in the absorption capacity of the root. A typical case 2 is the uptake of nitrate from a fertile soil, or absorption of Cl and Br . For most essential nutrients, the limitation lies in their mobility in the soil. These cases may be treated as ‘‘zero-sink’’ cases, i.e., the gradient from the soil into the root is identical to what is expected toward a zero concentration, like flow of water from an open pipe to a pool. The level of water in the pool does not affect the flow rate out of the pipe. C.

Figure 3 Phosphorus relative concentration (fraction of initial concentration) in the soil solution near the root surface, after 1, 3, and 10 days of uptake, predicted by the Barber-Cushman model. The values of the soil and plant parameters used in the simulation are listed in the legend. (From Oates and Barber, 1987.)

Imax Cl E Km þ Cl

Uptake by the Whole Root System

The root system is assumed to comprise initial (already existing) root length (L0 ) and additional root length that grows at the rate of @L=@t. The simplest assumption is that all parts of the root system have a uniform radius and the same uptake characteristics. Total uptake (T) by the whole root system during the simulation period (i.e., between t ¼ 0 and t ¼ tm) may be

Simulation of Ion Uptake

calculated as (Barber and Cushman, 1981; Barber, 1984): ð ð tm ð tm dL tm t T ¼ 2r0 L0 Jrðr0 ;sÞ ds þ 2r0 0 0 dt 0 ð12Þ Jrðr0 ;sÞ ds dt where t is time of root growth and s is time of uptake: Whenever a new root segment is initiated (as a function of time t), it starts to absorb the nutrient from the initial concentration of the nondepleted soil, and the radial flux of the solute to that root segment starts from s ¼ 0. D.

The Outer Boundary and Root Competition

Assuming that the roots are randomly dispersed in a given soil volume V, the outer boundary of the soil cylinder should be: sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi sffiffiffiffiffiffiffiffiffi V 1 r1 ¼ ¼ ð13Þ  Lt  RLDt where Lt is root length and RLD is root length density (root length per unit soil volume, L2 ) at time t. Equation (13) indicates that there are inverse relationships between root density and the distance between adjacent roots. It determines the volume of soil that provides nutrients to the root. The parameter r1 is therefore a mean to quantify the competition between neighboring roots. The early models (Lambert and Penning de Vries, 1973; Baldwin et al., 1973; Claassen and Barber, 1976; Cushman, 1979 [Case I]) did not account for a restricted rhizosphere (‘‘Single-root’’ models). The extent of the outer boundary of the rhizocylinder with ptime ffiffiffiffiffiffiffiffi and the diffusion coefficient was estimated as De t (Brewster et al. 1976), assuming that the concentration at that distance is constant, and equals to that of the initial concentration in soil Cli . The Barber-Cushman model (Cushman 1979, Case II) assumed a constant value for r1 , allowing the rhizosphere to become depleted of the nutrient up to this distance without external supply. This assumption was reasonable for ions with low mobility, like P, but introduced an error in cases of high root densities or with mobile ions like Kþ or NO 3 (Barber and Silberbush, 1984). Yanai (1994) used a combined approach—namely, an extension of the depletion zone up to a finite distance determined by the root abundance. Abbes et al.

657

(1996) used a relative value for the radial distance, so the distance between r0 and r1 was transformed to 0–1, respectively, while r1 was updated according to equation (13). Hoffland et al. (1990) updated the value of r1 for the new fraction of roots each time the depletion zone of the former fraction reached the previous r1 . This correction improved the accuracy of the estimation of nitrate uptake as long as the limitation to uptake was due to the rate of supply by the soil. A similar correction was used by Buysse et al. (1996). E.

Uptake by Root Hairs and Mycorrhizal Hyphae

Mycorrhyzal hyphae and root hairs serve as adsorbing extensions of root surfaces. Yet, while root hairs usually grow perpendicular to the root surface, with rather uniform length and density, mycorrhizal hyphae may grow in any direction, to different distances, or even to form a sheath around the root. Uptake by root hairs was treated by several models, and in different approaches. Brewster et al. (1976) used the model of Nye and Marriott (1969) to explain the contribution of root hairs to the uptake of P. Itoh and Barber (1983) added a sink component to equation (7) to account for uptake by root hairs, but considered them as active for a limited period of time. They assumed that the radial flow to a root hair is by diffusion only, and that uptake by root hairs is affected by their abundance on the root surface and their length. A similar approach was used by Claassen and coauthors in the NST 3.0 model (Claassen, 1990; Claassen and Steingrobe, 1999). The same system was treated by Geelhoed et al. (1997b) as a 3-D model, with radial, longitudinal, and angle (around the root) dimensions. They considered one root with a soil cylinder of half the distance between roots, and a root hair with known length and radius, where half the distance is determined by root hair density (Fig. 4). The combined uptake is by the root surface (radial flow to the root) and by the root hair (radial flow to its surface). In both organs, influx term was treated as a zero-sink case. Aging of the individual root hair is accounted for by the decrease in its contribution to uptake with time, depending on root hair density (h1 ), radius (h0 ), and the soil buffer power. When it comes to modeling of uptake by mycorrhizae, the literature is even poorer. Although such symbiosis should result in improved uptake of compounds with low mobility (Barber and Silberbush, 1984; Clarkson, 1985), modeling of this system was unsuc-

658

Silberbush

in the two root zone fractions by specific parameters. These studies enabled them to recommend the optimal ratio between the enriched and the untreated root zones according to the soil characteristics (Kovar and Barber, 1989). G.

Figure 4 Illustration of a combined radial flow to a root (Jr) and to a root hair (Jrh); radius of the root (r0) and of the root hair (h0 ); half-distance between roots (r1 ) and between roothairs (h1 ); lrh: length of a root hair.

cessful. Using the Barber-Cushman model, Ernani et al. (1994) underestimated P uptake observed by plants infected by mycorrhizae. Their simulation result was only 36% of the actual uptake, a recovery level typical of simulation of P uptake from low-P soils. This last problem was partially solved by Geelhoed et al. (1999), who included in their model exudation (efflux) of protons and citrate by the root surface, which increased the estimate of the uptake of P sorbed on goethite. F. Uptake from Heterogeneous Soils Most uptake models assume uniform, isotropic soils. In reality, this is rarely the case: The topsoil is usually richer in nutrients owing to fertilization, decomposition of plant debris, and the weathering of mineral materials. The topsoil is often less compacted owing to cultivation and biological activities, so root may penetrate it relatively more easily. Nonuniform distribution of nutrients in the soil results in preferential root growth in richer patches that may compensate for the reduced nutrient supply from generally nutrient-poor soil (Robinson, 1994). For all these reasons, nutrients will be absorbed from various parts of the soil at different rates, and the overall uptake is an integration of supplies by the whole root system. Barber and coauthors (Barber 1984; 372–387; Kovar and Barber, 1989) used the Barber-Cushman and the Claassen-Barber models to predict uptake either from the untreated soil or from an enriched fraction (by placement of fertilizer). In these studies, they used measured root distribution patterns that were measured in the field, and characterized the soil parameters

Patchy Distribution and Ecological Implications

Williams and Yanai (1996) used the Yanai (1994) model for sensitivity analysis to evaluate ecological trends that may be beneficial to plants. They concluded that, in highly fertilized soils, an increase in Imax would not require a high investment of carbon and photosynthetic energy by a plant. Furthermore, an investment of N to increase Imax should result in high returns in nutrient uptake as a whole, so photosynthesis product may be invested more efficiently in the aboveground parts of the plant. Under low soil fertility, however, with low N concentration, the increase in Imax will bring about only little increase in nutrient uptake, so investment of carbon in extended root length would be more beneficial to the plant. The Yanai and the Barber-Cushman models were used to evaluate possible advantages of certain traits in plant adaptation to patchy nutrient distribution (Jackson and Caldwell, 1996; Ryel and Caldwell, 1998). Under patchy P and NO3 -N distributions, there appears to be a competitive advantage to root systems with a high plasticity regarding root abundance and uptake kinetics. Under such conditions, the plant as a whole should benefit more when its roots have the adaptive plasticity to change root abundance and uptake kinetics in accordance with the patchy distribution of soil fertility. H.

Uptake of Toxic Elements

Although uptake of toxic elements was studied quite intensively, little was done on modeling of uptake of interfering substances by roots of plants grown in soil. These effects were studied, to some extent, as exchange modeling between the soil and the root surfaces (Calba et al., 1999; Van Oene, 1998). However, Singh and Pandeya (1998) used the Baldwin model to simulate Cd uptake and toxicity to plant roots from soils treated with sludge. Their results showed that Cd mobility in the soil, as of other metals, is not in the mineral form, but as complexes with organic compounds (mainly of fulvic acids), which determine their solubility and diffusion in the soil.

Simulation of Ion Uptake

I.

Uptake of Microelements

Unlike major and macronutrient uptake, the number of publications on modeling of microelement uptake by roots from soils is rather small. This is apparently because the chemistry of these elements in the soil is complex, so the definition of the model parameters is more complicated. Sadana and Claassen (2000) employed the NST 3.0 model of Claassen (1990), a version of the Barber-Cushman model, to predict Mn uptake. Their measurements showed that this process is associated with low initial, nondepleted concentration Cli (see also Section III.D), but also with low b (unlike in the case of P, for example, where low Cli is usually associated with high buffer power). As a result, Mn uptake appeared to be very sensitive to Cli , Imax , and Km , more than to the soil supply parameters.

IV.

CONCLUSIONS

The early models for nutrient uptake by roots grown in soil were rather simple, based on simplified assumptions that rarely met the reality, especially under field conditions. When simulating extreme conditions, such as very low nutrient levels or interactions of ions, these models often failed to describe the real plant uptake accurately. Improvements adapted in the more recent models made them more realistic, taking into account processes such as exudation, pH changes at the soil–root interface, ion interaction, a realistic description of the buffer power, root morphology, and uneven distribution of minerals and water in the soil. There are attempts to use this approach to simulate the effects of toxic compounds, coexistence and competition between roots of different individual plants and/or different species in field studies or ecosystem evolution, soil variation in time and space, and plant adaptation. It is anticipated that this kind of modeling will be developed toward a more precise description of the soil–root interface, including the space occupied by root hairs. Another direction is to account for changes in root morphology and physiology (probably in a system with more than two species), root mortality and regeneration, subberization, root growth as affected by salinity and toxicity, and root herbivory (see Chapter 22 by Page`s in this volume). The obstacle will still lie with the combined effects of plant physiology and root functioning in the soil. In this respect, defining plant physiological performance under variable environ-

659

mental conditions (rather than in standard conditions as is done nowadays) may also improve the accuracy of root uptake modeling.

REFERENCES Abbes C, Robert JL, Parent LE. 1996. Mechanistic modeling of coupled ammonium and nitrate uptake by onions using the finite element method. Soil Sci Soc Am J 60:1160–1167. Acock B, Pachepsky YA. 1996. Convective-diffusion model of two-dimensional root growth and proliferation. Plant Soil 180:231–240. Baldwin JP, Nye PH, Tinker PB. 1973. Uptake of solutes by multiple root systems from soil. III. A model for calculating the solute uptake by a randomly dispersed root system developing a finite volume of soil. Plant Soil 63:621–635. Barber SA. 1984. Soil Nutrient Bioavailability. A Mechanistic Approach. New York; John Wiley & Sons. Barber SA, Cushman JH. 1981. Nitrogen uptake model for agronomic crops. In: Iskandar IK, ed. Modeling Wastewater Renovation Land Treatment. New York; John Wiley & Sons, pp 382–409. Barber SA, Silberbush M. 1984. Plant root morphology and nutrient uptake. In: Barber SA, Bouldin DR, eds. Root, Nutrient and Water Influx, and Plant Growth. Madison, WI: Spec Publ No 49, American Society of Agronomy, pp 65–87. Bar-Yosef B, Fishman S, Talpaz HA. 1980. Model of zinc movement to single roots in soils. Soil Sci Soc Am J 44:1272–1279. Behrendt H, Bruggemann R, Morgenstern M. 1995. Numerical and analytical model of pesticide root uptake model comparison and sensitivity. Chemosphere 30:1905–1920. Bhat KKS, Nye PH, Baldwin JP. 1976. Diffusion of phosphate to plant roots in soil. IV. The concentration distance profile in the rhizosphere of roots with root hairs in a low-P soil. Plant Soil 40:309–319. Bouldin DR. 1989. A multiple ion uptake model. J Soil Sci 40:309–319. Brewster JL, Bhat KKS, Nye PH. 1976. The possibility of predicting solute uptake and plant growth response from independently measured soil and plant characteristics. V. The growth and phosphorus uptake of rape in soil at a range of phosphorus and a comparison of results with the predictions of a simulation model. Plant Soil 44:295–328. Buysse J, Smolders E, Merckx R. 1996. Modelling the uptake of nitrate by a growing plant with an adjustable root nitrate uptake capacity. I. Model description. Plant Soil 181:19–23.

660 Calba H, Cazevieille P, Jaillard B. 1999. Modelling of the dynamics of Al and protons in the rhizosphere of maize cultivated in acid substrate. Plant Soil 209:57–69. Claassen N. 1990. Die Aufnahme von Na¨hrstoffen aus dem boden durch die ho¨here Pflanze als ergebins von Verfu¨gbarkeit und Aneignungsvermo¨gen. Go¨ttingen, Germany: Severin-Verlag. Claassen N, Barber SA. 1976. Simulation model for nutrient uptake from soil by a growing plant root system. Agron J 68:961–964. Claassen N, Steingrobe B. 1999. Mechanistic simulation models for a better understanding of nutrient uptake from soil. In: Rengel Z, ed. Mineral Nutrition of Crops: Fundamental Mechanisms and Implications. New York; Haworth Press, pp 331–371. Clarkson DT. 1985. Factors affecting mineral nutrient acquisition by plants. Annu Rev Plant Physiol 36:77–115. Cushman JH. 1979. An analytical solution to solute transport near root surfaces for low initial concentration. I. Equation development. Soil Sci Soc Am J 43:1087– 1090. Cushman JH. 1980. Analytical study of the effect of ion depletion (replenishment) caused by microbial activity near roots. Soil Sci 129:69–87. De Willigen P, Van Noordwijk M. 1994a. Mass flow and diffusion of nutrients to a root with constant or zerosink uptake. I. Constant uptake. Soil Sci 157:162–170. De Willigen P, Van Noordwijk M. 1994b. Mass flow and diffusion of nutrients to a root with constant or zerosink uptake. II. Zero-sink uptake. Soil Sci. 157:171– 175. Ernani PR, Santos JCP, Kaminski J, Rheinheimer DS. 1994. Prediction of phosphorus uptake by a mechanistic model in a low phosphorus highly weathered soil as affected by mycorhhizae inoculation. J Plant Nutr 17:1067–1078. Gardner WR. 1991. Modeling water uptake by roots. Irrig Sci 12:109–114. Geelhoed JS, Findenegg GR, Van Riemsdijk WH. 1997a. Availability to plants of phosphate adsorbed on goethite: experiment and simulation. Eur J Soil Sci 48:473–481. Geelhoed JS, Mous SLJ, Findenegg GR. 1997b. Modeling zero sink nutrient uptake by roots with root hairs from soil: comparison of two models. Soil Sci 162:544–553. Geelhoed JS, Van Riemsdijk WH, Findenegg GR. 1999. Simulation of the effect of citrate exudation from roots on the plant availability of phosphate adsorbed on goethite. Eur J Soil Sci 50:379–390. Greenwood DJ, Karpinets TV. 1997. Dynamic model for the effects of K-fertilizer on crop growth, K-uptake and soil-K in arable cropping. 1. Description of the model. Soil Use Mgmt 13:178–183. Gregory PJ. 1996. Approaches to modelling the uptake of water and nutrients in agroforestry systems. Agrofor Sys 34:51–65.

Silberbush Hagin J, Amberger A, Kruth G, Segall E. 1976. Outlines of a computer simulation model on residual and added nitrogen changes and transport in soils. Z Pflanzenern Bodenk. 4:443–455. Hoffland E, Bloemhof HS, Leffelaar PA, Findenegg GR, Nelemans JA. 1990. Simulation of nutrient uptake by a growing root system considering increasing root density and inter-root competition. In: Van Beusichem ML ed. Plant Nutrition—Physiology and Application. Amsterdam; Kluwer, pp 9–15. Harrison CB, Graham WD, Lamb ST, Alva AK. 1999. Impact of alternative citrus management practices on groundwater nitrate in the Central Florida Ridge. II. Numerical modeling. Trans Am Soc Agr Eng 42:1669– 1678. Helyar KR, Munns DN. 1975. Phosphate fluxes in the soilplant system: a computer simulation. Hilgardia 43:103–130. Hutson JL, Wagenet RJ. 1995a. The application of chemical equilibrium in solute transport models. In: Loepert RH, Schwab AP, Goldberg S, eds. Chemical Equilibrium and Reaction Models. Madison, WI: Spec Publ No 42, American Society of Agronomy, pp 97–112. Hutson JL, Wagenet RJ. 1995b. An overview of LEACHM: a process based model of water and solute movement, transformations, plant uptake and chemical reactions in the unsaturated zone. In: Loepert RH, Schwab AP, Goldberg S, eds. Chemical Equilibrium and Reaction Models. Madison, WI: Spec Publ No 42, American Society of Agronomy, pp 409–422. Ibrikci H, Ulger AC, Cakir B, Buyuk G, Guzel N. 1998. Modeling approach to nitrogen uptake by fieldgrown corn. J Plant Nutr 21:1943–1954. Itoh, S, Barber SA. 1983. A numerical solution of whole plant nutrient uptake for soil–root systems with root hairs. Plant Soil 70:403–413. Jackson RB, Caldwell MM. 1996. Integrating resource heterogeneity and plant plasticity: modelling nitrate and phosphate uptake in a patchy soil environment. J Ecol 84:891–903. Jardine PM, Wilson GV, Luxmoore RJ. 1988. Modeling transport of inorganic ions through undisturbed soil columns from two contrasting watersheds. Soil Sci Soc Am J 52:1252–1259. Jemison JM, Jabro JD, Fox, RH. 1994a. Evaluation of LEACHM. I. Simulation of drainage, bromide leaching, and corn bromide uptake. Agron J 86:843–851. Jemison JM, Jabro JD, Fox RH. 1994b. Evaluation of LEACHM. II. Simulation of nitrate leaching from nitrogen-fertilized and manured corn. Agron J 86: 852–859. Kage H. 1997. Is low rooting density of faba beans a cause of high residual nitrate content of soil at harvest. Plant Soil 190:47–60.

Simulation of Ion Uptake Kovar JL, Barber SA. 1989. Reasons for differences among soils in placement of phosphorus for maximum predicted uptake. Soil Sci Soc Am J 53:1733–1736. Lai CT, Katul G. 2000. The dynamic role of root-water uptake in coupled potential to actual transpiration. Adv Water Resour 23:427–439. Lambert JR, Penning de Vries FWT. 1973. Dynamics of water in the soil–plant–atmosphere system: a model named Troika. In: Hadas A, Swartzendruber D, Rijtema PE, Fuchs M, Yaron B, eds. Physical Aspects of Soil Water and Salts in Ecosystems. Ecological Studies. Analysis and Synthesis, Vol 4. Berlin; Springer-Verlag, pp 257–273. Leadley PW, Reynolds JF, Chapin FS. 1997. A model of nitrogen uptake by Eriophorum vaginatum roots in the field: ecological implications. Ecol Mon 67:1–22. Loepert RH, Schwab AP, Goldberg S, eds. 1995. Chemical Equilibrium and Reaction Models. Spec Publ No 42. Madison, WI: Soil Science Society of America. Mankin KR, Fynn RP. 1996. Modeling individual nutrient uptake by plants: relating demand to microclimate. Agric Syst 50:101–114. Nye PH, Marriott FHC. 1969. A theoretical study of the distribution of substances around roots resulting from simultaneous diffusion and mass flow. Plant Soil 30:459–472. Nye PH. 1992. Towards the quantitative control of crop production and quality. J Plant Nutr. 15:1129–1192. Nye PH, Tinker PB. 1977. Solute Movement in the Soil-Root System. Studies in Ecology, Vol 4. Oxford, U.K.: Blackwell Scientific Publications. Oates K, Barber SA. 1987. Nutrient uptake: a microcomputer program to predict nutrient absorption from soil by roots. J Agric Educ 16:65–68. Passioura JB. 1996. Simulation models: science, snake oil, education, or engineering? Agron J 88:690–694. Rao S, Mathur S. 1994. Modeling heavy metal (cadmium) uptake by soil-plant root systems. J Irrig Drain Eng ASAE 120:89–96. Reginato JC, Tarzia DA, Cantero A. 1990. On the free boundary problem for the Michaelis-Menten absorption model for root growth. Soil Sci 150:722–729. Richter J. 1987. The Soil as a Reactor: Modeling Processes in the Soil. Cremlington, Germany: Catena Paperback, Catena Verlag. Robinson D. 1994. The responses of plants to non-uniform supplies of nutrients. New Phytol 127:635–674.

661 Ryel RJ, Caldwell MM. 1998. Nutrient acquisition from soils with patchy nutrient distributions as assessed with simulation models. Ecology 79:2735–2744. Sadana US, Claassen N. 2000. Manganese dynamics in the rhizosphere and Mn uptake by different crops evaluated by a mechanistic model. Plant Soil 218:233–238. Siddiqi MY, Glass AKM. 1982. Simultaneous consideration of tissue and substrate potassium concentration in Kþ uptake kinetics: a model. Plant Physiol 69:283–285. Silberbush M. 1996. Simulation of ion uptake from the soil. In: Waisel Y, Eshel A, Kafkafi U, eds. Plant Roots: The Hidden Half. 2nd ed. New York; Marcel Dekker, pp 643–658. Silberbush M, Sorek S, Yakirevich A. 1993. K+ uptake by root systems grown in soil under salinity. I. A mathematical model. Transport Porous Media 11:101–116. Singh AK, Pandeya SB. 1998. Modelling uptake of cadmium by plants in sludge-treated soils. Bioresour Technol 66:51–58. Smethurst PJ, Comerford NB. 1993. Simulating nutrient uptake by single or competing and contrasting root systems. Soil Sci Soc Am J 57:1361–1367. So HB, Nye PH. 1989. The effect of bulk density, water content and soil type on the diffusion of chloride in soil. J Soil Sci 40:743–749. Somma F, Hopmans JW, Claustitzer V. 1998. Transient three-dimensional modeling of soil water and solute transport with simultaneous root growth, root water and nutrient uptake. Plant Soil 202:281–293. Timlin D, Pachepsky YA, Acock B. 1996. A design for a modular, generic soil simulator to interface with plant models. Agron J 88:162–169. Van Oene H. 1998. A mechanistic model for the inhibiting effects of aluminium on the uptake of cations. Z Pflanzenernahr Bodenk 161:661–670. Van Rees KCJ, Comerford NB, Rao PSC. 1990. Defining soil buffer power: implications for ion diffusion and nutrient uptake modeling. Soil Sci Soc Am J 54:1505–1507. Williams M, Yanai RD. 1996. Multi-dimensional sensitivity analysis and ecological implications of a nutrient uptake model. Plant Soil 180:311–324. Wu J, Zhang R, Gui S. 1999. Modeling soil water movement with water uptake by roots. Plant Soil 215:7–17. Yanai R. 1994. A steady-state model of nutrient uptake accumulating for newly grown roots. Soil Sci Soc Am J 58:1562–1571.

38 Soil Water Uptake and Water Transport Through Root Systems John S. Sperry, Volker Stiller, and Uwe G. Hacke University of Utah, Salt Lake City, Utah

I.

INTRODUCTION

cussed as such. We consider water extraction from drying soils, because it is under these water-limited circumstances that uptake and supply characteristics are most important. In this respect, this chapter departs from many treatments of the subject which ignore the complicating but critical effects of soil drought on water uptake. The focus on water uptake is not meant to ignore the importance of nutrient availability on rooting traits. To the contrary, by specifying root qualities necessary for optimal water use, we can identify deviant root systems where nutrient supply may be paramount. The twin axioms underlying the chapter are (1) that plant water use cannot exceed the supply capacity of the soil-to-leaf pipeline, and (2) that natural selection will result in plants that maximize their water supply per investment in transport tissue. By defining the supply capacity of this system and understanding how it is influenced by soil and root characteristics, we can analyze the efficiency of rooting patterns with respect to plant water uptake across the landscape. The supply characteristics of the pipeline are dictated by the nature of the transport mechanism.

Water loss is the price plants pay for obtaining CO2 from a dry atmosphere. The exchange rate is poor. To fix one molecule of CO2 the plant will typically transpire 200–400 molecules of water, depending on humidity and on stomatal conductance (Raven, 1984). That plants are generally more productive when grown in high humidity indicates that the cost of transpiration outweighs any direct benefits in terms of leaf cooling or nutrient transport (Salisbury and Ross, 1992). To obtain an adequate supply of water, plants invest a considerable portion of their resources in building and maintaining uptake and transport tissues that fix practically no CO2. The size of this investment depends on plant and habitat: In cool desert shrubs, root biomass can be as much as three to nine times the shoot biomass (Dobrowolski et al., 1990); on the other extreme, in submerged aquatic plants the root biomass may be as little as 6% of the shoot (Waisel and Agami, 1996). Within each habitat, a successful competitor will achieve maximum water supply for a given investment. In this chapter, we discuss how root system characteristics can influence plant water uptake from soils with different water supply characteristics. This leads to predictions for combinations of root and soil traits that should maximize the plant’s water supply for a given allocation of resources to water uptake. While soil and root features are emphasized, water uptake and transport are whole-plant processes and are dis-

II.

MECHANISMS OF WATER UPTAKE

As anyone knows who has watched cut flowers take up water from a vase, it is the aerial shoot that drives most water uptake, not the roots. According to the cohesion–tension mechanism (Fig. 1, left), evaporation 663

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Figure 1 Outline of topics. (Left) Leaves pull water from the soil, placing the water column under negative pressure, as if it were a hydraulic rope. (Top right) Water transport can be described by Darcy’s law, wherein E is the transpiration rate per leaf area, dC/dx is the gradient in the water potential component that is driving flow, and K is the hydraulic conductivity per leaf area. (Middle right) Throughout the continuum, K declines with increasingly negative Cw as shown in the KðCÞ plot. The KðCÞ behavior depends on soil texture, root tissue properties, and cavitation in xylem. When Cw drops low enough to drive K to zero, the hydraulic rope breaks, and leaves are disconnected from soil. (Bottom right) Darcy’s law describes built-in limits on E, which can be predicted from knowledge of the root-to-leaf area ratio (AR:AL), root depth profile, and soil water potential (Cm ) profile. The result is a prediction of the maximum hydraulically possible transpiration rate (Ecrit) as a function of Cm as shown by the Ecrit (Cm ) curve. This is referred to as a water use ‘‘envelope,’’ and it provides a mechanistic explanation for maximum water extraction capability (the area under the envelope) and the permanent wilting point (the Cm below which water uptake ceases).

from the cell walls of the leaf creates a capillary suction on the water in the wall pores. This negative pressure is transmitted through the wall pores to the lumina of the xylem conduits (vessels or tracheids), across the peripheral root tissues, and to the water in the soil (Pickard, 1981). The roots serve to conduct rather than drive the transpiration stream. Although a signif-

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icant portion of the transpiration stream moves through cells and membranes, the symplast does not create the negative pressure in the apoplast that drives transpirational flow—the suction results from evaporation and capillarity at the air–water interface of the apoplast. The contrasting water relations of apoplast and symplast in relation to the cohesion–tension mechanism are best explained in terms of water potential (Cw ) of plant tissue and its additive components of pressure (Cp ) and osmotic potential (Co ; Nobel, 1991). Transpiration causes a drop in Cw of the apoplast by lowering the Cp component via the cohesion–tension mechanism. The apoplastic Co is usually near zero, but regardless of its value, it does not participate in driving apoplastic flow which is driven by pressure alone. (It is a common error to assume water will always flow down a gradient in Cw , because for pressure-driven flow that does not cross an osmotic membrane, as within the xylem apoplast or within the phloem symplast, it is Cp rather than Co or Cw that drives movement.) The Cw of the symplast responds to the transpiration-induced drop in apoplastic Cw by a lowering of symplastic Cp (turgor) and Co . Turgor pressure remains positive as long as the cell Co is kept more negative than the apoplastic Cw . Transpiration-driven water uptake is accompanied by at least two other mechanisms. In growth-induced water uptake, the growing cells initiate the water movement by a plastic yielding of their walls that causes a drop in cell turgor and Cw (Kramer and Boyer, 1995). This draws water from the apoplast, and creates the same capillary suction in the cell walls as does transpirational flow (but from water movement to the cells rather than to the atmosphere). In the second mechanism—osmotic uptake—the accumulation of ions in the apoplast of the stele creates a Co gradient across the endodermis, drawing water into the root stele. This can create a positive Cp in the apoplast of the stele (‘‘root pressure’’; Pickard, 1981) that pushes water up the xylem, causing water to drip from foliar hydrathodes (guttation). Neither of these mechanisms contribute substantially to leaf water supply under transpirational conditions. Water moving through the plant by evaporational pull far exceeds cell volume growth during the day. Moreover, it also inhibits the accumulation of solutes in the stele (Kramer and Boyer, 1995). Root systems have a number of characteristics that suit their role as collectors and conductors of water. Chief among them are great length and surface area to enhance contact with soil water. These dimensions are best presented in relation to the area of leaves that are

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demanding the water. Root area to leaf area ratios (AR:AL) span a tremendous magnitude, from as low as 0.24 to > 10 (Rendig and Taylor, 1989; Tyree et al., 1998). Root surface area is commonly distributed in the top 1–3 m of soil, but can go below 60 m in some plants (Canadell et al., 1996). Root xylem is much more efficient at conducting water than stem xylem, with conductivities per unit xylem area over four times those of shoots (Sperry and Saliendra, 1994). This is due to longer and wider xylem conduits in roots (Kolb and Sperry, 1999b; Zimmermann and Potter, 1982; Sperry and Ikeda, 1997). This efficient transport allows roots to be relatively thin and exhibit minimal taper with length, thus minimizing their cost of production and maintenance. However, as we shall see (Section IV.B), this efficiency can be associated with greater susceptibility to drought-induced dysfunction of root xylem. Although mycorrhizae greatly enhance the uptake of nonmobile nutrients and indirectly increase water uptake by boosting the health of the root system, their direct participation in the water uptake process is debated (Davies and Linderman, 1993; Ebel et al., 1994; 1997; Subramanian et al., 1995). The root regulates solute uptake and selectively filters particulates by forcing much of the water across cell membranes as it passes from root surface to xylem (Fig. 2). Water can take a variety of paths as it travels

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across the primary root to the xylem. In the symplastic pathway (Fig. 2, A), water flows from epidermis to root xylem largely through living cells. The movement between cells can be entirely symplastic via plasmodesmata (Fig. 2, A1), or transcellularly through the membrane and wall (Fig. 2, A2). In the apoplastic pathway (Fig. 2, B), water moves through the cell walls. As long suspected by the early anatomists (Haberlandt, 1914), this apoplastic path is interrupted at the anticlinal walls of the endodermis by the Casparian bands of hydrophobic suberin and lignin; these apoplastic barriers are also found in the exodermis of some species (Peterson et al., 1993). In these layers most water is channeled through the symplast, allowing the plant some control over the composition of the incoming soil solution. The membrane filter is not perfect, and there can be significant apoplastic bypasses. Bypasses include the periderm of older roots where the endodermis is absent and water uptake still occurs (Macfall et al., 1990), lateral root junctions in younger roots where the endodermis is at least temporarily ruptured during lateral root development, or the Casparian bands themselves which can remain somewhat permeable to water (Fig. 2). Apoplastic bypasses can have a large effect on the selective uptake and retention of ions by the root system, but they may have less importance in determining

Figure 2 Pathways for water uptake across root tissues. The symplastic path (A) can occur through cell cytoplasm and plasmodesmata (A1, inset), or transcellularly across cell membranes (A2, inset). The apoplastic path (B) occurs through cell walls and intercellular spaces. It is interrupted by the Casparian bands at the endodermis. These hydrophobic regions force most apoplastic water through the endodermal symplast, although some may leak around the cells via apoplastic bypasses. (From Steudle and Frensch, 1996.)

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the overall hydraulic conductance of the root tissue, except perhaps in older roots (Steudle and Peterson, 1998).

III.

LIMITATIONS ON TRANSPIRATIONAL WATER SUPPLY

The supply characteristics of transpiration-driven transport can be formalized by Darcy’s law: E ¼ KðdC=dxÞ

ð1Þ

where E is the leaf area–specific transpiration rate, K is the leaf area–specific hydraulic conductivity of the flow path, and dC=dx is the (negative) gradient in the Cw component that is driving the flow. Within the plant apoplast and in the soil, the driving component is the negative Cp generated by capillary forces. In soil, Cp is usually lumped with electrostatic surface forces and termed ‘‘matric potential’’, Cm (Tyree and Karamanos, 1980). In transmembrane flow across root tissues (Fig. 2, A2), the driving force can also include Co modified according to the reflection coefficients for the various solutes (Steudle, 1994). The K term represents the conducting capacity of the flow path. Although it appears as a constant in Equation (1), it actually varies considerably with Cw components and other factors, as discussed at length in Section IV. With the appropriate modifications to handle these complexities as well as non-steady-state flow, Darcy’s law (or the equivalent Ohm’s law analogy) forms the basis of most quantitative descriptions of water uptake and transport. If Darcy’s law describes a ‘‘supply function’’ for water uptake, Fick’s law can be used to derive a ‘‘demand function’’ for water use by foliage: E ¼ gL
w

ð2Þ

where gL is the leaf-specific stomatal conductance to water vapor diffusion, and
w is the leaf–air mole fraction difference. Stomata regulate E via active changes in gL . Given that stomata control E, it may seem like putting the cart before the horse in stating that the key to understanding patterns of water use lies behind equation (1) and the properties of roots rather than behind equation (2) and the properties of stomata. However, in the same way that the characteristics of the cart limit the performance of the horse, the properties of K and Cw in roots limit the action of stomata. To illustrate this, consider that it is to the advantage of the plant to maximize gL by maximum stomatal opening thereby

maximizing CO2 uptake. Yet, plants frequently operate well below their maximum stomatal opening. According to current understanding, this prevents the demand for water from exceeding the supply necessary to avoid critically low Cw and dehydrative damage. Thus, the negative Cw range available to drive water uptake is limited. What defines the limit to plant Cw ? Most work has focused on the effects of negative Cw on cell function. However, this work faces the paradox that the most C-resistant vegetative plant cells are found in the algae and nonvascular plants (Oliver, 1996). Why should this valuable trait have been lost as plants evolved a vascular system and cohesion-driven water uptake? Perhaps because these novel traits brought with them a new set of Cw limitations that were more restrictive than those affecting cell physiology. In fact, K of the transport pathway can decrease quite abruptly with reductions in Cw components (Fig. 1, KðCÞ plot) of soil and plant, as detailed in the next section. The K in Darcy’s law needs to be represented not as a constant, but as a direct function of the Cw component driving flow (Fig. 1). The consequence of this behavior is that as the driving force for water uptake is increased with more negative Cw , a point of diminishing returns is reached owing to the consequent decline in K. Thus, E cannot be increased without limit because beyond some critical maximum (Ecrit), K will be driven to zero and the hydraulic rope between plant and soil will break at this ‘‘weak link’’ in the continuum (Tyree and Sperry, 1988; Sperry et al., 1998). Darcy’s law has built-in limitations that constrain stomatal regulation of water use. From the axiom that demand cannot exceed supply, important insights into plant water use can be gained by identifying the soil and plant factors that limit water supply. These insights are the focus of this chapter. The goals of the chapter are outlined in Fig. 1. The K(C) plot represents conductivity functions at different points in the soil–leaf continuum that are dependent on soil, tissue, and xylem properties (Section IV). To understand how these functions influence foliar water supply, they must be incorporated into Darcy’s law and integrated over the continuum. The architectural considerations that influence this integration include AR:AL, root depth, and soil Cm profiles. We restrict ourselves to nonsaline soils where Cm ¼ soil Cw . In Section V these traits are used to define a ‘‘water use envelope’’ which shows how the critical maximum transpiration rate, Ecrit, varies with Cm (Fig. 1, Ecrit (Cm )). Values of E outside this envelope result in the loss of hydraulic contact with bulk soil.

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Such an envelope is a useful description of the plant’s ability to extract soil water. The Cm at which Ecrit approaches zero is analogous to the soil’s permanent wilting point and defines the readily available soil water (Kramer and Boyer, 1995). The area under the envelope describes the maximum rate of water use as a function of Cm and it is referred to as the ‘‘extraction potential’’ of the soil–plant system. In sections VI–VIII we summarize how hydraulic characteristics of plant and soil interact to determine the water use curve, and discuss recent case studies.

IV.

BEHAVIOR OF HYDRAULIC CONDUCTIVITY

The KðCÞ functions along the soil–leaf continuum play a critical role in both the ability of plants to extract water from a soil during a drying cycle, and to maintain and recover that ability when soil water is replenished. Not only is the loss of K with declining Cw important, but also whether this function is hysteretic, namely, whether K increases as Cw rises with rewetting of soil. Although we focus on C-related changes in K, it is important to remember that K will also change with temperature (Sperry et al., 1988a), membrane composition (Tyerman et al., 1999), xylem sap composition (Ieperen et al., 2000), and development. Ultimately, the significance of a given KðCÞ function for limiting water uptake depends on how it influences the total bulk soil-to-leaf conductance (kt ). The kt represents the integral of K with respect to distance along the soil-to-leaf flow path. It is expressed as volume flow rate per
C from bulk soil to foliage, usually on a per-leaf area supplied basis (Tyree and Ewers, 1991). A large drop in K can have a minimal effect on kt if it occurs over a short distance and in a nonlimiting component of the continuum. An example of this is the double saw-cut experiment where overlapping cuts a short distance apart on opposite sides of a tree trunk can have little effect on tree transpiration. The reason is that even though the saw cuts drastically reduce the conductance for a short piece of the trunk, the initial conductance of the piece is high and its length is trivial compared to the total flow distance. Thus, reducing the conductance of this component has a negligible effect on kt for the tree (Sperry et al., 1993). In the following three sub sections (IV.A–C) we discuss the K(C) functions for the three components of the soil–leaf continuum that can have a significant

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influence on kt : soil, xylem, and the radial flow path across nonxylary root tissue. A.

Soil

In the soil, K is usually expressed as volume flow rate per dCm =dx per cross-sectional area of soil. Changes in soil K associated with drying have a direct link to soil matric potential (Cm ) that depends on soil porosity. As water is pulled from the soil by evaporation or by plant transpiration, the Cm drops, capillary forces fail, and water in the soil pores is replaced by air (Nobel, 1991). As the pores empty, fewer are available to conduct water, and K drops. The larger pores with weakest capillary suction empty first, followed by narrower ones as Cm becomes more negative. The response of K to declining Cm is immediate and depends on the distribution of pore sizes. Sandy soils with uniformly large pore channels have a high K when they are saturated that plummets rapidly with Cm . Finer soils with a wider range of pore sizes have lower saturated K but maintain it over a wider negative range of Cm. When Cm rises to zero after rewetting, there is hysteresis in the K(Cm) curve because the pore channels do not all immediately refill with water (Campbell, 1985). However, compared to more substantial hysteresis in plant K(C) behavior as described below, the K of the soil is unlikely to limit water uptake following rewetting. The soil-to-root pathway can be divided into two component conductances: rhizosphere and pararhizal (Newman, 1969). The rhizosphere conductance represents the flow path across a cylindrical sheath of soil of a few millimeters in thickness around the root. As illustrated in Fig. 3A, as water flows from bulk soil to the root surface along a distance X, it is forced through a diminishing soil area a-b-c. This results in a nonlinear drop in Cm with X from bulk soil to the root surface (Fig. 3B) since a steeper gradient is required to drive the same flow rate through a smaller area. The nonlinearity is enhanced because K also drops along the gradient. Consequently, the conductance across the rhizosphere can fall low enough that despite the short distances involved, it can significantly reduce kt and the supply of water to the foliage. As shown in Fig. 3B, several independent factors can increase the magnitude of the rhizosphere limitation. Coarse soils with their more sensitive KðCm Þ relationships promote more severe rhizosphere limitations than fine soils. Higher E causes steeper Cm gradients and lower conductance across the rhizosphere. Low root-to-leaf area ratios (AR:AL) also steepen the Cm

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The pararhizal conductance represents the flow path towards the rooting zone in the bulk soil. This would include, for example, the movement from a deep water table toward a shallower rooting zone. This movement does not involve the shrinking surface area constraint posed by cylindrical uptake of individual roots, and it occurs ‘‘upstream’’ in the soil flow path where Cm values are less negative. Nevertheless, it does involve much longer distances. This means that a relatively small change in soil K could translate into a significant effect on kt . However, little quantitative information is available on how pararhizal resistances may limit plant water uptake. When the soil is wet, the total bulk soil-to-root surface conductance is generally much higher than plant conductance, so kt is primarily determined by the plant (Blizzard and Boyer, 1980; Nobel, 1994). However, as described in Section VI, under transpirational stress and drought stress, soil conductance can be a limiting factor depending on the soil texture and AR:AL. B.

Figure 3 Factors influencing hydraulic conductance of the rhizosphere. (A) Cylindrical geometry of water uptake by roots results in decreasing soil area a-b-c as water flows along distance X from bulk soil to root surface. (B) This results in nonlinear gradient in the soil matric potential driving force (Cm ) with distance X from bulk soil to root surface. The nonlinearity is enhanced by reduced soil conductivity at low Cm , potentially resulting in rhizosphere conductance limiting plant water uptake. Factors that enhance the Cm gradient (solid curve) and rhizosphere limitation include: high soil porosity, high transpiration rate (E), and low root-to-leaf area (AR:AL). Conversely, low soil porosity, low E, and high AR:AL minimize the rhizosphere limitation (dashed curve).

gradient because the transpiration water is forced to cross a smaller surface area as it moves into roots. The rhizosphere limitation is exacerbated by any shrinkage of the root surface away from the soil to form a root– soil gap. This can occur if cortical cells do not osmotically adjust to decreasing Cw and lose water (Faiz and Weatherley, 1982; Nobel, 1994). These factors have different dynamics. Soil porosity is constant over time at a given depth, but different soils can be encountered as the root system grows. The AR:AL changes with development and season. The E is most dynamic, following a daily course with potentially rapid fluctuations.

Xylem

Xylem K is expressed as volume flow rate per Cp =dx per leaf area or per cross-sectional area of xylem (Tyree and Ewers, 1991). Changes in xylem K associated with decreases in Cp are similar in nature to the soil situation. In the xylem, the maximum K occurs when all xylem conduits are filled with water. As Cp drops, xylem conduits cavitate, emptying their water and becoming embolized and nonconductive. Cavitation occurs conduit by conduit, resulting in a continuous and nearly instantaneous decline in K with Cp (Fig. 4A). Cavitation occurs by air entry into the water-filled xylem conduits, so the physical basis for the decline in xylem K is analogous with soil. The specific point of air entry appears to be pits between functional and previously embolized conduits (Tyree and Sperry, 1989; Jarbeau et al., 1995; Sperry et al., 1996). The xylem of different plant species differs considerably in K(Cp ), with some species being fairly resistant to cavitation and others being quite susceptible. In general, more resistant xylem is found in species that experience lower Cp in their native habitat. Often (but not always), the more susceptible xylem is associated with a higher maximum K when expressed on a xylem area basis (Sperry and Ikeda, 1997; Ewers et al., 2000; Pockman and Sperry, 2000). Recently, changes in xylem K that are possibly not related to cavitation or temperature have been documented (Ieperen et al., 2000; Tsuda and Tyree, 2000), but little is known of the significance or mechanism of these changes.

Soil Water Uptake

Figure 4 (A) Hydraulic conductivity [K(Cp )] functions of Pinus taeda xylem as caused by xylem cavitation. Hydraulic conductivity is shown as the percentage reduction from the maximum value in the absence of cavitation. Stem xylem (squares) is substantially more resistant to cavitation than root xylem. Within the root system, the thinner roots (circles, xylem diameter 1.6–4.1 mm) were more vulnerable than medium-diameter roots (diamonds, xylem diameter 4.2–6.4 mm), which were more vulnerable than large-diameter roots (triangles, xylem diameter 8.2–13 mm). (From Hacke et al., 2000a.) (B) The Cp required to reduce xylem conductivity by 50% (C50 ) in stem xylem versus root xylem of the same species. Data on the 1:1 line represent xylem that was equally vulnerable to cavitation in roots and stems. In all cases, root xylem was more vulnerable than stem xylem, by an average of 2.3 MPa. (From Sperry and Saliendra, 1994; Alder et al., 1996; Hacke and Sauter, 1996; Sperry and Ikeda, 1997; Linton et al., 1998; Hacke et al., 2000a,b; Sperry et al., 1998; Kolb and Sperry, 1999b; S.D. Davis, unpublished data.)

Within woody plants, root xylem is often significantly more vulnerable to cavitation than stem xylem

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(Sperry and Saliendra, 1994; Hacke and Sauter, 1996; Mencuccini and Comstock, 1997). Figure 4A compares K(Cp ) functions between stems and roots of differentdiameter classes in Pinus taeda (Hacke et al., 2000a). Whereas roots of this species are completely cavitated by 3 MPa, stems remain functional to below 4 MPa. Within the root system, the smaller-diameter roots are more vulnerable to cavitation than the larger ones. This size difference has also been seen in other conifers and angiosperm shrubs (Sperry and Ikeda, 1997; Hacke et al., 2000b). There is some indication that deeper roots of a given size class can also be more vulnerable than shallower roots (W.T. Pockman, unpublished) but few species have been examined in this regard. Figure 4B compares the Cp causing 50% loss of conductivity (C50 ) in root versus shoot xylem of 23 woody species. The C50 of roots is less negative than in stems, by an average of 2.3 MPa. However, this was not observed in pot-grown Acer saccharinum (Tsuda and Tyree, 1997), and it is not known whether similar differences occur in herbaceous plants. However, roots of desert succulents can exhibit reductions in xylem conductivity of up to 98% by drought-induced cavitation (North and Nobel, 1991). The relationship between drought-induced cavitation and conduit size is complex. Within a given cross section of xylem, the larger-diameter earlywood conduits are generally more susceptible to cavitation than narrower latewood conduits (Sperry and Tyree, 1990; Salleo and Lo Gullo 1989a,b; Lo Gullo and Salleo, 1991; Hargrave et al. 1994). When the C50 across species and organs is plotted versus mean conduit diameter (often weighted by contribution to hydraulic conductivity), a trend exists for greater vulnerability in larger conduits, but the relationship is generally very poor, with r2 < .20 (Tyree et al., 1994; Sperry and Saliendra, 1994; Pockman and Sperry, 2000). Apparently, the air seeding properties of the xylem conduits are not tightly correlated with conduit diameter across species and organs. In contrast, there is an excellent relationship (r2 > .75) between C50 and the thickness of the conduit wall relative to conduit diameter (Hacke et al. 2001b). This is consistent with the need for the conduit wall to be sufficiently reinforced to counter the collapsing stress caused by negative water pressure inside the conduit. While the response of xylem K to decreasing Cp during soil drought or transpirational stress occurs within seconds (Pockman et al., 1995), there is significant hysteresis in the recovery of K with following relief of stress. For air-filled conduits to refill, the Cp must rise above –2T/r (relative to atmospheric) where

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T is the surface tension of water and r is the radius of the gas-filled conduits (Yang and Tyree, 1992). This means that the poststress K will remain at the minimum stressed value until Cp rises to within 30 kPa of atmospheric pressure, the exact value depending on conduit r. If this Cp is achieved, refilling of the conduits can occur within hours (Yang and Tyree, 1992). Root pressure plays an important role in the reversal of xylem cavitation as long as transpiration is low and soil Cw is not too negative (Tyree et al., 1986; Sperry et al., 1987, 1988b, 1994; Sperry and Sullivan, 1992). Recent results suggest that refilling can occur in some species and circumstances when total Cw is still far below 2T=r and root pressure is absent, but the mechanism remains unknown (Edwards et al., 1994; Salleo et al., 1996; Holbrook and Zwieniecki, 1999; Chapter 39 by Nardini et al., this volume). However, even if refilling occurs, the KðCp ) function can become much more sensitive to subsequent declines in Cp in some circumstances, suggesting that the xylem can be weakened by the cavitation process (Hacke et al. 2001a). In the absence of cavitation, the total resistance to flow in xylem can account for 10–40% of total soil-toleaf resistance. This fraction may be even higher in large plants. As plants increase in size, their kt decreases (Saliendra et al., 1995; Mencuccini and Grace, 1996). This is due to the increased length of the xylem flow path. It is not just aboveground size that is important, but also the depth of the root system. Given that some root systems can exceed 60 m in depth (Canadell et al., 1996), flow resistance in xylem could be considerable even for small shrubs. As Cp drops under water stress and cavitation occurs, the loss of xylem conductance is predicted to be a major factor in limiting foliage water supply (Section VI). C.

Radial Flow Path Through Root Tissue

Compared to soil and xylem conductivities, much less is known of the potentially more complex KðCÞ relationships in the nonxylary parts of the flow path between soil and root xylem. These changes involve both pressure and osmotic components of Cw , and include direct and rapid responses to Cw components as well as slower anatomical changes in response to chronically low Cw . The radial flow path from root surface to root xylem is usually described by a radial conductance rather than a conductivity. A conductance is volume flow rate per
C, and is dependent on the length of the flow path. As such, it is not surprising that the narrower the root cortex, the higher

tends to be the radial conductance (Rieger and Litvin, 1999). The radial conductance is often measured on relatively short excised root tips where the axial xylem conductance can be factored out (North and Nobel, 1991; Steudle, 1993), and the conductance is usually expressed per surface area of root. There can be pronounced diurnal changes in root radial conductance that occur in association with diurnal changes in Cw . Typically, the conductance increases from early morning to midday before falling off again later (Tsuda and Tyree, 2000). There are several interacting processes that may determine this pattern. The onset of transpiration in the morning shifts the driving force for uptake from osmosis to pressure. This can increase root conductance because pressuredriven flow occurs through apoplastic as well as symplastic pathways across the root (Fig. 2) whereas osmotic uptake is restricted to the symplastic path (Steudle and Peterson, 1998). Aquaporin contents can strongly influence root radial conductance (Tyerman et al., 1999). These membrane water channels appear to be concentrated in the endodermal cell membranes where symplastic flow is greatest (Barrowclough et al., 2000). Changes in aquaporin content during the day have been positively correlated with changes in root conductance (Henzler et al., 1999). The drop in conductance later in the day may also be influenced by the draining of water from the extracellular spaces in the root cortex as Cw becomes more negative. This reduces the area for apoplastic water flow (North and Nobel, 1991). Diurnal patterns in root uptake capability can also be influenced by the transpiration-induced buildup of solutes excluded by endodermal and exodermal tissues. As the solute levels increase in the rhizosphere and cortical apoplast, they can interfere with water uptake through solute drag and the reduction of the driving force by an opposing concentration gradient (Passioura et al., 1992; Stirzaker and Passioura, 1996). These effects would be most prominent in saline or hyperfertilized soils. Of more relevance to this chapter are the changes in root radial conductance associated with soil drought. Although less is known of this topic, in general the evidence suggests a decline in radial conductance with decreasing Cw during drought. Direct effects of Cw include the draining and collapsing of cortical intercellular spaces and the shrinkage of the cortex and resulting soil–air gaps (Nobel, 1994). The pattern of aquaporin activity during soil drought is unknown. Longer term, indirect Cw effects include reduction in permeability of the endodermis and the exodermis

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associated with suberin and lignin deposition in endodermal and exodermal cell walls (North and Nobel, 1991; Stasovski and Peterson, 1993; LoGullo et al., 1998; Steudle and Peterson, 1998), and ultimately, the drought-induced death of entire fine roots (Eissenstat and Yanai, 1997). These combined effects caused a threefold drop in excised root conductance of desert succulents as soil Cw fell from 0 to below 10 MPa (Fig. 5; Nobel, 1994). A more than sixfold drop in whole root system conductance was observed in Olea oleaster as leaf Cw fell below the turgor loss point by a factor of 1.6 (LoGullo et al., 1998). However, the contribution of xylem cavitation versus root tissue changes to this decline was not assessed. Recovery of K in the nonxylary root tissue with rising Cw following drought may involve substantial hysteresis that can increase with duration and severity of the stress (Passioura, 1988; North and Nobel, 1998) and limit recovery of poststress water uptake. Longer and more severe stress periods can enhance suberization and dieback of roots whose effects on K are primarily reversible by new root growth with lag times on the order of many hours to days, weeks, or seasons (North and Nobel, 1998, 1991; Eissenstat and Yanai, 1997; LoGullo et al., 1998). Within the well-watered plant the root system as a whole (xylem plus nonxylem components) can contribute from 40% to 80% of the total plant resistance. In woody plants, the value is often near 50% (Hellkvist et al., 1974; McGowan et al., 1987; Moreshet et al., 1987;

Figure 5 Hydraulic conductance of excised roots (Lp) of three desert succulents as a function of their ambient water potential (C) prior to excision and measurement. Species are Ferocactus acanthoides (triangles), Opuntia ficus-indica (squares), and Agave deserti (circles). (From Nobel, 1994.)

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Saliendra and Meinzer, 1989; Sperry and Pockman, 1993; Yang and Tyree, 1993). Much of the total plant resistance (60–90%) in small herbaceous plants is probably in the nonxylary portions of the flow path (Tyree, 1999). This means that the radial conductance of the root tissue is an important determinant of total plant conductance in herbaceous plants, and kt will be sensitive to root surface area and the area-specific K of the root tissue. Recent experimental studies have supported the traditional view (Passioura, 1988) that radial root conductance is largely determined by single cell layers of the mature endodermis and exodermis (Barrowclough et al., 2000). However, when these layers are not mature, the radial conductivity appears to be evenly distributed from epidermis to root xylem (Peterson et al., 1993).

V.

DEFINING WATER SUPPLY CAPACITY

In the plant, the components discussed above are integrated in a single functioning supply system with a defined capacity for conducting water to the leaves, represented by the EcritðCÞ function in Fig. 1. Given that stomatal control mechanisms have evolved in part to keep E below Ecrit, it is difficult to override this control and assess the Ecrit failure point empirically. This is where transport models incorporating KðCÞ relationships are useful. If such a model can be shown to predict E, it can be used to extrapolate to Ecrit and to dissect the interactions between soil and plant that define the supply capacity of the continuum. Early KðC) models either only incorporated soil KðCm ) and ignored the plant (Cowan, 1965; Bristow et al., 1984), or only modeled the shoot KðCp ) and ignored the root and soil (Tyree and Sperry, 1988; Jones and Sutherland, 1991; Alder et al., 1996). Recently, soil, root system, and shoot dynamics were included in a single model in an attempt to predict the Ecrit envelope and how it is influenced by soil and plant properties (Sperry et al., 1998). In the following sections, we discuss and evaluate predictions gained from this model as recently published (Sperry et al., 1998; Kolb and Sperry, 1999a,b; Ewers et al., 2000; Hacke et al., 2000a; Jackson et al., 2000). The basic structure of the model is a conventional network of conductance elements representing rhizosphere and plant flow paths (Fig. 6). Its most important feature is the incorporation of a K(C) function for each element. These functions represent different cavitation sensitivities in different parts of the shoot and root, and different soil textures. Xylem and nonxylem

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Figure 6 Structure of transport model designed to predict transpiration rate (E) and the hydraulic maximum transpiration (Ecrit) from plant and soil properties. Plant architecture is represented by shoot and root modules each consisting of two orders of branching representing axial versus lateral components. Each module services a layer of canopy or soil. Inputs (in rectangles) include root area and leaf area profiles, Cm profile in soil, and the KðCÞ functions for soil and plant components. Given an E or Cw profile for the canopy, the model solves for flow and pressure at each node in the continuum. Thus, the model can predict E from Cw data, and also be used to predict the maximum hydraulically possible E (Ecrit). (From Sperry et al., 1998; Hacke et al., 2000a.)

conductances in the terminal elements of roots and shoots are lumped (as they are when whole-plant conductance is measured), and given K(C) functions of xylem. The lack of a separate element for the nonxylem component is due to the difficulty of knowing the fraction of total plant resistance in the two paths, and the

lack of a generally applicable K(C) function for nonxylem flow. Root–soil gaps from root shrinkage are also not incorporated. Importantly, these omissions will only influence model predictions if the K(C) of the nonxylem portion is more sensitive than that in xylem or soil. Comparison of K(C) functions of

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xylem (Fig. 4A) with the K(C) relationships of excised roots from desert succulents (Fig. 5) suggests that the proportional decline in xylem K can be at least as great as for the radial flow path across the root tissue. When tested for well-characterized stands of Pinus taeda, the model predicted E through two growing seasons with an r2 of 0.85 (Fig. 7A), suggesting it had captured the most significant K(C) components

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of the continuum, and allowing it to be used to predict Ecrit. Figure 7B indicates how E closely approached the Ecrit envelope under drought conditions in P. taeda, suggesting that these trees fully exploited their ability to extract water during drought when hydraulic limitations become most severe (Hacke et al., 2000a). Comparable results were observed in Artemisia tridentata (Kolb and Sperry, 1999b). When soil water was abundant, both of these species maintained a larger safety margin from Ecrit than under drought conditions. By being adapted to the extremes of drought, these species may be ‘‘overbuilt’’ from the hydraulic supply standpoint when soil water is readily available. In contrast, safety margins from Ecrit were minimal even under wet soil conditions in Betula occidentalis, which is an obligate riparian tree that is not adapted to drought (Sperry et al. 1998). These studies of the water use envelope support the axiom that water use as regulated by stomata will track water supply capacity— particularly when that supply becomes limited under drought conditions.

VI.

Figure 7 (A) Predicted transpiration rate (E) from the transport model in Fig. 6 versus measured E in Pinus taeda trees for two growing seasons. The r2 was 0.85, and the 95% confidence limits encompass the 1:1 line over the measured E range. (From Hacke et al., 2000a.) (B) Transpiration rate (E) per ground area versus Cm for P. taeda over two growing seasons that included significant droughts. Measured E are solid symbols, predicted E is the solid line, and maximum hydraulically possible E (Ecrit) is the dashed line. The E approaches Ecrit under water-limiting conditions, suggesting that the supply capacity of the soil–leaf continuum limits stomatal regulation of E. (From Hacke et al., 2000a.)

PREDICTING THE WEAK LINK IN THE CONTINUUM

The model can be used to predict the limiting conductance in the continuum where the hydraulic rope snaps as Ecrit is exceeded. The point of failure depends on several factors, most basic of which is the AR:AL. If this is too low, hydraulic failure occurs in the rhizosphere owing to an overly steep Cm gradient (Fig. 3). Rhizosphere failure cuts off access to bulk soil water which can still be at relatively favorable Cm . This narrows the Ecrit envelope and reduces the water extraction potential, defined as the area under the envelope (Figs. 1, 8). As the AR:AL is increased, the extraction potential increases to a plateau because hydraulic failure is shifted progressively from the rhizosphere to the plant xylem (Fig. 8). Once failure is occurring in the xylem, there is no longer any advantage to increasing AR:AL because the rhizosphere is not the limiting component. These results suggest that there is a threshold AR:AL required to maximize water extraction from a drying soil (Sperry et al., 1998; Hacke et al., 2000a). The AR:AL threshold is highly dependent on soil type. The coarser the soil, the higher the AR:AL threshold (Fig. 8; compare solid versus dashed lines for loam versus sand soil). This is because a coarse soil has a sensitive K(C) function, and a much higher root surface area is required to keep the Cm gradient in the rhizosphere from triggering critically low soil–root

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Figure 8 Percentage maximum extraction potential (defined as the area under the Ecrit(Cm ) curve) versus root-to-leaf area ratio (AR:AL) modeled for Pinus taeda growing in loam (solid symbol and line) versus sand (open symbol, dashed line). Extraction potential is shown relative to the value at AR:AL ¼ 40, a liberal estimate for a maximum obtainable by woody plants. Low AR:AL reduces extraction potential because of hydraulic failure in the rhizosphere when soil is still relatively wet. As AR:AL is increased, extraction potential increases until hydraulic failure is shifted to the plant xylem. Beyond a threshold value, increasing AR:AL has little effect on extraction potential because the rhizosphere is no longer limiting water uptake. The AR:AL threshold is shifted to higher values in a coarse (sand) soil compared to a fine (loam) soil because the more sensitive K(Cm ) behavior of coarse soils increases the severity of the rhizosphere limitation. Actual AR:AL in P. taeda on the two soils was near the threshold (symbols) as expected for the plant to maximize water extraction ability at minimum root biomass investment. (From Hacke et al., 2000a.)

conductance. The main effect of soil texture is in the sand-to-loam end of the soil spectrum. Much less of a difference in AR:AL threshold is seen from loam to clay (Sperry et al., 1998; Hacke et al., 2000a). Using the model we can test the assumption that root and shoot growth is balanced to maximize water supply for a given root biomass. To achieve this goal, the AR:AL should be near the threshold value. A lower AR:AL would result in less water supply during drought, owing to hydraulic failure in the rhizosphere. A higher AR:AL would do nothing to increase water supply because failure is occurring in the xylem. In support of this, the AR:AL for P. taeda was near this threshold in both loam and sand soil, suggesting that AR:AL was adjusted in response to the different hydraulic properties of the soil (Fig. 8; Hacke et al., 2000a).

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As long as AR:AL is at the threshold or beyond, the weak link in the continuum that defines Ecrit is predicted to be the xylem. Whether the failure occurs in the root or shoot xylem depends on the K(Cp ) functions along the xylem flow path. If this function is the same throughout the flow path, failure will occur in the leaf xylem where Cp is lowest. However, the K(Cp ) function is frequently much more sensitive in the root system than the shoot system (Fig. 4). In this case, the point of failure in the xylem appears to depend on the Cm of the bulk soil. When Cm is less negative under wet conditions, failure may occur in the shoot system. However, when Cm becomes more negative under drought, failure can shift to the root xylem (Kolb and Sperry, 1999b). Root cavitation as a limiting factor in water supply under drought has been indicated in the P. taeda study cited above, in desert shrubs (Mencuccini and Comstock, 1997; Kolb and Sperry, 1999b; Hacke et al., 2000b), and in arid land trees (Alder et al., 1996; Linton et al., 1998). The above studies suggest that hydraulic failure in the roots can define the minimum Cm for water extraction—essentially the plant’s permanent wilting point. For example, in P. taeda growing in loam soil, the lower Cm limit for water uptake was predicted to be –2.2 MPa (Fig. 7). This corresponded to the xylem pressure required to cause near complete loss of hydraulic conductivity in the xylem of small roots (Fig. 4A). In contrast, the desert shrub Artemisia tridentata had a much lower ‘‘permanent wilting point’’ of Cm ; 4 MPa. The difference between the two species was not in soil type, which was very similar, but in the cavitation sensitivity of the root xylem and the AR:AL. The lower extraction limit in A. tridentata was associated with loss of conductivity in root xylem being held off until a Cp between 4 and 5 MPa, and a predicted AR:AL of ca. 10 versus 2 in P. taeda (Kolb and Sperry, 1999b; Hacke et al., 2000a). These results point out the considerable variation in permanent wilting points, which for common field crops and temperate plants have generally been considered to be in the Cm ¼ 1:5 to 2 MPa range (Kramer and Boyer, 1995), and emphasize the importance of AR:AL and root cavitation in their determination. Roots may be acting as a hydraulic fuse: restricting failure in an overloaded hydraulic circuit to a cheap, replaceable unit. This would be particularly appropriate if failure is restricted to the smaller roots, which as far as is known appear to be most vulnerable to cavitation (Fig. 4A). If the drought stress is prolonged, these cavitated roots may senesce and die, but can be

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regrown when the soil rewets (Vartanian, 1996). If the drought stress is brief and roots stay alive, rewetting of soil would bring root xylem Cp above the –2T/r limit for refilling (cf. Section IV.B). Furthermore, failure in the smaller roots would shift water uptake to wetter parts of the root system without interrupting major transport arteries. In contrast, if failure occurred in the major roots and branches, the foliage could be prematurely cut off from remaining soil water, and these structures would be more expensive and timeconsuming to regrow and more difficult to refill given their greater distance from soil water. These ideas extend Zimmermann’s segmentation hypothesis (Zimmermann, 1983) which proposed that failure in shoots was localized to the minor branches and leaves given their lower Cp and sometimes more sensitive K(Cp ) functions (Tyree et al., 1993). Like fine roots, these peripheral parts are more easily replaced and, by triggering a pattern of patchy dieback in the canopy, have the effect of reducing water demand and improving the water status of the remaining canopy (Tyree and Sperry, 1988). It is possible that failure occurs in coordination in fine roots and minor branches.

VII.

CAVITATION RESISTANCE, ROOTING DEPTH, AND SOIL TEXTURE

Recently, the modeling approach has been used to predict interactions among cavitation resistance, root depth distribution, and soil texture in influencing the water use envelope (Jackson et al., 2000). In this analysis, three K(C) functions for the plant were used to represent sensitive, medium, and insensitive cavitation responses (Fig. 9A). Three root depth distributions were used to reflect the range seen in global root data bases (Fig. 9B). The water use envelope was predicted for various combinations of root depth and cavitation resistance for soils drying progressively from the top down, as represented by the set of Cm profiles in Fig. 9C. The entire profile is represented in the water use curves shown in Fig. 9D–G by the Cm at 50 cm depth. In all simulations, AR:AL was held constant at 10, which appears to be at the upper end of observed values for woody plants. Figures 9D and E show the interaction between cavitation resistance and soil texture at one rooting depth in determining water availability. In a loam or in a finer soil (Fig. 9E), greater cavitation resistance confers a substantial increase in water availability by broadening the water use envelope. However, the benefit is largely absent in a coarse soil (Fig. 9D) because

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this soil loses most of its water and conductivity at less negative Cm . Even if AR:AL is doubled from 10 to 20, there is little gain in water availability for more cavitation resistant xylem because the rhizosphere conductance was so limiting. This observation suggests that plants adapted to coarse soils should be more uniformly vulnerable to cavitation than plants in finer soils, since they derive little benefit from cavitation resistance and it costs the plant to build such xylem in terms of increased wood density (Hacke et al., 2001a). In support of this prediction, P. taeda roots and stems were more vulnerable to cavitation in sand than in loam soil (Hacke et al., 2000a). Figures 9F and G show the interaction between rooting depth and soil texture for one cavitation resistance. Obviously, a deeper root system increases water availability because it enhances access to the soil water at less negative Cm . However, the advantage is less in a fine than a coarse soil (compare Fig. 9F versus G). This is because the fine soil holds more water at more negative Cm , and as long as the plant has sufficient AR:AL and cavitation resistance to ‘‘mine’’ that water, there is less advantage to send roots deeper. Conversely, in a coarse soil, the only water to be had is in the deeper layers at less negative Cm , and deep roots are essential. This predicts that where surface soil drying is common, plants in coarse soils will have deeper root systems than plants in fine soils, particularly if plants in fine soils were also more resistant to cavitation, allowing them to extract more of the water in the shallow soil. Analysis of global root data sets for water-limited systems support this prediction with the depth capturing 95% of root biomass being from 0.6 to 0.8 m deeper in sandy than in loam or clay soils (Jackson et al., 1996).

VIII.

INTERACTIONS BETWEEN NUTRIENT AND WATER UPTAKE

Nutrient availability strongly influences AR:AL and root distribution independently of soil moisture characteristics (Haynes and Gower, 1995), and can have interactions with cavitation resistance (Harvey and Driessche, 1997). Fertilization can reduce root density (Linder and Axelsson, 1982; Linder et al., 1987), possibly shrinking the water use envelope and making plants more sensitive to drought. This could be particularly problematic in sand soils where high AR:AL is needed to maximize water supply (Fig. 8). A recent study analyzed the effects of fertilization on the water use envelope in P. taeda (Ewers et al., 2000). Fertilization of P. taeda trees in sand caused a

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Figure 9 Predicted interrelationships between plant K(C) behavior, rooting depth, soil texture, and water uptake from a drying soil. (A) Representive K(C) functions based on cavitation resistance in woody plants adapted to wet habitats (‘‘sensitive’’ curve, from Betula occidentalis; Sperry and Saliendra, 1994), mesic habitats (‘‘intermediate’’ curve, from Acer negundo; Sperry et al., 1998), and xeric habitats (‘‘insensitive’’ curve, from Artemisia tridentata; Kolb and Sperry, 1999a). (B) Root fraction per 25 cm depth based on the empirical model Y ¼ 1  d where Y is cumulative root fraction from soil surface to depth d in cm;  values of 0.95 (shallow), 0.97 (medium) and 0.98 (deep) were based on a typical range of rooting depths from a global root database (Jackson et al., 1996). (C) Soil matric potential (Cm ) profiles used in model runs to compare plant responses to drying soil. Dotted line indicates the use of Cm at 50 cm depth to represent the entire profile in panels D–G. (D,E) Relative water use rate as a function of soil moisture profile for plants of medium rooting depth but variable K(C) curves for loamy sand (D) and loam (E). (F,G) Water use versus soil moisture for plants with intermediate K(C) curves but variable rooting depth for loamy sand (F) and loam (G). (From Jackson et al., 2000.)

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50% reduction in AR:AL over several years. However, in nonirrigated trees this was mostly compensated for by a 50% increase in cavitation resistance of root xylem, resulting in only a 10% reduction in water extraction potential. In contrast, irrigated trees showed no adjustment in cavitation resistance and showed a 25% reduction in extraction potential. Should irrigation be interrupted, these trees should be very sensitive to drought, perhaps experiencing significant dieback. In general, more studies are needed on potential antagonism in root system allocation to nutrient versus water supply. While much work has focussed on the response of root systems to heterogeneity in nutrient availability in soil (Caldwell, 1994), few have examined the associated trade-off with water uptake. Isolating root properties essential for water uptake is a necessary step in being able to measure these trade-offs.

IX.

SUMMARY

Based on our twin axioms that water use cannot exceed supply, and that plants will maximize water supply at minimum cost, we have developed several predictions, most of which have at least some empirical support, and all of which should stimulate further research: 1.

2.

3.

4.

5.

6.

Plants in seasonally dry habitats will have an AR:AL near the threshold required to avoid rhizosphere failure. Because the AR:AL threshold is higher in coarser soils, plants in these soils should have higher AR:AL than plants in finer soils. Hydraulic failure under drought may occur in the root system by xylem cavitation, particularly in the finer roots. Plants of drier habitats and of finer soils should have xylem more resistant to cavitation than plants in wetter habitats or coarser soils. For habitats where surface drying is prominent, plants in coarser soils will have deeper root systems than plants in finer soils. To the extent that fertilizing reduces AR:AL, it can reduce water uptake from drying soils and increase drought sensitivity.

These hypotheses define some of the factors that determine the permanent wilting point for plants and the maximum rate of water uptake. By integrating effects of soil, root system, and shoot, through a drying cycle, it resolves many conflicting views concerning the location of limiting conductances in the continuum. Confusion on this point is at least partly because

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there is no single limiting conductance: it can shift from rhizosphere to the root or to the shoot depending on AR:AL, cavitation resistance, soil texture, and soil moisture. Only by integrating all of these aspects can the system as a whole be analyzed. This integrative approach is limited by our understanding of the hydraulic properties of the components of the soil–leaf continuum. While we can represent the K(C) function of xylem and soil reasonably well (hence their emphasis in our treatment), the same cannot be said of the more complex response functions of nonxylary root tissues. At the scale of the individual root, there are complex interactions between radial and axial (xylem) conductances that influence the pattern of uptake (Fowkes and Landsberg, 1981; Doussan et al., 1998a,b). How these interactions are affected by the shape of the K(C) curve and the age and position of the roots awaits further study. With the exception of the investigations of Nobel and colleagues on desert succulents (Fig. 5; Nobel, 1994; Chapter 53 by Nobel in this volume), we know little of how radial root conductance responds to transpirational and drought stress. Similarly, the representation of the hydraulic architecture of the root system in most transport models is very crude. As the exceptionally well-characterized model of Doussan and colleagues (1998a,b) has shown, the distribution of xylem and radial root tissue conductances with branch order and age can have significant implications for the pattern of water uptake. Total root system conductances on a per-unit root surface area basis have been measured (e.g., Tyree et al., 1998), but how this varies with root order, age, and depth is poorly understood even in the well-studied maize root system (Frensch and Steudle, 1989). To the extent that plants rely on different water sources, the hydraulic architecture of the root system may be adjusted to maximize the hydraulic conductance to that source. The simplest example is relatively deeprooted plants that obtain a significant fraction of their water from wet soil layers at depth. In both Banksia prionotes and Juniperus ashei the deeper roots can have larger xylem conduits and more efficient xylem transport than shallower roots (Pate et al., 1995; Pockman et al., in review). In J. ashei, these differences were associated with more sensitive K(C) functions for deep than for shallow root xylem (Pockman et al., in review). This pattern is not universal, however, because in the shrub Retama raetam the shallow lateral roots had larger-diameter conduits than the deeper-penetrating roots (Fahn and Cutler, 1992), suggesting a different pattern of water use where efficient uptake of deep

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water is not required. There is no information on the extent to which radial root conductances might be adjusted with depth, but a high aquaporin abundance has been shown to compensate for low root surface areas in Arabidopsis (Kaldenhoff et al., 1998). In the soil part of the system, a more explicit consideration of pararhizal soil conductances may reveal how plant water use, vegetation type, and local hydrology are coupled. In addition, rhizosphere dynamics are relatively easy to model, but much more difficult to substantiate empirically. There is a need for clever empirical approaches and technical advances such as magnetic resonance imaging for evaluating the importance of rhizosphere limitations in coarse-textured soils (Macfall et al., 1990). We have emphasized water extraction from a drying soil, but equally important is the response of root systems to the rewetting of soil following drought. Prolonged hysteresis in the plant K(C) functions can account for impaired water uptake from wet soil following a severe drought. For example, in P. taeda, the extensive loss of hydraulic conductance in the root system during drought was apparently not restored following rain. As a result, water use was less late in the growing season than initially, even though the soil was equally wet (Hacke et al., 2000a). Other plants such as desert succulents are much more responsive to soil rewetting (North and Nobel, 1991), and more needs to be learned of mechanisms by which plants reverse cavitation, control root growth, and restore radial conductance in root tissues (LoGullo et al., 1998). Our predictions of how the plant may control failure to optimize water extraction for a given investment in transport are very simplistic, and will no doubt be modified as more is learned about the behavior of the continuum. While we predict that hydraulic failure under drought may occur in the fine roots, it could in some circumstances be occurring in the rhizosphere, or in other plant components, with positive consequences for the plant. It is important to continue efforts to integrate processes throughout the continuum (Nobel, 1994; Doussan et al., 1998a,b), refining them as more is learned about the hydraulic behavior of root and soil. Ultimately, a systems-level approach will be necessary to understand and predict more complex phenomena such as competitive interactions between between neighboring root systems, the significance of soil water distribution via hydraulic lift (Jackson et al., 2000), and how plant water use may respond to climate change.

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679 tive aquaporins in the roots of Lotus japonicus. Planta 210:50–60. Holbrook NM, Zwieniecki MA. 1999. Embolism repair and xylem tension: do we need a miracle? Plant Physiol 120:7–10. Ieperen WV, Meeteren UV, Gelder HV. 2000. Fluid ionic composition influences hydraulic conductance of xylem conduits. J Exp Bot 51:769–776. Jackson RB, Canadell J, Ehleringer JR, Mooney HA, Sala OE, Schulze ED. 1996. A global analysis of root distributions for terrestrial biomes. Oecologia 108:389– 411. Jackson RB, Sperry JS, Dawson TE. 2000. Root water uptake and transport: scaling physiological processes for global predictions. Trends Plant Sci (in press). Jarbeau JA, Ewers FW, Davis SD. 1995. The mechanism of water-stress-induced embolism in two species of chaparral shrubs. Plant Cell Environ 18:189–196. Jones HG, Sutherland R. 1991. Stomatal control of xylem embolism. Plant Cell Environ 14:607–612. Kaldenhoff R, Grote K, Zhu JJ, Zimmermann U. 1998. Significance of plasmalemma aquaporins for watertransport in Arabidopsis thaliana. Plant J 6:187–199. Kolb KJ, Sperry JS. 1999a. Differences in drought adaptation between subspecies of sagebrush (Artemisia tridentata). Ecology 80:2373–2384. Kolb KJ, Sperry JS. 1999b. Transport constraints on water use by the Great Basin shrub, Artemisia tridentata. Plant Cell Environ 22:925–935. Kramer PJ, Boyer JS. 1995. Water Relations of Plants and Soils. San Diego, CA: Academic Press. Linder S, Axelsson B. 1982. Changes in carbon uptake and allocation patterns as a result of irrigation and fertilization in a young Pinus sylvestris stand. In: Waring RH, ed. Carbon Uptake and Allocation: Key to Management of Subalpine Forest Ecosystems, Internal Union Forest Research Organization Workshop. Corvallis, OR: Oregon State University, pp 38–44. Linder S, Benson ML, Meyers BJ, Raison RJ. 1987. Canopy dynamics and growth of Pinus radiata L. I. Effects of irrigation and fertilization during a drought. Can J For Res 17:1157–1165. Linton MJ, Sperry JS, Williams DG. 1998. Limits to water transport in Juniperus osteosperma and Pinus edulis: implications for drought tolerance and regulation of transpiration. Func Ecol 12:906–911. LoGullo MA, Salleo S. 1991. Three different methods for measuring xylem cavitation and embolism: a comparison. Ann Bot 67:417–424. LoGullo MA, Nardini A, Salleo S, Tyree MT. 1998. Changes in root hydraulic conductance (KR) of Olea oleaster seedlings following drought stress and irrigation. New Phytol 140:25–31. Macfall JS, Johnson GA, Kramer PJ. 1990. Observation of a water-depletion region surrounding loblolly pine roots

680 by magnetic resonance imaging. Proc Natl Acad Sci USA 87:1203–1207. McGowan M, Hector D, Gregson K. 1987. Water relations of temperate crops. Proceedings of International Conference on Measurement of Soil and Plant Water Status. Logan, UT: University of Utah Press, pp 289– 297. Mencuccini M, Comstock J. 1997. Vulnerability to cavitation in populations of two desert species, Hymenoclea salsola and Ambrosia dumosa, from different climatic regions. J Exp Bot 48:1323–1334. Mencuccini M, Grace J. 1996. Hydraulic conductance, light interception and needle nutrient concentration in Scots pine stands and their relations with net primary productivity. Tree Physiol 16:459–468. Moreshet S, Huck MG, Hesketh JD, Peters DB. 1987. Measuring the hydraulic conductance of syobean root systems. Proceedings of International Conference on Measurement of Soil and Plant Water Status. Logan, UT: Utah State University Press, pp 221–228. Newman EI. 1969. Resistance to water flow in soil and plant. I. Soil resistance in relation to amounts of root: theoretical estimates. J Appl Ecol 6:1–12. Nobel PS. 1991. Physicochemical and Environmental Plant Physiology. San Diego, CA: Academic Press. Nobel PS. 1994. Root–soil responses to water pulses in dry environments. In: Caldwell MM, Pearcy RW, eds. Exploitation of Environmental Heterogeneity by Plants. San Diego, CA: Academic Press, pp 285–304. North GB, Nobel PS. 1991. Changes in hydraulic conductivity and anatomy caused by drying and rewetting roots of Agave deserti (Agavaceae). Am J Bot 78:906–915. North GB, Nobel PS. 1998. Water uptake and structural plasticity along roots of a desert succulent during prolonged drought. Plant Cell Environ 21:705–713. Oliver MJ. 1996. Desiccation tolerance in vegetative plant cells. Physiol Plant 97:779–787. Passioura JB. 1988. Water transport in and to roots. Annu Rev Plant Physiol Plant Mol Biol 39:245–265. Passioura JB, Ball MC, Knight JH. 1992. Mangroves may salinize the soil and in so doing limit their transpiration rate. Func Ecol 6:476–481. Pate JS, Jeschke WD, Aylward MJ. 1995. Hydraulic architecture and xylem structure of the dimorphic root systems of southwest Australian species of the Proteaceae. J Exp Bot 46:907–915 Peterson CA, Murrmann M, Steudle E. 1993. Location of the major barriers to water and ion movement in young roots of Zea mays L. Planta 190:127–136. Pickard WF. 1981. The ascent of sap in plants. Prog Biophys Mol Biol 37:181–229. Pockman WT, Sperry JS. 2000. Vulnerability to cavitation and the distribution of Sonoran desert vegetation. Am J Bot 87:1287 (in press).

Sperry et al. Pockman WT, Sperry JS, O’Leary JW. 1995. Sustained and significant negative water pressure in xylem. Nature 378:715–716. Raven JA. 1984. Physiological correlates of the morphology of early vascular plants. Bot J Linn Soc 88:105–126. Rendig VV, Taylor HM. 1989. Principles of Soil–Plant Interrelationships. New York; McGraw-Hill. Rieger M, Litvin P. 1999. Root system hydraulic conductivity in species with contrasting root anatomy. J Exp Bot 50:201–209. Saliendra NZ, Meinzer FC. 1989. Relationship between root/ soil hydraulic properties and stomatal behavior in sugarcane. Aus J Plant Physiol 16:241–250. Saliendra NZ, Sperry JS, Comstock JP. 1995. Influence of leaf water status on stomatal response to humidity, hydraulic conductance, and soil drought in Betula occidentalis. Planta 196:357–366. Salisbury FB, Ross CW. 1992. Plant Physiology. Belmont, CA: Wadsworth. Salleo S, LoGullo MA. 1989a. Different aspects of cavitation resistance in Ceratonia siliqua, a drought-avoiding Mediterranean tree. Ann Bot 64:325–336. Salleo S, LoGullo MA. 1989b. Xylem cavitation in nodes and internodes of Vitis vinifera L. plants subjected to water stress. Limits of restoration of water conduction in cavitated xylem conduits. In : Kreeb KH, Richter H, Hinckley T, eds. Structural and Functional Responses to Environmental Stresses. The Hague, Netherlands: Academic Publishing, pp 33–42. Salleo S, LoGullo MA, De Paoli D, Zippo M. 1996. Xylem recovery from cavitation-induced embolism in young plants of Laurus nobilis: a possible mechanism. New Phytol 132:47–56. Sperry JS, Ikeda T. 1997. Xylem cavitation in roots and stems of Douglas-fir and white fir. Tree Physiol 17:275–280. Sperry JS, Pockman WT. 1993. Limitation of transpiration by hydraulic conductance and xylem cavitation in Betula occidentalis. Plant Cell Environ 16:279–287. Sperry JS, Saliendra NZ. 1994. Intra- and inter-plant variation in xylem cavitation in Betula occidentalis. Plant Cell Environ 17:1233–1241. Sperry JS, Sullivan JEM. 1992. Xylem embolism in response to freeze–thaw cycles and water stress in ring-porous, diffuse-porous, and conifer species. Plant Physiol 100:605–613. Sperry JS, Tyree MT. 1990. Water-stress-induced xylem embolism in three species of conifers. Plant Cell Environ 13:427–436. Sperry JS, Adler FR, Campbell GS, Comstock JP. 1998. Limitation of plant water use by rhizosphere and xylem conductance: results from a model. Plant Cell Environ 21:347–359. Sperry JS, Alder NN, Eastlack SE. 1993. The effect of reduced hydraulic conductance on stomatal conductance and xylem cavitation. J Exp Bot 44:1075–1082.

Soil Water Uptake Sperry JS, Donnelly JR, Tyree MT. 1988a. A method for measuring hydraulic conductivity and embolism in xylem. Plant Cell Environ 11:35–40. Sperry JS, Donnelly JR, Tyree MT. 1988b. Seasonal occurrence of xylem embolism in sugar maple (Acer saccharum). Am J Bot 75:1212–1218. Sperry JS, Holbrook NM, Zimmermann MH, Tyree MT. 1987. Spring filling of xylem vessels in wild grapevine. Plant Physiol 83:414–417. Sperry JS, Nichols KL, Sullivan JEM, Eastlack SE. 1994. Xylem embolism in ring-porous, diffuse-porous, and coniferous trees of northern Utah and interior Alaska. Ecology 75:1736–1752. Sperry JS, Saliendra NZ, Pockman WT, Cochard H, Cruiziat P, Davis SD, Ewers FW, Tyree MT. 1996. New evidence for large negative xylem pressures and their measurement by the pressure chamber method. Plant Cell Environ 19:427–436. Stasovski E, Peterson CA. 1993. Effects of drought and subsequent rehydration on the structure, vitality and permeability of Allium cepa adventitious roots. Can J Bot 71:700–707. Steudle E. 1993. Pressure probe techniques: basic principles and application to studies of water and solute relations at the cell, tissue and organ level. In: Smith JAC, Griffiths H, eds. Water Deficits: Plant Responses from Cell to Community. Oxford, U.K.: Bios Scientific Publishers, pp 5–36. Steudle E. 1994. Water transport across roots. Plant Soil 167:79–90. Steudle E, Frensch J. 1996. Water transport in plants: the role of the apoplast. Plant Soil 187:67–79. Steudle E, Peterson CA. 1998. How does water get through roots? J Exp Bot 49:775–788. Stirzaker RJ, Passioura JB. 1996. The water relations of the root–soil interface. Plant Cell Environ 19:201–208. Subramanian KS, Charest C, Dwyer LM, Hamilton RI. 1995. Arbuscular mycorrhizas and water relations in maize under drought stress at tasselling. New Phytol 129:643–650. Tsuda M, Tyree MT. 1997. Whole-plant hydraulic resistance and vulnerability segmentation in Acer saccharinum. Tree Physiol 17:351–357. Tsuda M, Tyree MT. 2000. Plant hydraulic conductance measured by the high pressure flow meter in crop plants. J Exp Bot 51:823–828. Tyerman SD, Bohnert HJ, Maurel C, Steudle E, Smith JAC. 1999. Plant aquaporins: their molecular biology, biophysics and significance for plant water relations. J Exp Bot 50:1055–1071.

681 Tyree MT. 1999. Water relations of plants. In: Baird AJ, Wilby RL, eds. Ecohydrology. London; Routledge, pp 11–38. Tyree MT, Ewers FW. 1991. The hydraulic architecture of trees and other woody plants: Tansley review No. 34. New Phytol 119:345–360. Tyree MT, Karamanos AJ. 1980. Water stress as an ecological factor. In: Grace J, Ford ED, Jarvis PG, eds. Plants and Their Atmospheric Environment. Oxford, U.K.: Blackwell, pp 237–261. Tyree MT, Sperry JS. 1988. Do woody plants operate near the point of catastrophic xylem dysfunction caused by dynamic water stress? Answers from a model. Plant Physiol 88:574–580. Tyree MT, Sperry JS. 1989. Vulnerability of xylem to cavitation and embolism. Annu Rev Plant Physiol Plant Mol Biol 40:19–38. Tyree MT, Cruiziat P, Sinclair B, Ameglio T. 1993. Droughtinduced leaf shedding in walnut—evidence for vulnerability segmentation. Plant Cell Environ 16:879–882. Tyree MT, Davis SD, Cochard H. 1994. Biophysical perspectives of xylem evolution: is there a tradeoff of hydraulic efficiency for vulnerability to dysfunction? IAWA Bull 15:335–360. Tyree MT, Fiscus EL, Wullschleger SD, Dixon MA. 1986. Detection of xylem cavitation in corn (Zea mays) under field conditions. Plant Physiol 82:597–599. Tyree MT, Velez V, Dalling JW. 1998. Growth dynamics of root and shoot hydraulic conductance in seedlings of five neotropical tree species: scaling to show possible adaptation to differing light regimes. Oecologia 114:293–298. Vartanian N. 1996. The drought rhizogenesis. In: Waisel Y, Eshel A, Kafkafi U, eds. Plant Roots: The Hidden Half. 2nd ed. New York: Marcel Dekker, pp 471–482. Waisel Y, Agami M. 1996. Ecophysiology of roots of submerged aquatic plants. In: Waisel Y, Eshel A, Kafkafi U, eds. Plant Roots: The Hidden Half. 2nd ed. New York; Marcel Dekker, pp 895–909. Yang S, Tyree MT. 1992. A theoretical model of hydraulic conductivity recovery from embolism with comparison to experimental data on Acer saccharum. Plant Cell Environ 15:633–643. Yang S, Tyree MT. 1993. Hydraulic resistance in Acer saccharum shoots and its influence on leaf water potential and transpiration. Tree Physiol 12:231–242. Zimmermann MH. 1983. Xylem Structure and the Ascent of Sap. Berlin; Springer-Verlag. Zimmermann MH, Potter D. 1982. Vessel length distribution in branches, stem, and roots of Acer rubrum. IAWA Bull 3:103–109.

39 Ecological Aspects of Water Permeability of Roots Andrea Nardini and Sebastiano Salleo University of Trieste, Trieste, Italy

Melvin T. Tyree U.S. Department of Agriculture Forest Service, South Burlington, Vermont

I.

resistance (K ¼ 1=R). The nature of the driving force can be rather complex, and readers are referred to Tyree (2000) for a more detailed discussion, but it is often adequate to approximate the driving force by the hydrostatic pressure difference,
P, across the resistance or conductance. Hence, we can write

INTRODUCTION

According to the cohesion–tension (C-T) theory, water is transported in plants in a metastable state under negative pressure, i.e., under tension (Dixon and Joly, 1894). Negative pressure develops by surface tension effects at the air–water interface because of the evaporation of water from leaf cell walls. The negative pressure is transmitted through the xylem down to the root system and ultimately to the root surface where it drives water uptake from the soil. Water uptake can also be driven by osmotic gradients between root xylem sap and soil, which occur in some species under low transpiration conditions that favour solute accumulation in the stele (Kramer, 1983). Water moves from roots to leaves to replace water evaporated from the leaf surface. Although this current view has been challenged in recent years (Canny, 1995, 1998; Zimmermann et al., 1994), experimental and theoretical evidence suggests that the C-T theory represents the most convincing and parsimonious explanation of water transport in plants (Tyree, 1997; Comstock, 1999; Stiller and Sperry, 1999; Wei et al., 1999). Water moving along the soil-to-leaf pathway have to overcome frictional resistances, R, in the soil, root, and stem (Boyer, 1985). R can be defined by the ratio of the driving force to the flow (F in kg s1 ) across the resistance. Conductance (K) is defined as the inverse of

R ¼
P=F

ð1AÞ

K ¼ F=
P

ð1BÞ

or

Equations (1A) and (1B) are appropriate in situations of high flow rate in roots, but they become increasingly inaccurate as F declines to zero because osmotic effects become more important. A rigorous treatment of water and solute flow through roots requires the use of equations from irreversible thermodynamics to explain all effects (Tyree, 2000). In the remainder of this chapter we will consider root hydraulic conductance values measured under high flow rate conditions that occurs during most daylight hours in plants. If we treat the soil, root, and shoot as conductances in series, then for the soil–plant continuum we can write: Psoil  PL ¼ ð1=Ksoil þ 1=KR þ 1=KS ÞF

ð2AÞ

Where subscripts L, R, and S refer to leaf, root, and shoot, respectively. In nonsaline soil, the difference in water potential, , between soil and leaf is approxi683

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mately equal to
P ðPsoil  PL ffi soil  L Þ It is usually assumed that Ksoil >> KR and KS except in dry soils so 1=Ksoil can be ignored. Hence leaf water potential is approximated by: L ffi soil  ð1=KR þ 1=KS ÞF

ð2BÞ

Or if we wish to express equation (2B) in terms of leaf area and average evaporative flux density (E) we have: L ffi soil  ð1=KR þ 1=KS Þ AL E

ð3Þ

This equation can be rewritten so that root and shoot conductances are scaled to leaf surface areas, i.e., to give leaf specific shoot and root conductances, KS =AL ð¼ KSL Þ and KR =AL ð¼ KRL ), respectively: L ffi soil  ð1=KRL þ 1=KSL Þ E

ð4Þ

Meristem growth and gas exchange are maximal when water stress is small, i.e., when L is near zero. From equation (4) it can be seen that the advantage of high KRL and KSL is that L will be closer to soil . Equation (4) is sometimes referred to as the Ohm’s law analog of water flow in plants. On the basis of the Ohm’s law analog, the vertical water flow in plants per unit leaf area is governed by two factors: (1) the driving force for the flow, i.e., the water potential difference between leaves and soil; and (2) the hydraulic conductance of the entire flow path. In particular, water uptake from the soil depends crucially on the hydraulic conductance of the root system (KR ), sometimes referred to as ‘‘root permeability.’’ Root systems are considered to account for 40–60% of the total hydraulic resistance of a plant (e.g., Tsuda and Tyree, 1997; Nardini and Pitt, 1999; Nardini and Tyree, 1999) and may therefore represent a significant ‘‘hydraulic bottleneck’’ in the plant. Most of our current understanding of water transport in the root is derived from studies by Steudle and coworkers (see Chapter 38 by Sperry et al. in this volume and Tyree, 2000). Based mostly on root pressure probe experiments (see below), these authors developed the so-called root composite transport model (Steudle and Peterson, 1998). According to the model, water flows from soil to the stele through an apoplastic (low-resistance) or a cell-to-cell (high-resistance) pathway. The former would predominate under active transpiration, i.e., when the driving force is mainly hydrostatic in nature while the latter would be favored under low transpiration, i.e., when osmotic forces prevail (see reviews by Steudle and Heydt, 1997, and Steudle and Peterson, 1998). Tyree (2000) has recently questioned the validity of the composite transport model as interpreted by Steudle’s group, but the

argument is beyond the scope of this chapter. Although the composite transport model has been developed on the basis of measurements performed on single excised young roots, it helped us greatly to extend our understanding of the basic processes involved in water transport across the root. In spite of this, our present knowledge of the behavior of whole root systems growing under natural conditions is rather poor. As McCully (1995) properly pointed out, ‘‘root systems are not just multiples of seedling roots. Field-grown roots have features unknown from solution culture. . . .’’ It follows that a thorough understanding of the ecological significance of root permeability requires extensive studies of the hydraulic properties of root systems of field-grown plants (Williams and Eamus, 1997). The present chapter deals with: (1) measurements of root hydraulic conductance in soil-growing plants, and (2) how root permeability is related to the adaptation of plants to the environment, between-species competitiveness, and plant ability to cope with different abiotic stresses.

II.

MEASURING THE HYDRAULIC CONDUCTANCE OF WHOLE ROOT SYSTEMS

Extensive measurements of the hydraulic conductance of whole root systems of plants were first performed by Fiscus (1975, 1977) using the pressure chamber technique. Here, detached root systems of plants growing in hydroponic solutions were inserted into a pressure chamber with the cut stem protruding outside the chamber. Positive gas pressure was applied, and the resulting flow could be measured by collecting the sap exuding from the stump base (Fig. 1A). Although similar measurements can be performed with potted plants (i.e., soil-grown roots), it is not possible to control the external ionic environment to the extent done by Fiscus. Pressure bomb studies using potted plants have proved to be very useful for measuring hydraulic properties of whole root systems (cf. Colombo and Asselstine, 1989; Gallardo et al., 1996; Nardini et al., 1998b; Huxman et al., 1999). The main disadvantage of Fiscus’ technique is that only relatively small root systems of potted plants can be measured, although some giant pressure chambers have been constructed to measure root permeability of small trees growing in pots (e.g., Tyree et al., 1995). Another problem with the pressure chamber technique is that measurements are best performed when roots are in

Ecological Aspects of Water Permeability of Roots

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Figure 1 Schematic view (not to scale) of three most common instrumentations for measuring root hydraulic conductance. Note that the root pressure probe and the high-pressure flow meter drive a flow in the antiphysiological direction.

aqueous solutions or when soil is relatively wet. When the water content of the soil declines, the high hydraulic resistance of the soil becomes a significant limitation for root conductance, thus confounding the interpretation of the measured root hydraulic conductance (Kramer, 1983; Lo Gullo et al., 1998). The pressure probe technique (Fig. 1B) has permitted measurement of water and solute transport parameters of excised root systems (Steudle and Jeschke, 1983). Although, in principle, the root pressure probe can be connected either to an excised root segment growing in a nutrient solution or to a soilgrown root system (Steudle, 1993), the former experimental setup has been most commonly used in root water relations studies (cf. Steudle and Frensch, 1989; Melchior and Steudle, 1993; Gunse` et al., 1997). As in the pressure chamber technique, measurements of the hydraulic properties of detached root systems using the root pressure probe are limited to small seedlings either potted or growing in hydroponics (e.g., Ru¨dinger et al., 1994; Steudle and Mescheryakov, 1996). Moreover, this method requires the root system

to be connected to the instrument many hours before measurements (often a day or more) in order to allow the roots to develop stable internal pressures and to dissolve all their air bubbles. Although the root pressure probe has important advantages over other methods, because it can be used to measure crucial parameters involved in solute and water transport (Steudle, 1993; Tyree, 2000), it is less useful in ecological studies because (1) only small root systems can be measured; (2) roots growing in confined environments (e.g., pots) would behave quite differently from those growing freely in the soil (Ray and Sinclair, 1998); and (3) measurements are critically temperature dependent because of the small water and oil volumes involved. This makes this instrumentation not suitable for field recordings. Field measurements of detached root systems of plants (up to 50 mm in diameter at the base of the trunk) growing in the field (Fig. 1C) have been made possible since the introduction of the high-pressure flow meter (HPFM; Tyree et al., 1995). The instrument was developed to provide dynamic and steady-state

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measurements of roots’ and shoots’ hydraulic conductance. In this case, the root system is connected to the HPFM via a rubber seal. Positive pressures are applied and water is forced to flow from a captive air tank into the root system (i.e., opposite the physiological direction). The instrument is designed to measure accurately the pressure applied and the resulting flow so that the hydraulic conductance of the root system can be calculated (see below). The same technique can also be used to measure the hydraulic conductance of leafy shoots or of parts of them (e.g., leaf blades, petioles, etc.). In the case of root systems, the hydraulic conductance is typically measured in the transient mode, i.e., by increasing the applied pressure rapidly (3–12 kPa/s) and measuring the corresponding flow every 2 s. The hydraulic conductance is then calculated by the regression of the straight line relating flow to pressure. The transient mode is more suitable for measurements on root systems than the steady-state mode. In the steady-state mode pressure is held constant and the resulting flow is measured. In this case, solute accumulation may occur at the root tips, generating an osmotic flow opposite the flow induced by the HPFM (Tyree et al., 1994b). This might result in inaccurate measurements of the root hydraulic conductance. A possible inconvenience of this method is that the high pressures applied are likely to compress and dissolve air bubbles during measurements, leading to possible overestimation of the actual root hydraulic conductance. This is one of the limits of the HPFM technique that is likely to give unreliable estimates of root hydraulic conductance when the root xylem is embolized (Sperry and Ikeda, 1997; Nardini and Tyree, 1999). In conclusion, the HPFM technique is most useful for measuring maximum KR s most depending on root anatomy and, ultimately, on ontogenetic and developmental changes. A further major advantage of the HPFM technique is that it allows measurements of large root systems of plants growing in the field.

III.

SCALING ROOT HYDRAULIC CONDUCTANCE

In an ecological context, we often want to compare root conductance between plants, but KR varies with species as well as with root size and type (see also Chapter 9 by Waisel and Eshel in this volume). Large roots are more conductive than small roots, so KR values need to be normalized to some measure of root or plant size. A natural measure of plant size

evident from equation (4) is leaf area, so we might compare leaf-specific root conductances (KRL ). But other useful measures of plant size might be total root surface area (AR ), total root length (L), or root dry weight (DWR ). Scaling by AR is justified by an analysis of axial versus radial resistance to water flow in roots. In the radial pathway, water flows from the root surface to the xylem vessels through nonvascular tissue. In the axial pathway, water flow is predominantly through vessels. The resistance of water flow through the radial path is usually more than through the axial path (cf. Tyree, 2000). Most water uptake is presumed to occur in fine roots (< 2 mm diameter), and fine root surface area is usually >90% of the total root surface area (personal observation). Thus, root uptake of water would appear to be limited by root surface area and hence it is reasonable to divide KR by AR yielding a measure of root efficiency (KRR ¼ KR =AR ). Division of KR by total root length (L) is not as desirable, but is justified because AR and L are highly correlated and L can be estimated by a low-cost, line intersection technique rather than a high-cost, image analysis technique. Scaling by root mass is justified by consideration of the cost of resource allocation. Plants must invest much of their assimilates in roots to grow and to maintain them. The benefit derived from this carbon investment is enhanced scavenging for water and mineral nutrient resources. Total root dry weight (DWR ) represents a major component of carbon investment into roots. Thus the carbon efficiency of roots can be measured in terms of KR =DWR specific root conductance (kg s1 g1 ), AR =DWR specific root area (m2 g1 ), or L=DWR specific root length (m g1 ). Scaling by DWR provides information that is more of ecological than physiological importance. Ideally it is best to scale KR by all possible measures of root or plant size (AL , AR , L; DWR ) to be sure that all possible relationships are discovered. In practice, it is laborious to recover quantitatively and to measure root size (AR , L, DWR ) in potted plants and generally impossible to do so in field-grown plants. Hence often we must limit our scaling to AL . Some people might argue that AR is the only valid means of scaling KR , but such reasoning overlooks important relationships. For example, Tyree et al. (1998) compared KR in five species of tropical tree seedlings. Two were young forest trees (light-demanding), fast-growing pioneers, and three were old forest trees (shade-tolerant), slow-growing species. Scaling KR by AR revealed no patterns of ecological adaptation, whereas significant differences were found in KR

Ecological Aspects of Water Permeability of Roots

scaled to AL and DWR . Pioneer species had much higher KR =AL and invested less carbon in roots to achieve high KR than old forest species; i.e., pioneers had higher KR =DWR . Pioneer species with low KR =AR compensated by having more root surface area with very little carbon investment in the roots, i.e., having high values of L=DWR and AR =DWR :

IV.

ROOT HYDRAULIC CONDUCTANCE AND ADAPTATION TO THE ENVIRONMENT

Water availability is one of the major factors influencing the distribution of plants throughout the world. Water is available to the plant when (1) soil water potential is higher than that of the root, and (2) the resistance interposed between soil and leaves can be overcome at water potentials less negative than those causing substantial cavitation-induced drops in the hydraulic conductivity of xylem. Since water uptake is positively related to root hydraulic conductance, this parameter is likely to be an important factor determining the adaptation of plants to their environment and, therefore, influencing their distribution range.

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The principal adaptive advantage of high KRL is evident from equation (4). If we consider two plants with the same average evaporation rate from leaves, E, and growing at the same soil , then the plant with high KRL and/or KSL can transpire water with a more favorable water balance (less negative L ). Since the rates of growth and of net carbon gain are both negatively impacted by low (more negative) L , the plant with higher KRL may grow faster. Fast-growing pioneers often invest less carbon in roots and achieve high KRL (Tyree et al., 1998), so there would appear to be little ‘‘down side’’ to this strategy. But there are costs involved. Pioneers generally have short life spans and suffer more herbivory since less carbon and nitrogen are invested in plant defenses. Plants are continually adjusting KRL during the growth season and sometimes experience large rapid increases in KRL , as shown in a seasonal study of root and shoot conductance in Acer saccharum (Fig. 2A and B; Tyree, unpublished). KR =AL increased by >100% between July 5 and 15, 1995, and similar changes were observed on the same dates in 1994 (data not shown) in Acer saccharum. This change could not be accounted for by changes in leaf area or soil temperature (Fig. 2C and D). Increases in KRL can be at the cost of increased

Figure 2 Seasonal trends in hydraulic conductance of Acer saccharum shoots and roots measured on 4-year-old saplings growing in the field. All measurements were made with an HPFM in the field. All values are shown as means (n ¼ 6) and error bars are 1 SEM. (A) Shoot conductance scaled by leaf area measured by the steady-state (closed symbols) and transient (open symbols) methods. (B) Root conductance scaled by leaf area. (C) Mean leaf area of the six saplings harvested on date shown on X-axis. (D) Midday soil temperatures measured at 20 cm depth. Open circles highlight the values on the sample dates for HPFM measurements.

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water use because E is a function of leaf conductance to water vapor (gL ), and gL is sometimes strongly dependent on L (Yang and Tyree, 1993). The 120% change in KRL observed in Acer saccharum in July can cause L to increase from the typical value of 1:5 MPa to 0:9 MPa, which in turn will make gL and hence E increase by 80% (assuming all other factors are the same). Few studies have appeared in the literature attempting to relate KR to ecological indices representative of the different distribution ranges and/or habitat preferences of different species. Nardini et al. (1998b) tried to relate the continentality index (Ci) first introduced by Nimis and Bolognini (1990) to the root hydraulic conductances scaled by leaf surface area (KRL ) of four different forest species (Fig. 3). This index is based

Figure 3 Root hydraulic conductance normalized for the supplied leaf surface area (KRL ) versus the continentality index (dimensionless). Vertical bars are SD (standard deviation), unless eclipsed by the symbol. (From Nardini et al., 1998b.)

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on the phytoclimatic subdivision of Europe into geographical zones characterized by different continentality gradients (Jaeger, 1968; Ellenberg, 1988). A grid of operational geographic units (OGU; Crovello, 1981) reporting the presence of a given species within each unit can be superimposed on the geographic map, and the Ci for each species is calculated as: X X pi ð5Þ Ci ¼ pi qi =N where p is the presence of a species in a given OGU with continentality class qi. In this form, Ci ranges from 0 (maximum continentality preference of a species) to 1 (maximum oceanicity preference of a species). When KRL (as measured in May 1997 in well-irrigated potted plants) was plotted versus Ci, a negative relationship existed (r ¼ :88) between these two variables. In other words, species adapted to oceanic, humid climates, with good water availability (lower Ci) showed the highest KRL (e.g. Fagus sylvatica) and vice versa. The correlation of KRL to Ci became weaker when measured in August (r ¼ :77) and November (r ¼ :67Þ. We suggest that the correlation is higher in the spring because interspecific differences in root permeability are largest during this season, when most species are actively growing. An exception is represented by Quercus suber, which grows most actively in the early autumn (see below). At first sight, the lower KRL s measured in species adapted to more arid environments were somewhat surprising. We expected that these species, which grow where soil water is more limiting, would be favored by higher water absorption and transport efficiency; i.e., they would be expected to have high KRL s. Notwithstanding this assumption, several studies have confirmed that root permeability is intrinsically higher in species adapted to humid climates compared to that of species adapted to arid environments. This was shown to be the case for different Quercus spp. (Steudle and Meshcheryakov, 1996; Nardini et al., 1999; Nardini and Tyree, 1999) as well as for other trees (Engelbrecht et al., 2000; Nardini et al., 1998c), shrubs (Huxman et al., 1999), and crop species (Gallardo et al., 1996). When seven different Quercus spp. were compared for KRL and KSL (shoot and root hydraulic conductance scaled by leaf surface area), it appeared that species from drought-prone habitats (like Q. suber, Q. pubescens, or Q. petraea) showed lower hydraulic conductances than species adapted to somewhat more humid environments, like Q. alba, Q. cerris, Q. robur, or Q. rubra (Fig. 4).

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Figure 4 Hydraulic conductance of roots (KR ) and shoots (KS), both normalized for the supplied leaf surface area (KRL and KSL) measured in June using the high-pressure flow meter, in 3-year-old potted plants of different Quercus species—Q. suber (Q su), Q. pubescens (Q pu), Q. petraea (Q pe), Q. alba (Q al), Q. cerris (Q ce), Q. robur (Q ro), and Q. rubra (Q ru). Vertical bars are SD. (From Nardini and Tyree, 1999.)

This paradoxical root behavior can be explained in different ways. As an example, selective pressure for high KRL might be stronger in plant species growing in habitats with sufficient water and nutrient availability. Here, light is probably the major limiting factor for plant survival and growth. In turn, high growth potential is likely to be the best strategy for successful competition for light. Rapid growth is promoted by large plant hydraulic conductances (e.g. McIntyre, 1987; Ryan and Yoder, 1997; Castro-Diez et al., 1998). Therefore, high KRL s, as shown by species growing in humid habitats, might represent an adaptation to competition for light (Colombo and Asselstine, 1989). On the contrary, spatial expansion of plants growing in arid environments is mainly limited by low water availability. In this case, the ability to resist drought is likely to be more crucial to survival than an efficient water transport. Hence, plants growing in arid zones seem to develop less efficient water conducting systems (e.g., with narrower xylem conduits), but also less vulnerable to drought-induced cavitation and embolism (Zimmermann, 1983; Tyree et al., 1994a). In summary, high KRL s can be considered advantageous to plants for a more successful competition for light, provided high water availability is assured. Under these conditions, species with lower KRL s might survive in more arid environments, where the safety of water transport (i.e., lower vulnerability to cavitation) is more crucial for maintenance of a sufficient growth potential.

An alternative explanation for lower KRL s in plants growing in arid zones was proposed (Gallardo et al., 1996). According to these authors, low-KRL plants would absorb available water at a lower rate, thus depleting soil water, more slowly than high-KRL ones. Such a behavior might be of advantage to plants that have to face long drought periods. In other words, low KRL s in arid habitats might represent the expression of a ‘‘conservative’’ soil water use strategy (Richards and Passioura, 1989; Gallardo et al., 1996; Sperry et al., 1998). The weakness of this argument is that whole-plant water use is limited more by leaf conductance to water vapor (gL ) than by KRL . However, whole-plant conductance, which is partly limited by KRL , does seem to limit gL in many cases (Sperry et al., 1998). In recent years, increasing attention has been paid to seasonal changes of KRL as a possible factor in plant adaptation to the environment. Seasonal KRL changes have been measured in some temperate (Nardini et al., 1998b,c) as well as Mediterranean trees (Rambal, 1984; Pavel and Fereres, 1998; Nardini et al., 1999) and crop plants (Gallardo et al., 1996). Plants usually reach their maximum root hydraulic conductance during the active growth period. This coincides with the spring for most temperate and Mediterranean species (e.g., Acer campestre, Castanea sativa, Fagus sylvatica) but can be delayed for some Mediterranean evergreens, e.g., Quercus suber, to the early autumn, i.e., when temperatures decline and air is more humid.

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Significant seasonal differences in KRL have been recorded within and among species. In some cases, changes in root hydraulic conductance are related to the specific strategy adopted by the species to withstand periods of water shortage. This happens in drought-tolerating plants, which undergo large reductions of KRL during the summer drought (e.g., Olea oleaster, Nardini et al., 1998c; Quercus cerris, Nardini et al., 1999). In contrast, drought avoiders (water savers and water spenders sensu Levitt, 1980) appears to maintain constant KRL or even increase their KRL s under aridity conditions. These species include Fraxinus ornus, Ceratonia siliqua (Nardini et al., 1998c), Quercus pubescens (Nardini et al., 1998b; Nardini and Pitt, 1999), Quercus suber (Nardini et al., 1999), and Quercus ilex (Nardini et al., 2000a). Drought-avoiding water spenders (like C. siliqua) require high hydraulic efficiency of their water absorbing and conducting system (Lo Gullo and Salleo, 1988). In this view, the capability of maintaining a high root hydraulic conductance during adverse conditions might represent the basis of the typical drought avoidance strategy shown by this species (Lo Gullo et al., 1986). On the contrary, species suffering aridity-induced reductions of their KRL s are likely to be unable to compensate for water loss during drought periods. Hence, drought tolerance would become a necessity. This is the case of Olea oleaster (Lo Gullo and Salleo, 1988; Lo Gullo et al., 1998). On the basis of the above, the co-occurrence of different woody plants in the same habitat might be based on the complementarity of their drought resistance strategies. This was shown to be the case of Quercus suber and Quercus cerris plants co-occurring in Sicily (Fig. 5). These two species were found to adopt different drought resistance strategies—i.e., drought avoidance in the case of Q. suber, and drought tolerance in the case of Q. cerris (Nardini et al. 1999). The seasonal changes in root hydraulic conductance of the two species were different; i.e., the maximum annual KRL was recorded in the spring for Q. cerris and in late summer or early autumn for Q. suber. This indicates that no actual competition for water availability existed between the two oaks. Quercus suber continued to extract water from the soil even during the dry Mediterranean summer and maintained high leaf hydration (relative water content). On the contrary, Q. cerris reached maximum KRL in the spring but was unable to maintain high KRL through the summer and suffered dehydration. A similar behavior was described in the case of the drought-tolerant olive tree (Olea oleaster) co-occurring with the drought-

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Figure 5 Root hydraulic conductance normalized for the supplied leaf surface area (KRL ) measured using the highpressure flow meter in adult plants of Quercus suber and Q. cerris growing in the field in Sicily. Vertical bars are SD. (From Nardini et al., 1999.)

avoiding carob tree (C. siliqua) (Lo Gullo and Salleo, 1988). This suggests that the co-occurrence of woody plants in an area where water is a limiting factor can be explained on the basis of their different strategies of water use and, in particular, of different seasonal changes of KRL . This could be a promising starting point for further studies on the physiological adaptation of plants to environmental stresses. It would also probe into the ecophysiological meaning of the concept of ‘‘plant community’’ on which most of phytosociological studies are based.

V.

DROUGHT AND FREEZE STRESSINDUCED CHANGES OF ROOT HYDRAULIC CONDUCTANCE

Root systems have been reported to reduce their hydraulic conductance after exposure to different environmental stresses including drought (North and Nobel, 1992, 1998; Dubrovsky et al., 1998; Lo Gullo et al., 1998), freezing (Cui and Nobel, 1994; Nardini et al., 1998a, 2000a; Pavel and Fereres, 1998; Wan et al., 1999), salinity (Azaizeh and Steudle, 1991; Azaizeh et al., 1992; De Herralde et al., 1998), and flooding (Birner and Steudle, 1993). Among these stresses, frost and drought are common occurrences in temperate regions, characterized by marked seasonal fluctuations in temperature and water availability and have, therefore, noticeable ecological significance. In particu-

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lar, freeze and drought stress have been reported to produce similar effects in plant roots—i.e., a drop in root hydraulic conductance caused by structural and functional modifications of the root system. The most evident response to freeze and drought stress in the root is the increased suberization of cell layers outside the stele including the endodermis (North and Nobel, 1991, 1992; Lo Gullo et al., 1998; Nardini et al., 1998a). This increases the hydraulic resistance of the radial water pathway. However, the possibility that stress-induced changes in hydraulic conductance of roots are mediated by changes in membrane configuration, e.g., fluidity of the lipid bilayer and/or blockage of water channels, cannot be ruled out. The reduction of water flow through the root system if combined with high transpiration causes plants to undergo water stress (Pavel and Fereres, 1998; Terradas and Save`, 1992) and reduction of growth (Wan et al., 1999). The reduction of root hydraulic conductance, however, has been suggested to play an adaptive role in preventing water loss from the roots to the soil when the water potential in the soil is extremely low (North and Nobel, 1992). Global vegetation patterns are correlated with temperature and precipitation (Walter, 1979), and stress tolerance of plants often determines the boundaries of species distribution ranges (Woodward, 1988). We now know that among other factors, species distribution is limited by plant vulnerability to freezing- and/or drought-induced xylem cavitation (e.g., Cochard et al., 1992; Higgs and Wood, 1995; Brodribb and Hill, 1999; Pockman and Sperry, 1997). Much less is known about the importance of stress-induced reduction of root permeability for the plant’s adaptation and for its distribution range. Circumstantial evidence indicates that the distribution range of Quercus ilex might be limited by the vulnerability of its water-conducting system and especially of root system to freezing stress. Potted seedlings of this species exposed to naturally occurring below-zero air temperatures (as low as 88C) in northeastern Italy (Venezia Giulia), showed up to 90% reductions of KRL (Fig. 6) as a consequence of increased suberization of the endodermis (Nardini et al., 1998a). Large KR changes were also measured in Q. ilex plants growing in the field in two extreme areas of the Mediterranean Basin (Nardini et al., 2000a). A number of different parameters, including KRL , were measured on young plants growing in two natural sites in Italy, i.e., Sicily (a typical habitat of the species) and Venezia Giulia. The latter is considered to represent the northernmost distribution area of Q. ilex forests (Pignatti, 1982; Poldini, 1989). Plants growing in

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Sicily adopt an efficient water-saving strategy to withstand summer water shortage, and their KRL remained higher in fall and winter months (Fig. 7a), without any apparent xylem cavitation in the stem (Fig. 7b) as measured using the hydraulic method (cf. Cochard et al., 1992). On the contrary, plants growing in Venezia Giulia, which were winter freeze stressed, underwent large reductions in their stem and root hydraulic conductances from November to March, thus suffering a higher leaf dehydration. Vulnerability to drought stress can be expected to play a significant role in limiting plant distribution. Extensive studies in this regard were conducted with desert succulents (e.g., North and Nobel, 1991, 1998; Dubrovski et al., 1998; Chapter 53 by Nobel in this volume), which undergo large reductions of KRR (i.e., root hydraulic conductance scaled by root surface area) during drought periods but are capable of prompt recovery after rewetting. The KRR drop measured in these species was mainly due to oversuberization of cells, cell ruptures creating air lacunae in the cortex, root shrinkage leading to gaps at the root–soil interface, and xylem embolism. In desert habitats the

Figure 6 Hydraulic conductance of roots (KR ) and shoots (KS ) both normalized for the supplied leaf surface area (KRL and KSL ), measured using the high-pressure flow meter in 2year-old seedlings of Quercus ilex exposed to freezing stress and unstressed controls. Vertical bars are SD. (Modified from Nardini et al., 1998a.)

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Figure 7 (a) Root hydraulic conductance normalized for the supplied leaf surface area, of 10-year-old trees of Quercus ilex growing in the field in Sicily (SI) and in Venezia Giulia (VG). (b) Annual time course of the percentage loss of root hydraulic conductance (PLC) in SI and VG plants. Vertical bars are SD. (From Nardini et al., 2000a, with kind permission from Kluwer Academic Publishers).

soil can often fall to lower water potential than the roots, so the drop in KRR is thought to retard the loss of water from the succulents to the soil. Rapid recovery after rewetting is possible, in most cases, by the emergence of new lateral roots and renewed apical elongation. In some cases, however, the recovery of root permeability was not due to new root growth but to the development of roots, with higher than average hydraulic conductivity, in soil regions where moisture was still available during the dry period. Large drops of KRL after exposure to drought have been recorded in seedlings of Olea oleaster (Lo Gullo et al., 1998); again, the KRL drop was apparently due to the increased number of suberized cell layers of exodermis and endodermis. Recovery was made possible

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by the resumption of root growth and emergence of new lateral roots. It is reasonable to assume that the balance between drought-induced reduction of KRL and capability of recovering after rewetting might represent a crucial factor affecting the fitness of plants in their environment. Arid-zone plants might take advantage of large reductions of KRL during drought, thus preventing water losses from the plant to the soil. However, their exhibited recovery of high KRL as soon as water is available becomes more advantageous. Such behavior might confer a significant advantage to plants growing in arid environments as far as their competition with other species is concerned. In this regard, more studies are needed to clarify such an intriguing aspect of plant and plant community water relations. The impact of xylem cavitation and embolism on root water relations is still a matter of debate (see also Chapter 38 by Sperry et al. in this volume). A number of studies have shown that roots are more vulnerable to cavitation than other plant organs (Hacke and Sauter, 1996; Sperry and Saliendra, 1994; Mencuccini and Comstock, 1997; Sperry and Ikeda, 1997), but in some cases roots are less vulnerable than shoots (Tsuda and Tyree, 1997). Some authors have hypothesized that cavitation in roots may play a role in limiting gas exchange (Sperry and Ikeda, 1997). However, because roots are located at the beginning of the sequence of plant hydraulic resistances, these organs are likely to experience the highest water potentials in a plant (Mencuccini and Comstock, 1997). Root xylem embolism is expected to occur when soil water potentials approach the threshold for root xylem cavitation. However, embolism must be very severe to cause a significant reduction in KR because the hydraulic resistance of the radial water pathway from soil to root is much higher than the axial hydraulic resistance (Frensch and Steudle, 1989). Hence, a 50% reduction in conductance of a woody root segment could result in < 5% reduction of KR . Other effects may be more important; so, during soil drying, suberization of root tissues or formation of air gaps between roots and soil would limit the hydraulic conductivity of the radial water pathway and affect KR much more than xylem embolism. The impact of xylem embolism may be of very short duration because some roots seem to refill embolized vessels very quickly (Zwieniecki and Holbrook, 1998; McCully, 1999). A further consideration is that the higher vulnerability to cavitation of roots with respect to stem might be due to the fact that plants have not under-

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gone during their evolution a strong selective pressure addressed to develop roots with xylem vessels more resistant to cavitation. Kavanagh et al. (1999) reported that different populations of Douglas fir (Pseudotsuga menziesii) showed rather different root xylem vulnerabilities, indicating that the different ecotypes of a species can adjust this physiological feature to adapt to the type of stress prevailing in their habitats. This is likely to be the expression of the control of xylem conduit dimensions by plants growing in different environmental conditions. It has been suggested that roots are capable of efficient xylem refilling even during active transpiration, i.e., when xylem pressures are still substantially negative (Buchard et al., 1999; McCully, 1999). This is in accordance with previous studies conducted with stems and petioles of different species (Salleo et al., 1996; Canny, 1997; Tyree et al., 1999; Holbrook and Zwieniecki, 1999; Zwieniecki et al., 2000), showing that xylem refilling in stems is not an uncommon occurrence in plants even when xylem is under tension.

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SOME RECENT FINDINGS AND DIRECTIONS FOR FUTURE RESEARCH

have positive effects on root permeability (Safir et al., 1971), but some studies have reported negative effects of ectomycorrhizal symbiosis on root water uptake (e.g., Coleman et al., 1990). The HPFM was used to study how the ectomycorrhizal fungus Tuber melanosporum Vitt. influences the hydraulic conductance of Quercus ilex seedlings roots (Nardini et al., 2000b). Root hydraulic conductance was measured on infected (I) and noninfected (NI) seedlings and scaled by leaf area. KRL was significantly higher in I than in NI seedlings (Fig. 8). Interestingly, when KR was scaled by root surface area (KRR ), it appeared that the unit surface area of I seedlings was 2.5 times less efficient in water uptake than that of NI seedlings. I seedlings, however, compensated for low KRR by investing more carbon in root biomass (more roots), thus increasing the overall KRL and allowing higher stomatal conductance and CO2 fixation. Results from this preliminary study suggest that mycorrhizal plants would suffer from lower root hydraulic conductances caused by the fungal sheaths, which might influence the drought resistance of mycorrhizal plants. On the other hand, mycorrhizal infections improve plant nutrient balance and enhance the carbon gain of plants, which otherwise would be invested in the production

Our current knowledge of root water relations of plants growing in the field is still limited, and more studies are needed to confirm the validity of some of the ideas outlined in the present chapter. Studies of root systems growing in the field have to include the possibility that symbiotic associations (e.g., mycorrhizae) can significantly modify the hydraulic behavior of roots, with respect to that described under laboratory conditions. Most plant species have mycorrhizal roots when growing in natural conditions, and this symbiosis improves mineral nutrition of plants (e.g., Medeiros et al., 1994; Sharma et al., 1996) and hence their growth capacity (Lu et al., 1998; Gaur and Adholeya, 1999). The possible effect of mycorrhizae on root permeability is still a matter of speculation. Most experiments have been conducted on VAM (vescicular-arbuscular mycorrhizae). The classical view that VAM would improve host water relations and root permeability (Safir et al., 1971; Sands et al., 1982; Huang et al., 1985) has been more recently questioned (Andersen et al., 1988; Steudle and Heydt, 1997). Even more uncertainty exists about the role of ectomycorrhizae in the water relations of host plants. Ectomycorrhizae have been classically interpreted to

Figure 8 Hydraulic conductance of roots (KR ) and shoots (KS ) normalized for the supplied leaf surface area (KRL and KSL ) or for the surface area of fine roots (KRR ), i.e., of roots < 2 mm in diameter. Measurements of seedlings of Quercus ilex infected (I) or not infected (NI) with Tuber melanosporum. Vertical bars are SD. (From Nardini et al., 2000b.)

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of more roots. The majority of studies on mycorrhizal plants have been conducted with young seedlings, some weeks (or months) after infection. In our opinion, more studies are needed using adult mycorrhizal plants in the field, on a seasonal time scale in order to evaluate the impact of mycorrhizal symbiosis on plant water relations both under good water availability and under drought stress. Recent evidence suggests the possible role of water channels (aquaporins), found in root cells, in water relations of plants. The existence of water-conducting protein components in living cell membranes has been demonstrated both in animals and in plants (see Maurel, 1997). Plant aquaporins might play a significant role in improving water permeability of the cellto-cell radial pathway in roots. The endodermis, which is considered to represent a significant barrier to the apoplastic water flow was shown to be a site of high expression of aquaporins (Schaffner; cited in Maurel, 1997). Experiments with mercurials, which are thought to block aquaporins selectively, have shown that root permeability of many species is significantly reduced upon blockage of water channels (e.g., Wan and Zwiazek, 1999; Quintero et al., 1999). It was also suggested that the well-known reduction of root permeability induced by salinity or nutrient stress might be mediated by blockage or degradation of aquaporins (Carvajal et al., 1999; Clarkson et al., 2000). Of particular interest is that water flow mediated by aquaporins can be regulated by the plant through aquaporin phosphorylation (Eckert et al., 1999) or changes in the aquaporin expression (Henzler et al., 1999). We think that this research area deserves greatest attention and that the regulation of aquaporin expression might be one of the mechanisms involved in some observed phenomena. Aquaporins may be involved in: (1) the stressinduced reduction of KR , (2) the subsequent KR recovery, and (3) the diurnal changes of KR . It would be of interest also to assess whether differences between species in terms of root hydraulic efficiency are related to different kinetics and/or levels of the aquaporin expression. In a recent study, Zwieniecki et al. (2000) successfully blocked the refilling of embolized xylem conduits by treating petioles of different species with HgCl2 , thus suggesting a role for aquaporins in refilling of cavitated xylem conduits. This hypothesis was first advanced by Holbrook and Zwieniecki (1999) and considered as plausible by Tyree et al. (1999). Interestingly, xylem parenchyma cells (which were suggested to be involved in xylem refilling by Salleo et al. [1996] and by Tyree et al. [1999]) are also a site

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with a high aquaporin expression (Kammerloher et al., 1994; Yamada et al., 1995). The possibility that aquaporins play a role in xylem refilling has noticeable implications in the interpretation of the adaptation of plants. However, because of predictable technical difficulties, field studies of the role of aquaporins both in regulating root permeability and in xylem refilling will represent a major challenge to plant ecophysiologists. In conclusion, root permeability and its changes play a major role in the adaptive behavior of plants. In particular, root permeability appears to be related to plant adaptation to the environment, resistance to environmental stresses, competition between species, and relationships between plants and different soil-living organisms like mycorrhizal fungi. Several new ideas on root permeability as mediated by aquaporins have been developed in recent years. The elucidation of the ecological relevance of these new findings awaits future investigators.

ABBREVIATIONS AND SYMBOLS Total leaf surface area (m2) Total root surface area (m2) Continentality index (dimensionless) Root dry weight (kg) Transpiration rate (kg s1 m2 ) flow (kg s1 ) Leaf conductance to water vapor (mmol m2 s1 ) HPFM High-pressure flow meter K Hydraulic conductance (kg s1 MPa1 ) KR Root hydraulic conductance (kg s1 MPa1 ) Root hydraulic conductance scaled by leaf KRL surface area (kg s1 m2 MPa1 ) Root hydraulic conductance scaled by root KRR surface area (kg s1 m2 MPa1 ) Shoot hydraulic conductance KS Shoot hydraulic conductance scaled by leaf KSL surface area (kg s1 m2 MPa1 ) Ksoil Soil hydraulic conductance (kg s1 MPa1 ) L Total root length (m) P Hydrostatic pressure (MPa) PL Leaf hydrostatic pressure (MPa) Soil hydrostatic pressure (MPa) Psoil Leaf water potential (MPa) L Soil water potential (MPa) soil R Hydraulic resistance (MPa s kg1 ) AL AR Ci DWR E F gL

Ecological Aspects of Water Permeability of Roots

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695 Cui M, Nobel PS. 1994. Water budgets and root hydraulic conductivity of opuntias shifted to low temperatures. Int J Plant Sci 155:167–172. De Herralde F, Biel C, Save´ R, Morales MA, Torrecillas A, Alarco`n JJ, Sa`nchez-Blanco MJ. 1998. Effect of water and salt stresses on the growth, gas exchange and water relations in Argyranthemum coronopifolium plants. Plant Sci 139:9–17. Dixon HH, Joly J. 1894. On the ascent of sap. Phil Trans R Soc Lond Series B 186:563–576. Dubrovsky JG, North GB, Nobel PS. 1998. Root growth, developmental changes in the apex, and hydraulic conductivity for Opuntia ficus-indica during drought. New Phytol 138:75–82. Eckert M, Biela A, Siefritz F, Kaldenhoff R. 1999. New aspects of plant aquaporin regulation and specificity. J Exp Bot 50:1541–1545. Ellenberg H. 1988. Vegetation Ecology of Central Europe. Cambridge, U.K.: Cambridge University Press. Engelbrecht BMJ, Velez V, Tyree MT. 2000. Hydraulic conductance of two co-occurring neotropical understory shrubs with different habitat preferences. Ann Forest Sci 57:201–208. Fiscus EL. 1975. The interaction between osmotic- and pressure-induced water flow in plant roots. Plant Physiol 55:917–922. Fiscus EL. 1977. Determination of hydraulic and osmotic properties of soybean root systems. Plant Physiol 59:1013–1020. Frensch J, Steudle E. 1989. Axial and radial hydraulic resistance to roots of maize (Zea mays L.). Plant Physiol 91:719–726. Gallardo M, Eastham J, Gregory PJ, Turner NC. 1996. A comparison of plant hydraulic conductances in wheat and lupins. J Exp Bot 47:233–239. Gaur A, Adholeya A. 1999. Mycorrhizal effects on the acclimatization, survival, growth and chlorophyll of micropropagated Syngonium and Draceana inoculated at weaning and hardening stages. Mycorrhiza 9:215–219. Gunse` B, Poschenrieder C, Barcelo J. 1997. Water transport properties of roots and root cortical cells in protonand Al-stressed maize varieties. Plant Physiol 113:595– 602. Hacke U, Sauter JJ. 1996. Drought-induced xylem dysfunction in petioles, branches, and roots of Populus balsamifera L. and Alnus glutinosa (L.) Gaertn Plant Physiol 111:413–417. Henzler T, Waterhouse RN, Smyth AJ, Carvajal M, Cooke DT, Schaffner AR, Steudle E, Clarkson DT. 1999. Diurnal variations in hydraulic conductivity and root pressure can be correlated with the expression of putative aquaporins in the roots of Lotus japonicus. Planta 210:50–60. Higgs KH, Wood V. 1995. Drought susceptibility and xylem dysfunction in seedlings of 4 European oak species. Ann Sci Forest 52:507–513.

696 Holbrook NM, Zwieniecki MA. 1999. Embolism repair and xylem tension. Do we need a miracle? Plant Physiol 120:7–10. Huang RS, Smith WK, Yost RS. 1985. Influence of vescicular-arbuscular mycorrhiza on growth, water relations, and leaf orientation in Leucaena leucocephala (Lam) De Wit. New Phytol 99:229–243. Huxman KA, Smith SD, Neuman DS. 1999. Root hydraulic conductivity of Larrea tridentata and Heliantus annuus under elevated CO2. Plant Cell Environ 22:325–330. Jaeger E. 1968. Die Pflanzengeographische Ozeanitaetsgliedurung des olarktis und die Ozeanizit-aetsbindung der Pflanzenareale. Feddes Rep 79:157–335. Kammerloher W, Fischer U, Piechottka GP, Scha¨ffner AR. 1994. Water channels in the plant plasma membrane cloned by immunoselection from a mammalian expression system. Plant J 6:187–199. Kavanagh KL, Bond BJ, Aitken SN, Gartner BL, Knowe S. 1999. Shoot and root vulnerability to xylem cavitation in four populations of Douglas-fir seedlings. Tree Physiol 19:31–37. Kramer PJ. 1983. Water Relations of Plants. Orlando, FL: Academic Press, pp 220–225. Levitt J. 1980. Responses of Plants to Environmental Stresses. New York; Academic Press. Lo Gullo MA, Salleo S. 1988. Different strategies of drought resistance in three Mediterranean sclerophyllous trees growing in the same environmental conditions. New Phytol 108:267–276. Lo Gullo MA, Salleo S, Rosso R. 1986. Drought avoidance strategy in Ceratonia siliqua L., a mesomorphic-leaved tree in the xeric Mediterranean area. Ann Bot 58:745– 756. Lo Gullo MA, Nardini A, Salleo S, Tyree MT. 1998. Changes in root hydraulic conductance (KR ) of Olea oleaster seedlings following drought stress and irrigation. New Phytol 140:25–31. Lu X, Malajczuk N, Dell B. 1998. Mycorrhiza formation and growth of Eucalyptus globulus seedlings inoculated with spores of various ectomycorrhizal fungi. Mycorrhiza 8:81–86. Maurel C. 1997. Aquaporins and water permeability of plant membranes. Annu Rev Plant Physiol Plant Mol Biol 48:399–429. McCully M. 1995. How do real roots work? Plant Physiol 109:1–6. McCully M. 1999. Root xylem embolism and refilling. Relation to water potentials of soil, roots, and leaves, and osmotic potentials of root xylem sap. Plant Physiol 119:1001–1008. McIntyre GI. 1987. The role of water in the regulation of plant development. Can J Bot 65:1287–1298. Medeiros CAB, Clark RB, Ellis JR. 1994. Growth and nutrient uptake of sorghum cultivated with vesciculararbuscular mycorrhiza isolates at varying pH. Mycorrhiza 4:185–191.

Nardini et al. Melchior W, Steudle E. 1993. Water transport in Onion (Allium cepa L.) roots. Plant Physiol 101:1305– 1315. Mencuccini M, Comstock J. 1997. Vulnerability to cavitation in populations of two desert species, Hymenoclea salsola and Ambrosia dumosa, from different climatic regions. J Exp Bot 48:1323–1334. Nardini A, Pitt F. 1999. Drought resistance of Quercus pubescens as a function of root hydraulic conductance, xylem embolism and hydraulic architecture. New Phytol 143:485–493. Nardini A, Tyree MT. 1999. Root and shoot hydraulic conductance of seven Quercus species. Ann Forest Sci 56:371–377. Nardini A, Ghirardelli L, Salleo S. 1998a. Vulnerability to freeze stress of seedlings of Quercus ilex L.: an ecological interpretation. Ann Sci Forest 55:553–565. Nardini A, Lo Gullo MA, Salleo S. 1998b. Seasonal changes of root hydraulic conductance (KRL ) in four forest trees: an ecological interpretation. Plant Ecol 139:81– 90. Nardini A, Salleo S, Lo Gullo MA. 1998c. Root hydraulic conductance of six forest trees: possible adaptive significance of seasonal changes. Plant Biosystems 132:97–104. Nardini A, Lo Gullo MA, Salleo S. 1999. Competitive strategies for water availability in two Mediterranean Quercus species. Plant Cell Environ 22:109–116. Nardini A, Salleo S, Lo Gullo MA, Pitt F. 2000a. Different responses to drought and freeze stress of Quercus ilex L. growing along a latitudinal gradient. Plant Ecol 148:141–149. Nardini A, Salleo S, Tyree MT, Vertovec M. 2000b. Influence of the ectomycorrhizas formed by Tuber melanosporum Vitt. on hydraulic conductance and water relations of Quercus ilex L. seedlings. Ann Forest Sci 57:305–312. Nimis PL, Bolognini G. 1990. The use of chorograms in quantitative phytogeography and in phytosociological syntaxonomy. Fitosociologia 25:69–87. North GB, Nobel PS. 1991. Changes in root hydraulic conductivity and anatomy caused by drying and rewetting roots of Agave deserti (Agavaceae). Am J Bot 78:906– 915. North GB, Nobel PS. 1992. Drought-induced changes in hydraulic conductivity and structure in roots of Ferocactus acanthodes and Opuntis ficus-indica. New Phytol 120:9–19. North GB, Nobel PS. 1998. Water uptake and structural plasticity along roots of a desert succulent during prolonged drought. Plant Cell Environ 21:705–713. Pavel EW, Fereres E. 1998. Low soil temperatures induce water deficits in olive (Olea europea) trees. Physiol Plant 104:525–532. Pignatti S. 1982. Flora d’Italia. Bologna, Italy: Edagricole.

Ecological Aspects of Water Permeability of Roots Pockman WT, Sperry JS. 1997. Freezing-induced xylem cavitation and the northern limit of Larrea tridentata. Oecologia 109:19–27. Poldini L. 1989. La vegetazione del Carso isontino e triestino. Trieste, Italy: Lint. Quintero JM, Fournier JM, Benlloch M. 1999. Water transport in sunflower root systems: effects of ABA, Ca2+ status and HgCl2. J Exp Bot 50:1607–1612. Rambal S. 1984. Water balance and pattern of root water uptake by a Quercus coccifera L. evergreen scrub. Oecologia 62:18–25. Ray JD, Sinclair TR. 1998. The effect of pot size on growth and transpiration of maize and soybean during water deficit stress. J Exp Bot 49:1381–1386. Richards RA, Passioura JB. 1989. A breeding programme to reduce the diameter of the major xylem vessel in the seminal roots of wheat and its effect on grain yield in rainfed environments. Aust J Agric Res 40:943–950. Ru¨dinger M, Hallgren SW, Steudle E, Schulze ED. 1994. Hydraulic and osmotic properties of spruce roots. J Exp Bot 45:1413–1425. Ryan MG, Yoder BJ. 1997. Hydraulic limits to tree height and tree growth. BioScience 47:235–242. Safir GR, Boyer JS, Gerdemann JW. 1971. Mycorrhizal enhancement of water transport in soybean. Science 172:581–583. Salleo S, Lo Gullo MA, De Paoli D, Zippo M. 1996. Xylem recovery from cavitation-induced embolism in young plants of Laurus nobilis: a possible mechanism. New Phytol 132:47–56. Sands R, Fiscus EL, Reid CPP. 1982. Hydraulic properties of pine and bean roots with varying degrees of suberization, vascular differentiation and mycorrhizal infection. Aust J Plant Physiol 9:959–969. Sharma MP, Gaur A, Bhatia NP, Adholeya A. 1996. Growth responses and dependence of Acacia nilotica var. cupriciformis on the indigenous arbuscular mycorrhizal consortium of a marginal wasteland soil. Mycorrhiza 6:441–446. Sperry JS, Saliendra NZ. 1994. Intra- and inter-plant variation in xylem cavitation in Betula occidentalis. Plant Cell Environ 17:1233–1241. Sperry JS, Ikeda T. 1997. Xylem cavitation in roots and stems of Douglas-fir and white fir. Tree Physiol 17:275–280. Sperry JS, Adler FR, Campbell GS, Comstock JP. 1998. Limitation of plant water use by rhizosphere and xylem conductance: results from a model. Plant Cell Environ 21:347–359. Steudle E. 1993. Pressure probe techniques: basic principles and application to studies of water and solute relations at the cell, tissue and organ levels. In: Smith JAC, Griffiths H, eds. Water Deficits: Plant Responses from Cell to Community. Oxford, U.K.: Bios Scientific Publishers, pp 5–36.

697 Steudle E, Jeschke WD. 1983. Water transport in barley roots. Planta 158:237–248. Steudle E, Frensch J. 1989. Osmotic responses of maize roots. Water and solute relations. Planta 177:281– 295. Steudle E, Meshcheryakov AB. 1996. Hydraulic and osmotic properties of oak roots. J Exp Bot 47:387–401. Steudle E, Heydt H. 1997. Water transport across tree roots. In: Rennenberg H, Eschrich W, Ziegler H, eds. Contributions to Modern Tree Physiology. Leiden, Netherlands: Backuys Publishers, pp 239–255. Steudle E, Peterson CA. 1998. How does water get through roots? J Exp Bot 49:775–788. Stiller V, Sperry JS. 1999. Canny’s compensating pressure theory fails a test. Am J Bot 86:1082–1086. Terradas J, Save` R. 1992. The influence of summer and winter stress and water relationship on the distribution of Quercus ilex L. Vegetatio 99–100:137–145. Tsuda M, Tyree MT. 1997. Whole-plant hydraulic resistance and vulnerability segmentation in Acer saccharinum. Tree Physiol 17:351–357. Tyree MT. 1997. The cohesion–tension theory of sap ascent: current controversies. J Exp Bot 48:1753–1765. Tyree MT. 2000. Hydraulic properties of roots. In: Bloom CWPM, Visser EJW, eds. Root Ecology. Berlin; Springer-Verlag. Tyree MT, Davis SD, Cochard H. 1994a. Biophysical perspectives of xylem evolution: is there a tradeoff of hydraulic efficiency for vulnerability to dysfunction? IAWA J 15:335–360. Tyree MT, Velez V, Dalling J. 1998. Growth dynamics of root and shoot hydraulic conductance in seedlings of five neotropical tree species: scaling to show possible adaptation to differing light regimes. Oecologia 114:293–298. Tyree MT, Yang S, Cruiziat P, Sinclair B. 1994b. Novel methods of measuring hydraulic conductivity of tree root systems and interpretation using AMAIZED. Plant Physiol 104:189–199. Tyree MT, Patin˜o S, Bennink J, Alexander J. 1995. Dynamic measurements of root hydraulic conductance using a high-pressure flowmeter in the laboratory and field. J Exp Bot 46:83–94. Tyree MT, Salleo S, Nardini A, Lo Gullo MA, Mosca R. 1999. Refilling of embolized vessels in young stems of laurel. Do we need a new paradigm? Plant Physiol 120:11–21. Yang S, Tyree MT. 1993. Hydraulic resistance in Acer saccharum shoots and its influence on leaf water potential and transpiration. Tree Physiol 12:231–242. Walter H. 1979. Vegetation of the Earth and Ecological Systems of the Geo-Biosphere. New York; SpringerVerlag. Wan X, Zwiazek JJ. 1999. Mercuric chloride effects on root water transport in aspen seedlings. Plant Physiol 121:939–946.

698 Wan X, Landha¨usser SM, Zwiazek JJ, Lieffers VJ. 1999. Root water flow and growth of aspen (Populus tremuloides) at low root temperatures. Tree Physiol 19:879– 884. Wei C, Tyree MT, Steudle E. 1999. Direct measurements of xylem pressure in leaves of intact maize plants. A test of the cohesion-tension theory taking hydraulic architecture into account. Plant Physiol 121:1191–1205. Williams J, Eamus D. 1997. Plant ecophysiology: linking pattern and process—a review. Aust J Bot 45:351–357. Woodward FI. 1988. Temperature and the distribution of plants species. In: Long SP, Woodward FI, eds. Plants and Temperature. Cambridge, U.K.: Company of Biologists, pp 59–75. Yamada S, Katsuhara M, Kelly WB, Michalowski CB, Bohnert HJ. 1995. A family of transcripts encoding water channel proteins: tissue-specific expression in the common ice plant. Plant Cell 7:1129–1142.

Nardini et al. Zimmermann MH. 1983. Xylem Structure and the Ascent of Sap. Berlin; Springer-Verlag, pp 66–80. Zimmermann U, Meinzer FC, Benkert R, Zhu JJ, Schneider H, Goldstein G, Kuchenbrod E, Haase A. 1994. Xylem water transport: is the available evidence consistent with the cohesion theory? Plant Cell Environ 17:1169–1181. Zwieniecki MA, Holbrook NM. 1998. Diurnal variation in xylem hydraulic conductivity in white ash (Fraxinus americana L.), red maple (Acer rubrum L.) and red spruce (Picea rubens Sarg.). Plant Cell Environ 21:1173–1180. Zwieniecki MA, Hutyra L, Thompson MV, Holbrook NM. 2000. Dynamic changes in petiole specific conductivity in red maple (Acer rubrum L.), tulip tree (Liriodendron tulipifera L.) and northern fox grape (Vitis labrusca L.). Plant Cell Environ 23:407–414.

40 Inorganic Carbon Utilization by Root Systems Michael D. Cramer University of Stellenbosch, Stellenbosch, South Africa

I.

INTRODUCTION

the soil conditions, species, growth form, and environmental conditions (e.g., stress). Plant roots are normally exposed to high concentrations of IC. Several mechanisms by which root zone IC could influence terrestrial plant growth have been identified (Enoch and Olesen, 1993). Those include: (1) influence on soil nitrification, (2) Reduction in soil pH and consequent nutrient leaching from the soil, (3) contribution (<5%) to the plant C budget, and (4) influence on the production of plant hormones. Enoch and Olesen (1993) concluded that since the contribution made by DIC taken up by the roots to the overall C budget of the plant was small, it promoted growth through its influence on the plant hormones. In contrast, considerable evidence exists relating the effects of IC on growth and yield to the uptake and incorporation of IC in plant roots. Several aquatic species have been shown to use DIC taken up from the sediment, where DIC concentrations may be several orders of magnitude higher than in the bulk-phase water. One of the features of aquatic plants that utilize sedimentary CO2 is that they often have large leaf and root lacunae, suggesting a function in gas transport (Farmer, 1996; Chapter 42 by Armstrong and Drew in this volume). Although CO2 may be transported from the roots in the aerenchyma and is the main source of CO2 in submerged shoots or culms, it plays only a minor role in emergent parts of aquatic plants (Singer et al., 1994). Furthermore, on the basis of measurements of the activities of carboxylating enzymes in roots, Farmer (1996) concluded that root

Inorganic carbon (IC) concentration in soils has important effects on root physiology and plant growth that are the cumulative results of a number of biological, chemical, and physical processes. IC in the soil exists not only as gaseous CO2 but also as a pH-dependent combination of dissolved CO2 (i.e., H2 CO3 ), 2 HCO 3 , and CO3 , collectively referred to as dissolved inorganic C (DIC). The impact of soil IC concentrations on plant growth has been a topic of research since Birner and Lucanus (1866; cited in Enoch and Olesen, 1993) reported a response of oat plant growth to irrigation water enriched with IC. The aim of this chapter is to detail the physiological interactions between plant roots and IC and how shoot physiology and plant growth responds to uptake of IC by roots. The source and chemistry of root zone DIC has been briefly considered (Section III). The uptake of IC by roots of aquatic plants is more extensively reviewed by Farmer (1996).

II.

UPTAKE OF INORGANIC CARBON BY TERRESTRIAL AND AQUATIC PLANTS

There are special cases of terrestrial plants (e.g., Stylites andicola) that lack stomata and depend entirely on CO2 taken up from the root zone for photosynthesis (Keeley et al., 1984). The extent to which other terrestrial plants use root zone IC varies with 699

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incorporation of DIC into organic products in aquatic plants was also of negligible importance for the C budget. However, some species (e.g., Lobelia dortmanna) obtain most of the CO2 used in leaf photosynthesis from the root medium. Thus, the extent of dependence on root-derived DIC varies greatly among species and also depends on the concentration of DIC in the sediment (Nielsen et al., 1991).

III.

INORGANIC CARBON IN THE ROOT ZONE

Inorganic C exists as CO2 in the gas phase of soils and as DIC in the soil solution. The concentration of DIC depends on the concentration of CO2 in the gas phase with which the soil solution is in equilibrium and on the solubility of CO2 in the soil solution. Solubility in the soil solution is additionally determined by the pH, temperature, and ionic composition. The concentration of the dissolved CO2 component of DIC is not influenced by pH, but with increasing pH DIC solubility increases owing to the increasing proportion of the 2 total DIC comprised by HCO 3 and CO3 (Eshel and Beer, 1986). The concentration of CO2 in soil air spaces is affected by its production and consumption within the soil and by diffusion. CO2 readily accumulates in soils to between 0.2% (v/v) and 0.5%, but can accumulate to 20% under special circumstances (Norstadt and Porter, 1984). The concentration of CO2 within soil air spaces depends on the vegetation type and activity and thus varies seasonally (Sotomayor and Rice, 1999). In a variety of soils from forest communities (Fernandez et al., 1993; Fang and Moncrieff, 1998) and agricultural lands (Russell, 1950; Sotomayor and Rice, 1999), the average CO2 concentrations were found to increase with depth. The increase in CO2 concentration with depth may be attributed to in situ microbial activity and advection of DIC in soil water from shallower depths. Advective transport is likely to be more important in transporting DIC deeper into the soil profile when the soil is close to water saturation (Fang and Moncrieff, 1999). In soils where organic matter accumulates in the surface layers, the CO2 profile may be reversed, with greater CO2 concentrations closer to the surface owing to microbial activity in the organic layers (Alvarez et al., 1995). Sources of IC in the soil include respiration of microbes, micro- and mesofauna, and roots. The relative importance of these sources varies with soil type, season, vegetation type, and activity (Johnson et al.,

1994). Microbial activity may be of minor importance in some soils that are low in organic matter (Sotomayor and Rice, 1999). Howard and Howard (1993) found that microbial IC production was linearly correlated with increased temperature in a wide variety of soils, but was maximized at intermediate soil moisture contents. Agricultural practices such as tilling which change the availability of organic matter in the soil and increase the availability of O2 in the soil result in temporarily increased IC production and increased microbial activity in the soil (Rochette and Angers, 1999; Ellert and Janzen, 1999). Decades of intensive tillage induces increases in soil CO2 production that eventually deplete soil organic C reserves (Dao, 1998), although this detrimental effect of tillage on soil organic C may not be as significant in semiarid systems (Ellert and Janzen, 1999). Removal of IC from the root zone is due to diffusion vertically and laterally, advection, dissolution, and the formation of complexes with ions such as Ca2þ . Autotrophic CO2 utilization in the top layers of the soil may be high under some circumstances. However, this is not likely to influence IC concentrations much below the surface because it depends on light. Although plant roots incorporate DIC through the activity of phosphoenolpyruvate carboxylase (PEPc) and IC may be translocated from the root to the shoot, the net flux of IC is outward from roots and it is unlikely that most plant roots would act as net sinks for IC for prolonged periods. Diffusion of IC in the soils depends on the porosity of the soil, which is controlled by the soil particle size, organic matter content, and the soil water content (Bouma et al., 1997b). Most IC is lost from the soil through the soil surface accounting for 0.1–4 mole CO2 m2 d1 (Russell, 1950; Franzluebbers et al., 1995) but varying extensively between seasons (Fang et al., 1998). Restriction of such a loss of CO2 from the soil, e.g., by mulching, results in an increase of CO2 content in the soil even with moderate rates of CO2 release (Baron and Gorski, 1986). The conversion of dissolved CO2 to HCO 3 and CO2 3 results in the acidification of the soil solution increasing the dissolution of ions such as Ca and Mg (Van Lierop, 1990) and promoting their leaching (Enoch and Olesen, 1993). In some circumstances CO2 has been used to reduce the pH of highly alkaline irrigation water (personal observation), although such a pH control is only sustained for as long as CO2 remains in solution. Under the high concentrations of CO2 found in the soil, the buffering capacity of DIC is a strong determinant of soil pH. However,

Inorganic Carbon Utilization

solutions with high DIC concentrations rapidly lose CO2 when exposed to the low CO2 concentration of the atmosphere, especially when the pH of the solutions is low. In weakly buffered soil solutions, release of CO2 increases the pH of the solution resulting in the 2 stabilization of HCO 3 and CO3 content of the solution. The presence of various ions modifies the concentration of the components of DIC. To calculate the concentration of the components of DIC in solution, the activities of all the ions of the solution should be considered. The weak acid produced by dissolved CO2 is important in solubilizing a variety of mineral elements. Cations such as Ca2þ readily react with DIC to form complexes of varying solubility. For instance: Ca3 (PO4 Þ2 þ 2H2 CO3 $ 2CaHPO4 þ CaðHCO3 Þ2 and Na2 SiO3 þ 2H2 CO3 $ 2NaHCO3 þ H2 SiO3 (Enoch and Olesen, 1993). Thus DIC concentrations may increase weathering of soils while liberating ions for uptake by plants. On the other hand, complexing of cationic mineral nutrients by DIC may also result in a decrease in their availability. For example, bicarbonate-induced Zn deficiency (Yang et al., 1994) may be partially the consequence of formation of DIC complexes with Zn and the adsorption of Zn to CaCO3 (Marschner, 1995). Both positive and negative effects of IC on the growth and metabolism of bacteria, fungi, and protozoa have been reported (Santruckova and Simek, 1997). Fungi have been shown to have a requirement for IC for growth; however, high concentrations of IC may be inhibitory (Tabak and Cook, 1965). Such ICinduced modifications of microbial activity may influence the growth of higher plants through their effect on mycorrhizae (see Chapter 50 by Kottke in this volume), N2 fixation, and other, less direct effects on soil properties and plant nutrition. IV.

INORGANIC CARBON IN ROOT TISSUE

A.

Transport of Dissolved Inorganic Carbon

Despite the fact that roots supplied with DI 14 C readily incorporate this into 14 C-labeled complexes, the normal and overwhelming flux of DIC is out of the root down the DIC gradient created by respiration (Cramer and Lewis, 1993). The majority of DIC incorporated by plant roots is likely to be derived from respiratory DIC, but the accumulation of intracellular DIC is determined by both the internal and external environments (Miller, 1960). The exogenous IC concentration

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determines the gradient of DIC between the root tissue and the root medium and therefore the tissue DIC concentration. The forms of DIC in plant cells depend on the pH of the subcellular compartment, the activity of carbonic anhydrase (CA), and the activities of the various sources and sinks for DIC. The main source of DIC in root cells is the mitochondrion, the matrix of which is normally alkaline. In this compartment DIC will exist as HCO 3 and may be transported out of the mitochondrion as such. However, most DIC is thought to leave the mitochondrion as an almost unidirectional flux of CO2 (Raven and Newman, 1994). A variety of carboxylases exist in plant cells that incorporate DIC, the most important of which is PEPc which utilizes HCO 3 . Thus the forms of DIC in root tissue vary with the compartment and activity of that compartment. Transport of DIC across membranes is of interest because the form of DIC transported has implications for ion transport across the membrane since transport of HCO 3 may alter the transmembrane potential and pH. Furthermore, the pHs of the subcellular compartments differ and thus partial gradients of the different species of DIC may differ from the total DIC gradient. At a nominal pH of 6 for the soil solution, HCO 3 comprises 31% of total DIC and 69% is in the form of dissolved CO2 . CO2 readily diffuses through cell membranes and available evidence indicates that CO2 is the major form of DIC translocated across the plasmalemma of roots (Raven and Newman, 1994). Although CO2 readily diffuses across the plasmalemma, there is evidence in animal systems that CO2 transport may be facilitated by aquaporins (Reuss, 1998). Transport of HCO 3 may be important at high DIC concentrations and at alkaline soil pHs (Alhendawi et al., 1997). The requirement of a carrier for the transport of HCO 3 has resulted in the postula tion of HCO 3 : NO3 exchange in plants (Ben-Zioni et al., 1971; Barneix et al., 1984). Much accumulated evidence indicates that NO 3 is transported as a symport with Hþ , but it is difficult to discriminate empirically   þ between an antiport for HCO 3 : NO3 and a NO3 : H symport with associated CO2 diffusion, since the pH and membrane potential consequences are identical. Several carrier systems for NO 3 are now known  (Logan et al., 1997), and an HCO 3 : NO3 antiport may still be described. B.

Apoplastic and Symplastic pH

While the pH of the bulk phase of the soil solution is undoubtedly important for root physiology, the pH

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perceived and controlled by the root is the apoplastic pH, which can be very different from that of the bulk solution (Kosegarten et al., 1999). The interconversion 2 of dissolved CO2 , HCO contributes to 3 , and CO3 buffering of the apoplastic pH, especially at the high CO2 partial pressures to which roots are exposed in many soils. The apoplastic pH of maize roots has been shown to remain remarkably constant (pH 5– 5.7) while bulk phase pH ranged between 5 and 8.6. Apoplastic pH is increased by exposure to 10 mM  HCO 3 in the presence of NO3 . Thus, it was concluded  þ that HCO3 neutralized H efflux, resulting in high þ cotransport apoplastic pH due to NO 3 : 2H (Kosegarten et al., 1999). DIC is also an important component of the pH buffering system within plant cells. Not only does the 2 equilibrium between dissolved CO2 , HCO 3 , and CO3 buffer pH changes (Gerenda´s and Schuur, 1999), but the incorporation of HCO 3 into organic acids through the activity of PEPc and subsequent sequestering of organic acids in the vacuole are possibly involved in functioning as a ‘‘pH stat’’ (Smith and Raven, 1979). Gerenda´s and Schuur (1999) concluded that pH depends on the ‘‘strong ion difference,’’ which is the net positive charge on fully dissociated ions. It also depends on the total concentration of weak ions and on the partial pressure of CO2 . Gerenda´s and Schuur (1999) have also shown the importance of the partial pressure of CO2 in determining the pH in xylem and phloem sap. C.

Westhuizen and Cramer (1998) reported that CA activity contributed to the incorporation of DI 14 C supplied to roots. This has indicated that CA was important for refixation within the roots. However, this may only be true for tissues exposed to low extracellular DIC concentrations, and consequently low in internal DIC. At high tissue DIC concentrations the uncatalyzed equilibrium is likely to be sufficient to supply processes requiring DIC (Van der Westhuizen and Cramer, 1998). Acetazolamide is a membrane-impermeable inhibitor of CA that inhibits extracellular CA activity (Amory et al., 1991). In tomato roots inhibition of extracellular CA activity by acetazolamide decreased incorporation of DI 14 C from the nutrient solution into organic 14 C and inhibited respiratory O2 consumption. It also stimulated NO 3 uptake, but did not influence net DIC release (Van der Westhuizen and Cramer, 1998). CO2 would be the predominant species of DIC in the apoplast since the pH of the root solution and that of the apoplast are normally acidic (Kosegarten et al., 1999) and transplasmalemma flux of DIC is likely to be mostly in that form (Raven and Newman, 1994). Inhibition of extracellular CA may increase extracellular CO2 , thus limiting respiratory O2 consumption and limiting incorporation of exogenous DI 14 C. Since DIC is an important determinant of inter- and extracellular pH (Gerenda´s and Schuur, 1999), the absence of CA-catalyzed hydration of CO2 to HCO 3 may result in changes in extracellular pH and in membrane potential.

Significance of Carbonic Anhydrase Activity D.

Carbonic anhydrase (CA) catalyses the hydration of CO2 to HCO 3 , the latter being the inorganic substrate for PEPc. Raven and Newman (1994) state that the biological demand for various forms of DIC in nongreen tissue often exceeds the uncatalyzed rate of interconversion. In roots, CA is likely to function in maintaining the equilibrium between HCO 3 and CO2 to facilitate the diffusion of CO2 out of the root. High CA activity found in N2 -fixing root nodules may be the consequence of a requirement for low gas (O2 ) permeability of the tissue resulting in preferential transport of HCO 3 over CO2 (Coba de la Pen˜a et al., 1997). The activity of CA in maize root tips is high relative to that of PEPc exceeding the activity of PEPc by 200-fold (Raven and Newman, 1994). Thus, the CA activity in root tissue may function to facilitate DIC transport as well as ensuring a supply of HCO 3 for PEPc. Using ethoxyzolamide as an inhibitor of CA activity, Van der

Role of PEPc

Classically, the TCA cycle is considered to have a catabolic role and is a point of convergence for many intermediate metabolites. In plants the TCA cycle fulfills the catabolic role, but is also a point of divergence of anabolic metabolism (Hill, 1997). The consequence of the anabolic role is that intermediates of the cycle are constantly removed, and continued functioning of the cycle requires ‘‘topping up’’ or ‘‘anaplerotic’’ replacement of OAA by PEPc-catalyzed carboxylation of PEP (Fig. 1). In addition, PEPc may function in NAD(P)H generation, in refixation of respiratory DIC (Vuorinen and Kaiser, 1997), as a pH stat or for the provision of carboxylates to maintain ionic balance in the xylem sap (Schweizer and Erismann, 1985; Arnozis et al., 1988). Incorporation of DIC by the combined activity of CA and PEPc is central to many of the effects of DIC on root physiology.

Inorganic Carbon Utilization

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Figure 1 Pathway of anaplerotic carbon into the TCA cycle in plant roots. C enters the TCA cycle through pyruvate and through OAA or malate, which may be derived from carboxylation of PEP. OAA may be also transaminated to yield aspartate. The TCA cycle provides citrate for synthesis of glutamate.

Dark incorporation of HCO 3 occurs in the roots of most plants owing to the presence of ubiquitous PEPc (Jackson and Coleman, 1959) that, in combination with CA, has a high affinity for CO2 (Makino et al., 1992; Wullschleger et al., 1994). PEPc readily utilizes exogenous DIC in roots (Splittstoesser, 1966; Vapaavuori and Pelkonen, 1985; Vuorinen et al., 1992; Cramer and Lewis, 1993), and reassimilation of respiratory DIC is also likely to occur (Cramer et al., 1995). Organic acids (especially malate, citrate, and malonate) comprised the largest fraction (70–90%) of 14 C incorporated by plant roots with amino acids being more important than sugars (Bedri et al., 1960). After initial labeling of willow twigs with DI 14 C, the label was also detected in protein and in various insoluble components (Vuorinen et al., 1992). The activity of PEPc measured in vivo as 14 C incorporation in roots was
10-fold higher in the roots of plants grown under a high ambient root-zone CO2 (0.5%) than in plants under an atmosphere of 0.036% CO2 (Cramer and Lips, 1995). Similar enhancement of PEPc activity by elevated DIC was reported for willow and barley (Vuorinen and Kaiser, 1997).

The fate of H14 CO 3 fixed by PEPc is partially determined by the form of N supplied. PEPc activity of several species was four times higher in NHþ 4 - than in -fed plants (Schweizer and Erismann, 1985; NO 3 Arnozis et al., 1988). The capacity of the plant to provide C skeletons through dark fixation of DIC by roots may determine its capacity to assimilate NHþ 4 , especially under conditions in which the supply of 2-oxoglutarate is limiting. The proportion of 14 C diverted  into amino acids is greater in NHþ 4 - than in NO3 -fed plants in which organic acids are preferentially synthesized (Cramer et al., 1995). Amino acids containing C incorporated through the activity of PEPc are synthesized by transamination of both OAA and 2-oxoglutarate. Root zone DIC concentration does not strongly influence in vitro PEPc activity (Cramer et al., 1996, 1999). Vuorinen and Kaiser (1997) have shown that the ‘‘potential’’ in vivo activity of PEPc measured at high DIC concentrations does not change greatly with DIC concentration. Furthermore, the in vitro activity of root PEPc was at least an order of magnitude greater than the in vivo activity of this enzyme in willow, bar-

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ley (Vuorinen and Kaiser, 1997), tomato (Cramer and Lips, 1995), and lupin root nodules (Christeller et al., 1977). This indicates that the level of PEPc is not a limiting factor for the assimilation of DIC from the root medium. The activity of this enzyme is either regulated or limited by other processes (Christeller et al., 1977; Cramer et al., 1999), such as pH and metabolites (Murray, 1997). Stimulation of dark DI 14 C fixation by þ NHþ 4 suggests that NH4 or its assimilation products may contribute to the control of PEPc activity in the root, possibly through the protein kinase mechanism that was described for leaves by Van Quy and Champigny (1992) and Champigny and Foyer (1992).

V. INFLUENCE OF ROOT-ZONE INORGANIC CARBON ON ASPECTS OF PHYSIOLOGY A.

Root Respiration

Root respiration is an important component of the C budget of the plant. More than 50% of carbohydrates translocated to the roots are respired by that organ (Lambers et al., 1996, Chapter 32 in this volume). The rate of ATP production in roots has to match the maintenance, growth, and ion uptake requirements (Johnson, 1990). Effects of ambient CO2 concentration on the rate of respiration were found in several species (Bunce and Ziska, 1996). These effects may be mediated by alterations of intracellular pH, by refixation of respired DIC, by suppression of respiratory enzyme activity, and by diversion of electron transport to the alternative oxidase (Qi et al., 1994). Respiration rates of roots and isolated mitochondria have been reported to decrease (Thomas and Griffin, 1994; Johnson et al., 1994; Ziska et al., 1991; Bunce and Ziska, 1996; Ziska and Bunce, 1994; Qi et al., 1994; Palta and Nobel, 1989), to increase (Azcon-Bieto et al., 1994; Gonza`lez-Meler et al., 1996; Thomas and Griffin, 1994; Wullschleger et al., 1994), or to be unchanged (Bouma et al., 1997a,b) with elevated IC. The problem with several reports on respiration is methodological: CO2 efflux rates, and not O2 consumption rates, were used as an indication of respiration (Wullschleger et al., 1994). Furthermore, Bouma et al. (1997b) suggested that experimental artifacts might arise from the use of IC concentrations outside the range normally experienced by the roots, and thus account for these contradictory results. Furthermore, variability in respiration rates due to environmental factors other than IC concentration may also be important.

Decreased DIC release rates in maple roots at elevated external DIC were attributed to ‘‘direct’’ effects that were not related to DIC fixation (Burton et al., 1997). In contrast, Cramer and Lewis (1993) concluded from large decreases in RQ at elevated DIC levels that DIC flux was reduced due to refixation of DIC through the activity of PEPc in tomato roots. Furthermore, incorporation of DI 14 C from root solutions into acid-stable organic products occurred at rates sufficient to explain the decrease in respiratory DIC loss. The anaplerotic contribution of dark incorporation of DIC to organic acids synthesis may account for increased O2 consumption at moderate DIC concentrations because of promotion of alternative pathway activity (Van der Westhuizen and Cramer, 1998). At high DIC concentrations O2 consumption may be inhibited through inhibition of cytochrome oxidase, cytochrome c oxidase, and succinate dehydrogenase activity (Gonza`lez-Meler et al., 1996; Gonza`lez-Meler and Siedow, 1999). Thus, the influence of DIC on O2 consumption depends on the balance between the inhibition of electron transport by DIC and the stimulation of alternative oxidase activity by DIC-derived organic acids. The complete oxidation of hexose to CO2 and H2 O yields a respiratory quotient (RQ) of 1, irrespective of the pathways of oxidation (Lambers, 1997). However, the synthesis of intermediates from the TCA cycle and the use of NADH for metabolism not related to respiration have consequences for the flux of both DIC and O2 , especially when anaplerotic C is utilized. For instance, the synthesis and excretion of citrate, which is important for phosphate uptake and resistance to Al toxicity (Zheng et al., 1998a), would result in a partial RQ of 0 for this component. The synthesis of 1 malate from hexose results in a partial RQ of 0.7, while the synthesis of two malates from a hexose results in the net incorporation of DIC. When amino acid synthesis is taken into account it is unlikely for the RQ of the root to be 1 during active DIC incorporation and N metabolism. In particular NO 3 reduction increases the RQ while amino acid synthesis reduces the RQ (Cramer and Lewis, 1993).

B.

Root Exudation

Root exudation, including exudation of organic acids, is an important component of the C balance of plant roots and of soils (Marschner, 1995). The exudation of low-molecular-weight compounds, such as organic acids, is important for the uptake of several ions and

Inorganic Carbon Utilization

has also been implicated in the resistance to toxic metals such as Al (Zheng et al., 1998a,b; Chapter 43 by Hagemeyer and Breckle in this volume). This exudation may partially depend on the provision of anaplerotic C. Carboxylic acids, especially citrate, are important in mobilizing P. P-deficient lupin roots were found to exude large amounts of malate and citrate derived from dark incorporation in proteoid roots (Johnson et al., 1996). The exudation of organic acids was much greater than the total plant organic acid content and therefore is likely to be an active process. Cramer and Van der Westhuizen (2000) reported between three- and fivefold higher exudation of 14 C from tomato roots grown with adequate P at root-zone CO2 concentrations of 0.4% compared to 0.036% CO2 . This indicates that root zone DIC concentrations could be important in anaplerotic C provision for organic acid production for mobilization of P and for immobilization of other elements.

C.

Interaction with Ethylene

Treatment of plant tissues with elevated IC modifies the levels of several plant hormones (Arteca et al., 1980). At low concentrations CO2 promotes ethylene synthesis in fruits while at higher concentrations ethylene synthesis is inhibited through modification of the activities of the enzymes responsible for ethylene synthesis (Murray, 1997). CO2 has also been found to be a competitive inhibitor of ethylene binding, causing inhibition of many ethylene-mediated processes (Abeles, 1973; Chapter 27 by Hussain and Roberts in this volume). Furthermore, CO2 can mimic the effect of ethylene on some processes, although it is possible that this is due to independent mechanisms rather than through the same signal transduction pathway. Govindarajan and Poovaiah (1982) described the abolition of ethylene-induced inhibition of photosynthesis by 20% CO2 in the root zone and suggested that this could be a factor responsible for increased growth of plants exposed to high root zone IC concentrations. CO2 has been shown to retard geotropic response of pea roots, which may represent a specific inhibition of ethylene control on geotropism (Chadwick and Burg, 1970). Thus, it seems that CO2 may stimulate ethylene synthesis at low concentrations and inhibit both synthesis and binding/action at high concentrations. Both situations would affect root growth and development.

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D.

Plant Mineral Nutrition

1.

Nitrogen Nutrition

a.

NO 3 Uptake

Nitrate uptake in barley plants grown with NO 3 (induced) is biphasic with a high-affinity, saturable system (HATS), at low concentrations (
<1 mM) and a low-affinity nonsaturable system (LATS) at higher concentrations (Siddiqi et al., 1990). Four different transporters have been identified including constitutive and inducible systems and it is likely that additional transporters will be found (Crawford and Glass, 1998). Stimulation of NO 3 uptake by elevated DIC has been reported for barley and tomato resulting in increased tissue N contents (Bialczyk, 1994; Cramer and Lips, 1995). Alhendawi et al. (1997) reported reduced tissue NO 3 concentration and increased efflux of NO 3 in barley, sorghum, and maize at concentrations of HCO 3 between 5 and 20 mM. The discrepancy between these results may be ascribed to the use of nutrient solutions buffered at different pHs—8 (Alhendawi et al., 1997) and 5.5–6 (Cramer et al., 1996, Van der Merwe and Cramer, 2000). The positive influence of enriched DIC concentrations on the growth of salanized tomato plants (Cramer and Lips 1995) was ascribed both to increased availability of anaplerotic C and to enhanced uptake of  NO 3 . The increase in uptake of NO3 with enriched DIC at pH 7 was the same as at pH 5, indicating that CO2 was the extracellular form of DIC that influenced NO 3 uptake (Cramer et al., 1996). The biggest response of NO 3 uptake to root zone DIC enrichment was at low external NO 3 (0.5 mM) concentrations (Van der Merwe and Cramer, 2000). Possibly this is due to a differential response of the HATS and the LATS to root zone DIC enrichment. The influence of DIC on NO 3 uptake may include a more direct component than only provision of anaplerotic C. This is implied by the following observations: 1. Stimulation of NO 3 uptake by DIC was similar in plants differing in PEPc activities (Van der Merwe and Cramer, 2000). 2. NHþ 4 uptake was not stimulated by root zone DIC enrichment in tomatoes (Van der Merwe and Cramer, 2000) and barley (Cramer et al., 1996). 3. NO 3 uptake in both wild-type and NR-deficient barley plants was similarly increased by elevated DIC (Cramer et al., 1995). 4. DIC stimulated NO 3 uptake by uninduced tomato roots indicating a lack of requirement for significant NRA (Van der Merwe and Cramer, 2000).

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Thus, the influence of DIC on NO 3 uptake is at least partially independent of the reduction of NO 3 and assimilation into amino acids. NO 3 uptake has previously been linked to an exchange with HCO 3 (Ben-Zioni et al., 1971). This was interpreted as an indication that 10–15% of CO2 released from the root was associated with  HCO 3 : NO3 exchange (Barneix et al. 1984). It is possible, however, that the alkalization of the root medium and CO2 release observed by these authors were independent processes. Stimulation of NO 3 uptake upon inhibition of apoplastic CA activity by acetozolamide could be due to a lower external HCO 3 con : HCO exchange (Van centration facilitating NO 3 3 der Merwe and Cramer, 2000). These authors also reported that ethoxyzolamide, which inhibits both symplastic and apoplastic CA activity, inhibited NO 3 uptake, linking cytoplasmic HCO 3 formation with NO 3 uptake. None of this evidence, however, directly  exchange rather than demonstrates HCO 3 : NO3  þ NO3 : H symport with coincidental CO2 release. The steady-state membrane potential of barley roots perfused with CO2 -free buffer (pH 6.0) was found to be reduced upon perfusion of the roots with buffer equilibrated with 0.4% CO2 (Cramer and Miller, unpublished). This may indicate that the influence of DIC on NO 3 uptake is mediated through changes in the membrane potential or pH gradient which could be sensitive to inhibition of apoplastic and symplastic CA activity. Further research is required to elucidate the interaction between DIC concentration/buffering and NO 3 uptake. b. NHþ 4 Uptake Uptake of neutral NH3 across the plasmalemma may occur at alkaline pHs and at high temperatures. However, under normal conditions the concentration þ of NH3 is low relative to that of NHþ 4 . At low NH4 concentrations, uptake is through a HATS (Ninnemann et al., 1994) while between 0.5 and 1 mM NHþ 4 a LATS has been described (Kronzucker et al., 1996). Exposure of tomato roots to 0.4 mM NHþ 4 resulted in depolarization of the roots followed by gradual repolarization (Ayling, 1993). Uptake of NHþ 4 may be through a facilitated diffusion or a symþ port of NHþ 4 with H (Ninnemann et al., 1994; Logan et al., 1997). The depolarization caused by NHþ 4 could through a uniport or be due to the entry of NHþ 4 translocation with a Hþ . The repolarization of the membrane potential may be due to stimulation of the plasma membrane Hþ ATPase, resulting in acidification of the apoplast. The well-known acidification of

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the root medium by plants supplied with NHþ 4 nutrition is due to ATPase activity rather than a direct consequence of NHþ 4 uptake per se (Ninnemann et al., 1994). Enhanced anaplerotic provision of C for amino acid synthesis at high root zone IC concentrations may be expected to contribute to NHþ 4 uptake. However, Cramer et al. (1996) found that aeration with air containing 0.65% CO2 inhibited NHþ 4 uptake by barley roots. In contrast, Van der Merwe and Cramer (2000) found no influence of 0.5% CO2 on NHþ 4 uptake by tomato roots. The reason for the discrepancies in these results is not known. However, the lack of uptake takes place despite stimulation of NHþ 4 increased PEPc activity due to NHþ 4 nutrition in the roots of many species (Schweizer and Erismann, 1985; Arnozis et al., 1988; Cramer et al., 1993). It is speculated that synthesis of aspartate and asparagine from PEP-derived OAA could lead to diversion of organic acids into the synthesis of these amino acids and a restriction of TCA cycle activity. The basis for that speculation is the decline in O2 consumption at elevated DIC with NHþ 4 nutrition (Van der Westhuizen and Cramer, 1998). This may interfere with the incorporation of NHþ 4 into glutamate and glutamine and thus inhibit NHþ 4 uptake. c.

Nitrogen Metabolism

Inorganic N taken up by the root may be assimilated in the root tissue or transported to the shoot. The proportion of NO 3 reduced in the root has been reported to be negatively correlated with the NO 3 concentration in the external medium (Smirnoff and Stewart, 1985; Andrews et al., 1992). With elevated DIC these authors found increased translocation of reduced N in the xylem sap, indicating that DIC influences both inorganic N uptake and assimilation by altering the contribution of the root to this process. Increased NRA of barley at elevated DIC concentrations may be due to increased uptake of NO 3 and the accumulation of tissue NO 3 (Cramer et al., 1995), However, it is possible that NRA is modified by other controls such as the phosphorylation of the enzyme or binding of inhibitor proteins (Kaiser and Huber, 1997). Acidification of the cytosol has been shown to activate NR by stimulating protein phosphatase and inhibiting protein kinase activity (Kaiser and Brendle-Behnisch, 1995), and it may be speculated that absorbed DIC can cause cytoplasmic pH changes which may alter NRA. Low NO 3 uptake rates measured in plants treated with ethoxyzolamide may be partially the result of decreased NRA due to decreased pH control (Van

Inorganic Carbon Utilization

der Merwe and Cramer, 2000) resulting from inhibition of the pH stat function of PEPc induced by inhibition of CA activity. The influences of DIC on the activities of other enzymes of N metabolism have not been extensively studied. Cramer et al. (1999) reported significant increases in leaf and root glutamine synthetase (GS) activity with elevated DIC. Elevated DIC also elicited an increase in root glutamine:2-oxoglutarate aminotransferase (GOGAT) activity in salt-treated plants. The changes were consistent with the synthesis of aspartate and asparagine from oxaloacetic acid (OAA). The changes in GS and GOGAT activity were small and it seems likely that they were due to changes in the uptake of N or in the availability of C skeletons, rather than the consequence of an influence of DIC on the enzyme activity. 2.

Mineral Nutrition (Other Than Nitrogen)

When excess cation over anion accumulation occurs, the charge balance is restored by organic anions synthesized from DIC. This has led to the assumption that cation accumulation is enhanced by elevated IC. Decreased cation (Kþ , Ca2þ , Mg2þ ) accumulation in soybeans supplied with NHþ 4 was reversed by exposure of the plants to 16 mM HCO 3 (Bhan et al., 1960). However, uptake by roots of various nutrients (Zn, Fe, K, P, Mg, and Mn) and subsequent translocation to the shoot are inhibited by high concentrations of HCO 3 (Dogar and Hai, 1980; Yang et al., 1993; Marschner, 1995). Furthermore, tissue K in barley, sorghum, and maize was decreased and Ca was increased by HCO 3 treatments between 5 and 20 mM (Alhendawi et al., 1997). Zinc deficiency symptoms associated with high IC concentrations in soils were associated with inhibition of root growth in ‘‘Zn-inefficient’’ cultivars (susceptible to Zn deficiency on alkaline soils), but a stimulation of growth in the ‘‘Zn-efficient’’ cultivars (Yang et al., 1994). Inhibition of growth in the Zn-inefficient cultivar was ascribed to superfluous accumulation of organic acids as compared with a much smaller accumulation in the Znefficient cultivars. Thus, although organic acids may play an important role in cation–anion balance, there may also be some specific effects of DIC incorporation. Accumulated organic acids may chelate cations and thus reduce their availability. Iron deficiency (‘‘lime-induced chlorosis’’) is common on calcareous soils with free CaCO3 in which increased DIC leads to the formation of Ca(HCO3 Þ2 (Marschner, 1995). For many years lime-induced

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chlorosis was known to be associated with high levels of HCO 3 (Brown, 1960) and with the accumulation of organic and amino acids (Rhoads and Wallace, 1960). Qualitative differences exist among plant genotypes in their deficiency-induced adaptation mechanisms. These differences are associated with Fe acquisition with plants classified as either ‘‘Strategy I’’ (nongraminaceous species) or ‘‘Strategy II’’ (graminaceous species). Strategy II plants, which depend on phytosiderophore release, are only slightly influenced by HCO 3 concentration in the soil. This has been attributed to their decreased root elongation at high HCO 3 concentrations (Alhendawi et al., 1997). Strategy I plants, in which Fe uptake is associated with Hþ excretion, are sensitive to high HCO 3 . At high pH, DIC reduces the ability of plants to acquire Fe by Strategy I plants because of the operation of several mechanisms including reduced Fe solubility, pH buffering, reduced release of phenolics, lowered reduction of Fe3þ at the plasmalemma, and increased synthesis of organic acids resulting in formation of complexes with Fe and sequestration in the vacuole (Marschner, 1995). 3.

Nitrogen Fixation and Mycorrhizal Symbionts

In N2 -fixing legume root nodules, DIC incorporated by PEPc activity contributes to the production of dicarboxylic acids available to the bacteroids (Raven and Newman, 1994). A significant proportion of the organic C derived from PEPc activity may be involved in producing C skeletons for export of N. Dark incorporation of DIC by root nodules is correlated with PEPc activity resulting in the formation of organic acids, glutamate, aspartate, and asparagine (Lawrie and Wheeler, 1975; Vance et al., 1983). Nodule PEPc activity in alfalfa was sufficient to provide
25% of the C required for assimilation and transport of symbiotically fixed N. This is equivalent to the utilization of 1 mole of OAA derived from PEPc activity per mole of dinitrogen fixed (Christeller et al., 1977). In soybean nodules, which transport fixed N in the form of ureides, increased acetylene reduction with elevated CO2 concentration (1.5–3%) was ascribed to the utilization of 1–3.4 mole of DIC per mole of dinitrogen incorporated into glutamate and aspartate. The latter may in turn be used for the synthesis of ureides (Coker and Schubert, 1981). Root systems of almost 80% of angiosperms grow in associations with root fungi, forming a mycorrhizal symbiosis (see Chapter 50 by Kottke in this volume). Since mycorrhizae supply the host plant with mineral

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nutrients in exchange for host-derived carbohydrates, the presence of symbiosis imposes an additional belowground sink for C. The importance of IC in the physiology of fungi is well known (Tabak and Cook, 1965). In the mycelium of the ectomycorrhizal fungi, there are several potential avenues for the metabolic use of DIC. The major pathway of DIC incorporation appears to be through replenishment of the dicarboxylic acids of the TCA cycle, which are used for amino acid synthesis (Wingler et al., 1996). These authors showed that phosphoenolpyruvate carboxykinase was considerably more active than pyruvate carboxylase while PEPc activity was not detected in the fungal symbiont. Interestingly, mycorrhizal infection results in reduced root PEPc activity indicating a shift in anaplerotic C supply from the root to the fungal symbiont. Other biosynthetic routes in the fungus that require DIC fixation include fatty acid biosynthesis, in which CO2 reacts with acetyl coenzyme A (coA) to produce the vital intermediate malonyl CoA (Gitterman and Knight, 1952). In in vitro studies using root tissue extracts in petri dishes, CO2 has been shown to stimulate hyphal growth of arbuscular mycorrhizal spores (Becard and Piche, 1989). Spore growth depends on generation of acetyl coA from lipid catabolism, and without anaplerotic C derived from incorporation of DIC, anabolic functioning would be inhibited. Extensive symbiotic infection and vesicle formation were reported at 0.5% CO2 in comparison to CO2 -free treatments (Saif, 1984). However, high CO2 concentrations (e.g., 5% for Glomus mosseae) inhibited mycorrhizal infection. The optimal CO2 concentration for the fungal symbiont is thus likely to depend on the fungal species and on environmental conditions. Mycorrhizal plants were shown to release more CO2 to the root zone than uninfected plants due to the respiratory activity of the fungal symbiont (Snellgrove et al., 1982; Knight et al., 1989). Furthermore, the plant P uptake was significantly greater by mycorrhizal plants and soil solution P content was highly correlated with the CO2 concentrations of the soil atmosphere. Knight et al. (1989) suggested that mycorrhizae partially contribute to P nutrition of the host by solubilizing Ca-phosphate through elevated soil atmospheric CO2 concentrations. E. Stress Physiology Salinity and water stress inhibit NO 3 uptake (Aslam et al., 1984; Peuke and Jescke, 1999) and partially shift the site of NO 3 reduction and assimilation from the

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shoot to the root (Smirnoff and Stewart, 1985). Apart from an inhibition of root DI 14 C assimilation by salinity, the products of DI 14 C incorporation are diverted from organic acids to amino acid synthesis (Cramer et al., 1993). Thus, in salanized plants, DIC incorporation provides anaplerotic C for assimilation of reduced NO 3 into amino acids, while in nonsalanized plants the products serve for maintenance of ionic balance in the cells and in the xylem sap. Salinity-stressed tomato seedlings accumulated a higher dry weight and assimilated more N when the roots were supplied with CO2 -enriched aeration (Cramer and Lips, 1995). Plants grown under increased salinity and an enriched DIC environment also had higher rates of NO 3 uptake. Such plants translocated more NO 3 and reduced N in the xylem sap than did comparable plants grown under ambient root zone DIC concentrations. Other aspects of the influence of DIC on plant susceptibility to stress are referred to later. F.

Influence of Root Zone Inorganic C on Shoot Physiology

The concentration of bicarbonate in plant tissues is many times greater than that of free CO2 due to interactions with alkalizing ions (e.g., P, Ca, Mg). This results in carbonates comprising between 0.03% and 0.5% of the dry weight of plants (Berthelot and Andre´, 1887, cited in Miller, 1960). Plants rooted in IC-rich media are able to translocate DIC from the root to the shoot. However, this transport of DIC normally accounts for a small proportion of the total C transported to the shoot (Heij, 1985; Vuorinen et al., 1989). In plants supplied with very high root zone CO2 concentrations (10–100%), however, translocation of DIC to the shoot does influence photosynthesis. Furthermore, organic C derived from root incorporation and transported in the xylem sap represented up to 10% of the C acquired by photosynthetic assimilation in plants supplied with elevated root zone DIC concentrations (Cramer and Richards, 1999). Rootderived organic acids translocated in the xylem could be decarboxylated in the shoot and the CO2 reassimilated by photosynthesis (Arteca and Poovaiah, 1982). Xylem-derived C may be especially important under stress conditions when photosynthesis is restricted owing to reduced stomatal conductance (Cramer and Richards, 1999). CO2 is transported from the roots to the shoots, through aerenchyma, in wetland species, in rice, and in wheat. In wheat with well-developed aerenchyma, photosynthetic rates, leaf chlorophyll contents, and

Inorganic Carbon Utilization

shoot dry matter production were increased by root zone CO2 concentrations of 10% (Huang et al., 1997). Rice grown under submerged conditions develops an extensive aerenchyma. Such plants absorb, translocate, and incorporate more DIC than wheat or dryland rice. Respiratory inhibitors and osmotically active solutes did not influence translocation of 14 C from the root to the shoot of submerged rice, indicating that rice translocated CO2 through the aerenchyma by a nonmetabolic system (Higuchi et al., 1984). VI.

INFLUENCE OF ROOT ZONE INORGANIC CARBON ON GROWTH

A.

Postgermination Radicle Growth

Reduced growth was observed immediately after radicle emergence of soybean and maize seedlings grown under 30% CO2 (Grable and Danielson, 1965a). However, stimulation of early postgermination growth of maize and soybean, at CO2 concentrations <20%, was also reported. Maize radicle extension was stimulated by
40% by elevated CO2 concentrations of up to 5% (Unger and Danielson, 1964). Coleoptile growth of Avena sativa kept in the dark was stimulated by 40% under 2% CO2 , but it was inhibited at CO2 concentrations >10% (Harrison, 1965). Thus, the influence of CO2 on postgermination radicle growth is stimulatory at low concentrations and inhibitory at high concentrations with the concentration at which CO2 becomes inhibitory being species specific. B.

Plant Growth and Crop Yield

Varying levels of IC have been applied to root systems to assess the influence of IC on growth. Such experiments have been conducted repeatedly with varying degrees of rigor and using different application systems. DIC supplied in irrigation water and in nutrient solutions has been reported to have both positive and negative effects on growth and yield. However, in a review of an extensive collection of data, Enoch and Olesen (1993) found that CO2 -enriched irrigation water increased crop productivity by an average of only 2.9%. Such an average is somewhat spurious, however, since the data included negative and positive effects and it is known that the effects of elevated IC depend on the concentration of IC, the species tested, soil pH, and a variety of other factors. Thus, it seems that identification of the specific genotypes that are sensitive to IC and the conditions required for this response are required for further

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understanding. With much of the research it is difficult to discriminate between the utilization of DIC by roots and the release of CO2 into the atmosphere and consequent beneficial effects on growth through photosynthetic incorporation. Unintended atmospheric enrichment may be the main reason for the beneficial effect of CO2 -enriched water on plant growth (Enoch and Olesen, 1993). In numerous reports, extremely high CO2 concentrations (up to 80%) have been applied to root systems. Such high CO2 concentrations make it extremely difficult to eliminate the possibility of leaks of CO2 into the atmosphere around the shoot, especially since many plastics are permeable to CO2 . Another source of contradictory reports is the fact that soils are enriched with IC owing to biological and chemical processes, and thus application of elevated IC treatments to such soils may meet with varying results owing to the natural enrichment of the soil. For instance, the amount of IC available from soil respiration exceeded the application of carbonates in some experiments by >10-fold (Stolwijk and Thimann, 1957). Research on the influence of IC on growth can be divided into that conducted at low pH or targeting calcifuge species and that at high pH related to calcicole species. This introduces considerable confusion since in many studies plants grown at low pH (
5) without added HCO 3 are compared with those grown at high pH (
8) with added HCO 3 (e.g., White and Robson, 1990), making it difficult to isolate pH and DIC effects. Growth of many species is inhibited by HCO 3 at high pHs, with calcifuge species being more susceptible to inhibition than calcicole species (Lee and Woolhouse, 1969a). Root growth of calcicole species is stimulated by concentrations of HCO 3 (1 mM) that inhibit root growth in calcifuge species (Lee and Woolhouse 1969a). Inhibition of growth of roots is associated with inhibited elongation of root cells, rather than inhibition of cell division. Decreased growth was also attributed to accumulations of organic acids and depletion of glycolytic intermediates (Lee and Woolhouse, 1969b). Resistance of calcicole species to high concentrations of HCO 3 is associated with removal of malate (Lee and Woolhouse, 1971). Alhendawi et al. (1997) reported that both shoot and root growth of calcifuge species, e.g., barley, maize, and sorghum (Strategy II), at pH 8 was inhibited by 5–20 mM HCO 3 . They suggested that such an inhibition of growth might also be associated with inhibited root respiration, accumulation of organic acids, and reduced uptake and translocation of Fe.

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Several reports describing the effects of soil IC have claimed to have eliminated or limited shoot atmospheric enrichment. Results of some of these are presented as examples: 1. Increased productivity found for eggplants grown in pots under plastic mulch, in which atmospheric enrichment was limited, were related to elevated (up to 14%) root zone CO2 concentrations (Baron and Gorski, 1986; Abney and Russo, 1997). 2. Potato plants grown with an enriched CO2 of the root zone (45%) accumulated more shoot and root mass and produced more and bigger tubers (Arteca et al., 1979). Using 14 CO2 labeling these authors showed that products of root incorporation (mostly malate) are translocated to the shoot and that 18% of the dry matter increase was derived from root incorporation. 3. Maize and soybean growth was stimulated by CO2 concentrations of up to 20% in the root zone (Grable and Danielson, 1965b). 4. Potted cotton grown with CO2 -saturated irrigation water accumulated 1.6-fold more dry weight and boll dry weight than controls exposed to the same atmospheric CO2 concentrations (Mauney and Hendrix, 1988). 5. Citrus dry weight accumulation was increased as soil CO2 concentrations were enriched up to 12% (Lababanauskas et al., 1971). Thus, enrichment of the root zone with CO2 increases growth and yield of some plants, but in all these cases the amount of CO2 supplied was high (10– 45%). At these high IC concentrations the amount of DIC transferred through the xylem sap may make a significant contribution to the C budget of the shoots. IC enrichment of the root zone may be better controlled in hydroponics. Hydroponic enrichment experiments conducted with a variety of plant species yielded an average increase in biomass accumulation of
30% (Bergquist, 1964; Vapaavuori and Pelkonen, 1985). Beneficial effects of increased root zone DIC were obtained for tomato seedling, especially in plants subjected to either salinity stress or high temperatures combined with high light intensities (Bialczyk, 1994; Cramer and Lips, 1995; Cramer and Richards, 1999). Bialczyk et al. (1996) reported 1.3-fold increased fruit yield and quality parameters of hydroponically grown tomato plants supplied with nutrient solutions enriched with HCO 3 in the absence of specially induced stress. Roots are clearly adapted to relatively high soil IC concentrations. It is likely that IC concentrations outside the range to which the plants are adapted will have

Cramer

negative implications for growth and yield. Although high levels of soil IC are essentially a byproduct of plant and microbe respiration, plants have evolved to withstand and to utilize IC for a variety of purposes. Exposure of roots to DIC-poor hydroponic solutions or to low-IC soils with high porosity and low organic matter contents is likely to interfere with growth for a variety of physiological reasons. On the other hand, high soil IC concentrations may be toxic to plants, particularly to calcifuges, i.e., to plants that are not adapted to these physiological conditions. Therefore, from an agricultural management perspective, it is necessary to establish what levels of soil IC are optimal for a crop species and to manage soil IC levels accordingly.

VII.

AGRICULTURAL SIGNIFICANCE OF ROOT ZONE INORGANIC CARBON

Soil IC concentrations are indisputably of ecological and agricultural importance. ‘‘Lime-induced’’ chlorosis is one of the major problems in production of sorghum and soybean in the Great Plains of the United States due to the calcareous nature of the soils (Marschner, 1995). At the other end of the spectrum, plants may be exposed to low root zone IC concentrations when grown in well-aerated hydroponics or when porous, organic matter-poor soils are used. ‘‘Sustainable’’ agronomic systems in which chemical inputs to the soil are replaced by biologically derived amendments are usually associated with significant pools of soil organic C (Paul et al., 1999). However, continual plowing can deplete both organic and inorganic soil C, and utilization of chemical fertilizers eliminates some of the organic material associated with natural recycling of mineral nutrients. Redressing low soil IC concentrations therefore requires retention or supplementation of soil IC using plastic or other mulches. There have been several attempts to enrich soil IC by irrigating the soil with CO2 -enriched water on a commercial agricultural basis. Of the systems used commercially for root zone IC enrichment, the most realistic appears to involve the use of drip irrigation supplied under plastic mulch which delivers the IC to the root zone with minimal losses. Crops with low, dense canopies may be especially suitable for application of this enrichment strategy since their leaves may be able to trap CO2 released from below the mulch. Artificial enrichment of agricultural soils requires large amounts of CO2 and sophisticated dosing systems, which renders the prospect of economically profitable enrichment of the root zone IC dubious.

Inorganic Carbon Utilization

VIII.

CURRENT ISSUES

It is important that plant physiologists be aware of the fact that roots are exposed and adapted to high concentrations of IC and that these concentrations may vary among soil types and with other environmental factors. The role of DIC in determining both extraand intracellular pH and pH buffering is also of crucial importance and underappreciated (Gerenda´s and Schuur, 1999; Chapter 33 by Gerenda´s and Ratcliffe in this volume). A considerable body of literature exists on the influence of soil IC on root physiology. Despite over a century of research, many questions on the influence of IC on root physiology remain unanswered. One of the complexities of working with the influence of IC on plant physiology is that DIC exists in several pH-dependent forms. This makes it difficult to discriminate between the effects of the various components of DIC and those of pH. Systems allowing the supply of out-of-equilibrium solutions containing CO2 or HCO 3 have been employed in animal systems (Zhao et al., 1995), but are yet to be employed in plant biology. Many physiological processes are dependent on the supply of anaplerotic C. Of these only the assimilation of N has been extensively studied. Interactions with the uptake of P under P-deficient circumstances and the amelioration of Al toxicity are still to be fully explored. The reasons for the variable effects of DIC on the uptake of N are still to be elucidated. This applies to þ the differential effects of DIC on NO 3 and NH4 uptake, but also to the fact that in some cases NO 3 uptake is stimulated and in others it is inhibited by elevated DIC. These questions are probably best addressed by techniques allowing assay of transporter activity (e.g., electrophysiology). Another important issue is the elucidation of the mechanisms responsible for the control of partitioning of DIC- derived C between organic and amino acids. Phosphorylation appears to be important in control of the enzymes of N metabolism, but the interaction between DIC and protein phosphorylases/kinases still has to be elucidated. Finally, the current confusion in understanding of the agricultural importance of root zone IC needs to be addressed. Agricultural application of CO2 gas and the use of plastic mulch may sometimes be illinformed choices, which need to be viewed against the desirability of returning organic residues to the soil.

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ACKNOWLEDGMENTS Thanks to Owen Lewis and Heidi Hawkins for commenting on the manuscript and to Aleysia Viktor for technical assistance.

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715 Vuorinen AH, Vapaavuori EM, Lapinuoki S. 1989. Timecourse of uptake of dissolved inorganic carbon through willow roots in light and in darkness. Physiol Plant 77:33–38. Vuorinen AH, Vapaavuori EM, Raatikainen O, Lapinjoki SP. 1992. Metabolism of inorganic carbon taken up by roots in Salix plants. J Exp Bot 43:789–795. Vuorinen AH, Kaiser WM. 1997. Dark CO2 fixation by roots of willow and barley in media with a high level of inorganic carbon. J Plant Physiol 151:405–408. White PF, Robson AD. 1990. Response of lupins (Lupinus angustifolius L.) and peas (Pisum sativum L.) to Fe deficiency induced by low concentrations of Fe in solution or by addition of HCO 3 . Plant Soil 125:39–47. Wingler A, Wallenda T, Hampp R. 1996. Mycorrhiza formation on Norway spruce (Picea abies) roots affects the pathway of anaplerotic CO2 fixation. Physiol Plant 96:699–705. Wullschleger SD, Ziska LH, Bunce JA. 1994. Respiratory responses of higher plants to atmospheric CO2 enrichment. Physiol Plant 90:221–229. Yang X, Romheld V, Marschner H. 1993. Effect of bicarbonate and root zone temperature on uptake of Zn, Fe, Mn and Cu by different rice cultivars (Oryza sativa L.) grown in calcareous soil. Plant Soil 155/156:441–444. Yang X, Romheld V, Marschner H. 1994. Effect of bicarbonate on root growth and accumulation of organic acids in Zn-inefficient and Zn-efficient rice cultivars (Oryza sativa L.). Plant Soil 164:1–7. Zhao J, Hogan EM, Bevensee MO, Boron WF. 1995. Out-ofequilibrium CO2 /HCO 3 solutions and their use in characterizing a new Kþ /HCO cotransporter. 3 Nature 374:636–639. Zheng SJ, Ma JF, Matsumoto H. 1998a. Continuous secretion of organic acids is related to aluminium resistance during relatively long-term exposure to aluminium stress. Physiol Plant 103:209–214. Zheng SJ, Ma JF, Matsumoto H. 1998b. High aluminium resistance in buckwheat: Al-induced specific secretion of oxalic acid from root tips. Plant Physiol 117:745– 751. Ziska LH, Hogan KP, Smith AP, Drake BG. 1991. Growth and photosynthetic response of nine tropical species with long-term exposure to elevated carbon dioxide. Oecologia 86:383–389. Ziska L, Bunce JA. 1994. Direct and indirect inhibition of single leaf respiration by elevated CO2 concentrations: interaction with temperature. Physiol Plant 90:130– 138.

41 Temperature Effects on Root Growth Bobbie L. McMichael and John J. Burke U.S. Department of Agriculture–Agricultural Research Service, Lubbock, Texas

I.

INTRODUCTION

ses. The water-holding capacity of these soils routinely exposes seedlings to water stresses through either lack of available soil water because of rapid drainage or by excess soil water associated with poor water movement through the soil resulting in the anaerobic conditions common to flooded fields. The formation of a soil crust following rain is an all-too-common physical barrier to seedling emergence, resulting in either delayed emergence or seedling death. Of all the stresses associated with germination, plant establishment, and subsequent plant development, temperature stress is probably the most common. Soil temperature affects seed germination and seedling development with both low- and high-temperature stresses commonly being observed in the seed bed. Plant productivity and economic yields are therefore impaired since these are directly related to plant establishment. In general, soil temperatures are lower than that of the air, with some exceptions such as in instances where the surface temperature can approach or exceed air temperature. Seasonal fluctuations in soil temperature are also evident (Fig. 1), with lower average temperatures occurring early and late in the season with the peak about day 200 (July 19). Baver et al. (1972) and Russell (1977) have shown similar patterns for different soil types and where different crops are growing. Kaspar and Bland (1992), in a review of soil temperature and root growth, point out seasonal changes in both air and soil temperature (Fig. 2), which emphasize not only the temporal changes in soil temperature but spatial (depth) changes as well. Rendig and Taylor

Soil temperature changes have a significant impact on the growth and development of plant root systems and ultimately on overall plant productivity. Numerous reviews have been written on the relationships of low and/or high temperatures on specific plant functions ranging from nutrient uptake and utilization to photosynthesis and carbon partitioning. Many of these topics were covered in the first edition of this book (Bowen, 1991). In our previous chapter (McMichael and Burke, 1996) we discussed the concepts of optimal temperature for root growth, genetic diversity in the response of plant roots to temperature, the temperature stresses encountered in soil environments, and the way these relate to the changes in root metabolic activity and ultimate crop yields. In the present chapter we shall update those topics as well as discuss some aspects of the impact of temperature on the interactions of plant roots with soil organisms, specifically with mycorrhizal associations.

II.

SOIL TEMPERATURE DYNAMICS

Usually crops are planted across a broad range of soil types, each with its own nutrient and water holding capacities. These different soil characteristics often result in either nutrient limitations that reduce plant development or in excess nutrient availability, resulting in toxicity to the plant and inducing salt or metal stres717

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sion, and latent heat of evaporation. Any property of the soil surface that affects these processes will have an impact on soil temperature. Most important in this respect are soil cover by a crop or crop residue, moisture content of the surface layers, and roughness of the surface. Therefore, management practices such as tillage and irrigation can impact the soil temperature and subsequently affect root growth and development. Barber and Kovar (1991) showed differences in soil temperature with depth for conventional versus ridge-tilled plots (Fig. 3). They indicated that although there were no significant differences between the tillage treatments, the soil temperature of the ridge-tilled plots tended to be higher. Irrigation or changes in soil water Figure 1 Seasonal changes in soil temperature at 10 cm for Lubbock, TX, 1994. (From Upchurch and Burke, unpublished data.)

(1989) also discuss this aspect in their review of soil temperature effects on root development. These spatial changes impact root growth by indicating particular zones favorable for root development and elongation during the season. Exchange of energy at the soil surface is the basis for soil heating or cooling. The main components of this energy balance are radiation absorption and emis-

Figure 2 (Top) Spring and summer warming of air temperature (5-year average of daily means for Temple, TX). (Bottom) Simulated soil thermal regime for the same location. (From Kaspar and Bland, 1992.)

Figure 3 Soil temperature at 2.5, 10, and 30 cm depths in maize plots during crop growth with conventional and ridge tillage. Values are the daily averages of measurements made every 5 min at four locations. (From Barber and Kovar, 1991.)

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content can also impact soil temperature changes and subsequently root growth. Average soil temperature from the surface down to 50 cm was significantly reduced as a result of early irrigation treatments (Wanjura, personal communication; Fig. 4). When the early nonirrigated plot was irrigated later in the season, a significant drop in soil temperature was recorded. The presence of cover crops can also influence soil temperature patterns. For example, when cotton was planted into wheat residue, the soil temperatures from the surface to 10–15 cm depth were warmer, and seeds germinated at a faster rate than when the cotton was planted in the presence of no residue (McMichael et al., unpublished). Many factors influence changes in soil temperature. Since other reviews have pointed out the overall impact of changes in root temperature on growth of plants in general, we shall concentrate in this chapter on other approaches to characterizing root temperature responses in relation to plant productivity. Specifically, we shall characterize the genetic diversity in temperature response both among and within species along with screening tools that can be used to evaluate such genetic differences. We shall also discuss root metabolic responses to changes in soil temperature in terms of enzymatic relationships. Finally, we shall discuss the interaction of soil microorganisms

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with plant root systems in terms of temperature responses specifically for mycorrhizal associations.

III.

GENETIC DIVERSITY IN TEMPERATURE RESPONSE

The growth of the root systems of plants is under genetic control. This genetic diversity is expressed in terms of plant response to changes in temperature both among and within species. In general, a temperature optimum for root growth for each species can be defined on the basis of changes in elongation rates, biomass production, and branching as well as of water and nutrient uptake characteristics and microbial interactions. A list of species and their optimum temperatures for root elongation and biomass accumulation presented by Glinski and Lipiec (1990) points out the genetic diversity among species (Table 1). The influence of changes on both root and shoot development of different species was described by Cooper (1973). McMichael and Quisenberry (1993) showed changes in the rooting dynamics of both cotton and sunflower as a function of changes in root temperatures (Fig. 5). There is also evidence that indicates genetic diversity within a species in the response of root systems to changes in soil temperature. Brar et

Figure 4 The effect of irrigation on changes in soil temperature during the 1994 season. T1 (solid symbols), nonirrigated early in season; T2 (solid symbols), irrigated early in season; T1 (open symbols), irrigated late in season; T2 (open symbols), continued irrigation late in season. (From Wanjura, unpublished data.)

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Table 1 Optimum Temperature for Root Growth

Plant

Optimum temperature (8C)

Direction of change

Sunflower (Helianthus sp.) Loblolly pine (Pinus taeda) Pecan (Cayra Illinoensis) Tomato (Lycopersicon esculentum Mill.) Maize (Zea mays)

Root Root Root Root Root

elongation elongation elongation elongation elongation

rate rate rate rate rate

20 20 30 30 30

Cotton (Gossypium hirsutum) Soybean Oats (Avena sativa)

Root elongation rate Taproot extension rate Root mass

Kentucky bluegrass (Poa pratensis) Strawberry (Fragaria sp.) Rape (Brassica napus) Oil palms (Elaeis guineenis Jacq.) White oak (seedlings) (Quercus alba) White oak (Quercus alba) Lolium perenne Rice (Oryza sativa) Maize (Zea mays) Agave deserti

Root mass Root mass Root extension Root growth Root growth Root elongation rate Dry weight Root growth Root mass Root elongation

33 25 5 (at maturity) 15 10–20
23 30–35 24
17 17 25–37 26 30

Reference Galligar (1938) Barney (1951) Woodroof (1934) White (1937) Anderson and Kempar (1964); Blacklow (1972) Arndt (1945) Stone and Taylor (1983) Neilsan and Humphries (1966) Brown (1939) Brouwer (1962) Moorby and Nye (1984) Agamuthu and Broughton (1986) Larson (1974) Teskey and Hinckley (1981) Clarkson et al. (1986) Kar et al. (1976) Walker (1969) Jordan and Nobel (1984)

Source: Modified from Glinski and Lipiec (1990).

al. (1990) showed that the optimum temperature for growth of the root systems of a number of forage legumes was significantly different among the genotypes evaluated. McMichael and Quisenberry (1986) have shown that there are differences in the response of seedling root systems of a number of cotton genotypes for both primary and lateral root growth. Quisenberry et al. (1981) showed similar results in more mature cotton plants. Thus, genetic diversity for temperature response as described for a number of species can provide the opportunity for improvement of plant growth under a wide range of environments. A.

Low-Temperature Response

If the temperature of the soil drops significantly from the optimum, then the structure and function of the root system may be altered. Root systems grown at temperatures lower than the optimum are generally smaller (Brower and Hoagland, 1964). The uptake of water is generally reduced and nutrient uptake may be affected as well (Neilsen et al., 1960; Nielsen, 1974). Cooper (1973) has shown that root extension rates of tomatoes, for example, are very low at 108C while root extension rates of other species such as pea are signifi-

cantly higher at the same temperature. Misra (1999) showed that roots of Eucalyptus globulus were more sensitive to temperature than those of E. nitens. In their work with cotton and sunflower seedlings, McMichael and Quisenberry (1986) showed that sunflower roots grew better at low temperatures (15–208C) than cotton (Fig. 5). Root systems grown at low temperatures are also less branched (Brower and Hoagland, 1964). Using magnetic resonance imaging (MRI) techniques for in situ measurements of root systems, McMichael et al. (unpublished data) observed significantly less branching in root systems of young cotton plants grown at 188C than at 288C (Fig. 6). Little information is available concerning genetic diversity in terms of differences within a species regarding response to freezing temperatures. However, work with individual species such as Scots pine have indicated that root freezing tolerance may be related to electrolyte leakage from the roots, but the methods used for measuring such leakage difference have limitations (Sattin and Lindstrom, 1999). Models have also been developed to assess freezing injury in alfalfa (Medico sativa) which incorporated effects of temperature but also of soil moisture and plant density (Kanneganti et al., 1998). In other studies, plant hormones such as ABA and certain ABA analogs have

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B.

High-Temperature Response

When root systems are exposed to temperatures that are higher than the optimum, both elongation rates (Arndt, 1937) and enzymatic activity of the root systems can be affected (Nielsen, 1974). Elongation rates of tomato roots were reduced at soil temperatures >308C, while elongation rates of white pine roots have increased at the same temperature range (Cooper, 1973). Sattelmacher et al. (1990) showed that in both heat-tolerant and heat-sensitive clones of potato (Solanum tuberosum) the size of the root system was reduced by what they referred to as supraoptimal temperatures (308C). They indicated that the size reduction was due to decreased number of lateral roots and to their reduced length. They also showed that the first indication of heat damage was a reduction in the rate of cell division, followed by a reduction of root elongation. A differential response to the production and growth of lateral roots in several cotton genotypes when grown at high (35–408C) temperatures was observed (McMichael unpublished data). Less difference in the growth of the primary or ‘‘tap root’’ was observed at the same high temperatures. The duration of exposure to the high temperature also appears to have an impact on the growth and development of root systems. Pardales et al. (1991) showed that the longer the roots of sorghum (Sorghum bicolor) remained at 408C, the greater was the inhibition of root growth when the plants were returned to 258C. Eidsten and Gislerod (1986) showed that a relatively short (30 min) exposure to temperatures >308C retarded the growth of roots of curly parsley and that a low daily average root zone temperature did not compensate for the damage caused by the short exposure to high temperatures. C. Figure 5 Development of root systems of 10-day-old cotton and sunflower seedlings as a function of temperature. (From McMichael and Quisenberry, 1993.)

been shown to be implicated in freezing tolerance of winter rye (Secale cereale) seedlings when applied to the roots of the seedlings.

Screening for Genetic Diversity

Many techniques have been developed to screen germplasm for diversity in response to changes in root zone temperatures. McMichael and Quisenberry (1991) found differences in root branching of 60-day-old cotton plants of several genotypes grown in the greenhouse. McMichael et al. (1987) demonstrated variability in root growth in 10-day-old cotton seedlings grown in polyethylene pouches under controlled conditions. This system is now utilized extensively as a screening tool for the impact of temperature on early plant development (Fig. 7A) (Stafford and McMichael,

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Figure 6 MRI images of sections of 30-day-old cotton plants grown at two different root temperatures. Images A and B represent side views of plants grown at 28 and 188C, respectively. Images C and D represent top views of the same plants. (From McMichael, MacFall, and Burke, unpublished data.)

1990). McMichael et al. (2000) evaluated the influence of root temperature on several cotton genotypes using root zone-controlled environment cabinets to control soil temperatures (Fig. 7B). These cabinets are capable of controlling root temperature independent of shoot temperature and may be utilized in a greenhouse or as an integral part of a larger, controlled-environment facility. Other screening tools have been utilized to address specific questions concerning genetic variability in, for example, cold tolerance. McMichael et al. (2000) adapted the screening procedures for relating cold tolerance as measured by differences in seedling emergence under different temperature regimens developed by Schulze et al. (1997) and Deusterhuas et al. (1999), to differences in cotton root develop-

ment grown under different temperatures. They showed a positive relationship between root length in a number of commercial cotton genotypes and the cold tolerance index; this is an index derived from relative emergence information when plants are subjected to both cold (58C) and warm (308C) temperatures during imbibition when plants were grown at either optimum temperatures (288C) or low temperatures (188C) (Fig. 8). In this case the relationship (changes in the root length relative to the index) was stronger for plants grown at 188C, suggesting a differential expression of the cold tolerance traits. The concepts developed for this technique should also be applicable to screening other species for genetic diversity in temperature response for root development.

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Figure 7 Methods utilized to evaluate genetic variability of root systems in response to changes in root temperature. (A) Growth pouch technique. (B) Root zone cabinet technique.

IV.

METABOLIC RESPONSE TO CHANGES IN SOIL TEMPERATURE

Cotton and sunflower seedlings produce maximal root numbers and root length development at different temperatures. One question that comes to mind is whether the temperatures reported to be optimal for seedlings are the same for root development at later stages of plant growth. The limited information from the studies by McMichael et al. (1986) and Quisenberry et al. (1981) suggests that the genotypes of cotton that had

Figure 8 Relationship between development of root length and cold tolerance index for 10 cotton genotypes. Rlexp1 represents plants grown for 10 days at 288C. Rlexp2 represents plants growth at 188C for 10 days. (From McMichael, Hopper, and Zak, unpublished data.)

improved root development at low temperatures at the seedling stage also performed well under low temperatures under field conditions when the plants were older. Studies with plants such as cucumber have suggested that although a metabolic process may have a specific temperature optimum, the range of temperatures over which this process occurs can be broadened by increasing the availability of substrate. It appears that the additional substrate helps to overcome identified temperature limitations associated with enzyme– substrate interactions. During the first 10 days following germination, large changes occur in available substrates, derived from the mobilization of seed reserves, for root growth. This is demonstrated by differences in the temperature sensitivity of the respiration rates, i.e., by in vivo reduction of 2,3,5-triphenyltetrazolium chloride (TTC) by cotton root tips between 3 and 7 days after planting (Fig. 9). More TTC reduction occurred <208C and >358C in the root tips of seedlings 3 days after planting, while similar rates of TTC reduction were observed at 25 and 308C (McMichael and Burke, 1994). The measurements of root development shown in Fig. 6 represent a composite value derived over the first 10 days following germination. The ability of the cotton roots at 3 days after planting at higher temperatures than 7-day-old roots suggests that the temperature optimum for root development is lower that determined from measurements of root length or root numbers during the first 2 weeks following germination. Other studies by Burke et al. (1988), Burke (1990), Mahan et al. (1990), and Ferguson and Burke (1991)

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Comparison of the reported thermal kinetic window for cotton of 23.5–328C (Burke et al., 1988) with the measured rates of TTC reduction at 3 and 7 days after planting (McMichael and Burke, 1994) shows that the level of TTC reduction in cotton roots of 7-day-old seedlings remained high at the temperatures within the TKW but fell at temperatures above or below the TKW (Fig. 9). Such data support the suggestion that elevated substrate levels, during the period of rapid seed reserve mobilization, provided an evolutionary advantage by broadening the temperature range for metabolism. If the root temperature is to be maintained under favorable conditions for longer periods of time for optimum root system development and for plant growth, then strategies that deal with changing the soil temperatures during the season or altering the temperature characteristics of the roots must be addressed. A. Figure 9 Effect of seedling age on the reduction of TTC in cotton roots exposed to various temperatures. (A) Threeversus 7-day-old seedlings. Vertical bars about each mean = 95% confidence limits. (B) TTC reduction in 7-day-old seedlings expressed as a percentage of the 3-day-old seedlings. (From McMichael and Burke, 1994.)

established a concept known as the thermal kinetic window for optimum enzyme function in plants. Specifically, the thermal kinetic window (TKW) is defined as the temperature range over which the apparent Km of glutathione reductase, an enzyme implicated in temperature stress response, is within 200% of the minimum Km for the enzyme. The purpose of the TKW was to provide a general indicator of the range of temperatures in which the optimal temperature for metabolism was located. The temperature ranges comprising the TKWs for wheat and cotton for enzyme activity were 17.5–238C and 23.5–328C, respectively. Although the TKWs in this case were 5–88C in breadth, it was shown that these plants were only within the optimal temperature range of their respective TKWs for at least a fraction of the growing season (Burke et al., 1988). These observations called for a reevaluation of our understanding of the temperature stresses experienced by plants in the field. Therefore temperature characteristics of cotton root metabolism were investigated by measuring root length, root numbers, and determination of the level of TTC reduction as previously described (McMichael and Burke, 1994).

Alteration of Soil Temperature in the Field

Alteration of soil temperature to coincide more favorably with the temperature characteristics of the plants is difficult to achieve on a field scale. Several approaches have been utilized in the past. However, attempts to achieve such a goal depends on whether one desires to cool or warm the soil. One approach has involved the use of both plant remains and artificial material such as plastics as a mulch to change the soil temperature. Indeed, some of the early work conducted with mulches showed that straw mulches, for example, tended to lower the soil temperatures (Cooper, 1973). On the other hand, plastic mulches tended to raise the soil temperature as well as to reduce the large fluctuations in temperature at various depths depending on the season. Mbagwu (1991), working with cassava plants, showed that yields were increased when plants were grown under plastic mulches and that the magnitude of the increases was cultivar dependent. Ham et al. (1993) also showed that soil temperatures were higher under mulches with high-shortwave absorbance, such as black plastics. Wein et al. (1993) also showed that root development in tomatoes was enhanced when plants were grown under plastic mulches. Other approaches to changing the soil temperature have included the use of power plant waste water to heat the soil by sprinkling the water on the surface (Rykbost et al., 1974) and various tillage practices (Gupta et al., 1982, 1983). Planting at various row spacings has also been utilized to induce changes in

Temperature Effects on Root Growth

soil temperature. In a study of the effects of cotton row spacing on soil temperature (Upchurch and Burke, unpublished data), it was found that the canopy closed much earlier in the narrow rows, thus shading the soil and reducing its temperature. The narrow rows may also be beneficial from the standpoint of reduced water loss from the soil surface. However, this advantage may be offset by the reduction of root growth as a result of lower soil temperatures. B.

Alteration of Soil Temperature in the Greenhouse or Soilless Culture

The alteration of soil temperatures under the more controlled conditions of a greenhouse or in soilless or hydroponic culture has been accomplished in a number of ways. McMichael (1998) and Zak and McMichael (2000) have used specially constructed cabinets that use forced air of a given temperature to control the temperature of roots growing in soil independent of the aboveground temperature. The cabinets were capable of controlling soil temperatures across a wide range of temperatures, depending on the research protocol. The chambers have also been used to control the temperatures of roots growing in polyvinylchloride tubes filled with nutrient solution. C.

Alteration of Root Temperature Characteristics

There are differences between species and cultivars in root growth characteristics and response to temperature. Significant differences were observed in root system development between a number of cotton genotypes grown under similar conditions (McMichael and Quisenberry, 1991; Table 2). The strategy for modification of the characteristics of the root system to overcome adverse soil temperatures can take at least two approaches. One way for reducing the inhibition of root growth by soil temperature stress is by growing a larger and more branched root system. Another approach is to alter genetically the function of the root system to be more tolerant to low soil temperatures. This may be accomplished by changing the lipid composition of the root cell membranes. Regardless of how changes in the temperature characteristics of the root system are realized, it is evident that much more research has to be done in this area of root physiology before germplasm developed to fit the prevailing soil temperature conditions will be available.

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V.

ROOT–SOIL–ORGANISM INTERACTIONS

There are many soilborne organisms that interact with plant root systems. These organisms can be broadly categorized into saprophytic organisms, such as bacteria and fungi, that can alter the availability of nutrients; pathogens such as Rhizoctonia, etc.; or symbiotic organisms such as Rhizobium or mycorrhizal fungi. The response of AM mycorrhizae (see Chapter 50 by Kottke in this volume) to changes in soil temperature will be addressed in the following section. Information on the impact of soil temperature on AM colonization is somewhat limited. In terms of low temperature response, Addy et al. (1977) showed that some fungal hyphae are capable of colonizing roots following soil freezing. They also showed (Addy et al., 1998) apparent acclimation of the fungi to low soil temperatures since the colonization was greater

Table 2 Average Root Dry Weights for 25 Cotton Genotypes Grown in Soil Under Greenhouse Conditionsa Genotype T184 T141 T252 T283 T1 T171 T256 T461 T25 T115 T15 T1236 T185 T80 T45 Paymaster 145 Deltapine 61 G. herbaceum Coker 5110 T151 T50 Tamcot CAMD-E T169 Pima S-5 (G. Barbadense) Lubbock Dwarf LSD (0.05)

Root dry weight (g) 3.95 3.86 3.30 3.12 3.11 3.08 3.07 2.83 2.80 2.79 2.74 2.73 2.47 2.45 2.36 2.16 2.15 2.15 2.05 2.03 2.01 1.91 1.87 1.77 1.63 0.47

Plants were
60 days old when harvested. Source: Modified from McMichael and Quisenberry (1991).

a

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when the soil was allowed to cool slowly. AM colonization was generally inhibited at soil temperatures <158C (Menge, 1984). McMichael and Zak (unpublished data) showed that AM colonization of cotton was reduced when plants were grown at 188C as compared to 288C. Borges and Chaney (1989) also showed that response to inoculation of Fraxinus pennsylvanica was delayed when root temperatures were 158C as opposed to the response at root temperatures of 25 or 358C. In general, higher temperatures up to the optimum temperature result in greater colonization of AM mycorrhizae (Daniels-Hetrick, 1984). It was that maximum colonization of soybean roots by mycorrhizal fungi occurred near 308C (Schenck and Schreder, 1974). However, in some species the highest level of colonization may occur at the lower temperatures (158C vs. 278C) (Forbes et al., 1996). Colonization was increased at higher temperatures only after a period of cold stress (Ferguson and Woodhead, 1982). Since it has been shown that improving root development in plants by manipulating soil temperature or genetically altering the temperature response of plants may improve plant performance under adverse conditions, it would appear that a similar scenario could apply to mycorrhizal colonization. However, in a preliminary study, Zak et al. (2000, unpublished data) found that AM colonization of different cotton genotypes that differed in cold tolerance when grown at low soil temperatures (188C) were less mycorrhizal than genotypes that were rated highly cold tolerant. The mechanisms for these effects have not been determined, and further research is needed to investigate these relationships.

VI.

IDENTIFICATION OF PROBLEM AREAS AND FUTURE RESEARCH NEEDS

Roots develop and function in dynamic thermal environments that change diurnally and seasonally. Because of the fluctuations in soil temperatures experienced in the field, roots seldom function under optimal and constant conditions. Attempts to alter root function in response to temperature result in small, incremental changes in growth or function. It is unlikely that a single genetic modification will result in improved plant development and plant function across the broad range of temperatures. Rather, it is more reasonable to assume it will impact one end of the temperature spectrum experienced by roots. Future efforts that combine genetic modifications of existing germplasm

with appropriate management procedures should help alleviate some of the thermal stress currently experienced by roots of field crops.

REFERENCES Addy AD, Miller MH, Peterson R. 1977. In effectivity of the propagules associated with extrafadical mycelia of two AM fungi following winter freezing. New Phytol 135:745–753. Addy AD, Boswell EP, Koide RT. 1998. Low temperature acclimation and freezing resistance of extraradical VA mycorrhizal hyphae. Mycol Res 102:582–586. Arndt CH. 1937. Water absorpson in the cotton plant as affected by soil and water temperature. Plant Physiol 12:703–720. Barber SA, Kovar JL. 1991. Effect of tillage practice on maize (Zea mays L.) root distribution. In: McMichael BL, Perrsson H, eds. Plant Roots and Their Environment. New York; Elsevier, pp 402–409. Baver LP, Gardner WH, Gardner WR. 1972. Soil Physics. 4th ed. New York; John Wiley and Sons. Borges RG, Chaney WR. 1989. Root temperature affects mychorrhizal efficacy in Fraximus pennsylvania Marsh. New Phytol 112:411–417. Bowen GD. 1991. Soil temperature, root growth, and plant function. In: Waisel Y, Eshel A, Kafkafi U, eds. Plant Roots: The Hidden Half. New York; Marcel Dekker, pp 309–330. Brar GS, Gomez JF, McMichael BL, Matches AG, Taylor HM. 1990. Root development of 12 forage legumes as affected by temperature. Agron J 82:1024–1026. Brouwer R, Hoagland A. 1964. Responses of bean plants to root temperatures. II. Anatomical aspects. Meded Inst Biol Scheid Onderz Landb Gewass 236:23–31. Burke JJ. 1990. Variation among species in the temperature dependence of the reappearance of variable fluorescence following illumination. Plant Physiol 93:652– 656. Burke JJ, Mahan JR, Hatfield JL. 1988. Crop-specific thermal kinetic windows in relation to wheat and cotton biomass production. Agron J 80:553–556. Burke JJ, Oliver MJ, 1993. Optimal thermal environments for plant metabolic processes (Cucumis sativa L.). Plant Physiol 102:295–302. Cooper AJ. Root Temperature and Plant Growth. Slough, U.K.: Commonwealth Agricultural Bureaux. Daniels-Hetrick BA. 1984. Ecology of VA mychorrhizal fungi. In: Powell CL, Bagyaraj DJ, eds. VA Mychorrhizal. Boca Raton, FL: CRC Press, pp 35–55. Duesterhaus B, Hopper N, Gannaway J, Jividen GM. 1999. Development of a laboratory screening test for the evaluation of cold tolerance in cotton seed germination. Proc Beltwide Cotton Prod Res Conf, National Cotton Council, Memphis, TN, 1:621–623.

Temperature Effects on Root Growth Eidsten IM, Gislerod HR. 1986. The effect of root temperature on growth of curled parsely. Plant Soil 92(1):23– 28. Ferguson DL, Burke JJ. 1991. Influence of water and temperature stress on the temperature dependence of the reapperance of variable fluorescence following illumination. Plant Physiol 97:188–192. Ferguson JJ, Woodhead SH. 1982. Production of endomycorrhizal inoculum: increase and maintenance of vesicular arbuscular mycorrhizal fungi. In: Schenck NC, ed. Methods and Principles of Mychorrizal Research. St. Paul, MN: American Phytopathology Society, pp 47–54. Forbes PJ, Ellison CH, Hooker JE. 1996. The impact of arbuscular mycorrhizal fungi and temperature on root system development. Agronomie 16:617–620. Glinski J, Lipiec J. 1990. Soil Conditions and Plant Roots. Boca Raton, FL: CRC Press. Gupta SC, Radke JK, Larson WE, Shaffer MJ. 1982. Predicting temperatures of bare- and residue-covered soils from daily maximum and minimum air temperatures. Soil Sci Soc Am J 46:372–376. Gupta SC, Larson WE, Linden DR. 1983. Tillage and surface residue effects on soil upper boundary temperatures. Soil Sci Soc Am J 47:1212–1218. Ham JM, Kluitenberg GJ, Lamont WJ. 1993. Optical properties of plastic mulches affect the field temperature regime. J Am Soc Hort Sci 118:188–193. Kanneganti VR, Rotz CA, Walgenbach RP. 1998. Modeling freezing injury in alfalfa to calculate forage yield. I. Model development and sensitivity analysis. Agron J 90:687–697. Kaspar TC, Bland WL. 1992. Soil temperature and root growth. Soil Sci 154:290–299. Mahan JR, Burke JJ, Orzech KA. 1990. Thermal dependence of the apparand Km of glutathione reductases from three plant species. Plant Physiol 93:822–824. Mbagwu JCS. 1991. Influence of different mulch materials on soil temperature, soil water content and yield of three cassava cultivars. J Sci Food Agric 54:569–577. McMichael BL. 1998. The influence of seed treatments on early root growth in cotton under different environmental conditions. Proc Beltwide Cotton Prod Res Conf, National Cotton Council, Memphis, TN, 2:1410–1410. McMichael BL, Burke JJ. 1994. Metabolic activity of cotton roots in response to temperature. Environ Exp Bot 34:201–206. McMichael BL, Burke JJ. 1996. Temperature effects on root growth. In: Waisel Y, Eshel A, Kafkafi U, eds. Plant Roots, The Hidden Half. 2nd ed, revised and expanded. New York: Marcel Dekker, pp 383–396. McMichael BL, Quisenberry JE. 1986. Variability in lateral root development and branching intensity in exotic cottons. Proc Beltwide Cotton Prod Res Conf, National Cotton Council, Memphis, TN, p 89.

727 McMichael BL, Quisenberry JE. 1991. Genetic variation for root–shoot relationships among cotton germplasm. Environ Exp Bot 31:461–470. McMichael BL, Quisenberry JE. 1993. The impact of the soil environment on the growth of root systems. Environ Exp Bot 33:53–61. McMichael BL, Quisenberry JE, Upchurch D. 1987. Lateral root development in exotic cottons. Environ Exp Bot 27(4):499–502. McMichael BL, Batson B, Boman R, Blasingame D, Colyer P, Edminston K, Roberts B, Sumner D. 2000. Cotton root health work group: preliminary determination of effects of seedling disease control strategies on crop maturity. Proc Beltwide Cotton Prod Res Conf, National Cotton Council, Memphis, TN, 1:592. McMichael BL, Hopper N, Zac JC, Duesterhaus B. 2000. Genetic variability for earlyroot growth, root development, and mychorrhizal association in cotton. Proc Beltwide Cotton Prod Res Conf, National Cotton Council, Memphis, TN, 1:589. Menge JA. 1984. Inoculum production. In: Powell CL, Bagyaraj DJ, eds. VA Mycorrhiza. Boca Raton, FL: CRC Press, pp 187–204. Misra RK. 1999. Root and shoot elongation of rhizotrongrown seedlings of Eucalyptus nitens and Eucalyptus globulus in relation to temperature. Plant Soil 206:37–46. Nielsen KF. 1974. Roots and root temperature. In: Carson EW, ed. The Plant Root and Its Environment. Charlottesville, VA: University of Virginia Press, pp 293–335. Nielsen KF, Halstead RL, Maclean AJ, Holmes RM, Bourgest SJ. 1960. The influence of soil temperature on the growth and mineral composition of oats. Can J Soil Sci 40:255–263. Pardales JR, Yamauch A, Kono Y. 1991. Growth and development of sorghum roots after exposure to different periods of a hot root-zone temperature. Environ Exp Bot 31:397–403. Quisenberry JE, Jordan WR, Roark BA, Fryrear DW. 1981. Exotic cottons as genetic sources for drought resistance. Crop Sci 21:889–895. Rendig VV, Taylor HM. 1989. Principles of Soil–Plant Interrelationships. New York; McGraw-Hill. Russell RS. 1977. Plant root systems—their function and interaction with the soil. London; McGraw-Hill. Rykbost KA, Boersma L, Mack HJ, Schmisseur WE. 1974. Crop responses to warming soils above their natural temperatures. Special Report 385. Corvallis, OR: Oregon State University Agriculture Experiment Station. Sattin E, Lindstrom A. 1999. Influence of soil temperature on root freezing tolerance of Scots pine seedlings. Plant Soil 217:173–181. Schulze D, Hopper N, Gannaway J, Jividen G. 1997. Evaluation of chilling tolerance in cotton genotypes.

728 Proc Beltwide Cotton Prod Res Conf, National Cotton Council, Memphis, TN, 2:1383–1385. Stafford RE, McMichael BL. 1990. Primary root development in guar seedlings. Environ Exp Bot 30:27–34. Sattlemacher B, Marschner H, Kuhne R. 1990. Effects of the temperature of rooting zone on the growth and development of roots of potato (Solanum tuberosum). Ann Bot 65:27–36. Schenck NC, Schroder VN. 1974. Temperature response of Endogone mycorrhiza on soybean roots. Mycologia 66:600.

McMichael and Burke Wien HC, Minotti PL, Grubinger VP. 1993. Polyethylene mulch stimulates early root growth and nutrient uptake of transplanted tomatoes. J Am Soc Hort Sci 118:207–211. Zac JC, McMichael BL. 2000. Effect of low soil temperature and soil disturbance on early root growth and mycorrhizal association in cotton. Proc Beltwide Cotton Prod Res Conf, National Cotton Council, Memphis, TN, 1:589–589.

42 Root Growth and Metabolism Under Oxygen Deficiency William Armstrong University of Hull, Hull, England

Malcolm C. Drew Texas A & M University, College Station, Texas

I.

chemical adaptations which enable plants to cope with the consequences of excess soil wetness. Particular emphasis is placed on the O2 concentration gradients in root and rhizosphere. Other reviews which might be consulted for a wider picture include Grable (1966), Armstrong (1979), Crawford (1992), Drew (1990, 1997), Armstrong et al. (1994a), Allen (1997), Jackson and Armstrong (1999), Jackson and Ricard (2001), and Greenway and Gibbs (2001a,b).

INTRODUCTION

All higher plants require water to be freely available for their establishment and survival. On the other hand, water can be a very effective barrier to gas exchange. In conjunction with oxygen-consuming processes it can, to varying degrees, help create oxygen deficiency in soils and roots and be detrimental to many plant processes, disturbing growth, nutrient and water uptake, and hormonal balances. Further to this, the total disappearance of oxygen from soils is often the prelude to microbially mediated anaerobic transformations of mineral and organic compounds with the creation of pools of often highly phytotoxic materials such as sulfides and the lower monocarboxylic acids. Excess water in the soil environment can thus prove harmful or even lethal for land plants, and in many parts of the world agricultural production is adversely affected by heavy seasonal rainfall and poorly draining soils. In Western Australia alone it has been estimated that winter waterlogging costs farmers tens of millions of dollars each year in reduced cereal yields and pasture production (Ayling, 1990). Indeed, much practical agriculture is associated, directly or indirectly, with ensuring adequate drainage and optimizing soil pore size distribution for plant roots (Russell, 1977). In this chapter we review the conditions which can lead to oxygen deficiency in roots including the potential for oxygen stress in nonsaturated as well as flooded soils, and examine the various morphological and bio-

II.

HOW ROOTS BECOME OXYGEN DEFICIENT

Any lowering of oxygen concentration to below ambient, whether in root or soil, requires some consumption and some impedance to flow. Water, which preferentially displaces air from soil pores, is the single most effective impedance to flow. There are two reasons for this: firstly, it slows the diffusion of oxygen to 1/10,000 of that in air, and secondly, it reduces its concentration to about 1/32 of that in air. The net result is an effective resistance to flow which is around 320,000 times greater in saturated soils than that of air. It should be noted, however, that the partial pressure of oxygen does not significantly alter at an air–water interface; consequently, there is often a preference to express data in terms of oxygen partial pressures to avoid air–water interface singularities. In soils there are further impedances to oxygen transport such as 729

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the solid matrix which occludes the diffusion path; also, the pore spaces through which the oxygen travels may be tortuous. Tortuosity lengthens the diffusion path and hence the impedance. Currie (1965) was able to show that because of low porosity and high tortuosity the effective gas diffusivity in dry soil crumbs could be as low as 1/40 of that in air, and hence if the crumbs were saturated their effective impedance to oxygen diffusion could be 12.8 million times that in air. How might the above translate into the development of oxygen deficiency in roots? To answer this it has to be appreciated that roots usually have two major sources of oxygen: (1) that which diffuses radially from the soil, and (2) that which diffuses along the intercellular gas space of the cortex from the shoot; other factors such as pressurized gas flow in roots or dissolved oxygen entering through water uptake are probably of minor importance (Armstrong, 1979; Jackson and Armstrong, 1999). Whether a root suffers some oxygen deficiency depends upon the efficacy and balance in the transport from these two sources (Armstrong, 1979; Laan et al., 1990; Visser et al., 1997). Radial oxygen profiles have been obtained (Armstrong et al., 1994b; Darwent, 1997) which show oxygen being simultaneously supplied to the epidermal–hypodermal cylinder of a maize root both from the cortex and from the rooting medium. Also, it is necessary to consider what constitutes an oxygen deficiency and be aware that oxygen deficiencies within roots may vary in their distribution and significance; e.g., a mildly oxygen-deficient meristem might well be of much greater significance for the functioning of a root than some stelar anoxia in subapical parts. In some plant organs, viz., legume nodules, oxygen deficiency may be a desirable property (Witty et al., 1987). Although in this chapter we do not consider these special cases, recent modeling work in this field is exciting and worth recording (Thumfort et al., 1994; Thumfort et al., 2000). Oxygen deficiency in a root arises when supply fails to satisfy fully the potential demand at some locus, and the tissue oxygen concentration at which deficiency is first experienced (the ‘‘respiratory’’ critical oxygen pressure: COPR) will depend upon the oxygen affinity of certain enzyme systems. In whole roots or even excised segments it is extremely difficult to determine exactly the tissue oxygen concentration at which the root suffers a deficiency: since cytochrome oxidase is generally the major respiratory enzyme and has a very high affinity for oxygen (Km 0.14 mmol m3 —partial pressure
0.01 kPa), reduced rates of oxygen consumption will not usually be detectable until tissue

Armstrong and Drew

oxygen concentrations are very low. The difficulty in determining critical oxygen pressures is further compounded by (1) the asymptotic nature of the consumption versus concentration curve; (2) the activity of other enzymes with higher Kms, but which might contribute only to a small degree to the total rate of oxygen consumption; (3) the experimental procedures which dictate that oxygen concentrations are normally monitored at some point remote from the experienced deficiency; (4) the volume of tissue experiencing the deficiency—only a small cylindrical core of tissue might be affected; and (5) the contribution to root oxygen supply made by the longitudinal oxygen movement from the shoot, which can be quite significant even in nonaerenchymatous roots (Armstrong, 1979; Armstrong et al., 1983; Visser et al., 1997). Regarding (3) (4), and (5), it is important to keep in mind that in experimental measurements of COPR, oxygen concentration is usually monitored outside the root in a bathing solution, whereas oxygen deficiency can be expected to occur first at the core of the root, e.g., in the meristem or at the center of the stele. Furthermore, the oxygen in the bathing solution is usually finite in amount, and in experiments with intact plants the lower this concentration becomes, the more likely it is that oxygen in the cortex supplied from the shoot will preferentially supply some of the oxygen demand previously obtained from the rooting medium. The rate of consumption from the external medium will therefore decline (Greenwood, 1968; Laan et al., 1990; Visser et al., 1997), and the prevailing oxygen concentration at which this starts could be interpreted by the experimenter as the COPR. However, in reality, there may be no overall change in oxygen consumption by the root. Because of these considerations, the literature presents a very confusing picture regarding critical oxygen pressures. A.

Radial Path and Unsaturated Soils

Despite the above qualifications, it is interesting to explore the potential for roots to become oxygen deficient and to consider the relative importance of the various parameters that may bring this about. A useful starting point is to assume that a root has grown beyond the length to which internal transport from the shoot can make a significant contribution. This is certainly ensured in respirometers where segments of excised roots are employed. For the isolated root obtaining all of its oxygen by radial transport the development of oxygen deficiency will depend upon the oxygen demand and oxygen diffusivities external

Oxygen Deficiency

to it and across it. In their pioneering work on COPR, Berry and Norris (1949) demonstrated how increasing the oxygen demand of excised onion root segments by raising temperature, increased the COPR for respiration as measured in the bathing medium. A rise of the temperature from 20 to 308C reduces the density of air, decreases oxygen solubility, and increases diffusion coefficients. The net result is that the effective impedance of a water path will be reduced by
10.5% and the potential oxygen flux increased by
6%. A counter to this, however, and far outweighing it, is the approximate doubling (Q10 ¼ 2; Atkin et al., 2000) of the respiratory oxygen demand. Oxygen deficiency in roots will thus be more likely to occur in warm soils than in cooler ones. Modeling has proved to be a useful way of illustrating the efficacy of radial oxygen transport to roots and predicting the likelihood of oxygen deficiency (Kristensen and Lemon, 1961; Lemon, 1962 a,b; Armstrong, 1979; DeWillegan and Van Noordwijk, 1984, 1989; Armstrong and Beckett 1985; Armstrong et al., 1991; Beckett and Armstrong, 1992); the models are equally applicable to isolated roots, roots in aerated soils or solution culture, or root segments in the bathing fluids of respirometers. Armstrong and Beckett extended the earlier approaches by treating the root–rhizosphere system as a series of concentric cylinders having different diffusive and oxygen demand characteristics: (1) the water-filled rhizosphere (RH) which could be partially occluded by solid matter; (2) the epidermal–hypodermal tissue cylinder (EH), which generally lacks gas space and where cell walls may be variously thickened, thus increasing impedance to oxygen diffusion; (3) a cortical cylinder (C) in which the presence of intercellular gas spaces having some degree of radial and circumferential connection very much minimize the radial impedance to oxygen transport; and (4) one or two stelar cylinders—e.g., an outer zone, nonporous and highly active, comprising the pericycle, phloem, and xylem elements, and an inner, less active but sometimes porous central parenchymatous cylinder (medulla). The total oxygen deficit,
T , developed radially across the root and rhizosphere is shown to be the sum of a series of deficits developed inwardly across each of the various cylinders such that
T ¼
RH þ
EH þ
C þ
S . For each cylinder, however, there are usually two components to the oxygen deficit, the first caused by the oxygen consumption within the cylinder itself, the second caused by the total oxygen consumed by any other cylinders lying closer to the center. For example an oxygen deficit

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across the cortex will arise not only because of cortical oxygen consumption along the cortical impedance (in situ deficit) but also because of oxygen consumed by the stele that must first pass across the cortex (throughflow deficit). Some predicted deficits dependent upon the dimensions of root and rhizosphere, and on oxygen demand in the root, are shown in Table 1. For simplicity, the rhizosphere has been modeled only as a water film, having resistance but no respiratory activity, and the stele has been treated as a single cylinder. Also, for simplicity, it has been assumed that the rate of oxygen consumption at any locus is constant until the point of oxygen extinction; this means that the COPR becomes the oxygen partial pressure at the edge of the boundary layer when anoxia first arises in the root—most likely at its centre. Provided there is a significant boundary layer resistance, results obtained by this oversimplification might not differ markedly in terms of COPR from those incorporating Michaelis-Menten kinetics and low enzyme Km values. On the other hand, the simplified modeling predicts the development of anoxic cores in the root, whereas with Michaelis-Menten models absolute anoxia is never predicted (Beckett and Armstrong, unpublished). There are several striking points to emerge from this type of analysis: 1. The radii of the various tissue cylinders and any boundary layer are of great importance: a stele of only 0.025 cm radius and respiratory demand of 15 nmol O2 cm3 s1 could develop alone an oxygen deficit of 8 kPa, but at double the radius the deficit would rise to 32 kPa, i.e., 1.5 times atmospheric levels. 2. Boundary layers, even without a microbe population, have considerable potential to limit the oxygen supply to the root, and a fall of nearly 8 kPa is forecast for a boundary layer thickness of 160
m around a 1-mm-diameter root. However, with a microbe population, a similar and additional in situ deficit would be easily attained. 3. Nonporous epidermal–hypodermal layers are also predicted as being very effective at reducing oxygen flow: the in situ deficit for a 225-
mthick cylinder could be as much as 12 kPa even without any of the extra cell wall thickening which can be a feature of these cell layers. The equivalent throughflow deficit across the same tissue could be as high as 14 kPa. However, this latter figure could never be realized in conjunction with the former, since again

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Table 1 Oxygen Deficits (
C) that Might Develop in the Stele (s), Cortex (c), Epidermis-Hypodermis (eh) and the Boundary Layers (BL) of Roots When Respiratory Demand Is Dependent Upon Radial Oxygen Diffusion from the Soil or Other Bounding Medium

Data derived from simple mathematical models in which the root is treated as a series of concentric cylinders within each of which the oxygen diffusion coefficient (D) and respiratory demands (Q) are considered as uniform and where oxygen consumption is constant at any locus until the point of extinction; the radius reh is the root radius. At 208C and normal pressure the fractional volume of oxygen in dry air is approx. 0.21 and the partial pressure 21.27 kPa; this would equate with an oxygen concentration in air-saturated water of 290 mmol l1 . Examples in parentheses from an associated series which could be added to any of the BL deficits.

Oxygen Deficiency

4.

they add up to a value which is greater than ambient. The cortex offers the least impedance to radial flow and cortical oxygen deficits can be expected to be very small in porous roots, although the respiratory demand contributes significantly to the deficits developed across the epidermal–hypodermal cylinder and any boundary layer.

Overall, it is clear from this analysis that even a small impedance to flow introduced on the root boundary might be sufficient to create hypoxia, or possibly something approaching anoxia, at the root center, and that the dimensions and activity of the root are also of the utmost importance. It follows that an unbounded root which was just totally aerobic would be likely to become anoxic at its center if any boundary layer whatsoever was imposed upon it. The radius of such a root has been described as its critical radius; similarly there would be a critical stelar radius, critical cortical thickness, boundary layer thickness, etc.; an increase in thickness of any of the tissue cylinders, an increase in temperature, or a lowering of the external oxygen concentration would result in the development of a severely O2 -deficient core. Consequently, among other things, increasing amounts of stress proteins that are indicative of severe oxygen deficiency (Sachs et al., 1980) will be detected. When this situation applies to the apical meristem, where oxygen consumption per unit volume of tissue is greatest, there is inhibition of respiratory metabolism and of dependent growth processes (Saglio et al., 1984). The modeling predictions accord with the findings of Saglio et al. (1984) that, at 258C, nutrient solution vigorously bubbled with air was scarcely enough to sustain aerobic respiration throughout excised maize root tips. In older parts of the root and coinciding with a much reduced oxygen demand, the critical oxygen pressure (COP) was approximately half that of the tip. It is evident, therefore, that the soil does not need to become anaerobic before there may be some detriment to root growth and function. On the other hand, it also follows from what has been said above that the thinner the root, the less likely it may be to suffer from oxygen deficiency. For example, a threadlike root 200
m in diameter and with a respiratory demand of 1.5 nmol cm3 root tissue s1 would not have an anoxic core even when separated from the air by a water cylinder as great as 6:9  1010 m thick, whereas if the root diameter were 2 mm, the equivalent water thickness would be reduced to
30
m (Armstrong, 1979).

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We see what may be evidence for this in the way in which plants that are unable to produce aerenchyma in their roots can tolerate waterlogged soils by means of shallow rooting with narrow roots (Justin and Armstrong , 1987). Improvements in microtechnique have recently allowed direct measurement of O2 concentrations in the root vicinity (Hojberg and Sorensen, 1993; Bidel et al., 2000) and in the root itself (Armstrong et al., 1994b; Ober and Sharp, 1996; Gibbs et al., 1998a) which also confirm the potential for root oxygen deficiency to occur in unsaturated soils. Hojberg and Sorensen (1993) quantified the concentration of O2 in the rhizoplane and rhizosphere of single roots of barley growing in a transparent gel, as well as the localized rates of O2 consumption using an oxygen microelectrode with a tip diameter 3–5
m. When the root was in a sterile medium the partial pressure of dissolved O2 at the root surface declined to about 9 kPa. With nonsterile medium, following inoculation with a soil extract, O2 concentrations at the root surface became low (2–4 kPa O2 ) because of the intense respiratory use by bacteria, especially close to the root surface, where respiration rates increased by a factor of 30–60, relative to the bulk medium. Although the diffusion characteristics of the medium were not identical to those of a well-drained soil, the results suggest that considerable gradients of O2 could occur in the rhizosphere and in the root itself. This has now been confirmed by a number of oxygen microelectrode studies. The oxygen profile shown in Fig. 1 is for an excised maize root (Gibbs et al., 1998a) in which the respiration is being supplied only by a radial inflow of oxygen from a bathing glucose-enriched nutrient solution streamed past the root. In this example the oxygen concentration in the bathing medium had been set to 4 kPa and had resulted in the development of an ‘‘anoxic’’ core with the stele. Where the oxygen concentration in the bathing medium was at air saturation (>20 kPa) oxygen partial pressure in the stele was everywhere > 10 kPa. Although no microbes had been added to the nutrient solution, it can be seen that there was a very significant drop in oxygen concentration in the 150
m of boundary layer close to the root caused by root oxygen consumption alone. Further to this, there is the relatively large decline across the nonporous epidermal–hypodermal zone as forecast by the modeling work, and an imperceptible decline across the cortex. It is interesting to note, however, that the pericycle of the stele remained aerobic but with its inner fringe at or near the Km for oxygen uptake. Separate experiments (Gibbs et al., 1998b)

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Figure 1 ‘‘Radial’’ O2 profile across an excised primary root of maize lying in a nutrient medium that was streamed lengthwise past it at a velocity of 50 mm min1 . The O2 concentration in the medium was 0.054 mol m3 (4.3 kPa); the profile was 75 mm from the apex and total root length was 135 mm. (Modified from Gibbs et al., 1998a.)

showed that this was associated with a 45% reduction in root pO2 and a concomitant decrease in net radial energy-dependent ion transport into the xylem. In unsaturated soils the soil air connects directly with the aerial atmosphere, but because there are particulate matter and water to impede transport, oxygen concentrations in the soil air decline with distance from the soil surface in response to the oxygen demands of the roots, the soil fauna, fungi, and microbes. In general, the oxygen deficits developed in the soil air itself should not be very great ( 5 kPa, i.e.,
5% O2 by volume; Currie, 1962). Nevertheless, it is obvious that this might in itself be critical, as might the presence of the soil matrix, and water and microorganisms in the immediate vicinity of roots. In aggregated soils the microporous structure may be water filled and the aggregates low in oxygen or even anoxic (Currie, 1961, 1962; Greenwood and Goodman, 1967; Smith, 1980; Sierra et al., 1995). Where root surfaces are directly in contact with the solid soil matrix, they are not exposed to the soil gases or soil solution and do not receive an oxygen flux. Such a situation would aggravate oxygen deficiency within roots, particularly those that lack an extensive volume of gas-filled space (Fig. 2) (De Willigen and Noordwijck, 1984; Armstrong and Beckett, 1985; Armstrong et al., 1991). Armstrong and Beckett (1985) concluded that nonporous roots ‘‘will require exceptionally well drained soils to function adequately.’’

Figure 2 Modeled E-W oxygen concentration profiles across a porous (upper) and nonporous root (lower) demonstrate the likely effects of circumferential blocking of root surfaces by impermeable soil materials on oxygen concentrations and distribution in roots. b ¼ length of blockage/total circumference. The blockage is centered on the left-hand yaxis (W). Note that where b ¼ 0:9 (upper) and b ¼ 0:25 (lower) the boundary conditions of the model have been breached and the profile diagrams exaggerate the extent of the anoxic region. (Modified from Armstrong and Beckett, 1985.)

Bacteria and fungi are probably the major consumers of oxygen along the radial diffusion path in the immediate vicinity of the root. However, the potential for root hairs to make a significant impact on root

Oxygen Deficiency

aeration should not be overlooked. In addition to providing microorganisms with respirable substrate, they are themselves a sink for oxygen, and, moreover, in some circumstances, they may play a role in causing soil wetness. McCully (1995) has reported that in normally dry sandy soils the rhizospheres of some plants saturate at night as a result of water exudation driven by the hydraulic lift of more deeply penetrating roots. Some indication of the enormous influence that this might have on root aeration can be deduced from Fig. 3. This figure shows the radial oxygen profile across a 1300-
m-thick root hair zone of an excised banana root lying in a moving stream of aerated culture solution. Although there was no impeding soil matrix and no significant numbers of microorganisms, there was an 11-kPa fall in oxygen partial pressure across the tomentum of root hairs. One effect of the root hairs was to increase the thickness of the unstirred boundary layer around the root, and oxygen consumption by the root itself was a large contributor to the 11kPa drop. However, if the zone had also been occupied by microorganisms to contribute to the oxygen demand and by a soil matrix which even only doubled the impedance, it is likely that the root would have

Figure 3 Radial O2 profile 28 mm from the tip of an excised adventitious root of banana (L ¼ 165 mm) lying in a stream of aerated nutrent solution (oxygen concentration 0.27 mol m3 , partial pressure 21 kPa) flowing past lengthwise at a velocity of 50 mm min1 . E þ H ¼ epidermis plus hypodermis. (Armstrong, Aguilar and Turner, unpublished data.)

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become seriously oxygen deficient. Even in dry soils, therefore, the diurnal development of oxygen deficiency in roots could perhaps be a common phenomenon. B.

Saturated Soils

Understanding how roots might become oxygen deficient in a fully saturated soil necessitates an understanding of the workings of the internal oxygen path from shoot to root and the peculiar characteristics of flooded soils. That roots should be more likely to experience oxygen deficiency in waterlogged as opposed to non-waterlogged soils seems obvious: flooding drives out most of the air from soils, and apart from surface layers, any oxygen remaining will soon be depleted by the respiration of roots and soil micro-organisms. Total depletion may require only a few hours (Turner and Patrick, 1968; Meek et al., 1983) to several days (Blackwell and Ayling, 1981; Blackwell, 1983), depending on soil temperature and the respiring biomass (roots and microorganisms). However, the situation in waterlogged soils is more complex than this, and the roots of wetland plants such as rice might be less likely to become oxygen deficient in flooded soils than those of most nonwetland species do in soils which might be temporarily wet but not waterlogged. The reason for this lies in the much enhanced gas space provision that can develop in most wetland plants. It is not uncommon in the subapical parts of wetland plant roots for as much as 60% of the root volume to be gas space and this provides a particularly low resistance path for diffusion (and to some extent convection) of oxygen from the shoot. The resistance offered to radial inflow of oxygen across the 80-
m nonporous epidermal–hypodermal cylinder of a 5-mm segment of root 1.1 mm in diameter could equate with a path length of
76 cm along the cortex of a 60% porous root of similar dimensions. Pioneering work on rice by Van Raalte (1941, 1944) and Barber et al. (1962) highlighted the potential of this gas space to enhance internal oxygen transfer from shoot to root oxygen transport; Van Raalte also showed that the ability of rice roots to oxidize methylene blue and to raise the redox potential of an anerobic rooting medium was dependent upon this internal oxygen transport and was probably the result of radial oxygen loss (ROL) to the rhizosphere. It eventually became possible to quantify locally these rates of ROL using a new type of polarographic electrode, the sleeving electrode (Armstrong, 1964). This

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showed that oxygen release tended to be greatest in the immediately submeristemic parts of the root, that rates were highest in plants most tolerant of reducing conditions and, in the case of rice (Armstrong, 1969), highest in those varieties more resistant to some physiological diseases thought to be caused wholly or partly by reduced soil conditions (Baba, 1963). Nevertheless, the fully saturated soil can be a particularly hostile environment even for wetland plants since oxygen depletion from the bulk soil sets in motion a series of chemical and microbiological activities which (reflected in the lowering of soil redox potential) results sequentially in nitrate reduction to nitrogen gas, manganese, and iron solubilization (Turner and Patrick, 1968), sulfate reduction to various sulfide species (Starkey 1966), and some hydrogen-dependent microbial reduction of carbon dioxide to methane (Rothfuss and Conrad, 1993). Additionally, organic matter is anaerobically decomposed, yielding among other things a whole suite of organic acids including the lower volatile monocarboxylic acids—formic, acetic, propionic, butryic, caproic, and valeric—which are highly toxic to plants, particularly at low pH (Rao and Mikkelsen, 1977; Armstrong and Armstrong, 1999), and methane (Rothfuss and Conrad, 1993). Reduced iron, manganese, and sulfides act as phytotoxins (Pezeshki et al., 1988; Snowden and Wheeler, 1993, 1995; Furtig et al., 1996; Armstrong et al., 1996b) but also as chemical sinks for oxygen (Begg et al., 1994; Saleque and Kirk, 1995). Thus, if root surfaces are permeable to oxygen, these reduced chemicals will act as a competitive drain on the oxygen supply from shoot to root. Oxygen leaking from the root into the rhizosphere can also stimulate aerobic microbes (Emerson et al., 1999) which participate in the transformation of toxins to less noxious products; methanotrophic microbes may also be supported (Gilbert and Frenzel, 1998). Some microbes may even increase the root’s permeability to oxygen (Ueckert et al., 1990). ROL by one species might help maintain the presence in waterlogged soil of less flood-tolerant species (Setter and Belford, 1990; Callaway and King, 1995). To summarize: ROL to the rhizosphere may render a root more likely to oxygen deficiency but, at the same time might be necessary to help afford protection from phytotoxins. The ROL-dependent chemical and microbially mediated reactions in the rhizosphere which afford the plant protection against phytotoxins are extremely complex, and our knowledge of them is still very rudimentary. Iron immobilization has received the most attention, and results have revealed consequences

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which extend far beyond a simple protective process. These include the lowering of rhizosphere pH and increased availability of phosphorus, zinc, and arsenic, and complexing reactions with other ions (Begg et al., 1994; Kirk and Bajita, 1995; Saleque and Kirk, 1995; Wang and Peverly, 1999; Zhang et al., 1999; Batty et al., 2000). However, experimentation is difficult and unless conditions are such as to allow the natural development of an oxygenated rhizosphere, results may have to be treated with the utmost caution. Where the root becomes overwhelmed by the phytotoxins, the consequences can be far reaching. Diseases of rice variously attributable to reduced iron and manganese, as well as to sulfide and the lower volatile fatty acids, have been extensively documented (Hollis et al., 1967, 1975); aluminum can also be involved (Jugsujinda and Patrick, 1993). Similarly, die-back of Spartina species in the United Kingdom and the United States and of Phragmites australis in several parts of northern Europe have been attributed to phytotoxins, the former to sulfide toxicity (Mendelssohn and McKee, 1988), the latter to a variety of causes and linked to sulfide and/or organic acids (Armstrong and Armstrong, 2001a). The exact mechanisms which bring about the death or decline of the plant in highly reducing soils are not fully worked out, but each of the phytotoxins can inhibit or kill root tips and variously interfere with other functions of the plant including photosynthesis. Sulfide is of course a potent respiratory poison while the lower monocarboxylic acids in their undissociated forms cause membranes to become leaky. Both effects will limit nutrient uptake and help increase water stress, as will stunted root growth. Other effects may be equally important, however. Studies on rice and Spartina patens Muhl. reveal a complex relationship between ROL soil redox potential, growth, and photosynthesis (Kludze and DeLaune, 1995). ROL increased with decreasing redox potential until a threshold was reached beyond which O2 release decreased in response to oxygen demand; photosynthesis was also depressed at the lowest redox potential. The authors attributed the effects to physiological and morphological changes including oxygen conductance by the plants. Results from experiments on Phragmites (Armstrong and Armstrong, 1999) and rice (Armstrong and Armstrong, 2001b) might explain these reductions in ROL and photosynthesis: sulfide and the volatile organic acids, both of which are associated with low redox potentials, induced (1) abnormal suberisation and lignification of hypodermal cells in the extension zone of the roots (normally the most oxygen-perme-

Oxygen Deficiency

able region), (2) internal callus which blocked the root aerenchyma, and (3) vascular blockages. Water uptake in Phragmites was severely curtailed, buds died, and the shoot systems senesced. It has been suggested that this train of events might become cyclic leading to extensive die-back in perennial vegetation (Armstrong et al., 1996a). As in an unsaturated soil, the likelihood of a root becoming oxygen deficient in a saturated soil depends chiefly upon the degree and distribution of two factors: physical resistances to transport and the oxygen demands. In the saturated soil, however, since the major oxygen source will normally be the shoot, the physical resistance is a function of root length as well as path tortuosity, path lengths, and diffusion coefficients in the various tissues of the root and in the soil; the oxygen demands take the form of oxygen scavenging by root tissues and by the chemical and microbial sinks in the rhizosphere. The major route for shoot-toroot transport of oxygen is the cortical gas space, and movement from this space is chiefly radial into the stele or into the epidermal–hypodermal layers and into the soil. Lateral roots provide an additional drain on the oxygen resources of a parent root (Armstrong et al., 1983); their presence also increases the resistance to gas phase diffusion down the parent root. Since all of the oxygen transported to the root must pass though it, the root–shoot junction might also be a significant impedance to oxygen transport (i.e., the throughflow deficits could be large) as may the shoot system, particularly if it is partially submerged (as is often the case with rice) or buried (as is the case with the rhizomes of rice and other species). More work is required to analyze the magnitude of these influences, but there is probably some gaseous continuum through the root–shoot junction in most species. Reports to the contrary should be examined very critically (e.g., Justin and Armstrong, 1983); it is usually possible to demonstrate the continuum by the bubbling of gases from the cut ends of submerged roots if the shoot has been cut and a pressurized gas supply attached to the cut end. As with the unsaturated soil condition, models have also been used to assess the likelihood of oxygen deficiency development in roots in flooded soils. The latest of these accommodates the multicylindrical anatomy of the root (Armstrong and Beckett, 1987; Sorrell et al., 2000; Beckett and Armstrong, 2001). Not surprisingly, they reveal that at some critical root length, hypoxic/anoxic cores will begin to develop at the root tip; such anoxic cores have since been detected experimentally (Thomson and Greenway, 1991; Gibbs et al., 1995). However, it can also be deduced

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that, because of increasing resistance to radial transfer to the stele due to the gradual lignification of the endodermis and stelar tissues, hypoxic/anoxic cores may develop in more basal regions even before they do so at the apex (Armstrong and Beckett, 1987). The models also show that, because of the high surface-to volume ratio of lateral roots and their relatively low porosities, the oxygen sink imposed by the soil may be very limiting to their extension growth. There is much experimental evidence for root oxygen deficiencies in flooded soils, ranging from the obvious limitations to extension growth, to reduced energy charges and the production of stress proteins, byproducts of anaerobic metabolism, lipid damage, hormonal changes, leakiness to solutes, and ultrastructural changes (Vartapetian and Jackson, 1997; Drew, 1997; Greenway and Gibbs, 2001a,b). III.

OXYGEN DEFICIENCY AND ROOT GROWTH, NUTRIENT AND WATER UPTAKE, AND ROOT–SHOOT SIGNALING

A.

Growth

Although some rhizomes are capable of anaerobic growth, roots, with few exceptions, very soon stop growing if the tip becomes anoxic, and this applies even to a species like rice (Webb and Armstrong, 1983). The critical oxygen concentration at which root extension is first affected (COPE), as with COPR, very much depends upon where the oxygen is being sampled (Armstrong and Gaynard, 1976; Armstrong and Webb, 1985). The values detected could be very high (and with intact roots, be sometimes erroneous) for the same reasons that apply to measurements of COPR. What might be responsible for the first slowing of root extension in response to lower oxygen concentrations is still uncertain and the reasons could be quite complex; e.g., it could perhaps be due to (1) a reduction in cytochrome oxidase-dependent respiration toward the center of the root tip/meristem, which might affect growth in various ways, e.g., cell division or water uptake and turgor (Amoore, 1961; Gibbs et al., 1998b); (2) the inhibition of another oxidase with a higher Km; or (3) an ethylene effect. Oxygen at below ambient levels is known to stimulate ethylene production. Ethylene in turn is known to stimulate aerenchyma production in some roots and can also affect root growth. Brailsford et al. (1993), who employed a new and highly sensitive photoacoustic laser technique to study the effects of oxygen pressure

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on root extension and ethylene generation in intact maize seedlings, concluded that enhanced ethylene production was not linked to the COPR. With the coleoptiles in air, and the roots isolated in a separate chamber in which the oxygen concentration was varied and ethylene evolution monitored, extension rate at 12.5 kPa was
75% of that in air, but ethylene production was not stimulated until the oxygen pressure was reduced to 5 kPa at which point roots become swollen and plagiotropic. At 1 kPa oxygen there was still some growth but no enhancement of ethylene production or growth abnormalities. Brailsford et al. (1993) suggested that the higher Km of IAA oxidase might have been responsible for the obviously high COPE but, as has been pointed out, an anoxic/hypoxic core could exist with an oxygen partial pressure of 12.5 kPa at the root surface and hence the COPE could still have been linked to cytochrome oxidase. Shoot-to-root gas exchange in these experiments was probably exceedingly limited because of the resistance imposed by the coleoptile sheath (Thomson and Armstrong, 1990) and impeded ethylene loss via the shoot could have contributed to the growth abnormalities when exposed to 3–5 kPa oxygen. The results are exceedingly interesting, however, in revealing that ethylene could have such major effects on growth at intermediate oxygen concentrations, and clearly there is much to be learned about where the stimulus for increased ethylene production or its precursors is located. Using the photoacoustic laser technique, it has been shown for two Rumex species, one aerenchymatous the other nonaerenchymatous, that the apparent COPE (monitored in the root chamber) can be significantly lower if the shoot remains in air and the oxygen is reduced only around the root (Visser et al., 1997). It was concluded also that aerenchyma in R. palustris roots allows the rapid escape of endogenously produced ethylene and prevents the accumulation of growth-inhibiting levels, while in the nonaerenchymatous R. thyrsiflorus the trapping of endogenously produced ethylene might be as inhibiting to growth as the low oxygen per se. The authors concluded that high CO2 concentrations in the roots and rooting medium (up to 10 kPa) did not greatly affect elongation. An alternative means of measuring both COPR and COPE is to bathe the roots of intact plants in an anaerobic medium and to monitor ROL from the roots using sleeving polarographic electrodes 2–7 mm behind the tip (i.e., avoiding the root cap) (Armstrong and Gaynard, 1976; Armstrong and Webb, 1985). The oxygen regimen in the root can be controlled by exposing the shoot to appropriate gas mixtures, and root exten-

Armstrong and Drew

sion can be followed by means of a traveling microscope. These electrodes are a very efficient and quantifiable sink for oxygen, provide accurate values of oxygen partial pressure on the root surface, and, if allowance is made for the oxygen deficits developed radially out across the epidermal–hypodermal cylinder, tolerable estimates of the oxygen partial pressure within the root cortex are possible. For rice the COPE estimated in this way was revealed to be extremely low ( 0.8 kPa); effectively it is the critical oxygen pressure of the stele/meristem rather than a value which encompasses the deficits developed across the epidermal–hypodermal cylinder and any boundary layer external to the root. If the oxygen had been supplied only radially, it was estimated that the COPE would have appeared to have been > 3.5 kPa. Rice roots generally have a narrower stele and thinner epidermal–hypodermal cylinders than maize and hence would normally be expected to have a lower COPE. It has been suggested that growth below the COPE may involve metabolic cooperation in the form of symplastic transport of energy-rich compounds between cells receiving adequate oxygen supply and cells in anoxic zones (Gibbs et al., 1995). B.

Nutrient and Water Uptake

Low oxygen concentrations in the root environment strongly inhibit ion uptake by roots and transport to the shoot (Drew, 1988). In consequence, soil-grown plants usually show marked depression of N, P, and K concentration in the foliage (Letey et al., 1961, 1962, 1965; Leyshon and Sheard, 1974). Such changes in concentration of mineral nutrients in leaves were detected within 48 h, long before the concentration of nutrients in the waterlogged soils had changed (Drew and Sisworo, 1979; Trought and Drew, 1980a,b). A comparable inhibition of ion transport to shoots occurs in deoxygenated nutrient solutions (Trought and Drew, 1980c), which induces a precocious redistribution of phloem-mobile elements from older to younger leaves, with early chlorosis or senescence of the older leaves. However, nutrient starvation of leaves is not always the case: in potted tomato, transfer of phosphate in the xylem sap increased during flooding of the root system (Else et al., 1995), perhaps through release of P from O2 -starved, injured cells of roots and submersed stem base. The link between oxygen supply and ion transport is principally through respiration and the generation of ATP to drive transport. Anaerobic metabolism does not maintain energy metabolism at a level that will

Oxygen Deficiency

drive primary active transport via the Hþ -translocating ATPase in the plasma membrane. Some ion transport can take place anaerobically, but this is thermodynamically passive, comprising the ‘‘downhill,’’ inward movement of cations in response to the membrane potential (Cheeseman and Hanson, 1979). Reduced transpiration flux under anaerobic conditions would also reduce ion transport to the shoot through mass flow reduction. Among the cations, sodium is unusual in that its concentration or ion activity within root cells of glycophytes is usually lower than expected on the basis of a passive equilibrium with Naþ in the outer solution (Higinbotham et al., 1967; Jeschke, 1984). Since plasma membranes are permeable to Naþ , the low intracellular activity of Naþ must imply that energy is used in an outwardly directed, active transport of Naþ . Evidence of a Kþ -dependent efflux of Naþ is consistent with that assumption (Jeschke, 1984). Low concentrations of O2 thus inhibit the active efflux of Naþ , and the ability of roots to exclude this ion from the leaves is lost. Transport of Naþ to the leaves of maize is greatly enhanced under such conditions, while active transport of Kþ is inhibited, so that the ratio Naþ /Kþ can increase by a factor of 100 or more compared with aerobic plants (Drew and Dikumwin, 1985; Drew and Lauchli, 1985). Over the concentration range 1.0–50 mol m3 NaCl in the nutrient solution, the ratio of Naþ /Kþ translocated to shoots of maize increased by a factor of 860 for anoxic roots, a clear measure of their inability to control fluxes of Naþ (Drew et al., 1988). Such an abnormally large accumulation of Naþ with simultaneous depression of Kþ is likely to cause interference in stomatal regulation (Devitt et al., 1984). Lack of aeration thus can exacerbate the problems of regulation of ion fluxes to the shoots in saline media. Field and greenhouse observations bear out this expectation: Naþ and C1 concentrations in foliage become greater with poor root aeration (Letey et al., 1962, 1965; West and Taylor, 1984), and plant growth is slowed and shows obvious signs of damage by excess salts (Aubertin et al., 1968; West and Black, 1978; West and Taylor, 1980, 1984). Whereas the drastic effects of root anoxia on solute transport are relatively well understood, the effects of hypoxia may be more subtle, especially in relatively large-diameter roots where gradients of O2 concentration can establish. Maize roots were made hypoxic by exposure to gas mixtures containing 4% or 1.5% O2 , and concentration of dissolved O2 across the radial pathway to the xylem was determined by O2 microelectrode (Gibbs et al., 1998a). The cortex remained

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sufficiently oxygenated to support oxidative phosphorylation while the stele became anoxic. Under these hypoxic conditions, energy-dependent transport of 36  Cl to the xylem sap was reduced to 61% and 28% of the normoxic controls, at the two lower concentration of O2 , respectively. Accumulation of 36 Cl by the root tissue, composed mainly of cortical cells, was less markedly inhibited by hypoxia, but when the whole root was made anoxic, 36 Cl transport to the xylem and accumulation by root tissue were abolished completely. Such observations underline the importance of the oxygenation and energy status of the xylem parenchyma (xylem border) cells in solute transport to the xylem and thence to the shoot. Wilting is a common observation in plants with inadequate soil aeration. Laboratory investigations of the hydraulic conductivity of whole root systems under conditions of extreme O2 deficiency have shown a rapid initial decline (Mees and Weatherly, 1957; Everard and Drew, 1987, 1989), which might account for the restricted supply of water to leaves. Hypoxia has a less drastic effect, with only relatively minor restrictions to water transport in excised roots with hydrostatic or osmotic driving forces, followed by recovery after 4–6 h (Gibbs et al., 1998b). A large component of water flux across root membranes is mediated by aquaporins (Chrispeels et al., 1999; Johannson et al., 2000). Daily (circadian) variation in hydraulic conductivity in normoxic roots of Lotus japonicus (Regel) K. Larsen accords with daily variation in levels of both aquaporin mRNA and aquaporin protein (Henzler et al., 1999). The participation of aquaporins and their apparent rapid turnover provide an explanation of how inhibition of protein synthesis under anoxia might quickly limit water flux in roots.

C.

Root Oxygen Deficiency and Root– Shoot Signaling

Coordinated growth of shoots, roots, and other organs depends on an interchange of signals (hormones and other metabolites), although the details of how they are transduced and coordinated on the scale of a whole plant are poorly understood. It is well recognized that oxygen deficiency inhibits synthesis of indolylacetic acid (IAA), gibberellins, and cytokinins by roots (Reid and Bradford, 1984), while the concentration of abscisic acid (ABA) in the xylem sap increases (Zhang and Davies, 1987). In this section we examine evidence of a role for some of these changes in regulating whole-plant responses to oxygen stress.

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A rise in the concentration of free ABA in leaves has been detected in a range of species following flooding of the rooting zone (Hiron and Wright, 1973; Wadman–Van Schravendijk and Van Andel, 1985; Jackson and Hall, 1987; Chapter 26 by Hose et al. in this volume). This increase offers a ready explanation for the declines in stomatal conductance and leaf growth that take place with flooding (Pereira and Kozlowski, 1977; Jackson et al., 1978; Sojka and Stolzy, 1980; Bradford and Hsiao, 1982). However, there is less unanimity about the process by which ABA levels increase. In Phaseolus vulgaris, a major increase in free ABA coincided with decreases in leaf water potential and turgor (Wadman–Van Schravendijk and Van Andel, 1985), suggesting that a decline in leaf water status was a possible trigger for ABA release and for stomatal closure. However, in Pisum sativum (Zhang and Davies, 1987; Jackson and Hall, 1987) and in Lycopersicon esculentum (Bradford and Hsiao, 1982), stomatal conductance and leaf growth were inhibited even when the leaf water status remained unchanged. It is widely recognized that increases in leaf ABA originate both from a release of ABA sequestered in chloroplasts and from increased concentration of ABA in the xylem sap delivered to the leaves by the roots when the latter are under stress (Taiz and Zeiger, 1998). Bulk measures of ABA provide a poor index of the significance for stomatal regulation, which is sensitive to the ABA concentration in the apoplast adjoining the guard cells (Zhang and Davies, 1987; Wilkinson and Davies, 1997). Some researchers have focused on the absolute rate of delivery of ABA from roots to leaves, which in flooded tomato declines because of the slowing of water uptake and transpiration (Else et al., 1995, 1996; Chapter 26 by Hose et al. in this volume). However, guard cell regulation may be more in tune with the current concentration of ABA supplied to the leaf apoplast, which is increased by root flooding. One uncertainty is the influence of xylem sap pH during root flooding. In water-deficient Commelina communis, xylem sap pH increases, favoring a greater buildup of apoplastic ABA around guard cells, with concomitant stomatal closure (Wilkinson and Davies, 1997), and it would be interesting to know if comparable changes occur with root flooding. One of the best-characterized examples of rootsourced hormonal modification of shoot morphology is that of leaf epinasty (Jackson, 1985; Jackson, 1997). Oxygen deficiency stimulates ACC production by roots, and the ethylene precursor is carried in the transpiration stream to the leaves. Here, the availability of

Armstrong and Drew

O2 (required for the conversion of ACC to ethylene), together with enhanced activity of ACC oxidase in the leaf petiole, accelerates ethylene production (English et al., 1995; Else and Jackson, 1998; Chapter 27 by Hussain and Roberts in this volume). The extra ethylene enhances cell expansion on the upper (adaxial) side of the petiole so that the leaf is forced to bend downward, thereby minimizing interception of light and transpirational losses.

IV.

COMBATING OXYGEN DEFICIENCY

Some of the reactions to oxygen deficiency and ways of accommodating and combating it have already been touched upon. In the following sections we consider (1) the part played by shoots in facilitating root and rhizosphere aeration in flooded soils, (2) the role of aerenchyma in helping roots avoid/minimize internal oxygen deficiency and combat the consequences of soil anaerobiosis, and (3) metabolic adaptations to help withstand oxygen deficiency and the damaging free radicals that can be produced where aerobic conditions are reestablished. A.

Improvements in Gas Exchange

1.

Shoot-Facilitated Exchanges

Except for special cases such as mangrove pneumatophores and emergent stilt roots (Andersen and Kristensen, 1988; Hovneden and Allaway, 1994), or where the surface layers of the soil may be aerated enough to support the growth of fine (Armstrong, 1979; Justin and Armstrong, 1987) and sometimes apogeotropic roots (Armstrong and Boatman, 1967; Ellmore, 1981; Periera and Kozlowski, 1977), the shoot system is essential for supplying oxygen to the roots if sediments are water saturated. For greatest efficiency the shoot should offer as little resistance as possible to gas flow, and this is often achieved by the presence of extensive aerenchyma in stems and leaves (e.g., rice); in some plants the root–shoot junction is especially porous (Armstrong and Armstrong, 1990), and in some the pith cavity of rhizomes and aerial shoots may play an important role in transport (Armstrong et al., 1996c). Shoot systems can also facilitate the escape of gases such as carbon dioxide, ethylene, and methane from the roots and, via the roots, the sediment (Dacey and Klug, 1979; Sorrell and Boon, 1994; Banker et al., 1995; Brix et al., 1996; Visser et al., 1997; Butterbach-Bahl et al., 1998; Yavitt and Knapp, 1998). Where diffusion predominates in shoot–root gas

Oxygen Deficiency

exchange, those pores just above the water line are likely to be the principal entry and exit points (Armstrong, 1979; Harden and Chanton, 1994). With convective gas flows, however, oxygen may be driven below ground by pressure flow from entry points well above the water line (Armstrong and Armstrong, 1991; White and Ganf, 2000). The chances of oxygen deficiency developing in roots as a consequence of the whole or partial submergence of shoot systems or, in the case of rhizomes, because of their belowground habit, may be partly offset by convective gas flows generated by exposed leaves, leaf sheaths, or shoots (Dacey 1981; Armstrong et al., 1992; Brix et al., 1992); by photosynthetic activities (Gaynard and Armstrong, 1987; Waters et al., 1989; Christensen et al., 1994; Connell et al., 1999); or (to a very small extent) by convective gas flows generated by the roots (Koncalova et al., 1988). Some convections generated in the shoots can have very marked effects on root aeration, often increasing the oxygen concentrations at the root– shoot junctions to near-ambient levels (Armstrong

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and Armstrong, 1990; Armstrong et al., 1992). However, the effects of nonthroughflow convections detected in deep-water rice have been shown to be relatively insignificant (Beckett et al., 1988), and pressurized gas flow into submerged tree roots (Grosse and Schroder, 1985; Grosse et al., 1992) is likely to be transitory and of no real significance in alleviating potential oxygen deficiency (Armstrong et al., 1994a). Throughflow convections from emergent leaves and shoots, and photosynthetic activities in the case of wholly or partially submerged plants, may lead to considerable diurnal fluctuations in the oxygen regimen within the roots and the rhizosphere (Gaynard and Armstrong, 1987; Waters et al., 1989; Pedersen and Sand-Jensen, 1992; Pedersen et al., 1995; Christensen et al., 1994; Connell et al., 1999). Depending upon a whole complex of factors, root tips might be close to anoxia at night with their growth ceasing (e.g., in submerged deep-water rice seedlings: Fig. 4), while in the daytime because of photosynthesis their may be no oxygen deficiency and root growth will resume. The differential solubilities of O2 and CO2 mean that

Figure 4 Photosynthesis-dependent diurnal variation in the oxygen regime in the root apices of intact rice plants submerged at 328C. The roots were in stagnant anaerobic medium and the water around the shoots was bubbled with gas mixture as shown. Upper two graphs show root extension; the lower two show root surface oxygen partial pressures 2–7 mm behind the tip dependent upon radial oxygen loss to a polarographic electrode sheathing the root. (From Armstrong and Setter in Armstrong et al., 1994 but see also Waters et al., 1989.)

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photosynthetically generated O2 is partially trapped in the plant. This increases the concentrations, and more oxygen is transmitted to the roots to alleviate any deficiencies. Thus, in flooded rice fields photosynthesis may be critical (Ram et al., 1999; Krishnayya et al., 1999). Krishnayya et al. (1999), found that the survival of a submergence-intolerant rice cultivar increased from 0% to 17% to 62% as pH was lowered from 8 to 7 and to 5 and as CO2 concentrations were accordingly increased from 0.02 to 0.3 to 1.0 mol m3 . The consequences of fluctuating oxygen regimens in root and rhizosphere need much more study. For normally emergent species it is essential that some connection with the aerial environment be maintained both to maintain a positive carbon balance and to avoid severe oxygen deficiency in belowground parts. Rhizome tip growth in some species, and early seedling development in others, can proceed anaerobically until the shoots reach better-illuminated and better-aerated zones close to the water surface and preferably above it (Brandle and Crawford, 1987; Pearce and Jackson, 1991; Summers and Jackson, 1994; Voesenek and Blom, 1999); however, underwater photosynthesis of normally emergent plants may remain critically low unless submerging waters are rapidly moving or the dissolved CO2 concentrations are high (Gaynard and Armstrong, 1987; SandJensen et al., 1992; Sand-Jensen and Christensen, 1999; Ram et al., 1999; Krishnayya et al., 1999). Some plants such as the deep-water rice varieties, and species of Rumex and other wetland genera, which are often subject to total submergence by rising water tables, can rapidly regain their connection to the atmosphere by a fast shoot or petiole elongation. Oxygen deficiency (rather than anaerobiosis) and impeded gas exchange brought about by the submergence have been identified as likely triggers for this response involving as they do an acceleration of ethylene production and accumulation as well as interactions with other hormones (Voesenek and Blom, 1999). 2. Aerenchyma, Root Permeability, and Rhizosphere Oxygenation a. Aerenchyma Formation A majority of plants, whether of wetland or nonwetland origin, have a continuum of gas space in the root cortex which usually arises within a few microns of the root–root cap junction, connects with the spaces in the shoot cortex at the root shoot junction and ultimately with the atmosphere through the stomata or lenticels of the shoot (Armstrong, 1979). Collected illustrations

Armstrong and Drew

of the whole gas space continuum of individual species are rare; examples are Eriophorum angustifolium Roth (Gaynard and Armstrong, 1987) and Phragmites (Armstrong and Armstrong 1988, 1990; Armstrong et al., 1996d). Useful papers dealing with integrity and maintenance of the gas space system are Raven (1996), Michael et al. (1999), and Soukopp et al. (2000). Enlargement of this gas space provision to form aerenchyma is one of the most important adaptations which help prevent the onset of oxygen deficiency in growing roots and can be both primary and secondary in origin (Arber, 1920; Justin and Armstrong, 1987; James and Sprent, 1999). Primary aerenchymas are the most common in roots and broadly fall into two types: lysigenous, where cells die and ultimately collapse radially or tangentially leaving large longitudinally running voids; and schizogenous, where particular patterns of growth, often with oblique division of cells, are accompanied by their separation but not collapse. Again this leads to the development of large longitudinally running voids. Seago et al. (2000) have suggested that the ‘‘honeycomb’’ aerenchymas found in plants such as Rumex and Nymphaea would be more accurately classified as ‘‘differential expansion aerenchyma’’ rather than schizogenous. The cell collapse which characterizes lysigenous aerenchyma is usually considered to result in or be coincident with the death of the collapsed/collapsing cells and possibly to be a consequence of programmed cell death. Those files of cells which fail to collapse have been shown to remain viable (Armstrong and Armstrong, 1993), but now Longstreth and Borkhsenious (2000) have obtained evidence that, in some species at least, even the collapsed cells can retain intact plasmalemmas and mitochondria for extended periods. Although we know more about the intitiation of the lysigenous types, both appear to be inducible, but to varying degrees. At the one extreme aerenchyma development can appear constitutive (Jackson et al., 1985a), but even in rice there are varieties which show some plasticity of response (Das and Jat, 1977; Justin and Armstrong, 1987; Colmer et al., 1998; Chapter 56 by Beyrouty in this volume). Other plant species are highly flexible, normally developing aerenchyma only when the roots are growing in stagnant media. Other species, including many nonwetland plants, may never form aerenchyma (Justin and Armstrong, 1987). Some plants which form aerenchyma in the primary root tissues may lack the ability to produce aerenchyma during secondary growth. It has been suggested that this excludes most woody species from wetland habitats (Justin and Armstrong, 1987). In plants that show flexibility in

Oxygen Deficiency

terms of aerenchyma formation, nonaerenchymatous roots produced under drained conditions tend to be replaced by new aerenchymatous roots after flooding (Laan et al., 1989; Thomson et al., 1990). Lysigenous aerenchyma formation in the root cortex of maize is triggered by enhanced internal concentrations of ethylene and provides a model system for the study of programmed cell death (Drew, 1997). Induction of cell death followed by lysis are triggered by a rise in internal concentrations of ethylene, resulting from its accelerated production under hypoxic conditions. Ethylene-dependent cell death can be blocked by inhibitors either of ethylene action (with low, nontoxic concentrations of Agþ ) or of ethylene biosynthesis (AVG) (Jackson et al., 1985b). The oxygendeficient environment associated with flooding, and the presence of exogenous, soil-derived ethylene (Smith and Restall, 1971), may both contribute. Hypoxia leads to high levels of activity of (ACC) synthase in root tips (He et al., 1994, 1996b) with additional ACC accumulation (Atwell et al., 1988), indicating that the biosynthetic pathway is stimulated. Cellulase activity in the tip region is also augmented, in response to the triggering affect of ethylene, and presumably is part of the subsequent lytic process in which cell walls as well as protoplasm disappear (He et al., 1994). Transduction of the ethylene signal appears to involve a phosphoinositide signaling pathway, cytosolic Caþ 2 , and phosphorylation by a protein kinase (He et al., 1996b; Drew et al., 2000). However, little is known about the mechanism by which low oxygen partial pressures stimulate the synthesis of enzymes of the ethylene biosynthetic pathway (see Chapter 27 by Hussain and Roberts in this volume). Because of the involvement of ethylene in the development of lysigenous aerenchyma, the mere presence of a barrier to the radial loss of endogenously produced ethylene might be sufficient to induce the initiation of aerenchyma. Ethylene is a very insoluble gas, so the stagnant water regime in a flooded soil might function in this way, as also might the high radial resistance to radial gas exchange which develops subapically in the hypodermal cells in many wetland plant roots including rice (Armstrong, 1971; Colmer et al., 1998) and Phragmites (Fig. 5). Gas space formation in hypoxic roots involves loss of many cortical cells. An impairment in root function as a result of loss of the usual pathway for radial ion transport might have been expected. However, direct measurements of radial transport of Kþ or of H2 PO 4 by roots with or without gas spaces reveals no inhibition of such function (Drew and Saker, 1986).

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Figure 5 Radial O2 profiles through an intact adventitious root of Phragmites at 100 mm (A), 29 mm (B) and 7 mm (C) from the apex. The root (L ¼ 160 mm) was secured horizontally 2–3 mm below the surface of an O2 -depleted fluid agar across which O2 -free nitrogen was gently streamed to create a constant oxygen sink; the leafy shoot was fully exposed to air; E þ H ¼ epidermis plus hypodermis (Modified from Armstrong et al., 2000.)

Relatively few files of intact cells and radial strands of walls of lysed cells apparently provide sufficient inflow of nutrient ions, and this function might be maintained by the presence of passage cell areas in the hypodermis in line with the strands (Armstrong et al., 2000). It thus seems that the aerenchymalike structure of maize roots improves internal aeration without sacrificing ion uptake function. However, the conductivity of such roots to water has not been tested experimentally. b.

Functional Role of Aerenchyma

The basic functional attributes of aerenchyma are well known—viz., its low resistance to diffusive or pressur-

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ized gas flow and its relatively low oxygen consumption compared to nonaerenchymatous, but still porous, tissues (Luxmoore et al., 1970; Armstrong, 1979). The essential role of aerenchyma in waterlogging tolerance of plants continues to be documented for more and more herbaceous and woody species worldwide (Thomson et al., 1992; Schlu¨ter et al., 1993; Moon et al., 1993; Kludze et al., 1994; Yamamoto et al., 1995a,b; Baruch and Merida, 1995; Loureiro et al., 1994; Stoecker et al., 1995; Shimamura et al., 1997; Gibberd et al., 1999; Bacanamwo and Purcell 1999; Visser et al., 2000a,b). Contradictory results for some Carex species have been reported recently (Moog, 1998; Moog and Bruggemann, 1998): roots grown anaerobically produced less aerenchyma than those grown aerobically. However, the anaerobic treatment used (constant N2 bubbling) is a particularly poor mimic of flooded soil conditions (Wiengeera et al., 1997) and may for a variety of reasons have inhibited aerenchyma formation (Drew et al., 1979). In a separate investigation with a range of Carex species, Visser et al. (2000a) found that aerenchyma was always present in the roots under natural or simulated soil flooding. It was once argued that the primary functional significance of aerenchyma was its lowering of the oxygen demand in plant organs and that its structural dimensions were in excess of those necessary for improving transport (Williams and Barber, 1961). However, modeling studies based on rice later showed that not only is this not so, but that in terms of oxygen, the transport function is of significantly greater importance than the reduced respiratory demand (Armstrong, 1979; Fig. 16). The functional significance of aerenchyma in roots must be considered against the background of a whole range of requirements. Water is plentiful in saturated soils, but for adequate nutrient absorption and anchorage it is an advantage if the roots can exploit more than just the surface layers of soil. This requires that aerenchyma development be sufficient to maintain at least the root tips of anchoring roots relatively free from oxygen deficiency until a sufficient rooting depth has been achieved. Maintenance of ROL around the absorptive regions of the root is also required to help minimize the chances of phytotoxin entry, among other things. A survey involving 91 plant species collected from wetland and nonwetland habitats in Britain revealed a strong correlation between fractional root porosity and rooting depth (Justin and Armstrong, 1987). There must also be a sufficiency of O2 supply to support the growth of lateral roots since these are important for nutrient and

Armstrong and Drew

water uptake. Nevertheless, a considerable amount of oxygen can leak to the soil from lateral roots (Armstrong, 1970; Armstrong et al., 1996d), and without aerenchyma development in parent roots only very shallow rooting could be expected. The benefits of aerenchyma in enhancing root aeration have been demonstrated in maize in two ways. Drew et al. (1985) grew maize plants under such conditions that the roots either did or did not produce aerenchyma. With the shoots exposed to air both types of root were subjected to an O2 -free N2 atmosphere for 30 min to help establish an internal gradient of oxygen partial pressure, and consequently gradients of aerobic respiration and energy metabolism. The presence of the aerenchyma resulted in higher levels of ATP and adenylate energy charge (AEC) at distances as great as 300 mm from the shoot. However, the respiration rates at the tips of the aerenchymatous roots were estimated to be only 30% of that in fully aerobic roots. This is perhaps not too surprising since as we have already seen some oxygen stress might be expected at the core of maize roots at cortical oxygen pressure of 5 kPa. In this particular experiment the O2 -free N2 atmosphere would have imposed very strong radial oxygen sink on the root’s oxygen supply from the shoot. Darwent (1997) used oxygen microelectrodes to measure directly the oxygen partial pressures in aerenchymatous and nonaerenchymatous roots embedded in solid agar (Fig. 6). Again the shoots were in air but, rather than acting as an oxygen sink, the agar simply offered a substantial impedance to radial oxygen flow to the root. It can be seen that at the tip of a 100-mmlong aerenchymatous root the cortical oxygen concentration was > 5 kPa, whereas in a nonaerenchymatous root of the same length it was < 1 kPa. In the aerenchymatous roots the partial pressure at the centre of the meristem was still above the Km for cytochrome oxidase, whereas in the nonaerenchymatous roots the values were <0.1 kPa. If the roots had been much longer, it is clear that some oxygen deficiency could have been expected at the apex even in the aerenchymatous roots and hence the results seem to be in good agreement with those of Drew et al. (1985). From theoretical considerations it is evident that provided aerenchyma formation is sufficiently high and respiratory demand and radial oxygen loss are sufficiently low, internal oxygen transport might readily support root growth to lengths >1 m (Armstrong, 1979) and adventitious roots of this length are not unknown. More commonly, however, wetland plants do not root so deeply. Rice, which is a very successful wetland species,

Oxygen Deficiency

Figure 6 Internal cortical oxygen concentrations along aerenchymatous and non-aerenchymatous primary roots ðL ¼ 102 and 95 mm and 6 and 5 days old respectively) of intact maize seedlings. The shoots were in air and the roots were embedded in a solid oxygen-depleted agar. The oxygen concentrations were measured by servo-driving Clark-type oxygen microelectrodes (tip diameter < 10 mm) through the agar and into and across the roots. On the x-axis, distance from the base ¼ distance from the root-shoot junction. (Modified from Darwent, 1997.)

normally roots to a depth of <300 mm; the high temperatures in rice fields will of course tend to limit rice root penetration by enhancing respiratory oxygen demand. Several ancillary features which act in conjunction with aerenchyma formation will delay the development of oxygen deficiency in the tips of growing roots, particularly those of wetland species. Foremost among these is the subapical decline in permeability to ROL (Armstrong, 1964, 1971; Luxmoore et al., 1970; Gaynard and Armstrong, 1987; Colmer et al., 1998), a feature which can coincide with the onset of aerenchyma development but the permeability of the stele to radial oxygen transfer from the cortex also declines as does respiratory demand in the stele and hypodermal cells (Armstrong et al., 1991). It may be that a narrow stele will also lead to better root tip aeration. In Phragmites australis the initial site of declining permeability to ROL has now been identified as the tangential walls of the outermost hypodermal cell layer (Armstrong et al., 2000), but as the roots grow older the other cell layers of the hypodermis must also act in this way judging by the degree to which they

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lignify. The effects of the developing impedance can be seen in the oxygen microelectrode profiles shown in Fig. 5: ROL across the nonporous epidermal–hypodermal cylinder at 7 mm from the apex (60 ng cm2 min1 Þ is consistent with a permeability coefficient close to that of water; at 29 mm the impedance was obviously much greater (ROL¼ 29 ng cm2 min1 Þ, whereas at 100 mm from the apex it was sufficiently large to prevent ROL altogether. It is interesting to note, however, that oxygen consumption within the epidermal–hypodermal cylinder contributed in preventing oxygen from leaking into the rhizosphere— there was not simply an infinitely high physical resistance. Nevertheless, the physical resistance probably increases much nearer to the base. In some species of Juncus there is a very thick lignified hypodermal cylinder; in other species, e.g., maize, the hypodermal cylinder is very thick near the root base but is little more than two cells thick at the apex of a 300-mm-long root. The hypodermis in rice is only of two or three cell layers but subapical permeability can nevertheless diminish substantially within a few centimeters of the tip (Armstrong, 1971), and it has been reported that at the flowering stage there is very little radial loss of O2 from the root system (Revsbech et al., 1998). It has been shown that a stagnant agar medium is all that is required to induce this impedance in some rice varieties (Colmer et al., 1998). Impedance to ROL and to radial transfer into the stele will of course increase the oxygen availability at the apex of a root (Armstrong and Beckett, 1987) and might improve rooting depths, but the functional significance of the wall thickenings in the hypodermal layer may be more complex than this, and the dynamics of the rhizosphere need to be taken into account. Where the permeability of a root does not decline, and this can be shown using artificial Si rubber roots (Armstrong, 1979), the oxygenated rhizosphere grows rapidly to begin with but later shrinks again presumably owing to the buildup of an aerobic microflora. The corollary of very narrow oxygenated rhizosphere is that the source concentrations of inwardly diffusing phytotoxins become closer, their concentrations in the rhizosphere increased, their residence time reduced, and the chances of phytotoxin damage increased. Provided the hypodermal thickenings resist phytotoxin entry, they may be as important in that role as in conserving oxygen. When adventitious roots of wetland species stop growing, even the tip can become impermeable. In the case of lateral roots, which are usually limited in their extension growth, there may be safety in numbers because of overlapping rhizo-

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Armstrong and Drew

spheres, and with several adventitous roots in close proximity much of the sediment may become oxidizing (Pedersen and Sand-Jensen, 1992; Armstrong et al., 1992) and presumably free of phytotoxins in basal regions, where laterals are most prolific. The dynamics and spatial geometry involved in these processes needs further study. B.

Metabolic Adaptations to Oxygen Deprivation

1. Biochemistry of Anoxia Tolerance Although many dryland species are sensitive to soil aeration, specialized wetland plants can tolerate or even thrive in flooded soil with extended periods of oxygen deficiency. The distinction sometimes made between flood-resistant and flood-sensitive species is convenient, but the distinction is imprecise in terms of the plant response mechanisms. The roots of the flood-resistant Glyceria maxima Holmberg are highly sensitive to oxygen deficiency and appear no less exacting in their need for O2 than do those of flood-intolerant Pisum sativum (Jenkin and ap Rees, 1983; ap Rees and Wilson, 1984): they are both anoxia intolerant. Many wetland species seem to possess a specialized metabolism that allows them to gain sufficient energy when there is not enough molecular oxygen to act as the terminal electron acceptor for the cytochromes. Of 20 wetland species tested in an anaerobic workbench with an atmosphere completely devoid of oxygen (Barclay and Crawford, 1982), 13 plants survived at least 7 days at 228C and regrew when exposed to air. Even an ability to grow (leaf extension) under strictly anaerobic conditions has been documented for rhizomes of Schoenoplectus lacustris, Scirpus maritimus, and Typha angustifolia (Barclay and Crawford, 1982), as well as Iris pseudacorus (Hanhijarvi and Fagerstedt, 1995). Cell extension can also take place anaerobically in embryos and coleoptiles of rice (Pradet and Bomsel, 1978; Bertani et al., 1980; Alpi and Beevers, 1983) and of Echinochloa phyllopogon (Rumpho and Kennedy, 1983a,b; Rumpho et al., 1984). We can regard all these tissues as being truly anoxia tolerant. However, the distinction between anoxia tolerance and anoxia intolerance is only relative. Probably all plant cells are able to survive anoxia for periods of some hours, when ATP has to be generated by anaerobic metabolism. In metabolically active cells like those of the maize root apical meristem, the ATP content would be sufficient for only 1–2 min in the absence of its regeneration (Roberts et al., 1985). Yet the root tip of maize can

suddenly be made anaerobic, and can be kept under oxygen-free conditions for 20–24 h at 258C before cell death occurs (Roberts et al., 1984a,b). Even if metabolism is rapidly slowed in anoxic cells, ATP will still be required for maintenance and for synthesis of various metabolites, including the proteins that are synthesized anaerobically (Sachs et al., 1980; Chang et al., 2000). For storage tissue of beetroot, survival under anoxia can extend to 150 h (Zhang and Greenway, 1994). The conclusion must be that ATP continues to be synthesized in the absence of oxygen, but at a much slower rate than aerobically. The biochemical basis for anoxia tolerance, as exemplified by organs of some wetland species, involves a combination of properties. During anaerobic respiration there has to be regeneration of NAD from the NADH produced by dehydrogenases, a net synthesis of ATP (plant cells have no major store of ATP or other high-energy phosphate bonds such as pyrophosphates), and the production of end products that either are compatible with metabolism or leak to the exterior where dilution renders them harmless. Additionally, dealing with metabolically generated protons is critical, because of the damage to metabolism that otherwise ensues at low pH in the cytoplasm. Formation of particular metabolites has been proposed as a means of consuming excess Hþ during anoxia, thereby offsetting cytoplasmic acidosis. Succinate is one such metabolite, and Menegus et al. (1989) found that leaves of anoxia-tolerant species (rice and Echinochloa crus-galli) have higher ratios of succinate to lactate in the cell sap than to anoxia-intolerant wheat and maize leaves. Formation of GABA from glutamate has also been suggested as a means of consuming protons and delaying cytoplasmic acidosis (Reid et al., 1985). The significance of such protonconsuming reactions to the ability of roots to survive anoxia has been little explored. In maize roots, succinate production was negligible (Roberts et al., 1992) and most production of GABA occurred late in anoxia, when cells were close to death. However, decarboxylation of malic acid to yield pyruvate, through the low pH activation of malate dehydrogenase (EC: 1.1.1.38), appeared to be consuming a significant quantity of protons during the early response to anoxia, with pyruvate being subsequently converted by transamination to alanine (Edwards et al., 1998). It is clear that roots do not possess a mechanism for long-term tolerance of anoxia, even those of wetland species. In rice and in Echinochloa phyllopogon, seedling survival eventually depends uon the emergence of the coleoptile above the saturated zone and into the

Oxygen Deficiency

air. Oxygen is then transferred through the coleoptile or first true leaf (the ‘‘snorkel effect’’) via the intercellular spaces to the embryonic root axis, which only then begins to elongate. The same O2 requirements are shown by roots of rhizomatous species which obtain O2 and switch from anaerobic to aerobic metabolism, once the terminal bud or shoot breaks the water surface. However, studies of energy metabolism and fermentation reveal that anoxia-intolerant roots can acclimate to some degree and become more resistant. Maize root tips are usually considered to be anoxia intolerant, and the sudden imposition of an anaerobic atmosphere rapidly lowers their content of ATP in a few minutes, simultaneously depressing the AEC. The cytosol of aerobic, vegetative cells usually stabilizes at an AEC of 0.90, but under sudden anoxia it falls almost at once. In excised maize root tips, values of AEC range between 0.2 and 0.6, depending on the content of soluble sugars in the roots. With the addition of exogenous sugars during anoxia, it remained close to 0.6 for as long as 23 h. The role of sugars under these conditions was to promote glycolysis and fermentation, which in turn were associated with higher-energy metabolism (Saglio et al., 1980). In nature, oxygen concentrations in waterlogged soil decline over periods of a few hours to several days, depending on temperature (Trought and Drew, 1982; Blackwell, 1983; Meek et al., 1983), so that root cells gradually experience oxygen deficiency; that is, they become transiently hypoxic before anoxic. Metabolic studies under laboratory conditions that often involve an abrupt transition from ambient air to anaerobic media do not allow acclimative responses to take place before conditions become lethal. Duration of survival of anoxia-intolerant tissues when suddenly exposed to anaerobic conditions is highly variable, depending on ambient temperature (hence the rate of respiration) and on the availability of substrates for respiration. The maximum duration of survival by unacclimated maize root tips is 15–24 h (Roberts et al., 1984a,b), and for rice roots observations range from 5–8 h (Webb and Armstrong, 1983) to 96–120 h (Bertani et al., 1980). In maize or pea roots, cell death under anaerobic conditions is closely associated with acidification of the cytoplasm. By following changes in 31 P chemical shifts, using noninvasive NMR techniques, the ratio of 2 H2 PO was used as an internal measure 4 to HPO4 of pH in cytoplasmic and vacuolar compartments (Roberts et al., 1984a,b, 1985, 1992). Protons initially originated from synthesis of lactic acid and alanine. But lactic dehydrogenase (LDH: EC 1.1.1.27) is inhib-

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ited by low pH, and fermentation soon switched to production of ethanol rather than to lactate. Further acidification of the cytoplasm continued because of leakage of protons from the vacuole, which is normally maintained at pH 5.8 compared with pH 7.4 of the cytoplasm. Proton-translocating ATPases in the tonoplast normally maintain this steep gradient, but with a lowering concentration of ATP within the cytosol, a threshold must be reached below which their proton pumping activity diminishes, while Hþ leaks passively to the cytoplasm (Roberts et al., 1984b, 1992). Changes in the fine structure of root meristemic cells with increasing duration of hypoxia include swelling and elongation of mitochondria, proliferation of endoplasmic reticulum, condensation of chromatin followed by its dispersion, and inactive Golgi apparatus (Aldrich et al., 1985). Whether the pattern of response observed in anoxic roots of maize and pea applies generally to all higher plant cells is doubtful. Ratcliffe (1995) points out the discrepancies that have been identified in other tissues between the decline in cytoplasmic pH and the early production of lactate. In several cases, lactate was either too much or too little to account for the observed change in pH. Ratcliffe (1995) also emphasizes that the initial acidification cannot be attributed to lactic acid production alone, but must include the release of protons in other metabolic steps, especially in the hydrolysis of ATP during energy-consuming reactions. Attempts to aggravate the early demise of anoxic cells by overexpression of LDH activity in transgenic tomato had almost no impact (Rivoal and Hanson, 1994). Production and export of lactate to the external medium were essentially the same. Apparently, LDH activity exerted only 1–2% of the total control of the rate of glycolysis and fermentation to lactate. Sudden changes in oxygen partial pressure immediately lower the energy status of cells and thus give little opportunity for induction of an alternative metabolism. Despite this, in anoxic roots of maize, there is a remarkable change in the pattern of protein synthesis detected by PAGE (Sachs et al., 1980; Chang et al., 2000). Some of these ‘‘anaerobic polypeptides’’ (ANPs) correspond to aerobic proteins that selectively continue to be made, but overall protein synthesis is reduced to 10–15% of the normoxic rate. Some 20 ANPs were distinguished in maize, including ADH, pyruvate decarboxylase (EC 4.1.1.1), glyceraldehyde3-phosphate dehydrogenase (EC: 1.2.1.12), and other enzymes involved in glycolysis and fermentation (reviewed by Sachs et al., 1996). All of these enzymes

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are essential to glycolysis and fermentation, but the significance of the ANPs relative to the ability of root cells to survive anoxia is obscure. Inhibition by cycloheximide of ANP synthesis during anoxia failed to modify cytoplasmic pH regulation or the ability of maize root tips to remain viable under anoxia (Chang et al., 2000). By contrast, in coleoptiles and leaves of rice, inhibition of protein synthesis during anoxia caused degradation of membranes of mitochondria and other organelles (Vartapetian and Poljakova, 1994). When intact maize or wheat seedlings are made hypoxic by subjecting them to a partial deficiency of oxygen (hypoxic pretreatment, or hypoxic acclimation), their subsequent ability to tolerate extended periods of anoxia is greatly improved (Saglio et al., 1988; Johnson et al., 1989; Waters et al., 1991; Xia and Roberts, 1994). The apical zones of intact maize roots usually die in < 24 h, but after hypoxic acclimation for a minimum of 2–4 h, the majority remained alive up to 96 h, retaining turgor and a healthy appearance and able to resume extension on reexposure to O2 (Johnson et al., 1989; Chang et al., 2000). This improvement in anoxia tolerance (acclimation) was associated with an ability to maintain a greater rate of glycolysis and ethanolic fermentation (Hole et al., 1992) as well as greater concentrations of ATP and total adenine nucleotides (Johnson et al., 1989; Xia et al., 1995), relative to unacclimated root tips, in which respiration and energy metabolism collapsed after a few hours (Hole et al., 1992; Xia et al., 1995; Xia and Roberts, 1996). In terms of gene expression, hypoxia increased transcript levels for ADH1, ADH2, enolase (EC: 4.2.1.11), aldolase (EC: 4.1.2.13), and pyruvate decarboxylase (EC: 4.1.1.1) (Andrews et al., 1993, 1994a,b) and also raised enzyme activity levels for those that were tested (ADH, pyruvate decarboxylase) (Johnson and Drew, unpublished). It is tempting to suggest that the induction of vigorous fermentation by hypoxia, dependent on changes in gene expression, was a contributory feature in improved energy status and viability under anoxia. However, using metabolic inhibitors to lower ATP levels within anoxic maize root tips, Xia et al. (1995) concluded that survival of anoxia is not closely dependent on the energy status of the cells. Hypoxically acclimated root tips survived anoxia and were better able to regulate cytoplasmic pH, whether or not ATP levels were depressed by metabolic inhibitors. These results are difficult to reconcile with the notion that ATP would be essential to energize Hþ -ATPases at the tonoplast to reverse the otherwise inevitable acid-

Armstrong and Drew

ification of the cytoplasm resulting from passive leakage of Hþ from the vacuole (Roberts et al., 1984a,b) unless the Km for ATP is lower than the ATP concentration found in anoxic cells. An alternative interpretation is that the experiments of Xia et al. (1995) applied mainly to the initial few hours of anoxia, where metabolic consumption of protons is the predominant mechanism of pH regulation, as distinct from H+ATPase activity. As discussed below, a great deal of evidence points to the importance of maintaining the energy status within viable cells during anoxia. Acclimation during hypoxia involves improved ability to transport lactic acid from the cytoplasm to the external medium. In maize roots that were hypoxically pretreated and then made anoxic, there were less acidification of the cytoplasm and a greater transport of lactic acid to the exterior than in unacclimated roots (Xia and Roberts, 1994, 1996; Xia et al., 1995). Tomato roots showed a similar increased ability to transport lactic acid following acclimation (Rivoal and Hanson, 1994). These results suggest that hypoxia induces formation of a lactate transporter at the plasma membrane and that the loss of lactic acid from the cytoplasm helps delay acidosis. So far, no direct evidence of increased synthesis of a lactate transporter has been presented. However, an ability to continue protein synthesis during the hypoxic pretreatment is crucial to the induction of anoxia tolerance. Many of the proteins synthesized in normoxic roots continue to be synthesized under hypoxia (Chang et al., 2000), and using cycloheximide to block protein synthesis at the stage of hypoxic pretreatment eliminated both the improvement in root tip viability under anoxia and the ability to regulate cytoplasmic pH. Of the hundreds of proteins that are synthesized under hypoxia (Chang et al., 2000), those that contribute critically to anoxia tolerance have yet to be identified. The ability of roots to tolerate anoxia seems to depend at least in part on an ability to continue glycolysis, fermentation, and ATP synthesis; one enzyme that is particularly critical in this is glucokinase (EC: 2.7.1.2). At a slightly acid pH (6.5), typical of the pH of the cytoplasm during a damaging period of anoxia, GK is strongly inhibited (Bouny and Saglio, 1996). No other enzyme of glycolysis or fermentation was found to be so pH sensitive. Additionally, the ATP concentration in anoxic roots was close to the Km for GK and potentially limiting. Finally, the activity of GK in extracts of root tissue was similar to the in vivo rate of glycolysis. Taken together, the evidence suggests that GK could be limiting to the rate of gly-

Oxygen Deficiency

colysis in anoxic roots, and account for the decline in anaerobic respiration rate observed in roots that had not been hypoxically acclimated. GK was strongly induced in roots that had been hypoxically pretreated (Bouny and Saglio, 1996; Ricard et al., 1998). Similarly, GK activity was enhanced in roots of the flood-tolerant Echinochloa phyllopogon (Fox et al., 1998) and in germinating rice (Ricard et al., 1991). Another critical enzyme induced by O2 deprivation is sucrose synthase (SuSy: EC: 2.4.1.13), which catalyzes the reversible reaction: sucrose þ UDP $ UDP-glucose þ fructose SuSy is strongly induced in hypoxic root tips of maize, along with HK (Ricard et al., 1998). Under hypoxia and anoxia, invertase (EC: 3.2.1.26) is greatly inhibited, at the levels of both transcription and enzyme activity (Ricard et al., 1998; Zeng et al., 1999), so that sucrose degradation and the continuation of glycolysis depend solely on SuSy activity. In double mutants of maize lacking appreciable expression of the two genes, Sh1 and Sus1 encoding SuSy, viability was quickly lost in root tips under anoxia, but this could be alleviated simply by supplying glucose (Ricard et al., 1998). Not all species follow the maize model. In roots of potato (Biemelt et al., 1999), SuSy activity was not found to be limiting to glycolysis under anoxia. Although transformed plants with antisense constructs to SuSy showed less tolerance to anoxia, levels of hexoses and other glycolytic intermediates were similar to those of wild-type plants, and remained high. The authors suggested that increased SuSy activity in potato might serve to provide UDP-glucose for the synthesis of cellulose and callose, acting as a carbon store. However, it is difficult to see how this might influence cell survival during anoxia, as distinct from acting as a carbon source during recovery following reoxygenation. A further complexity in assessing the importance of SuSy is a possible overlap between oxygen and sugar signaling systems (Koch, 1996; Zeng et al., 1999; Chapter 28 by Bacon et al. in this volume). The invertase genes and the SuSy genes in maize are both modulated by sugar levels as well as by hypoxia and anoxia. The overall effect can be to inhibit sucrose breakdown to the point where maintenance of root tip viability is compromised (Zeng et al., 1998). For the SuSy genes in maize roots, Sh1 is induced by anoxia or sugar deprivation, in contrast to Sus1, which is induced by hypoxia or sugar abundance. Sugar signal transduction is thus closely linked to the flow of carbon in

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glycolysis: low O2 availability can lead to an accumulation of sugars and other intermediates in the glycolytic pathway (Biemelt et al., 1999) or to a depletion of the same intermediates (Bouny and Saglio, 1996). Clearly, oxygen has the potential to affect carbohydrate-modulated gene expression (Koch, 1996; Koch et al., 2000). Seedling survival can be enhanced under laboratory conditions by death or removal of the tip of the primary root (Zeng et al., 1999; Ellis et al., 1999; Subbaiah et al., 2000). By minimizing the utilization of carbon in keeping the metabolically demanding apical meristem alive, seedlings are able to grow away more rapidly when reoxygenated. The role of the former apical meristem is then taken over by an emerging, dominant, lateral primordium. However, this new apical meristem becomes once more a major sink for carbon, and equally as vulnerable as the original root tip to a new cycle of oxygen deprivation, should that recur. Sacrifice of the root apical meristem, upon which the development of the entire root system depends, may be an adaptation of limited survival value. Although previous exposure to hypoxia improves the ability of roots of several graminaceous species to tolerate relatively short periods of anoxia, it is not yet clear whether this response is widespread. In Arabidopsis thaliana, hypoxic acclimation improved root and shoot tolerance of very low levels of O2 (0.1% O2 for 48 h) but not strict anoxia (0% O2 ) (Ellis et al., 1999). The mechanism of shoot acclimation to extreme hypoxia was independent of the root; for example, shoots of adh null mutants were able to acclimate, but not the roots. Other studies, however, have shown that expression of ADH in the shoots of Arabidopsis is induced by exposure of the roots to lowoxygen conditions (Chung and Ferl, 1999) and that a signal transduction pathway involving Ca2þ is implicated. Exposure of roots of intact barley seedlings to very low concentrations of oxygen increases the activity of LDH some 20-fold (Hoffman et al., 1986). Synthesis of lactate (like that of ethanol) would allow recycling of NAD and production of ATP. But in maize root tips, LDH is quickly inhibited by cytoplasmic acidosis (Roberts et al., 1984a,b). Such an inhibition is not evident in barley (Hoffman et al., 1986), where lactate is still a minor component compared with ethanol production. Differences in technique used in the two investigations could perhaps explain the apparent contrast between the findings for maize and barley root tips; excised maize root tips were maintained in a constantly

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replenished N2 atmosphere, whereas barley roots were investigated with intact plants. In such plants, oxygen would have diffused from the leaves to the roots in the intercellular spaces. Thus the internal concentration of oxygen would have been greater than that used to sparge the nutrient solution in which the roots were submersed. In fully vacuolated cells of maize adventitious roots, several centimeters behind the tip, there is appreciable fermentation during anoxia to yield lactate. With hypoxic pretreatment, a greater proportion of the lactate is transported to the external solution. Such mature zones are much more tolerant of anoxia than the apical zone, despite a continuous production of lactate (McAlpine, 1995). The response of these older zones of maize roots resembles that of roots of some species of the halophytic genus Limonium, where fermentation under anoxia leads to production of lactate rather than ethanol, with a continuous transport of lactic acid to the medium (Rivoal and Hanson, 1993), without which a lethal decrease in pH would occur. The authors have suggested that in intact, transpiring plants, lactate might be transported in the xylem to the leaves and be oxidized to pyruvate for further metabolism, but this remains to be shown. 2. Signaling and Regulation of Gene Expression Under O2 Deprivation It is evident that at the level of transcription, the response to oxygen shortage is rapid. In maize root tips, new transcripts of Adh1 were detectable within 60 min (Rowland and Strommer, 1986) and under anoxia they peaked at about 6 h (Andrews et al., 1993). The nature of the signal for induction of increased synthesis of mRNA for the anaerobic polypeptides is unknown. At the DNA level, several genes that are expressed strongly during O2 deprivation (Adh1, Adh2, Sh1, and aldolase) have concensus sequences within the promoter region, the anaerobic response element (ARE) (Springer et al., 1986; Walker et al., 1987; Dennis et al., 1988; Olive et al., 1991). This was presumed to be important in regulation of transcription under anoxia. More recent reports have raised doubts about the significance of the ARE, particularly the occurrence of this same nucleotide sequence in the promoter of genes, which are not anoxically inducible. (Russell and Sachs, 1989, 1991). Furthermore, comparison of the promoters of Adh1 and Adh2, which are similarly induced by O2 deprivation, revealed very little similarity in terms of functional ARE sequences or a common pattern of

Armstrong and Drew

binding of trans-acting factors (Paul and Ferl, 1991). More recently, Kohler et al. (1996) have isolated a promoter from a maize gene, cytosolic glyceraldehyde-3-phosphate dehydrogenase-4. A sequence within the promoter, comprising a 270-bp region between 191 and 461, was found to confer strong anaerobic induction, about a 1000-fold above background with GUS as reporter gene in transgenic tobacco seedlings. The above promoter sequence is found also in Adh1 and Adh2, but outside of the putative ARE. Posttranscriptional processes also regulate synthesis of the ANPs during anoxia. In anoxic maize root tips, aerobic as well as anaerobic mRNAs were present, but the aerobic messages were discriminated against by the translation system in vivo (Sachs et al., 1980; Fennoy et al., 1998). In an in vitro wheat germ translation system, both aerobic and anaerobic messages could be translated. Selective translation of anaerobic mRNAs occurs at the level of initiation and of elongation. Untranslated regions (UTRs) of the mRNA are involved in selectivity (both 50 and 30 UTRs) for maize Adh1 mRNA (Bailey-Serres and Dawe, 1996). Selective translation may be affected by the phosphorylation of the cap-binding protein eIF4A that takes place under anoxia, thereby influencing 50 cap-dependent initiation (Bailey-Serres, 1999; Manjunath et al., 1999). At the cell level, little is known about the signaling of O2 deprivation. Transcription of some of the ANPs is induced more strongly by hypoxia than anoxia (Andrews et al., 1993, 1994a,b; Chang et al., 2000; Koch et al., 2000), so it is evident that there is a sensing mechanism for O2 and that it is not necessarily anoxia per se that activates changes in gene expression. Sensing mechanisms that respond to a lowering of O2 concentration have been well characterized in bacteria (Hornberger et al., 1991; Monson et al., 1992; Compan and Touati, 1994; Fu et al., 1994) and yeast (Zhang and Hach, 1999). For higher plants there have been suggestions that an O2 -sensing system based on O2 binding to nonsymbiotic hemoglobin or on some aspect of energy metabolism might participate (Drew, 1997; Arredondo-Peter et al., 1998), but there is as yet no hard evidence. When bulky structures such as large-diameter plant roots are made hypoxic, a radial concentration gradient for O2 must develop so that cells in the root interior may be anoxic while those at the epidermis remain fully aerobic. Under these conditions, it is possible that ATP produced in the outer cells by oxidative phosphorylation is transported to the energy-starved interior that can only synthesize ATP by the much

Oxygen Deficiency

less efficient fermentive pathways. Tracer studies with a fluorescent analog of ATP support this possibility (Cleland et al., 1994). Thus, as discussed above, hypoxic roots must present a very heterogeneous population of cells in terms of O2 status, energy level, and gene expression. Whatever may be the precise conditions of O2 deprivation within individual cells, activation of a signal transduction pathway clearly involves transient changes in the concentration of Ca2þ in the cytosol (see also Chapter 28 by Bacon et al. in this volume). Subbaiah et al., (1994b) used Ruthenium Red (RR) to inhibit Ca2þ release from subcellular organelles in maize roots and found that it prevented anoxic induction of ADH1 and SH1 mRNAs as well as ADH enzyme activity. Seedlings treated with RR could not recover from as little as 2 h of O2 deprivation (maize seedlings usually survive this for up to 72 h), but these effects were blocked when Ca2þ was included with RR in the medium. Using fluo-3 AM as an intracellular fluorescent probe of free Ca2þ , maize cells in tissue culture showed a sharp rise in cytosolic Ca2þ within 1–2 min of the start of anoxia that was dependent on internal, not external, Ca2þ (Subbaiah et al., 1994a). Significantly, treatment with caffeine, which increased cytosolic Ca2þ under aerobic conditions, induced ADH activity, as if O2 deprivation had occurred. The question remains as to what leads to changes in cytosolic Ca2þ ; the rapidity of the Ca2þ response suggests that the signal transduction system is closely in tune with oxygen status, perhaps involving some aspect of energy metabolism which changes almost immediately on imposition of anoxia, but that is a speculation at present.

V.

CONCLUSIONS

A variety of media and techniques are used experimentally to expose plant roots to O2 deficiency, so it is sometimes impossible to define precisely the O2 status at the organ or cellular level. Measurements of dissolved O2 , and the use of microelectrodes to quantify O2 concentration gradients over very short distances are helping remove this uncertainty. Apart from the frequent failure to define the oxygen status of the media to which roots are exposed, there are different terminologies in the literature, so it is difficult to judge whether cells were aerobic, hypoxic, or anoxic, or even the precise meaning given by authors to those terms (Pradet and Bomsel, 1978). For those interested in signaling of low O2 , this seems a critical issue, especially

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where genes respond differentially to anoxia and hypoxia. Root dysfunction as a result of inadequate oxygenation can modify plant growth and development, and depress crop yield, through interference in water relations, mineral nutrition, and hormone balance. Conversely, wetland species have been successful in colonizing a habitat that is disadvantageous to many potential competitors. Some of the adaptations, structural as well as metabolic, that improve the fitness of wetland species in low-oxygen environments are now beginning to be appreciated. Future development of improved flood resistance in dryland species is likely to be through constitutive production of root aerenchyma to minimize oxygen deficit, as well as by engineering an efficient anaerobic metabolism to help the root system tolerate more extended periods of life, transiently without oxygen. REFERENCES Aldrich HC, Ferl RJ, Hils MH, Akin DE. 1985. Ultrastructural correlates of anaerobic stress in corn roots. Tissue Cell 17:341–348. Allen LH. 1997. Mechanisms and rates of oxygen transfer to and through submerged rhizomes and roots via aerenchyma. Soil Crop Sci Soc Fli 56:41–54. Alpi A, Beevers H. 1983. Effects of O2 concentration on rice seedlings. Plant Physiol 71:30–34. Amoore JE. 1961. Dependence of mitosis and respiration in roots upon oxygen tension. Proc R Soc B 154:109–129. Andersen FO, Kristensen E. 1988. Oxygen microgradients in the rhizosphere of the mangrove Avicennia marina. Mar Ecol Prog Ser 44:201–204. Andrews DC, Cobb BG, Johnson JR, Drew MC. 1993. Hypoxic and anoxic induction of alcohol dehydrogenase in roots and shoots of seedlings of Zea mays: Adh transcripts and enzyme activity. Plant Physiol 101:407– 414. Andrews DC, Drew MC, Johnson JR., Cobb BG. 1994a. The response of maize seedlings of different age to hypoxic and anoxic stress: changes in induction of Adh1 mRNA, ADH activity and survival of anoxia. Plant Physiol 105:53–60. Andrews DL, MacAlpine DM, Cobb BG, Johnson JR, Drew MC. 1994b. Differential induction of mRNAs for the glycolytic and ethanolic fermentative pathways by hypoxia and anoxia in maize seedlings. Plant Physiol 106:1575–1582. Arber A. 1920. Water Plants: A Study of Aquatic Angiosperms. Cambridge, U.K.: Cambridge University Press. Armstrong W. 1964. Oxygen diffusion from the roots of some British bog plants. Nature 204:801–802.

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Armstrong and Drew dicotyledonous wetland species with contrasting aerenchyma types. Plant Cell Environ 23:1237–1245. Visser EJW, Nabben RHM, Blom CWPM, Voesenek LACJ. 1997. Elongation by primary lateral roots and adventitious roots during conditions of hypoxia and high ethylene concentrations. Plant Cell Environ 20:647– 653. Voesenek LACJ, Blom CWPM. 1999. Stimulated shoot elongation: a mechanism of semiaquatic Plants to avoid submergence stress. In: Lerner HR, ed. Plant Responses to Environmental Stresses: From Phytohormones to Genome Reorganization. New York; Marcel Dekker, pp 431–448. Wadman–Van Schravendijk H, Van Andel OM. 1985. Interdependence of growth, water relations and abscisic acid level in Phaseolus vulgaris during waterlogging. Physiol Plant 63:215–220. Walker JC, Howard EA, Dennis ES, Peaock WJ. 1987. DNA sequences required for anaerobic expression of the maize alcohol dehydrogenase-1 gene. Proc Natl Acad Sci USA 84:6624–6628. Wang T, Peverly JH. 1999. Iron oxidation states on root surfaces of a wetland plant (Phragmites australis). Soil Sci Soc Am J 63:247–252. Waters I, Armstrong W, Thomson CJ, Setter TL, Adkins S, Gibbs J, Greenway H. 1989. Diurnal changes in radial oxygen loss and ethanol metabolism in roots of submerged and nonsubmerged rice seedlings. New Phytol 113:439–451. Waters I, Morrell S, Greenway H, Colmer TD. 1991. Effects of anoxia on wheat seedlings. 2. Influence of O2 supply prior to anoxia on tolerance to anoxia, alcoholic fermentation, and sugar levels. J Exp Bot 42:1437–1447. Webb T, Armstrong W. 1983. The effect of anoxia and carbohydrates on the growth and viability of rice, pea and pumpkin roots. J Exp Bot 34:579–603. West DW, Black JDF. 1978. Irrigation timing-its influence on the effects of salinity and waterlogging stresses in tobacco plants. Soil Sci 125:367–376. West DW, Taylor JA. 1980. The response of Phaseolus vulgaris L. to root zone anaerobiosis, waterlogging and sodium chloride. Ann Bot 46:51–60. West DW, Taylor JA. 1984. Response of six grape cultivars to the combined effects of high salinity and rootzone waterlogging. J Am Soc Hort Sci 109:844–851. White SD, Ganf GG. 2000. Flow characteristics and internal pressure profiles in leaves of Typha domingensis. Aquat Bot 67:263–274. Wiengweera A, Greenway H, Thomson CJ. 1997. The use of agar nutrient solution to simulate the lack of convection in waterlogged soils. Ann Bot 80:115–123. Wilkinson S, Davies WJ, 1997. Xylem sap pH increase: a drought signal received at the apoplastic face of the guard cell that involves the suppression of saturable abscisic acid uptake by the epidermal symplast. Plant Physiol 113:559–573.

Oxygen Deficiency Williams WT, Barber DA. 1961. The functional significance of aerenchyma in plants. In: Milthorpe FL, ed. Mechanisms in Biological Competition. London; Cambridge University Press, pp 132–144. Witty JF, Skøt L, Revsbech NP. 1987. Direct evidence for changes in the resistance of legume root nodules to O2 diffusion. J Exp Bot 38:1129–1140. Xia JH, Roberts JKM. 1994. Improved cytoplasmic pH regulation, increased lactate efflux, and reduced cytoplasmic lactate levels are biochemical traits expressed in root tips of whole maize seedlings acclimated to a low-oxygen environment. Plant Physiol 105:651–657. Xia JH, Roberts JKM. 1996. Regulation of H+ extrusion and cytoplasmic pH in maize root tips acclimated to a low-oxygen environment. Plant Physiol 111:227–233. Xia JH, Saglio PH, Roberts JKM. 1995. Nucleotide levels do not critically determine survival of maize root tips acclimated to a low-oxygen environment. Plant Physiol 108:589–595. Yamamoto F, Sakato T, Terazawa K. 1995a. Physiological, morphological and anatomical responses of Fraxinus mandshurica seedlings to flooding. Tree Physiol 1:713– 719. Yamamoto F, Sakato T, Terazawa K. 1995b. Growth, morphology, stem anatomy and ethylene production in flooded Alnus japonica seedlings. IAWA J 16:47–59.

761 Yavitt JB, Knapp AK. 1998. Aspects of methane flow from sediment through emergent cattail (Typha latifolia) plants. New Phytol 139:495–503. Zeng Y, Wu Y, Avigne WT, Koch KE. 1998. Differential regulation of sugar-sensitive sucrose synthases by hypoxia and anoxia indicate complementary transcriptional and posttranscriptional responses. Plant Physiol 116:1573–1583. Zeng Y, Wu Y, Avigne WT, Koch KE. 1999. Rapid repression of maize invertases by low oxygen. Invertase/ sucrose synthase balance, sugar signaling potential, and seedling survival. Plant Physiol 121:599–608. Zhang J, Davies WJ. 1987. ABA in roots and leaves of flooded pea plants. J Exp Bot 38:649–659. Zhang J, Schurr U, Davies WJ. 1987. Control of stomatal behaviour by abscisic acid which apparently originates in the roots. J Exp Bot 38:1174–1181. Zhang L, Hach A. 1999. Molecular mechanism of heme signaling in yeast: the transcriptional activator Hap1 serves as the key mediator. Cell Mol Life Sci 56:415– 426. Zhang Q, Greenway H. 1994. Anoxia tolerance and anaerobic catabolism of aged beetroot storage tissues. J Exp Bot 45:567–575. Zhang X, Zhang F, Mao D. 1999. Effect of iron plaque outside roots on nutrient uptake by rice (Oryza sativa L.): phosphorus uptake. Plant Soil 209:187–192.

43 Trace Element Stress in Roots Ju¨rgen Hagemeyer and Siegmar-W. Breckle University of Bielefeld, Bielefeld, Germany

I.

INTRODUCTION

rather rare. It is now one of the most widely distributed trace metals and it is more evenly distributed in the terrestrial ecosystems than before (Neite et al., 1992; Nriagu, 1992; Singh et al., 1997). The worldwide use of leaded gasoline, which began in 1923, has resulted in contamination of all ecosystems.

Heavy metals comprise a small part of the earth’s crust. Nevertheless, these elements play an important role in plant ecology and affect growth and performance of plant roots even in small quantities as trace elements. Trace elements can be divided into three groups. Some of them are rare and others are more abundant. The presence of both is not essential to plants. The third, smaller group includes those elements that are essential for some or for all organisms. The dose response curves of the effects of essential trace elements on organisms consist of three parts: (1) at very low concentration organisms suffer from deficiency exhibiting characteristic symptoms; (2) at a range of medium concentrations organisms grow normally; and (3) at concentrations above a critical level the elements are toxic (Berry and Wallace, 1981). High concentrations of trace elements in the rhizosphere of higher plants primarily damage the roots. Consequently, they also affect other plant parts. The threshold levels of deficiency and of toxicity differ widely for the various elements (Baker and Walker, 1989). In the nonessential elements, only normal and toxic concentration ranges (2) and (3) are found. In most ecosystems potentially toxic trace elements occur naturally in an active form only in very small quantities. Human mining activities have resulted in an ever-increasing contamination of the biosphere with potentially toxic trace metals (Nriagu, 1996). An outstanding example is lead. Once this element was

II.

ESSENTIAL, BENEFICIAL, AND TOXIC TRACE METALS

Several trace metals are essential for higher plants. This means that (1) plants cannot complete their lifecycle without the respective elements; (2) they cannot be replaced by other elements; and (3) they have a specific function in plant metabolism (Marschner, 1986). For such elements the concentration range between deficiency and toxicity varies widely. Currently six trace elements are considered essential for higher plants: boron, copper, iron, manganese, molybdenum, and zinc. Two additional candidates are chlorine and nickel (Marschner, 1986). Nickel seems to be an essential micronutrient, although the failure to complete the life-cycle without nickel has only been confirmed in few plant species (Gerendas et al., 1999). Some elements, like cobalt, iodine, sodium, silicon, and vanadium, are considered beneficial but not essential for plant growth (Marschner, 1986). All beneficial and essential elements cause toxicity symptoms when concentrations are high. Nevertheless, they are not regarded as toxic elements. 763

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Only those exhibiting no essentiality are regarded as toxic elements. Because of the variability in the responses of various plants to some elements, essentiality is sometimes difficult to ascertain. An insufficient supply of a plant with an essential element causes specific deficiency symptoms. Nonessential elements never cause deficiency symptoms, even at extremely low concentrations. However, in a number of studies, unexplained growth enhancements were observed in plants subjected to mild stress from toxic trace elements. For example, root growth stimulation of Betula pendula seedlings was induced by low concentrations of cadmium (Gussarsson, 1994). Enhanced root elongation, root biomass gain, and root hair formation were found in plants of a tolerant population of Silene vulgaris treated with lead chloride (Wierzbicka and Panufnik, 1998). Jiang and Liu (1999) reported stimulated root growth of Brassica juncea plants treated with low concentrations of lead nitrate.

III.

EFFECTS OF TRACE ELEMENTS ON EXTENSION GROWTH AND DIFFERENTIATION OF ROOTS

Trace element effects on plants also depend on whether the element is an essential nutrient or not. Inhibition of root extension growth can result from interference with cell division or with cell elongation. Trace elements were shown to affect both processes. Slowing down of the mitotic rate of root cells of Allium cepa, Zea mays, and Lupinus luteus was caused by high lead concentrations (Hammett, 1929; Przymusinski and Wozny, 1985). It was hypothesized that such inhibition is related to a lead-induced reduction of cell cycle proteins, like cyclin (Deckert and Gwozdz, 1999). Apparently lead has several effects. However, its main influence on root growth was on root cell elongation (Garland and Wilkins, 1981; Sieghardt, 1981). The elasticity of cell walls is so much reduced by lead or by cadmium that under mechanical stress they may break (Lane and Martin, 1982; Barcelo et al., 1986). A physiological explanation of root growth inhibition under lead stress suggests an increased level of free radicals and of reactive oxygen species, which exceeds the capacity of the antioxidant enzymes (Rucinska et al., 1999). This may result in reduced root growth. Lead nitrate (102 M) had no effect on the emergence of the seminal roots of maize (Zea mays) seedlings but did affect root elongation after emergence. At 103 M lead nitrate, the growth of primary and of other semi-

nal roots was slowed down as a result of partial inhibition of cell division and cell elongation (Obroucheva et al., 1998). Lead stress also had marked effects on root branching pattern and on root system morphology. Lead toxicity to Fagus sylvatica seedlings started at a concentration of 48
mol lead/kg soil. At lower lead concentrations, root growth was slightly enhanced (Breckle et al., 1988). Similar results were observed in a culture experiment with young Picea abies trees (Hagemeyer et al., 1994). Root growth increased at low soil concentrations of cadmium or zinc. However, at higher concentrations of both metals root growth of Picea and Fagus saplings was strongly inhibited (Hagemeyer et al., 1994). A slight growth enhancement by low metal concentrations depends on the specific metal and is known in general already since Sachs (1874). Lead caused reductions in root elongation of Allium cepa already at concentrations of 0:1
M lead nitrate (Liu et al., 1994a). The lead treatment caused irregularities in mitoses. Nickel sulfate treatments up to 10
M resulted in stimulated root growth, but higher concentrations inhibited it. At high nickel concentrations irregularly shaped nucleoli were observed in root cells (Liu et al., 1994b). Structural and ultrastructural effects of copper stress on Zea mays roots were reported by Ouzounidou et al. (1995). Seedlings grown at higher copper concentrations (80
m) exhibit damaged epidermal cells of their roots. In other root tissues the effects of such a copper level were varied. Cortical or stelar cells with disintegrated cytoplasm were observed next to cells with well-preserved structure. The root ultrastructure was less affected by copper than their morphology and physiology. The occurrence of healthy cells in copper-stressed roots indicated a varied response of the cells to such harmful conditions. Inhibition of root elongation of Picea abies seedlings grown in nutrient solutions containing mercury, cadmium, or zinc was reported by Godbold and Hu¨ttermann (1985). The order of toxicity was Hg > Cd > Zn. Toxicity symptoms of mercury in spruce seedlings, like decreased transpiration rates and lowered chlorophyll contents in needles, were attributed primarily to root damage (Godbold and Hu¨ttermann, 1988). Ultrastructural alterations of root cells of Cajanus cajan treated with zinc sulfate or with nickel sulfate and inhibition of radicle elongation in seedlings were described by Sresty and Madhava Rao (1999). Toxic effects of Zn and Ni were correlated with their concentrations. Extensive damage to root cells grown under

Trace Element Stress

metal stress was shown by EM. The nuclei of root tip cells showed condensed chromatin strands. Some cortical cells showed disruption and dilation of their nuclear membranes. Other toxicity symptoms were expressed by structureless cytoplasm, by disintegration of organelles, and by the development of vacuoles. Some cortical cells showed two nucleoli. The authors suggested that this might be a result of the stimulation of the nucleolus to increase the production of ribosomes and mRNA, which enhance the synthesis of new proteins involved in the trace metal tolerance. Effects of nickel sulfate ( 10
M) on root growth of Pisum sativum plants resulted in reductions of root extension growth and a reduction of potassium concentrations (Gabbrielli et al., 1999). The water content of roots was negatively correlated with the tissue nickel concentration. Roots under nickel stress had increased phenol contents and higher extracellular peroxidase activities. Such effects were indicative to a rapid senescence. A selective cell death of damaged tissues may be part of a defense strategy. The chemistry as well as the physiological effects of chromium are rather complicated (Barcelo and Poschenrieder, 1997; Mishra et al., 1997). Trivalent and hexavalent chromium had somewhat different effects on root growth of onion plants (Liu et al., 1992). At concentrations of 0.2–20
M hexavalent dichromates reduced root growth more than Cr3þ , mostly by inhibition of cell division. Chromium interfered with mitoses and caused chromosome aberrations. The effects of trace elements were also studied with soil-grown plants. Soil treatments are usually better comparable to natural or field conditions than nutrient solution experiments. However, such conditions are more difficult to control and direct observations of roots are restricted. A standardized ‘‘artificial soil’’ consisting of an ion exchange resin embedded in an inert sand matrix was proposed for studies of trace element effects on root growth (Ko¨hl, 1997). The metal ions are buffered by the ion exchanger and the sand provides the mechanical impedance like in natural soils. Implementation of this experimental technique should advance our understanding of the root–soil–trace element relationships. Under natural conditions plant roots are rarely exposed to stress from a single toxic element in the soil. Various ions usually affect plant growth simultaneously (Hagemeyer, 1999). The interactions of the effects of different metals can be described as independent, additive, synergistic, or antagonistic (Berry and Wallace, 1981; Wallace, 1982). Such interactions

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should also be considered in experimental studies. In combination treatments, cadmium and lead showed additive or even synergistic effects on root growth of Fagus sylvatica seedlings (Kahle, 1988; Breckle et al., 1988; Kahle and Breckle, 1989). For instance, in a treatment with 2.4 mmol Pb (kg soilÞ1 the roots had only 35% of the dry matter of the control. At 45
mol Cd (kg soilÞ1 , the root dry matter was 71% of the control. However, the two metals applied at the same time reduced root growth to 29% of the control. The combination of the two metals (269
mol Pb (kg soilÞ1 þ 178
mol Cd (kg soilÞ1 , ammonium acetate-extractable fraction) reduced the root mass of saplings significantly more than application of each of the ions separately. Another example for additive effects was found with saplings of Picea abies (Hagemeyer et al., 1994). Combined treatments with Cd þ Zn reduced root growth much more than either of the separate treatments. The effects of combinations of copper, cadmium, and zinc on root growth of Silene vulgaris exposed to single metals or binary combinations in hydroculture were nonadditive (Cu þ Zn; Cu þ Cd) or antagonistic (Zn þ Cd), when applied concentrations were rather low. The nature of the combination effects depended on the metal concentrations (Sharma et al., 1999). When one of the metals of the combinations was applied in concentrations above a critical toxicity level, synergism was the predominant interaction. Synergistic effects in seedlings of Sinapis alba, i.e., greater growth inhibition, were found in combinations of vanadium with nickel, molybdenum, or copper (Fargasova, 1999). Vanadium and manganese had mutually antagonistic effects. Manganese, molybdenum, and copper were antagonists of nickel. In some combinations no interactions of the metals were found. Many such interactions between trace elements are encountered in studies using soil or other complex substrates. Although they pose additional complicating factors, they should not be neglected in realistic studies. Reactions of plant roots to trace elements can be very sensitive. However, the outlined results demonstrate the large variability in effective critical concentrations, which cause toxicity symptoms. This can be observed even within the same species. Obviously the effects of a certain element depend largely on substrate conditions, particularly the presence of interacting ions. It therefore seems questionable to establish critical upper levels of toxic elements, which can be tolerated by plants.

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EFFECTS OF TRACE ELEMENTS ON PLANTS OF VARIOUS LIFE FORMS AND SYSTEMATIC CATEGORIES

Genotypic differences in resistance to trace elements were described for various wild plants (Ernst, 1982; Baker and Proctor, 1990; Macnair, 1993). It has been well known for a long time that the flora of mining areas is resistant to toxic levels of such mostly metallic elements. Those plants were named metallophytes (Duvigneaud and Denaeyer–De Smet, 1963). In some species or ecotypes resistance is restricted to one particular element; in others, cotolerance to two or several trace elements occurs (Cox and Hutchinson, 1979). Very few species of trees are found among metallophytes (Ernst, 1985). During their long life span, trees accumulate large amounts of toxic elements when growing on contaminated soils. They generally lack the morphological or physiological adaptations that regulate internal concentrations of toxic trace elements, which are found in various herbaceous plants. Some exceptions can be found among mangroves and other halophytic trees (Hagemeyer, 1990, 1997; Breckle, 2000). Therefore, most trees can survive only on less contaminated substrates, where trace element concentrations in their tissues do not exceed critical levels (Ernst, 1985). There are, however, a very few specialized tree species which thrive on metal-enriched soils. An outstanding example is Sebertia acuminata (Sapotaceae), a Ni hyperaccumulator from New Caledonia. It has a remarkable capacity for nickel accumulation (Jaffre et al., 1976; Sagner et al., 1998). The survival strategy of most trees on metal-rich sites seems to rely on the phenotypic plasticity, which enables tree root systems to avoid soil regions of high contamination (Dickinson et al., 1991; Turner and Dickinson, 1993). Plants vary in their response to trace elements not only among various life forms but also among various taxa. The subclass Caryophyllidae is a systematic group with many resistant members. A variety of trace element–resistant genera belong to the Caryophyllaceae and Plumbaginaceae. Some Brassicaceae are also typical metallophytes. Root growth can be used as an indicator of trace metal resistance. In this way the lead responses of 23 different plant taxa were compared by Wierzbicka (1999). Under the applied experimental conditions four groups of plants were distinguished: (1) species with the highest tolerance growing on mine waste heaps, like Silene vulgaris or Leontodon hispidus; (2) species with high constitutional tolerance, like Biscutella laevigata or Zea mays; (3) species with intermediate constitutional

tolerance, like Allium cepa grown from seeds, Triticum vulgare, Pisum sativum, or Secale cereale; and (4) species with low constitutional tolerance, like Brassica napus or Phaseolus vulgaris. Taxonomic relations are, however, not always reliable indications for the trace element resistance of a species. The necessary mechanisms have apparently evolved several times independently. The physiotype concept (Albert and Kinzel, 1973; Kinzel, 1982; Choo and Albert, 1999), proposed for the classification of halophytes, has not yet been applied to metallophytes, with the exception of cadmium (Kuboi et al., 1986). This concept of a chemical characterization of various groups of plants by distinct ion patterns deserves further consideration in the case of trace element–resistant plants.

V.

PLASTICITY OF ROOT DEVELOPMENT IN RESPONSE TO ENVIRONMENTAL CONDITIONS

Root development within a species is a result of a broad genetic disposition enabling the species to cope with a wide range of soil factors (Carlson and Bazzaz, 1977; Taylor and Allison, 1981; Stienen, 1985; Fitter, 1991; Chapter 2 by Fitter in this volume). In different horizons of the soil profile concentrations of nutrient and trace elements can show spatial as well as temporal variations. Such edaphic variations stimulated the evolution of the remarkable plasticity of root systems. The effects of different soil layers on the development of Fagus sylvatica roots were investigated using growth chambers. In homogeneous substrate, root development of beech plants was faster than that of roots growing through three horizontal layers of soils, which differed in metal concentrations (Fig. 1) (Breckle and Kahle, 1992; Breckle, 1996). Stressed roots (240
mol Pb (kg soilÞ1 ), after reaching a horizon of low heavy metal stress (48 or 14
mol Pb (kg soilÞ1 ), grew slightly faster than unstressed roots from the control growth chambers. This indicates an enhanced recovery growth. Characteristics of a distinct soil layer affect root growth and architecture. Also, the heterogeneity and patchiness of the soil profile have an effect. Different parts of the root system of a plant, i.e., tap roots, basal roots, or lateral roots, can react differently to metal toxicity. This is another aspect of the plasticity of root development under stress. In an experiment with aluminum the lower layer in containers with stratified soil had toxic concentrations, whereas the upper

Trace Element Stress

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Figure 1 Effects of lead-contaminated soil layers on elongation of primary roots of Fagus sylvativa seedlings grown for 110 days in root growth chambers with soils differing in lead contamination. The numbers at each curve indicate the contamination of the layer with lead (
mol/kg, extractable fraction). Triangles indicate the time, resp. root-length, when the root reaches the next layer. 14-14-14 treatment : control. The 14-48-240 treatment 2 indicates increasing lead availability; the 240-48-14 treatment 3 indicates decreasing lead availability. (From Breckle and Kahle, 1992.)

part of the soil was nontoxic (Bushamuka and Zobel, 1998). Recordings of root elongation of maize and soybean cultivars showed that in some aluminum-tolerant cultivars only part of the root system was able to grow in the aluminum toxic layer. Some cultivars increased lateral root production in the nontoxic topsoil layer, thus avoiding toxicity. It was concluded that individual parts of the root system could respond independently to aluminum stress in the soil. Therefore, measurements of total root production may produce misleading results in assays of metal tolerance of plant species. The root systems of metal hyperaccumulating plants (see Section IX) also show adaptive responses to uneven distributions of metals in the soil. Zinc-hyperaccumulating Thlaspi caerulescens plants were grown in transparent containers with different types of soils differing in zinc (Schwartz et al., 1999) or cadmium concentrations (Whiting et al., 2000). The distribution of the metal in the containers was either homogeneous, layered, or patchy. Root distribution in the soil profile depended on the type of soil and the contaminating metal. While in a zinc-contaminated soil roots were clustered in the 5–10 cm of the profile, in a lead-contaminated soil more vertical roots explored the 10–

15 cm layer. Nevertheless, in both treatments the mean lengths of the root systems were similar. When spots of zinc-contaminated soil were included into uncontaminated soil profiles, roots colonized the zinc-enriched zones strongly and almost exclusively. Unlike the roots of nonhyperaccumulating plants, the roots of Thlaspi caerulescens explored mainly zincenriched soil areas. The plants consistently allocated
70% of their total root biomass and length in the Zn-enriched soil (Whiting et al., 2000). Zinc availability in the soil was the main factor influencing the root architecture. This is of particular importance when plants are used for phytoremediation of contaminated soils (Saxena et al., 1999). The effects of low concentrations of trace elements on root growth depend also on the growth conditions. When comparing the effect of lead on Phaseolus vulgaris in aeroponic and hydroponic cultures it was found that seedlings grown in aeroponic chambers exhibited a significant decrease of growth with only 24
M lead in nutrient solution. The roots were the organ with the highest percentage of growth inhibition with values reaching only 55% of the controls (Engenhart, 1984). On the other hand, in hydroponics all organs had increased their biomass in the 24-
M

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lead concentrations of 5 mg L1 , the root hair length of the tolerant plants was somewhat smaller, but not as much as that of the nontolerant plants.

lead treatment, whereas with 48-
M lead root growth was slightly inhibited. Aeroponic plants had a stronger and larger root system with well-developed root hairs. In contrast, hydroponic plants had stronger and larger shoots and fewer roots with no root hairs. This can partly explain why aeroponic plants, which develop a larger root surface, were much more sensitive to lead (Christlieb and Weber, 1980; Engenhart, 1984).

VI.

VII.

DEVELOPMENT OF LATERAL ROOTS

It is generally accepted that the development of laterals determines the architecture of growing root systems. Adaptational changes of root architecture may be important for plants to survive on sites with a strong metal stress, where soil is a heterogeneous mosaic of patches with different chemical and physical properties. In seedlings of Picea abies grown for 4 weeks in solutions containing 0:5
M Pb, growth of primary, secondary, and tertiary roots was reduced (Godbold and Kettner, 1991). The initiation of lateral roots was more sensitive to lead than the growth of already established older roots. The development of laterals of second and third order in Fagus sylvatica roots was slightly stimulated by increased lead concentrations (Table 1). Such an increase can be explained by the decrease of the growth of the primary roots. The number of laterals of second and third order was higher. Therefore, the total length of all the roots of a system remained constant, although the primary root was shorter (Table 1). Dense branching of the root system is a typical response to damage of root tips. The architecture of root systems of beech trees was altered by lead from a loose to a more compact, branched structure. This was also demonstrated by experiments with cadmium and with combined applications of cadmium and lead (Bertels et al., 1989). In contrast, the density of lateral roots of maize decreased in a nutrient solution with 24
M Pb, but increased again to the original level in

DEVELOPMENT OF ROOT HAIRS

The development and turnover of root hairs play a major role in the establishment of an efficient waterand mineral-absorbing root system (Gilroy and Jones, 2000; Chapter 5 by Ridge and Katzumi in this volume). The root hair density of Raphanus sativus, when grown in hydroponic culture, decreased with increasing lead concentrations (Lane and Martin, 1980). The lower density coincided with their earlier collapse under relatively low concentrations of copper, nickel, or cobalt (Blaschke, 1977; Patterson and Olson, 1983). Thus, absorption capacity for the toxic element and effective absorption time of the root hairs were distinctly reduced. This was shown for various crop plants as well as for young Betula trees. At the same time, the lower root hair density will exert a negative effect on the absorption capacity of water and of nutrients (Engenhart, 1984). Plants of a lead-tolerant population of Silene vulgaris from southern Poland and of a nontolerant population were treated with 2.5 mg Pb L1 . After 10 days of treatment, roots of tolerant plants were covered with root hairs on 100% of their surface, while nontolerant plants had hairs only on 60–68% of the root surface (Wierzbicka and Panufnik, 1998). At higher

Table 1 Total Number of Lateral Roots of Various Orders per Plant, Percentage of Short Roots (< 2 mm long) Among All Laterals, and Length of the Unbranched Main Root of Fagus sylvatica Seedlings Grown for 40 Days in Root Chambers in Soil with Various Ammonium Acetate–Extractable Lead Concentrations Pb (
mol/kg)

Number of laterals first order second order third order Total number of lateral roots Percentage of short roots Length of main root from tip to first laterals (mm) Source: Breckle (1996).

14.4

48

115

211

1360

110 127 1.5 238 36.6 35.4

87 195 1.0 283 39.4 37.6

70 189 9.6 268 43.5 33.3

47 180 14.0 241 46.7 24.3

4.1 1.2 0.3 5.6 53.8 6.0

Trace Element Stress

a higher lead treatment (Malone et al., 1978). This was explained by the need for a minimum stimulus to activate dictyosomes to export lead ions actively out of the cells. Stimulated branching of roots under trace metal stress as a result of damage to the root tips by cadmium, lead, or zinc with effects on the formation of lateral roots in the herb Ocimum sanctum and in a metal-tolerant cultivar of the grass Festuca rubra was also reported by Cadiz and Davies (1997). In both species the metals at concentrations up to 10
M stimulated the formation of lateral root primordia. Zinc had the strongest inducing effect. The authors also observed a reduction in the size of the apical root meristem under metal stress. They suggested that this effect is similar to a chemical decapitation, which releases the dominance effect of the root apex and thus increases the number of lateral root primordia. An enhanced development of laterals was observed in various crop plants grown with increased but nontoxic copper levels (Blaschke, 1977). Under such conditions, the root systems showed a denser and more compact structure and the rates of water uptake had decreased. This was shown to occur in Fagus under lead stress (see above) and in Trifolium under manganese, copper, and zinc stress (Vogel, 1973). Indications for the same phenomenon were given by Rastin et al. (1985) showing an enhanced dieback of lateral roots in spruce under increased levels of trace metals in forest soils. To some extent such a dieback can be balanced by the growth of new laterals. Under strong lead stress (> 211
mol ðkg soilÞ1 ) an enhanced development of second-order laterals was observed (Breckle et al., 1988). Such flushes might cause periodic cycles of growth, development, and death as part of a survival strategy (Altgayer, 1979). Control plants and stressed plants apparently had different growth cycles. The density of laterals is not only determined by the number of developing primordia, but also by the elongation of the main roots. The architecture of the root systems of Zea mays was changed by solution of 1 mM lead nitrate. The growth and number of laterals was not altered, but their distribution along the roots was denser and the branching zone of the main root was shorter. This was caused by a reduced length of the mature cells in the primary roots. Thus, the leadinduced inhibition of primary root growth resulted in a more dense arrangement of laterals (Obroucheva et al., 1998), which is almost a general rule. Results obtained so far show that the root architecture of plants under trace element stress is altered to a more dense and compact structure. The metal-induced

769

stimulation of the development of laterals often leads to a more densely branched root system.

VIII.

UPTAKE AND ACCUMULATION OF TRACE ELEMENTS

Root uptake of lead, cadmium, or other trace elements depends on pH, the mobility of the trace elements, and the developmental stage of the plants. The binding capacity of the soil and, thus, the extent of the plantavailable fraction of an element is a soil characteristic, but the latter depends also on the uptake abilities of specific roots. An element of comparatively low mobility in soils and in plants is lead. Lead is passively absorbed into the root tip of seedlings of Zea mays, mainly by thin epidermal cell walls in the meristemic region (Tung and Temple, 1996). Only limited quantities of lead entered into protoplasts. As cells matured, accumulation of lead in cell walls increased. Both short- and long-distance transport of lead were apoplastic. When the conducting vascular tissue in the root center had differentiated, lead entered into the conducting systems. It was also absorbed from the water-absorbing zone of the roots, but these quantities remained in the root cortex. The Casparian strip was an effective barrier for lead, but transport through passage cells was possible. Such results underline the comparatively low mobility of lead in plant tissues. The same was observed in roots of Brassica juncea where considerable amounts of lead from the treatment solutions were accumulated in the roots, while only small quantities were transported into hypocotyls and shoots (Liu et al., 2000). The localization of trace metals in cells and tissues of plant roots can be determined with x-ray microanalysis and similar techniques. The distribution of metals in roots of water hyacinth (Eichhornia crassipes) showed distinct patterns (Vesk et al., 1999). Iron was found accumulated at the root surface, e.g., in root plaques known from wetland plants (see Section XV; Ye et al., 1997). Concentrations of iron decreased centripetally. They were higher in cell walls than within cells. In contrast, the trace metals copper, zinc, and lead were not found on the root surface. Their levels increased centripetally and were higher inside the cells than in the walls. Highest levels were found inside the cells of the stele. Although some distribution patterns of elements could be described, the authors caution against general interpretations, since the individual variability of the sampled plants was large.

770

The complicated solution chemistry and speciation of chromium also affects root absorption. Uptake and translocation of chromium by 11 species of common vegetable crop plants supplied with either Cr3þ or CrO2 4 were studied (Zayed et al., 1998). A speciation analysis indicated that in the roots of all tested plants CrO2 was converted to Cr3þ . The translocation of 4 chromium from roots to shoots was limited. Accumulation in roots was about 2 orders of magnitude higher than in shoots, regardless of the chromium species in the nutrient solution. Highest concentrations were found in crop species of the Brassicaceae family, e.g., cauliflower, kale, or cabbage. Silicon is a major inorganic constituent of many higher plants (grasses, conifers, Equisetum, etc.), but it is rarely considered in biological studies. It might turn out to react as a trace element in some plant groups. In nutrient solutions silicon absorption of the roots of Triticum aestivum was rapid (Rafi and Epstein, 1999). Nearly mature plants that were preloaded with silicon showed the same absorption rates as plants previously grown in solutions without silicon addition. The authors give two reasons for this observation: (1) About 90% of the absorbed silicon was transported to the shoots and the roots maintained a low silicon status which promoted further uptake. (2) The absorbed silicon is largely immobilized in insoluble form. Thus, there was no negative feedback from shoots to roots and the silicon uptake continued unabated. There is growing concern about the radioactive contamination of the environment, particularly after accidents in nuclear power stations and industries. Investigations of the mobility of radionuclides, e.g., isotopes of cesium or strontium, in ecosystems also have to consider root uptake and mobility of these isotopes in plants with respect to their alkaline earth or alkali metal chemistry (Carini and Lombi, 1997; Jones et al., 1998; Entry et al., 1999; Guivarch et al., 1999; Zhu et al., 1999). A comparative study of the root uptake of cesium-134 of 30 different plant taxa was presented by Broadley and Willey (1997). They found lowest accumulations in slow-growing Gramineae and highest accumulations in fast-growing Chenopodiaceae. This was observed after a short-term exposure of the plants. If radiocesium uptake of Chenopodiaceae is also high during long-term exposures, implications for food contamination and the potential of such plants for phytoremediation of contaminated soils should be considered. The uptake of trace elements into roots depends on the ionic milieu of the rhizosphere. With hydroponi-

Hagemeyer and Breckle

cally grown Sinapis alba plants interactions in the uptake of various trace metals were found (Fargasova and Beinrohr, 1998). Root accumulation of vanadium was inhibited by nickel, manganese, and copper, and the accumulations of nickel or manganese were both inhibited by copper. However, none of the tested metals inhibited copper accumulation. Many toxic effects of trace elements result from their replacement of calcium at vital sites of cell membranes in root cells. Absorption of Cd2þ by roots of Tamarix aphylla was markedly inhibited by increasing concentrations of Ca2þ in the solution (Hagemeyer and Waisel, 1989). Magnesium ions were less effective in reducing Cd2þ uptake. Monovalent ions (Naþ , Kþ , Liþ ) also reduced Cd2þ uptake, but to a lesser extent than divalent ions (Hagemeyer, 1990). An optimal supply of calcium or magnesium can considerably alleviate the toxicity of some trace elements (Wilkins, 1957; Wallace et al., 1980; Hagemeyer and Waisel, 1989; Hagemeyer, 1990; Skorzynska-Polit et al., 1998; Saleh et al., 1999). Not only the calcium status of a plant but also the phosphorus supply determines uptake of trace elements in a specific way (Wallace et al., 1978). Furthermore, the form of nitrogen supply can affect the toxicity of trace metals (Zornoza et al., 1999). The nickel tolerance of Helianthus annuus was lowest when grown with nitrate alone. Simultaneous supply with nitrate and ammonium reduced nickel toxicity.

IX.

RESPONSE TO LONG-TERM EXPOSURE OF PLANTS TO TRACE ELEMENTS FROM GEOGENIC SOURCES

A constant, long-term exposure of Artemisia vulgaris plants to slightly increased concentrations of lead along roadsides has given rise to the selection of lead-resistant ecotypes (Helming and Runge, 1979). Similar ecotypic variation was also found in Salix and Betula in biotopes affected by ore mining (Denny and Wilkins, 1987a–c). Ore deposits and similar remnants from mining operations have a rather high total content of various trace elements. However, the plant-available fraction is usually much lower (Wenzel and Jockwer, 1999). This is the reason that sometimes a great variety of plant species can grow on ore deposits rich in lead, zinc, or other trace elements. For example, in the area of Stolberg, an old mining site south of Aachen, Germany, birch trees are growing well on mounds of

Trace Element Stress

ore smelter ash with total lead concentrations reaching 10–20 g (kg substrateÞ1 . High zinc concentrations are often found on ore outcrops in mining areas. There are impressive genotypic differences in Zn resistance among certain species of the natural vegetation (Ernst, 1982; Macnair, 1993). The mechanisms of zinc resistance are in principle similar to those of copper resistance. Compartmentation plays a major role at the cellular, tissue, or organ level. At the cellular level, zinc is accumulated in the vacuoles (Harmens et al., 1993b). Serpentine is a common name for a number of rock types that contain ferromagnesian minerals. Owing to high concentrations of magnesium and iron, such rocks are called ultramafic (Proctor, 1999). After weathering, ultramafic rocks produce soils with naturally elevated levels of trace elements. They are found on all continents (Roberts and Proctor, 1992). Serpentine soils have increased concentrations of nickel, chromium, or cobalt as well as unfavorably low calcium/magnesium ratios (Menezes de Sequeira and Pinto da Silva, 1992; Rodenkirchen and Roberts, 1993; Shallari et al., 1998; Proctor, 1999; Ater et al., 2000). This poses severe problems to plant growth. A specialized flora with many endemic species has developed on ultramafic sites (Arianoutsou et al., 1993). Some species have a remarkable potential for trace element accumulation; nickel concentrations in their leaves can reach the 1% range. Plants are called hyperaccumulators if the concentration of a trace metal regularly exceeds 0.1% under natural conditions (Baker and Brooks, 1989; Greger, 1999). Many hyperaccumulating species were found in the genus Alyssum (Morrison et al., 1980). Some accumulating species of this genus showed root growth in solutions with nickel concentrations up to 1 mM, whereas root growth of a nonaccumulating species was inhibited even by traces of nickel. Two serpentine species with differing resistance strategies were studied by Gabbrielli et al. (1990). Silene italica limits its nickel uptake. Root growth was inhibited by a suppression of mitotic activity in root tips at 7:5
M nickel in the culture solution. The same concentration did not affect root growth in Alyssum bertolonii, which is a nickel accumulator. A calcium supply of 25 mM reversed the effects of nickel on root growth in Silene, but in Alyssum the addition of calcium reduced root growth. This finding demonstrates that Alyssum bertolonii is also adapted to low calcium concentrations in the substrate, which is typical for serpentine soils. Concentrations of nickel in roots and shoots of this species reached 0.3% and 1.6% of the dry weight, respectively (Pandolfini and

771

Pancaro, 1992). It is amazing that a plant can tolerate such high concentrations of a toxic element in its live organs. In several hyperaccumulating Alyssum spp. it was shown that exposure to nickel caused an increase of free histidine in the xylem sap (Kra¨mer et al., 1996). The authors suggested that nickel tolerance is based on an enhanced production of histidine, which serves as a chelator of nickel in the xylem sap. The transport of the toxic ions from the roots into the shoot is thus promoted. Hyperaccumulators of other elements, like cobalt, copper, or chromium, were also found (Baker and Brooks, 1989). For instance, Brassica pekinensis was identified as a hyperaccumulator of lead (Xiong, 1998). Such plants have a potential use in phytoremediation of metal-contaminated soils (Saxena et al., 1999). Furthermore, recent research demonstrated a defensive function of accumulated metal ions against herbivores and pathogens (Boyd and Martens, 1998; Sagner et al., 1998; Boyd and Moar, 1999).

X.

EFFECTS OF RARE EARTH ELEMENTS ON ROOT GROWTH

The rare earth elements (REE), or lanthanides, are often left out of the biological studies. This group includes the 14 elements with atomic numbers from 58 (cerium) to 71 (lutetium) as well as the elements lanthanum, scandium, and yttrium (Greenwood and Earnshaw, 1986). In spite of their name, most of these elements are not really rare in nature. For instance, in the earth crust cerium is five times more abundant than lead. The total concentration of the REE in the earth crust is
0:01% (Holleman and Wiberg, 1995). In general, the REE with even atomic numbers are more abundant than those with odd atomic numbers, a fact known as ‘‘Harkins rule’’ (Holleman and Wiberg, 1995). The physical and chemical properties of the different REE are similar. The ions of REE are mostly trivalent. There are > 100 minerals known to contain REE, and the different elements often occur in groups in the minerals (Greenwood and Earnshaw, 1986). In comparatively few studies root uptake and accumulation of REE in higher plants were investigated. One reason may be insufficient detection limits of older analytical methods, since the REE occur in plants in rather low concentrations (Markert, 1987; Breckle, 1997; Fu et al., 1998). Several studies were made in China, owing to widespread use of REE in

772

agriculture and industry in that country (Yang et al., 1999). The uptake of scandium (Sc3þ ) into root tips of three different cultivars of Sorghum bicolor was determined by short-term (10-min) uptake studies (Wilkinson and Duncan, 1992). In root tips of the acid soil stress sensitive cultivar accumulation of nonextractable (water, EDTA), scandium ions increased with decreasing pH of the growth medium. However, nonextractable scandium did not increase in root tips of the tested cultivars that were tolerant to acid soil stress. The study showed a strong effect of pH on scandium accumulation. The REE levels in Citrus tree samples in Florida were correlated with soil concentrations (Wutscher and Perkins, 1993). The highest concentrations were found in feeder roots, ranging from 4.6 to 585
g g1 . Accumulation of REE by sugarcane (Saccharum officinarum) was possible via the leaves when sprayed on leaf surfaces or via the roots after soil application (Chua et al., 1998). Root uptake was determined in plants grown on soil sprayed with a microelement fertilizer containing nitrates of various REE, including cerium and lanthanum. Root uptake showed a linear correlation with REE concentrations of the soil. The absorbed quantities of REE were mobile in the plants and were accumulated in all parts. The authors concluded that high concentrations of REE in the soil could result in harmful effects for humans consuming sugarcane products. The chemical speciation in the soil, chelation, binding forms, and bioavailability of REE were addressed in several studies. Sun et al. (1997) studied the effects of EDTA on the uptake of lanthanum, gadolinium, and yttrium from nutrient solutions by 2-week-old wheat (Triticum sp.) seedlings. Addition of EDTA to the nutrient solution reduced the accumulation of the elements in the roots, but increased their accumulation in stems and leaves. Roots showed a comparatively higher accumulation capacity than the aboveground parts. The REE concentrations in the roots were linearly correlated with concentrations in the nutrient solutions in the tested range of 0–4 ppm. This was found with both the ionic and the EDTA-complexed forms of the elements. A similar result was described for wheat (Triticum sp.) plants grown in soil (Yang et al., 1999). Bioaccumulation of all the elements (lanthanum, cerium, samarium, gadolinium, and yttrium) in roots was much higher than in stems and leaves. Uptake was increased by EDTA addition to the soil, which resulted from desorption of REE ions from soil compounds.

Hagemeyer and Breckle

A sequential extraction procedure was used to determine the distribution of REE in different chemical fractions of Mollisols in China. Samples of Zea mays and Oryza sativa plants growing on the soils were analyzed (Li et al., 1998). Concentrations in plant parts followed the order root > leaf > stem > grain. Total soil concentrations or the sum of all extracts of the sequential extraction procedure were not useful indicators of REE plant uptake. The acetate-extractable fraction of REE in the soil might play a role in controlling plant uptake. However, the authors concluded that a more accurate speciation analysis method is necessary to establish a relationship between REE speciation in soil and the bioavailability. Comparatively little is known about the effects of the REE on growth and development of roots of higher plants. An extensive study with roots of maize (Zea mays) and mungbean (Vigna radiata) seedlings revealed that the relative root elongation of both species was inversely correlated with lanthanum or cerium concentrations in the solution (Diatloff and Smith, 1995a). Mungbean was more sensitive than maize. Cerium was more toxic than lanthanum to mungbean. The concentration causing 50% reduction of mungbean root elongation was 0:9
M cerium and 3:1
M lanthanum. To maize roots lanthanum was more toxic. Concentrations causing 50% reduction of maize root elongation were 12:2
M cerium and 4:8
M lanthanum at pH 5.5. Lanthanum at concentration < 1
M enhanced elongation of maize and mungbean roots but not root dry matter accumulation. Transport of La to the shoot was blocked and it accumulated in the roots. Similar results were obtained for cerium (Diatloff and Smith, 1995b, c). The effects of REE were examined by REE spraying at the end of the tillering phase of sugarcane, Saccharum officinarum (Pan et al., 1993). It increased the activity of Na-K-ATPase in root cells. The vigor of root systems, the amount of rhizosphere bacteria and enzyme activities in the soil were increased. As a result, growth rate and yield of the plants were increased. The cytological effects of praseodymium oxide and neodymium oxide (1–5 ppb) on root tip cells of Vicia faba caused chromosomal aberrations and mitotic anomalies in various proportions (Singh et al., 1997). Depending on the concentration and duration of the treatment, abnormal meta- and anaphase cells were recorded. These effects were similar to aberrations induced by radiation and radiomimetic chemicals. Europium ions (Eu3þ ) influenced the contents and composition of anthraquinones in root cultures of the

Trace Element Stress

Chinese medicinal plant Cassia obtusifolia (Guo et al., 1998). Thus, the production of secondary metabolites in plants can be affected by REE. Roots of water hyacinth (Eichhornia crassipes) remove large quantities of europium(III) from water. This species is used for decontamination of polluted water. In the roots, intracellular europium ions are probably complexed by organic acids (Kelley et al., 2000). The varied effects of the different REE on root growth of plants require further attention. Research should particularly focus on synergistic or antagonistic effects of combinations of several REE on plant growth, since the naturally occurring REE minerals usually contain combinations of various elements.

XI.

PHYSIOLOGICAL BASIS OF TRACE ELEMENT TOLERANCE

The evolution of trace element resistance is still a matter of discussion. The basic question is whether the trace element resistance in higher plants is controlled by one or a few genes or by the combined action of many genes (polygenic control). Macnair (1983) presented evidence that the copper tolerance of Mimulus guttatus is controlled by a single major gene. Accordingly, he argued that there must be a single physiological or biochemical process which generates the tolerance required for the colonization of toxic soil. Other physiological differences between tolerant and nontolerant plants may occur which are manifestations of subsequent genetic changes to improve the degree of adaptation. A similar result was found for the copper tolerance of Silene vulgaris (Schat and Ten Bookum, 1992). With results from crossing experiments of Silene vulgaris plants from populations differing in copper tolerance it was suggested that copper tolerance of this species is under the control of two major genes (Schat et al., 1993), and the tolerance level seems to be controlled by two additional genes. The authors concluded that all these genes are involved in the control of an exclusion mechanism operating at the plasma membrane. In a review of the responses of higher plants to cadmium it was concluded that cadmium detoxification is probably a complex phenomenon under polygenic control (Sanita di Toppi and Gabbrielli, 1999). The results of earlier studies using grasses and herbs were taken as evidence for polygenic control of trace metal tolerance (Wilkins, 1960; Bro¨ker, 1963; Urquhart, 1971; Gartside and McNeilly, 1974).

773

Besides investigation of the genetic basis of trace element tolerance, it is also necessary to further elucidate the physiology of mechanisms that enable metallophytes to survive on contaminated substrates. Furthermore, it will be important to clarify the regulatory mechanisms involved in uptake and turnover of essential trace elements that are required in very low concentrations. Toxic effects of high internal concentrations can be alleviated by a number of physiological mechanisms. These depend on rates and mechanisms of uptake, on translocation along root tissues, and on the properties of the trace element. The biological effects of trace elements on water relations, especially the primary toxicity mechanisms of the different metal ions, may be as different as their chemical properties—e.g., valency, ion radius, redox potential, and stability of organic complexes (Barcelo and Poschenrieder, 1990; Poschenrieder and Barcelo, 1999). The metal ions can induce a sequence of biochemical and physiological alterations (Foy et al., 1978; Lepp, 1981) by damaging membranes and altering enzyme activities (Kennedy and Gonsalves, 1987). A multitude of secondary effects have been observed, such as disturbances of the hormone balance, deficiency of essential nutrients, inhibition of photosynthesis, or changes in carbon allocation. One mechanism of trace element resistance is to avoid toxic accumulations of such elements in sensitive plant parts, like meristems. In some plant species, this is achieved by reduced root uptake. Also, the translocation from root cortex to xylem vessels can be slowed down. Toxic elements are therefore sequestered outside the root symplasm or in specialized tissues outside the endodermis. In roots of Betula, Zn accumulated in cells of the endodermis when the roots were subjected to concentrations below the lethal threshold (Denny and Wilkins, 1987a,b). The distribution of cadmium in roots of Phaseolus vulgaris plants was investigated after cultivation in cadmium containing nutrient solutions (Vazquez et al., 1992). The accumulation of cadmium decreased from outer to inner parts of the root cortex. Only small amounts were detected in the endodermis. As the endodermis constitutes a barrier to ion transport, root cortex cells usually contain higher element concentrations than cells in the central vascular cylinder. A study of the ultrastructural localization of lead in roots of Allium cepa suggested protective mechanisms against lead in root tip cells (Wierzbicka, 1998). Plants were treated with lead added to nutrient solutions as chloride or nitrate. Lead accumulated in the apoplast of root tips. Based on ultrastructural observations, the

774

author hypothesized that there are two protective mechanisms against lead in the root tips of onion: (1) The amount of polysaccharides in the cell walls and the thickness of the walls increase, which allows a larger retention of lead outside the cytoplasm. (2) Lead is exported from the cells through plasmatubules to the root tip apoplast. In this way lead levels in the symplast are kept low. Another strategy of metal resistance found in certain cases is the accumulation of toxic ions in cell vacuoles. A zinc-malate shuttle mechanism was proposed for the transport of zinc ions through the cytoplasm into the vacuole (Mathys, 1977; Ernst et al., 1992). This hypothesis needs further investigation. Using isolated oat (Avena sativa) root tonoplast vesicles, it was found that ions of zinc, manganese, cadmium, or calcium could be transported by metal/Hþ antiport mechanism into the plant vacuole (Gonzalez et al., 1999). This antiport mechanism seems to be metal specific, since in the same system of oat root tonoplast vesicles no Ni/H antiport was found (Gries and Wagner, 1998). In this study the vacuoles were not a major compartment for nickel accumulation. Also, no heavy metal accumulation was found in leaf cell vacuoles of Silene vulgaris plants from a heavy metal–polluted mine dump (Bringezu et al., 1999). Plants can avoid detrimental effects on sensitive physiological processes in cells by immobilization and sequestration of toxic trace elements. Specialized molecules with large numbers of negatively charged groups capture trace element cations in the cytoplasm. Metallothioneinlike proteins were found in roots of plants, which are similar to metal-binding metallothioneins from animals and fungi (Tomsett and Thurman, 1988; Robinson et al., 1993; Prasad, 1999). In general, such molecules have large numbers of sulfur-containing amino acids, like cystein, which bind cations to –SH groups. A copper-binding thionein was found in roots of a copper-resistant strain of Agrostis gigantea (Rauser and Curvetto, 1980). In roots of maize a Cd binding protein appeared after subjecting the plants to cadmium stress. This protein contained 40% cystein (Rauser and Glover, 1984). Additionally, smaller metal-binding polypeptides were discovered in plants and named phytochelatins (Grill et al., 1985). In most cases they consist of only three different amino acids: glutamic acid, cystein, and glycine. The polypeptide chain is a repetitive sequence of poly(gamma-glutamylcysteinyl)glycine. Chain length and proportions of the constituents vary in different plants (Robinson and Jackson, 1986; Narender Reddy and Prasad, 1990; Rauser, 1990; Prasad, 1999).

Hagemeyer and Breckle

Such compounds were found in various members of the Fabales (e.g., Phaseolus vulgaris, Glycine max) and assumed to bind and immobilize toxic trace metals (Grill et al., 1986). Similar cadmium-binding polypeptides were observed in root tissue of six different plant species including sunflower, soybean, and potato grown under cadmium stress (Fujita and Kawanishi, 1987). The authors concluded that low-molecular-weight cadmium-binding complexes play a role in the trace element resistance of plant roots, the organ directly in contact with the noxious metals. Also, roots of pepper plants (Capsicum annuum) exposed to cadmium stress responded with increased phytochelatin concentrations (Jemal et al., 1998). Higher levels of phytochelatins were found in roots of cadmium stressed Silene vulgaris plants than in control plants. More than 60% of total cadmium in the roots was bound to polypeptides. It was suggested that the cadmium sequestration by polypeptides plays a role in cadmium resistance of the plants (Verkleij et al., 1990). However, this assumption was challenged by the findings of other authors. When subjected to copper stress, both sensitive and resistant genotypes of S. vulgaris produced phytochelatins in the root tip. Thus, the differential copper resistance of this species did not depend on differential phytochelatin production (Schat and Kalff, 1992). Also, the zinc resistance of S. vulgaris was not due to increased phytochelatin production (Harmens et al., 1993a). Under cadmium stress, sensitive strains of S. vulgaris produced more phytochelatins in root tips than resistant genotypes. Thus, phytochelatins may serve to chelate and detoxify cadmium. However, the cadmium resistance of some varieties did not depend on an increased production of such polypeptides (De Knecht et al., 1994). Synthesis and accumulation of phytochelatins in Phaseolus coccineus plants depended on the growth stage in which cadmium was added to the nutrient solution (Tukendorf et al., 1997). Only when cadmium was applied in an early growth stage was a high accumulation of phytochelatins observed. The contributions of trace element–binding polypeptides or proteins to metal resistance appear to be not fully understood. This problem should be tackled in future research.

XII.

TOXICITY TESTS USING ROOT GROWTH PARAMETERS

The growth of roots of higher plants is a sensitive indicator of trace element toxicity (Hagemeyer,

Trace Element Stress

1999). Effects of trace elements on different growth parameters of roots, like elongation or branching, are detectable at low substrate concentrations. Based on this observation, a number of short-term test systems for biotoxicity of metals were proposed (Ko¨hl and Lo¨sch, 1999). Wilkins (1957) described a method to determine the lead tolerance of different genotypes of Festuca ovina originating from soils with different lead concentrations. Tillers of the test plants were grown in hydroculture with added lead. Root elongation was measured at daily intervals. Differences in the growth rates indicated varied degrees of lead tolerance of the plants. More recently, a rapid screening method for chemicals involved in environmental hazards was developed (Liu et al., 1995). A test using roots of onion (Allium cepa) was proposed. It is based on a previously described ‘‘Allium test’’ (Fiskesjo¨, 1985). Onion bulbs are placed in the test liquids which are daily renewed. The bulbs are allowed to sprout and to produce roots for 24–96 h in beakers, protected from light. Then root samples are cut and fixed in Carnoy’s reagent. For the examination of chromosome and nucleus morphology the fixed roots are squashed in carbol-fuchsin solution. A silver-staining procedure is used to examine changes in the nucleoli. In order to examine the described Allium test technique, Liu et al. (1995) used salt solutions of 11 different trace elements in tap water at pH 6.5. The applied concentrations ranged from 107 to 101 M. The effects of the tested metals on cell division and nucleoli in root tip cells depended on the concentration and the duration of the treatment. The metals caused irregularities of chromosomes, nuclei and nucleoli, like c-mitosis, chromosome bridges, chromosome stickiness, or irregularly shaped nuclei and nucleoli to varying degrees. Based on the concentrations that caused serious toxic effects the metals could be divided into three groups: the first group of highest toxicity included mercury and cadmium (107 –105 M); the second group of medium toxicity included zinc, lead, copper, nickel, cobalt, aluminum, and chromium (104 –103 M); and the third group of low toxicity included manganese and magnesium (102 M). This test appears to be a simple and fast, but sensitive, screening technique. A biomonitoring method for the cadmium contamination of soil and water using growth parameters of mungbean (Phaseolus aureus) roots was devised by Geuns et al. (1997). The toxicity threshold value for root elongation was an internal cadmium concentration of 25
g=g root dry weight. Above this concentration sterol synthesis in roots was reduced.

775

Cadmium increased the stigmasterol-sitosterol ratio and induced a redistribution of sugars in roots. At internal cadmium levels of 100
g=g dry weight an increase of polyunsaturated fatty acids in roots was found. The polyamine synthesis was strongly affected. At a cadmium concentration of the medium of 100
M the synthesis of putrescine was sixfold increased. When mungbean seedlings are used as biomonitors of cadmium toxicity, the most sensitive parameters are the root growth reduction and the putrescine accumulation in the roots that occurs before any growth reduction is detectable. In situations where funds are limited and an extensive laboratory infrastructure is not available, the uncomplicated root tests can offer reasonable alternatives to microorganism-based toxicity tests.

XIII.

MYCORRHIZA AND TRACE ELEMENT RELATIONS OF PLANTS

The symbioses of plant roots and fungi, called mycorrhizae, are a widespread phenomenon (Varma and Hock, 1999; see also the Chapters 50 by Kottke and 49 by Sieber in this volume). Effects of mycorrhizae on trace element relations of plants have been repeatedly investigated (Leyval et al., 1997). Two aspects were intensively studied: effects of mycorrhizae on trace element resistance of higher plants, and enhancement of nutrient absorption by mycorrhizae. Ectomycorrhizae with Paxillus involutus could ameliorate Zn toxicity in some varieties of birch (Brown and Wilkins, 1985; Denny and Wilkins, 1987c,d). The mycorrhizal association of Pinus sylvestris with Paxillus involutus reduced the toxic effects of cadmium and zinc on root elongation (Hartley-Whitaker et al., 2000). The infection of the roots with the fungus decreased the transport of cadmium or zinc to the trees shoots. Grown on contaminated substrates, the mycorrhizal hyphae contained vacuoles with accumulated cadmium (Turnau et al., 1993). This is considered a detoxification mechanism that reduces the trace element burden. The localization of trace metals in roots of Picea abies seedlings colonized with the fungus Hebeloma crustuliniforme was studied with x-ray microanalysis (Brunner and Frey, 2000). Cadmium was found mainly in the Hartig net. Nickel was detected in the Hartig net and in cell walls of the cortex. Zinc occurred in the Hartig net, cortical cell walls, and the fungal mantle. In laboratory experiments ectomycorrhizae of various fungi with roots of Pinus sylvestris seedlings were

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investigated (Colpaert and Van Assche, 1993). Uptake of cadmium was highest in the nonmycorrhizal control; thus, a protective effect of the symbiosis against cadmium toxicity was concluded. In contrast, the mycorrhizal association of Corylus avellana roots with Tuber albidum did not prevent chromium accumulation in the roots (Strati et al., 1999). Agrostis capillaris showed a negative correlation between copper concentrations of the soil and the degree of mycorrhizal infection of the roots (Griffioen et al., 1994). However, in an area contaminated with cadmium and zinc the mycorrhizal fungi had evolved resistance to these metals and can thus play a role in the trace element resistance of this grass. The symbiotic ectomycorrhiza does not always reduce metal toxicity in forest trees. The amelioration depends on the species and strain of the ectomycorrhizae as well as on the metal (Godbold et al., 1998). Mycorrhizae can also stimulate the absorption of trace elements by plant roots (Ernst, 1985). The uptake of zinc by endomycorrhizal roots of Araucaria cunninghamii and by ectomycorrhizal roots of Pinus radiata was enhanced as compared to uninfected controls (Bowen et al., 1974). In this same way the symbiosis can enhance root acquisition of mineral nutrients in soils of poor nutrient availability (Dehn and Schu¨epp, 1989; Faber et al., 1990; Clark and Zeto, 1996; Caris et al., 1998; Clark et al., 1999). However, transport of cadmium and zinc to the shoots of lettuce was lower in mycorrhizal plants. The metal retention in roots was attributed to complexation by cystein-containing ligands of fungal proteins (Dehn and Schu¨epp, 1989). Such a mechanism can also support the trace element resistance of plants. The enhancement of plant metal uptake by mycorrhizae could improve the efficiency of the phytoremediation of contaminated soils. In the three grass species Paspalum notatum, Sorghum halpense, and Panicum virginatum, plants inoculated with the fungus Glomus sp. had higher radioisotope concentrations (137 Cs and 90 Sr) in the plant tissue (Entry et al., 1999). Plant bioconcentration ratios were higher in mycorrhizal plants than in uninoculated plants. The authors concluded that sites contaminated with radionuclides can be effectively cleaned with mycorrhizal plants. The outlined results show that there is no simple and straightforward interpretation of the effects of mycorrhizae on trace element relations of plants. Apparently, these effects depend on both partners of the symbiosis as well as on environmental conditions. The assumption that a plant gains additional resistance to toxic trace elements from the association with a

Hagemeyer and Breckle

symbiotic partner needs detailed investigation in each particular case.

XIV. EFFECTS OF PLANTS ON SOILS It is well known that trace elements in the soil affect the development of plant roots. On the other hand, the roots of plants can affect the concentrations of plantavailable trace metal fractions in the soil. Plant roots can mobilize or immobilize soil minerals (McCully, 1999; Chapter 36 by Neumann and Ro¨mheld in this volume). Consequently, root growth of all plants growing in such a soil is then influenced. For elements like iron and manganese the mobility and the availability for root absorption is controlled by the oxidation status of the soil. Plant roots that penetrate the soil layers can alter the oxygen partial pressure in the rhizosphere. Thus, the roots can affect the accumulation and the mobility of elements in their vicinity. This was shown for Spartina townsendii and Atriplex portulacoides effect on a wetland soil from a salt marsh in Ireland (Doyle and Otte, 1997). The salt marsh soils are effective sinks for trace elements in ecosystems. The soil pool of trace elements that is influenced by the studied plants can have considerable effects on the biogeochemistry, accumulation, and plant availability of the trace elements (Doyle and Otte, 1997). Another aspect of plant effects on soils concerns the interaction with rhizosphere bacteria (Wenzel et al., 1999). The role of such microorganisms in facilitating selenium and mercury accumulation in roots of the wetland plants Scirpus robustus and Polypogon monspeliensis was studied by De Souza et al. (1999). In a laboratory experiment the plants were treated with the antibiotic ampicilline to inhibit growth of rhizobacteria. The root uptake of selenium and mercury was significantly lower than in plants without ampicilline treatment. When axenic Scirpus plants were inoculated with bacteria isolated from the rhizosphere of fieldgrown plants the accumulation of selenium and mercury was significantly higher than in axenic controls. The authors concluded that rhizosphere bacteria can promote the accumulation of selenium and mercury in roots and shoots of wetland plants. The nature of the stimulating effect is as yet unknown. Several possible effects of the rhizobacteria were discussed: (1) stimulation of production of compounds like siderophores which facilitate metal absorption by roots; (2) increase of root surface area by stimulation of root hair growth; (3) transformation of the trace elements into more

Trace Element Stress

readily absorbable forms; (4) increase of selenium uptake by stimulation of the sulfate transport protein which also transports selenate; and (5) reduction of the rhizosphere pH, which enhances mercury absorption into roots. The stimulating effect of rhizobacteria on trace element absorption is important when plants are used for phytoremediation of contaminated soils (Wenzel et al., 1999; Saxena et al., 1999). Plant roots can also reduce the mobility of trace elements in the soil. In pots with Agrostis plants copper activities in the soil solution were 2 orders of magnitude lower than in bare pots without vegetation. It was concluded that the plant growth affects soil pH, the dissolved organic carbon concentration, and the calcium concentration of the soil solution to such an extent that the total dissolved copper concentration and the free metal activity in the soil were reduced. In this way potentially toxic metals can be immobilized in the soil by the action of plant roots (Ro¨mkens et al., 1999). The multiple interactions among plant roots, soil microorganisms, and the soil matrix are complex. Nevertheless, such relations should be studied in detail under realistic field conditions. This will improve our understanding of trace element effects on plant growth.

XV.

TRACE ELEMENT DEFICIENCIES

The key symptom of iron deficiency in leaves is chlorosis, caused by an inhibition of chloroplast development. In many plant species iron deficiency is also associated with inhibition of root elongation, increase in the diameter of apical root zones and an abundant root hair formation (Ro¨mheld and Marschner, 1981). It can also be connected with the development of a typical rhizodermal cell wall labyrinth as in other transfer cells. This seems to be part of a regulatory mechanism which enhances iron uptake (Kramer et al., 1980). It was found only in those plant species that can acidify the rhizosphere (Ro¨mheld and Kramer, 1983). Iron deficiency stress can stimulate the activity of the enzyme Fe(III)-reductase, which is associated with plasma membranes in root cells. The enzym catalyzes the reduction of Fe(III) to Fe(II), which is more readily absorbed by roots. This was shown for Pisum sativum plants (Grusak et al., 1993). The plasma membrane bound reductase system can also increase the uptake of other trace elements, like copper and manganese. It has been suggested that it plays a general role in cation absorption of roots (Norvell et al., 1993; Welch et al., 1993).

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Under conditions of iron deficiency graminoid species can release substances from their roots, which mobilize Fe(III) by chelation (Marschner et al., 1986, 1987; Ro¨mheld and Marschner, 1990; Chapters 35 by Jungk and 36 by Neumann and Ro¨mheld in this volume). The chemical nature of these substances, sometimes called low-molecular-weight organic acids, or phytosiderophores, varies with species. Compounds like mugineic acid or avenic acid were found in root exudates. Studies with iron-deficient barley (Hordeum vulgare) roots suggest that phytosiderophores of the mugineic acid family are secreted from roots as monovalent anions through anion channels (Sakaguchi et al., 1999). The Fe(III) chelates are transported into root cells (Takagi et al., 1984; Marschner, 1986). The release of phytosiderophores is stimulated by iron deficiency. They can also mobilize other trace elements, like copper, zinc, and manganese (Ro¨mheld and Marschner, 1990). In iron-deficient wheat plants the released phytosiderophores mobilized also zinc in the rhizosphere and in the root apoplast (Zhang et al., 1991). The same was found for cadmium (Cieslinski, et al. 1998). In different cultivars of wheat (Triticum turgidum), cadmium accumulation was proportional to the levels of low-molecular-weight organic acids in the rhizosphere. Apparently these substances have a role in the solubilization of cadmium in the soil solution. The phytosiderophore release of the efficient cultivar of wheat (with respect to zinc absorption efficiency) under conditions of zinc deficiency was higher than the inefficient variety (Cakmak et al., 1994). The principal compound was 2 0 -deoxyimugineic acid, which was also released under iron deficiency. The authors concluded that an enhanced release of phytosiderophores under zinc deficiency stress may be the reason for an efficient zinc uptake in certain gramineous species. While most authors report stimulation of metal uptake by secreted low-molecular-weight organic substances, Saber et al. (1999) reached a different conclusion. In hydroculture experiments with sunflower (Helianthus annuus), they found that aluminum and zinc ions stimulated the release of malic and citric acid from roots. Experimental additions of these organic acids into the growth medium to some extent alleviated inhibitory effects of the toxic ions on plant growth. The authors concluded that the tolerance mechanism of sunflower plants against toxic ions includes metal exclusion through secretion of organic acids into the rhizosphere, which reduces the uptake of the toxic ions. An additional mechanism is internal tolerance by chelation of toxic ions in the cytoplasm.

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Most metal ions are much more readily absorbed in a reduced form (Mn2þ , Fe2þ ; etc.) than in oxidized form. Apparently, most plants are able to reduce metal ions in the vicinity of their roots. This is facilitated by changes of pH of the rhizosphere, but also by exudation of reducing substances. Many questions remain open: How can plants achieve a balanced ion uptake to serve their particular needs with the help of rather unspecific chelators? In which way do the exuded chelating substances interact with ion-complexing compounds of the soil? Are there other plant groups with particular substances such as the iron-siderophores of grasses? For example, mugineic acid and avenic acid are typical phytosiderophores of all grasses, but not of members of other graminoid families, as Commelinaceae, Cyperaceae, and Juncaceae (Mino et al., 1983; Takagi et al., 1984; Marschner, 1986). The special iron mobilization of grasses is important under a normal or low supply of iron (see Chapter 36 by Neumann and Ro¨mheld in this volume). What happens under conditions of high iron concentrations, such as at low pH and under low O2 ? Whereas iron deficiency occurs more frequently, iron toxicity is rare. In habitats with periodically submerged soils, which are poor in O2 , iron and other metallic ions are present in a reduced state. Fe(II) is more soluble than Fe(III) and can thus cause toxicity symptoms. Such toxicity can appear in paddy fields. The roots of such plants are covered with thick crusts of brownish Fe(III) compounds (see also Chapter 56 by Beyrouty in this volume). Iron deposits on roots were shown to ameliorate toxic effects of excess copper (Greipsson, 1994). On the surfaces of roots and rhizomes of Spartina maritima plants of intertidal areas in Portugal metalenriched rhizoconcretions were found (Vale et al., 1990). They contained up to 11% iron, large amounts of manganese, and variable amounts of other trace elements. The ecological implications of trace element accumulations in the rhizospheres of some plant species deserve further investigation. Data on the effects of either deficiency or toxicity of Mn on roots are scarce. In Mn-deficient plants the formation of lateral roots ceased completely (Abbott, 1967). There was an increase in the number of small nonvacuolated cells in these roots. This would indicate that cell elongation is more sensitive to manganese deficiency than cell division. The formation of manganese oxide plaques was described for rice (Oryza sativa) plants in hydroculture (Crowder and Coltman, 1993). Special adaptations to high levels of manganese supply are not known.

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XVI. OPEN QUESTIONS An interesting but as yet unsolved question is how and why very low concentrations of toxic trace elements can stimulate plant growth to some extent. The described alleviation of trace element toxicity by calcium and other major nutrients also deserves a special attention. Some plant groups have evolved their own peculiar spectrum of ecological and physiological adaptations. For instance, grasses have developed effective regulatory mechanisms at the root level. In grass shoots, the element content, even on ore soils, tends to be much lower than in broadleaf herbs. The search for comparable, special adaptations, on the physiological or biochemical level, in other plant families should be intensified. In view of the widespread occurrence of mycorrhizae, the effects of such symbiosis on trace element relations of higher plants should be considered in all studies. For reasons of practicality, these phenomena are often neglected in laboratory experiments. Therefore, it seems necessary to conduct more experiments under realistic field conditions, which include such important factors. Owing to rapid developments in molecular biological studies, the investigation of the genetic control of trace metal tolerance of higher plants will certainly be pushed forward in the future. The major questions are still whether the tolerance mechanisms are under mono-, oligo-, or polygenic control and how these mechanisms have evolved.

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44 Root Growth Under Salinity Stress Nirit Bernstein Agricultural Research Organization, The Volcani Center, Bet-Dagan, Israel

Uzi Kafkafi The Hebrew University of Jerusalem, Rehovot, Israel

I.

INTRODUCTION

negative effect of such a change is the decreased ability of the shoot to supply assimilates to the roots and the growing tissues, which is likely to affect plant development and survival particularly under long-term salinization (Munns and Termaat, 1986; Cheeseman, 1993). Saline solutions impose on roots two types of stresses: osmotic stress resulting from lowered water potential in the root-growing medium, and ionic stress induced by changes in concentrations of specific ions in the root medium and inside the growing tissue. A better understanding of the mechanisms involved in root growth reduction would expand our understanding of whole-plant responses to saline conditions.

Salinity inhibits growth and development of most plants. Inhibition of shoot and root development is the primary response to the stress. Growth, morphology, anatomy, and physiology of roots are affected by salinity. Changes in water and ion uptake and production of hormonal signals that communicate information to the shoot might induce changes in development. Since root growth is usually less sensitive to salt stress than shoot growth, an increased root/shoot ratio is often observed when plants are subjected to saline conditions (Cheeseman, 1988; Rawson et al., 1988; Cruz and Cuatreno, 1990). Root growth of halophytes may be affected differently (Waisel, 1972, 1989). The underlying mechanisms involved in the inhibition processes of root growth are not clearly established. Restriction of root growth by salinity reduces the soil volume that can be explored by the root and hence the availability and uptake of water and essential minerals. The diminished supply of nutrients to the shoot may contribute to growth reduction (Delane et al., 1982; Pitman, 1984; Bernstein et al., 1993a,b; Lazof and Bernstein, 1998). The increase in root/shoot ratio under stress which might diminish the demand for element supply to the shoot has a potential to increase the ability of the root to supply those elements, and hence might present an adaptive advantage (Cheeseman, 1988). A potentially

II.

ROOT GROWTH UNDER SALT STRESS

Roots are in direct contact with the soil solution. As such, they are first to encounter the saline medium and are potentially the first site of damage or line of defense under salt stress. The shape and size of the root system are determined by extension growth of individual root tips as well as by the rate and location of lateral roots development. Salinity affects these root developmental processes differently. While root extension growth of many plants is severely inhibited by high concentrations of NaCl in the medium (Cramer et al., 1987; Zhong and La¨uchli, 1993a; Neumann et al., 1994, inter alia) lateral root formation is less affected 787

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(Waisel and Breckle, 1987), or might even be stimulated by the stress (Kramer, 1980). Growth of roots is restricted to a limited region of the root axis near its tip. Therefore, investigations into the physiological basis for root growth inhibition under stress involve examination of the root apical meristem and the adjoining elongation zone. Growth is not uniformly distributed throughout the apical growth region, but varies considerably along the root axis (Erickson and Sax, 1956; Chapter 7 by Silk in this volume). Similar to leaves, the response to salinity varies with location along the developing organ (Fig. 1; Zhong and La¨uchli, 1993a; Bernstein et al., 1993b; Neves-Piestun and Bernstein, 2001). Salinity imposes inhibition of root elongation rate by a combined effect on the size of the growth zone as well as the magnitude of localized tissue elongation (Fig. 1). The length of the elongation zone of maize (Table 1; Bernstein and Ioffe, unpublished; Pritchard, 1994), cotton (Fig. 1; Zhong and La¨uchli, 1993a), and sorghum (Koryo, 1997) roots was shortened under saline conditions. Reduction of localized growth intensity occurred throughout the elongation zone of cotton roots but only in the decelerating region of the maize roots. Growth in the most apical zone of maize-growing regions was unaffected by salt stress. The spatial effect on root elongation was

Figure 1 Spatial distribution of growth intensity over the apical 14 mm of cotton roots grown at 1 mM NaCl (1 Na) and 150 mM NaCl (150 Na). (Adapted from Zhong and La¨uchli, 1993b.)

Bernstein and Kafkafi

specific to NaCl and was not obtained with iso-osmotic concentration of manitol (Pritchard, 1994). The stress-induced inhibition of root elongation may result from effects on the rate of cell division, rate of cell expansion, duration of cell growth, or orientation of cell expansion. In principle, any of these processes could underlie the root’s growth response to salinity. Information about salt stress effect on root cell expansion is available from three types of indirect studies: 1. Salinity inhibited the elongation rate of small root tissue segments in the elongation zone (Fig. 1). The reduction of tissue growth intensity (relative elemental growth rate; REGR) in ‘‘elongation only’’ might result from inhibition of cell elongation rate. 2. Swift changes in root growth rate following salinization or removal of salt. Such changes are due to cell expansion and not cell division because newly dividing cells do not contribute to organ elongation before several hours or even days have passed (Clarkson, 1969; Powell et al., 1986). Plant organ growth rates were demonstrated to reduce rapidly by salinity (Cramer and Bowman, 1991) or increase rapidly after removal of the salt (Rawson and Munns, 1984), suggesting that salinity decreases cell expansion rate. 3. Cell length measurements suggested reduction in cell growth. Mature root cell size was reduced under salt stress (Kurth et al., 1986), and salinization resulted in shorter roots (Zidan et al., 1990; Azaizeh et al., 1992). However, differences in cell length do not necessarily reflect variations in cell elongation rates. Salinization may also affect cell length via an effect on the duration of cell elongation (Bernstein et al., 1993a,b; Lazof and Bernstein, 1998). Characterization of spatial and temporal aspects of cell displacement is required before conclusions can be drawn from elongating cell length data. Such information is not currently available for salt-stressed root cells. We have applied the methods of growth kinematics, to quantify the effect of salt stress on the rate of cortical cell elongation in maize roots (Bernstein and Ioffe, unpublished). The initial results, presented in Table 1, are the first direct demonstration of salinity induced restriction of cell elongation rate. The elongation rate of cells located 2 mm from the tip of the root was 37% lower under sallinity. Similar to cell expansion, only little evidence of salt effects on cell division rates have been reported. Salt stress inhibited cell number increase in roots (Samarajeewa et al., 1999) and apparent root cell pro-

Root Growth Under Salinity Stress

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Table 1 Effect of Salt Stress on Elongation Rate of Maize Roots, Length of Root Elongation Zone, and Duration and Rate of Cortical Root Cell Elongationa Treatment 1 mM NaCl 80 mM NaCl

Root elongation rate ðmm h1 )

Length of root elongation zone (mm)

Cell elongation rate (mm h1 )

Duration of cell elongation (h)

0:93  0:0002 0:66  0:0003

9.0 7.5

0:0078  0:00014 0:0049  0:00015

17.15 25.5

a Rates of cell elongation were calculated for cells located 2 mm from the root tip by combining results of cell elongation rates with their growth trajectory. Duration of cell elongation represents the length of time required for a cell initially located 0.5 mm from the root tip to be displaced past the end of the elongation zone. Data for first (3-day-old) adventitious roots of 12-day-old maize plants, 5 days following initiation of the salt treatment. Presented results are averages SE (n ¼ 5 roots), results from 20–40 cells were averaged to give each individual root average. Source: Ioffe and Bernstein, unpublished.

duction rate (Kurth et al., 1986; Zidan et al., 1990). It inhibited DNA synthesis during the S-phase of the cell cycle in cells growing in cultures (Belat et al., 1998) and induced nuclear deformation and degradation in meristemic root cells (Katsuhara and Kawasaki, 1996). The resulted cell death was suggested as a cause of root growth inhibition. Evidence from two studies suggests, indirectly, a relation between cell cycle regulation and salt growth tolerance. In Arabidopsis the gain of AtHAL3a function, a protein involved in cell cycle regulation, induced altered growth rate and improved salt tolerance (Espinosa-Ruiz et al., 1999). Trigoneline, a salt stress osmoregulator in legumes, was found to function as a cell cycle regulator (Tramontano and Jowe, 1997). The duration of root cell elongation in maize is lengthened under salt stress (Table 1). Cell growth kinematics analysis revealed that a nonstressed cell ceases elongation and reaches final length sooner than a salt-stressed cell in spite of the reduction of growth zone length under stress. The lengthened duration of cell elongation does not compensate for the reduction in cell elongation rate. The elongation rate of the root was therefore slower under stress (Table 1). The number of elongating cells along the growth zone is another important factor in determination of organ elongation rate. The number of elongating cells in the root at any given time is a function of cell production rate in the meristem and the duration of individual cell elongation. It is unknown how salt stress affects the number of elongating cells along the root. In addition to the effect on cellular expansion rates, salinity might also change the growth orientation (growth anisotropy) of cells. At present, no information is available concerning salt stress effects on cell growth anisotropy; however, some indirect evidence suggests that orientation of tissue growth is

affected. Salinization was shown to cause thicker but shorter roots (barley: Huang and Redman, 1995; cotton: Zhong and La¨uchli, 1993a) and to reduce cotton root elongation and change cell shape but not cell volume (Kurth et al., 1986). Salinity was also reported to increase cortex width and diameter of cortical cells in the stem of the halophyte A. Prostrate (Ewing, 1981). Finally, tobacco tissue culture cells adapted over many generations to grow in a highsalt medium expanded less anisotropically than unadapted cell under nonstress conditions (Chang et al., 1996). Alteration of cell growth anisotropy implies an effect of NaCl on the cytoskeleton (further discussed in Section VI.C). Root tissue development is also affected by salinity. Salt stress stimulated early development of cotton root endodermis and induced development of an exodermis (Reinhardt and Rost, 1995). In the halophyte Suaeda maritime the Casparian strip was developed closer to the root apex (Hajibagheri et al., 1985). These changes might be involved with altered regulation of water and solute transport under stress.

III.

POSSIBLE FACTORS AFFECTING ROOT GROWTH UNDER SALT STRESS

It is generally accepted that salt stress may reduce root growth by osmotic effects and/or by specific ion effects (toxicity, deficiency, or ion imbalance) (Greenway and Munns, 1980; Epstein, 1985; La¨uchli, 1990). The degree to which these factors affect root growth depends on the inherent plant sensitivity, the duration of exposure to the stress, the concentration and type of salts involved, and environmental variables (such as physical and chemical properties of the growing medium, air humidity and composition, soil and air temperature, and light intensity).

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A.

Bernstein and Kafkafi

Osmotic Effects

High salt concentrations in the root media result in low soil water potentials at the root zone and eventually may lead to water deficit (Maas and Nieman, 1977). Roots that are exposed to a sudden event of salt or water stress lose their turgor and respond in an immediate cessation of elongation (Itoh et al., 1987b). The resumption of growth after the cells have adjusted osmotically and their turgor has recovered led many investigators to emphasize the major role of turgor pressure in cell growth regulation and plant growth under osmotic stress. There is no evidence to support this prevailing dogma, although this has been the basis for many studies on water relations and osmotic adjustment (Munns, 1993). Experimental results demonstrated that it is not necessary for the original torgur to fully recover before root elongation can be recovered, but only for the turgor to exceed the wall ‘‘yield threshold’’ (which depends on cell wall properties that may change under stress). Changes in turgor alone cannot fully explain the effect of salinity on root elongation since the recovery of root elongation after salt stress induction, preceded the partial recovery of the turgor pressure (Itoh et al.,1987b). Cell pressure probe measurements have demonstrated that the turgor of nongrowing root cells is slightly lowered following exposure to 100 mM NaCl (Azaizeh et al., 1992). There are only a few measurements of turgor in growing tissues of salt-affected roots. Neuman et al. (1994) showed that the long-term inhibition in maize root growth following salinization did not involve a reduction in osmotic potential gradients, and turgor, in the toot tip tissues. They concluded that under longterm salinization root growth inhibition is not caused by any salinity-induced loss of capacity to maintain an osmotic potential gradient between the growing cells and the water source or by changes in turgor in the growing cells. These results are in agreement with cell pressure probe measurements (Pritchard, 1994) showing that turgor along the growing zone of maize roots was unaffected by salinization despite some 60% decrease in growth, and with results from intact roots of Aster trifolium (Zimmermann et al., 1992). On the other hand, they contradict results for Mesembryanthemum crystallinum roots (Rygol and Zimmermann, 1990) where the radial turgor pressure gradient in the root tip decreases with increasing salinity and turgor pressure decreases along the root axis upon salinization. The observation that turgor alone does not limit cell growth is not limited to root cells. A similar conclusion was reached for leaf cells (Termaat

et al., 1985). Thus, osmotic effects are not a simple mechanistic effect of reduction of turgor pressure in the plant. Osmotic adjustment involves the accumulation of inorganic salts in the vacuoles and an increased synthesis and accumulation of organic solutes, termed compatible solutes, in the cytoplasm. Apart from their function in recovery and maintenance of plant turgor, the compatible solutes are thought to stabilize the active conformation of cytoplasmic enzymes, therefore maintaining enzyme activity under high inorganic salt levels (Pollard and Wyn Jones 1979; Shomer-Ilan et al., 1986; Smirnoff et al., 1990). Biosynthesis of large quantities of organic osmolites required for osmotic adjustment is metabolically expensive and potentially limiting energy availability for growth. The alternative, accumulation of inorganic ions from the root medium, is energetically less demanding (Wyn Jones, 1981; Yeo, 1993) but may reduce root growth by ion toxicity effects. B.

Specific Ion Effects

Generally, macronutrient concentrations in roots are reduced under salinity whereas some micronutrient concentrations are increased (Izzo et al., 1991). Toxicity or deficiency of one or more ions may cause growth reduction under stress. The term specific means that the ion under consideration causes an additional depression of growth beyond what could be expected from the osmotic effect of its solution. 1.

Induced Toxicities

Considerable attention has focused on the hypothesis that specific ions can be toxic to root growth. It is well known that root elongation is restricted differently by specific cations (Hassan and Overstreet, 1952; Roundy, 1985). The alkali cations depress root elongation in a characteristic order of K > Na > Rb > Cs > Li. Inhibitory effect on root elongation rate was used by several investigators as a measure of toxicity of ions in the nutrient solutions (Devitt et al., 1984; Kinraide et al., 1985; Singer and Havill, 1985). Although much research focused on ion-specific effects under salinization, it is yet unknown whether Naþ , Cl , or other ions are the predominant growth-limiting factors—i.e., the ‘‘immediate cause’’ of growth reduction. The immediate causes of root growth inhibition have to be found in the processes, regulatory events, and metabolic changes that occur within the growing region (Lazof and Bernstein, 1998). Only a few mea-

Root Growth Under Salinity Stress

surements are available for deleterious ionic concentrations in the growing zone of roots, although such information is vital to the study of growth inhibition and maintenance processes. Information concerning the relationships between spatial and temporal distributions of cell elongation along an organ and concentration profiles of elements is needed to evaluate their possible role in growth reduction (Bernstein et al., 1995). Salinization increases Na content throughout the elongation region of cotton roots (see Fig. 1 for the growth profile). Interestingly, the highest Na content was found in the region of highest localized growth rate (Zhong and La¨uchli, 1994) and rates of sodium deposition were enhanced only in the apical 6 mm of the elongation zone, which is the region least affected by salinity. This may suggest that in maize roots, similar to leaves (Bernstein et al., 1995), sodium accumulation is not the main cause of root growth reduction under NaCl stress. Under salinization Naþ and Cl contents in root tissues often increase, while Kþ contents decrease. The maintenance of low levels of Naþ and high levels of Kþ in the cytoplasm is considered essential for the activities of many enzymes (Greenway and Munns, 1980). Potassium/sodium selectivity of the root plasma membrane is therefore an important factor in the tolerance of plants to salt (Kent and La¨uchli, 1985; Gorham 1993; Lazof and Bernstein, 1998; Volkmar et al., 1998). The gene locus controlling Naþ /Kþ discrimination has been identified for Triticum (Gorham et al., 1987; Dvorak et al., 1994); when introduced through recombination with related species, it induces tolerance (Gorham et al., 1991; Dvorak et al., 1992). While the permeability of the plasma membrane to Naþ is lower than the permiability to Kþ (Cheeseman, 1982), Naþ can be substituted for Kþ for uptake, with similar mechanisms of uptake for both ions are believed to be operating (Schroeder et al., 1994; further discussed in Section IV.B). NHþ 4 requires carbon skeletons for assimilation. Its assimilation is confined to the root. Any shortage of sugar in the root if combination with high ammonium concentration cause ammonia intoxication of the cytoplasm and root death. Salt stress, similar to high root temperatures (Ganmore-Neuman and Kafkafi, 1983), might reduce sugar content in the root due to increased respiration and therefore increased root sensitivity to ammonium. A significant part of the organic acids produced in the roots are probably used as carbon skeleton for transamination reactions (Sagi et al., 1997, 1998). Nitrogen metabolism changes under salinity (Sagi et al., 1998), with the increased activity of

791

nitrate reductase, phospohenolpyruvate carboxylase, and glutamine synthetase in roots constituting part of an adaptation strategy. 2.

Induced Deficiencies

Ionic imbalances which result from high concentrations of salts may retard root extension in soil (Roundy, 1985). This reduces the volume of soil that can be explored by the root system and therefore the quantities of nutrients available to the plant. In addition, salt ions that move in the soil by mass flow (such as Naþ and Cl ) may accumulate near the root surface and compete with nutritional ions for cell wall sorption, or membrane uptake sites. Saline environments may therefore induce suboptimal nutrient concentrations in plant tissues. High concentrations of Cl reduce NO 3 uptake (Guggenheim and Waisel, 1977; Kafkafi et al., 1982) and high concentrations of NO 3 reduce Cl (Bar et al., 1987, 1997; Xu et al., 2000) and phosphate uptake (Lamaze et al., 1987). High concentrations of Naþ inhibit Kþ influx into roots (Rains and Epstein, 1967; Lynch and La¨uchli, 1984, Cramer et al., 1987), and some Ca2þ concentrations reduce the influx and content of Naþ (Epstein, 1961; Waisel, 1962; Rains and Epstein, 1967; Cramer et al., 1987). Reduced Ca2þ and Kþ supply to the plant is often discussed in relation to salinity-induced growth inhibition. When roots are exposed to salinity, the rate of water and nutrient transport to the shoot is reduced. The effect of 50 mM NaCl salinity on the flow and ionic composition of exudates of tomato and pepper plants is presented in Table 2. Although the concentration of nutrient ions in the exudates increased by a factor of 2 or 3 following salinization, the much reduced exudates flow (reduction by a factor of 17–20) resulted in lowered supply of nutrients to the shoots. Nutrient supply in soil experiments may differ from that of solution culture experiments owing to different activities and mobilities of ions in these media at the root zone. High concentrations of CaCl2 , MgCl2 , and KCl did not cause deficiencies of phosphate in solution culture-grown plants (Kafkafi et al., 1982) while in soil-grown plants phosphate uptake was reduced under salinity (Champagnol, 1979; Kafkafi, 1984). This discrepancy could result from differences in phosphate concentration of the root medium (higher in the solution culture experiment), from the different salinities applied in these studies, or from the higher availability of phosphate to the roots in the solution culture experiments. In soil, inhibition of root elongation

792

Bernstein and Kafkafi

Table 2 Flow Rates and Composition of Root Exudation from 37-Day-Old Tomato and Pepper Plants Grown in Nutrient  2þ 2þ þ 2 Solution Containing 11 mM NO 3 , 1.5 m H2 PO4 , 4 mM Ca , 1 mM Mg , 6.5 mM K , and 1 mM SO4 , With and 3 Without 50 mol m NaCl, 27 Days After Beginning of Salinization Exudate flow

EC (dS/m) Salt Tomato  þ Pepper  þ

Ionic composition of exudate (mol m3 )

Solution

Exudate

(mL h1 )

Ca2þ

Mg2þ

Kþ

Naþ

NO 3

H2 PO 4

Cl

0.90 5.30

2.2 4.8

9.00 0.48

5.5 15.0

2.5 7.1

16.7 21.0

0.5 10.9

25.0 42.9

1.3 7.0

0.8 19.0

0.83 4.90

2.7 5.9

13.20 0.18

3.8 17.5

2.3 15.0

24.4 40.0

0.5 17.8

28.6 67.1

1.0 9.6

0.7 25.0

Source: Hampe, Meiri, and Kafkafi (unpublished data).

reduces the amount of phosphate that can reach the plant by diffusion, whereas in solution cultures the transport of phosphorous toward growing roots is not restricted. When the concentration of phosphorous in solution culture is lower, resembling soil solution, NaCl salinity reduces phosphorous uptake (Martinez et al., 1996). a. Reduced Ca Supply Calcium plays a unique role in the response of plants to saline conditions (reviewed by Rengel, 1992; Knight et al., 1997; Lazof and Bernstein, 1998; Cramer, 2001). NaCl-induced deficiency of Ca2þ supply to roots was demonstrated long ago to affect root growth beyond any Naþ effects on soil structure (Ratner, 1935). The importance of Ca2þ for the function of the plasma membrane under salinity is well accepted. High concentration of Naþ may replace Ca2þ on the root cell membranes and by that impair essential cell functions. NaCl salinity displace membrane-associated Ca2þ from plasmamembrane of cotton roots (Cramer et al., 1985), protoplasts of corn (Lynch et al., 1987) and barley (Brittisnich et al., 1989), and plasma membrane vesicles of melon (Yermiyahu et al., 1994), barley (Murata et al., 1998), and wheat (Kinrade, 1994). NaCl can also release Ca2þ from subcellular compartments that accumulate Ca2þ (Lynch et al., 1987). The reduction in cell growth when Ca2þ of the cell membrane is replaced by Naþ was attributed to potassium leakage from the cells (Leigh and Wyn Jones, 1984; Ben Hayyim et al., 1987). Elevated Ca2þ concentrations in the root zone have been long known to negate part of the salinity-induced plant growth inhibition (LaHaye and Epstein, 1971; Rengel, 1992; Bernstein et al., 1993a; Lazof and Bernstein, 1998 and references therein). Root growth

of different species and different cultivars respond differently to supplemented Ca2þ when salinized (Cramer, in press). Salt-tolerant genotypes (barley: Brittisnich et al., 1989; melon: Yermiyahu et al., 1997) demonstrate lower displacement of membrane associated Ca2þ by NaCl salinity than salt-sensitive genotypes. This indicates that the partial alleviation of NaCl-induced root growth inhibition by Ca2þ may in part be related to the quantity of membrane-associated Ca2þ as well as to the intensity of Ca2þ membrane association. b.

Reduced K Supply

High concentration of Kþ in plant cells is essential for growth and metabolic processes (Leigh and Wyn Jones, 1984). Maintenance of adequate Kþ concentrations and proper Kþ /Naþ ratios are necessary for normal cellular function under saline conditions (Greenway and Munns, 1980). Increasing concentrations of NaCl significantly inhibit Kþ influx into roots (Rains and Epstein, 1967; Lynch and La¨uchli, 1984; Cramer et al., 1987) and increase the loss of intracellular Kþ due to leakage (LaHaye and Epstein, 1969; Cramer et al., 1985). The growth of cultured Citrus root cells increased linearly with increased internal potassium concentration (BenHayyim et al., 1987), as did growth of mung bean (Vigna mungo) roots (Nakamura et al., 1990). Since root cells are the first to encounter variation in Naþ in the external medium, their growth also depends on their ability to prevent potassium leakage. Elevated concentration of Ca2þ has been shown to alleviate the inhibition of cells and root growth under salinization and to maintain high cellular concentration of Kþ (Ben-Hayyim et al., 1987; Nakamura et al., 1990). Thus, high external Ca2þ seems effective for mainte-

Root Growth Under Salinity Stress

nance of appropriate cellular concentration of potassium in the root and therefore supports the elongation of roots under stress. Only a few studies examined the effects of salinization on nutrient contents in growing zones of roots. Potassium concentration in the 5-mm apical region of mung bean roots was shown to decrease with increasing salinization, in correlation with suppression of elongation (Nakamura et al., 1990). Addition of Ca2þ to the external medium alleviated the inhibition of root elongation under NaCl stress and maintained high intercellular concentrations of Kþ in the elongating region of the roots. NaCl stress greatly reduced the deposition rates of Kþ and Ca2þ throughout the growing tissue of cotton roots (Zhong and La¨uchli, 1994). Supplemental Ca2þ greatly enhanced the selectivity of Kþ versus Naþ mainly in the apical 4-mm root region (Zhong and La¨uchli, 1994). Since this region corresponds closely with the region where most growth activity occurs (Fig. 1), and where cells are young and poorly vacuolated, it was concluded that supplemental Caþ2 alleviated the inhibitory effects of salinity by maintaining plasma membrane selectivity of Kþ over Naþ . In more mature cells further away from the root tip, the beneficial effects of Caþ2 diminishes since the selective properties of the tonoplast become more important. 3.

Cellular Compartmentation

Salinity affects ion concentrations in subcellular compartments of root cells. In some cases, the extent of stress-induced changes was found to differ in taxa differing in sensitivity to salinity. Hajibagheri et al. (1987, 1989) studied varieties of maize differing in resistance to salt. Na and Cl concentrations in the cytoplasm, cell wall, and vacuole were increased under stress. The salttolerant variety had a lower influx of Na and higher influx of K across the plasma membrane, and a lower increase in cytoplasmic Na and Cl under salinization. Sodium and Cl concentrations in the vacuoles of the salt-stressed salt-sensitive variety decreased radially inward from the epidermis cells. Salinization reduced cytoplasmic P, K, and Cl concentrations in rhizodermis cells of sorghum, but not in the salt-tolerant varieties (Koyro and Stelzer, 1988). Concentrations of Na, Cl, K, Mg, Ca, P, and S in vacuoles of growing root cells of the salt-tolerant grasses Puccinellia and Spartina are higher than those of salt-sensitive sorghum species. In vacuoles of mature cells of the halophyte Suaeda maritima the concentration of both Na and Cl were about four times the concentration in the

793

cytoplasm or cell walls, and 12–13 times those of K in the vacuoles (Hajibagheri and Flowers, 1989).

IV.

ROOT MEMBRANE PROPERTIES INVOLVED IN SENSITIVITY AND TOLERANCE

The ability of plants to regulate shoot ion composition relies, in part, on uptake and transport processes in their roots. Cell membranes are the major sites for controlling active and passive solute flux. Membrane characteristics of root cells are therefore of special interest in the study of growth regulation under saline stress. A.

Initial Responses

The plasma membrane is an important interface between the apoplastic space and the cytoplasm. Effects of salt on plant growth might involve interference with the membrane properties of the elongating root cells. The plasma membrane ATPase pumps generate and maintain an electrochemical gradient essential for ion and solute transport. The relationship between proton pump activity and cellular ion concentration is especially important in salt-stressed plants. The root outer membranes are major contributors to trans-root potential changes (Kennedy and Gonsalves, 1987); it is therefore expected that the specific effects of various salts and of the total salt concentration of the solution will influence first the polarization of such membranes. The reduction in trans-root potential and H+ gradients induced by total salt as well as by concentration of heavy metals in the external solution can be detrimental to the acquisition of nutrients that are transported into roots along these gradients. The membrane potential depolarizes rapidly under salinity (Cheeseman et al., 1985; Hoffmann and Bisson, 1988, 1990; Schubert and La¨uchli, 1988; Tufariello et al., 1988; La¨uchli and Schubert, 1989; Katsuhara and Tazawa, 1990; Reid et al., 1993) but in many cases recovers within minutes (Cheeseman et al., 1985; La¨uchli and Schubert, 1989; Katsuhara and Tazawa, 1990), suggesting that the change under salinity is usually transitory and in the long term, the membrane potential remains unchanged. B.

Membrane Transport

The root cell plasma membrane is an important control point for the exclusion, or selective uptake, of

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essential ions. Especially, Naþ /Kþ discrimination and Ca2þ and Cl uptake are discussed in relation to plant tolerance and sensitivity to salinity (Rengel, 1992; Gorham, 1993; Lazof and Bernstein, 1998, 1999). Sodium moves passively across the plasma membrane, down an electrical gradient, and is at equilibrium at internal concentrations of about 10 mol m3 Naþ and
0:2 mol m3 Ca2þ (Bush, 1995). There is evidence that Naþ can cross plant membranes through ion channels (Katsuhara et al., 1990; Maathius and Prins, 1990; Schachtman et al., 1991). Elevated root zone salinity increases the gradient that drives the passive movement of Naþ across the plasma membrane. Ion homeostasis under saline environments is likely to relay on Naþ evacuation from the cytoplasm to the vacuole or the cell wall apoplast. The action of a Naþ /H+ antiporter on the tonoplast and the plasma membrane reduces cytoplasmic Naþ and although it increases the gradient which drives passive movement of Naþ into the cell (Blumwald and Poole, 1985, 1987; Dupont, 1992) has an adaptive potential for salinity resistance. Overexpression of the vacuolar Naþ /Hþ antiport has indeed promoted sustained growth of Arabidopsis thaliana under elevated salinity (Apse et al., 1999); salt tolerance of Plantago was found to be associated with Naþ /Hþ antiport activity (Prins, 1995); and roots of salt-acclimated soybean had higher Naþ / Hþ activity than roots of nonstressed and nonacclimatized plants (Huang et al., 1998). The proton and electrical gradient generated by the plasma membrane Hþ -ATPase is the driving force for active secondary transport and the regulation of Naþ and Cl uptake (Niu et al., 1995). The sodium efflux from the cell via the Naþ /Hþ antiporter is coupled to outward movement of Hþ from the cell. The ability to compartmentalize Naþ may therefore result, in part, from stimulation of the Hþ -ATPases of the plasmalemma (PM-ATPase) and the tonoplast (V-ATPase). Salinity was reported to increase gene expression for PM-ATPase activity in the halophyte Atriplex nummularia roots (Niu et al., 1993), and to a higher extent in a salt-tolerant mutant of rice, than in the original cultivar (Zhang et al., 1999). Increased Hþ -ATPase activity was required for the establishment and maintenance of the electrochemical potential gradient across the plasma membrane of the epidermis of maize roots following salinization, for prevention of further Naþ and Cl apoplast transport to the xylem (Zhang et al., 1997, 1999). Salinity also increased PM-ATPase activity (but not V-ATPase activity) in cotton (Lin et al., 1997) and barley roots (Katsuhara et al., 1997). The inability of the V-ATPase in cotton to respond to

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increased levels of Naþ indicates that salt sensitivity may result, in part, from a lack of effective driving force for vacuolar compartmentation of Naþ . Plant roots might posses a passive component, as well as an active system for Cl uptake (Altman, 1973). The nature of the mechanisms of Cl exclusion by roots, which characterizes tolerance in some species (e.g., Citrus; Altman and Mendel, 1973) is not understood. The negative membrane potential was suggested to function as a thermodynamic barrier to Cl influx. Hence, chloride enters the cell against an electrical gradient by active processes. Sodium uptake into the cell may shift the electrochemical potential (e.g., depolarize the plasma membrane potential) to allow passive Cl entrance through specific anion channels (Skerrett and Tyerman, 1994; Niu et al., 1995). Chloride might also enter root cells through nonselective anion channels, or other ion carriers (Logan et al., 1997). ATPase-driven anion channels on the tonoplast were suggested to facilitate subcellular compartmentation of Cl in the vacuoles (Plant et al., 1994). Naþ and Cl can cross membranes by diffusion (Hille, 1992). There is evidence that salt stress–induced changes in lipid composition of the membranes can alter Naþ and Cl permeability (Leach et al., 1990; Vazquez-Duhalt et al., 1991); this effect is probably small relative to the other transport mechanisms available for these ions. Calcium may serve as a secondary messenger, signaling changes in external salt levels to initiate changes in mechanisms that control ion fluxes (Mendoza et al., 1994).

C.

Membrane Surface Charge and Surface Potential

Salt stress affects the electrostatic properties of the plasma membrane of root cells by reducing the membrane surface potential. The surface potential of tomato root cell plasma membrane became 48% more positive under saline conditions (Suhayda et al., 1990). The surface potential is a function of the ion activity in the external solution and the surface charge density. NaCl affects the surface potential directly by screening negative charges on the membrane, and indirectly, during longer exposure to salinity, probably by reducing the surface charge density of the membranes (Suhayda et al., 1990). Such a reduction in membrane charge density is bound to reduce the activity of cations at the outer surface of the plasma membrane, and hence affect rates of ion transport and growth.

Root Growth Under Salinity Stress

Based on Eisenman’s theory of sorption selectivity of monovalent cations (Eisenman, 1960; Eisenman and Horn, 1983), it was postulated (Kafkafi, 1991) that sensitivity of root elongation to the various alkali cations might correlate with high surface charge density on the root membranes. Roots with high surface charge density could presumably promote sorption of divalent cation, such as Ca2þ , to their membrane in competition with Naþ , and therefore retain better root membrane integrity in saline media. Analysis of sorption studies to vesicles extracted from plasma membrane of melon root cells ruled out the possibility that this trait of salt tolerance is a property of the plasma membrane (Yermiyahu et al., 1994). Membrane vesicles from cultivars differing in salt tolerance were found not to differ with respect to their surface potential, surface charge density, and binding affinity for Ca2þ , Naþ , and Mg2þ (Yermiyahu et al., 1995). Since root elongation of these cultivars responds to salinity and to Eisenman’s series (Kafkafi, 1991), it is possible that the sensitive site of Ca2þ to Naþ exchange might reside in the cell wall of the elongation zone. D.

Membrane Composition

Mineral imbalances of the root medium, common in saline environments, often affect the chemical composition and structure of root cell membranes (Kuiper and Kuiper, 1978; Ellouze et al., 1982; Diepenbrock, 1985; Chapter 9 by Waisel and Eshel in this volume). Salinization caused a decrease in root membrane fluidity that was attributed to changes in the weight ratio of lipids to proteins but not to modifications in lipid composition (cf. Chung and Matsumoto, 1989; Blits and Gallagher, 1990; Borochov-Neori and Borochov, 1991). The higher protein density may provide a change in membrane surface charge (Suhayda et al., 1990). The selectivity of biological membranes to ions is determined by membrane parameters like the size and charge of the polar head group, the length of the fatty acid chains, and the interaction of phospholipid with sterol (Blok et al., 1977). The fine structure and lipid composition of the membranes with their bound proteins are genetically encoded. The expression of that code changes to a certain degree under the influence of salts in the external solution. A specific phospholipid in a platelet arrangement was found to stimulates plant Hþ transport and its exchange with Kþ (Scherer, 1985). Thus, not only the composition but also the fine structure of the phospholipids are involved in ion selectivity by root cells. Salinity alters

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the metabolism and composition of lipids of root membranes (Kuiper, 1980, Stuiver et al., 1981; Cachorro et al., 1993; Lin et al., 1996; Yu et al., 1998). In the dicot halophyte Kosteletzkya virginica, the increase in free sterol/phospholipid ratio under salinization may be important in minimizing Kþ leakage (Blits and Gallagher, 1990). Ion permeability and ATPase activity are both strongly influenced by membrane lipid composition (Cocucci and Bellarin-Denti, 1981). Therefore, lipids have the capacity to regulate ion movement into the roots through their influence on both passive and active transport processes (Douglas and Walker, 1984). Root membrane composition was indeed found to effect Naþ and Cl uptake by the root and subsequent accumulation in the leaves of glycophytes (Kuiper, 1968, 1980, 1987). In cowpea (Vigna unguiculata) and the halophyte Suaeda maritime salt stress–induced changes in lipid composition altered the permeability of the membrane for Naþ and Cl (Vazquez-Duhalt et al., 1991; Leach et al., 1990). The ability of five varieties of grapes to tolerate salinity was negatively correlated with the solubility of chloride in the lipids of their membranes (Kuiper, 1968). Enrichment of these root cell membranes with phospholipid relative to their monogalactose diglyceride content limited chloride uptake. Salt-induced changes in the root lipid composition have been correlated with the relative abilities of different plant taxa to tolerate salinity or adapt to it (Erdei et al., 1980; Lin et al., 1996; Stuvier et al., 1981; Douglas and Walker, 1984; Zenoff et al., 1994).

V.

ROOT HYDRAULIC CONDUCTIVITY

Water moves through roots in response to a water potential gradient mostly generated by transpiration. The complex anatomical structure of roots dictates a complexity in the flow of water (Steudle and Peterson, 1998). The quantity of water moving from the root to the shoot, and the rate of movement determine the quality and concentration of substances arriving at the shoot (Markhart and Smith, 1990). Knowledge of the driving forces and the resistance that control the movement of water through the soil–plant continuum is important for understanding the effect of salinity on root function and its integration with shoot responses. The volume flow of water, Jv , between any two points in a diffusion-limited hydraulic system is related to the driving force by Jv ¼ Lp ð
P þ 
Þ

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where Lp is the hydraulic conductivity of the membrane,  is the reflection coefficient that relates the actual driving force due to the osmotic gradient to the magnitude of that gradient, and
P and
 are the differences in hydrostatic and osmotic pressures, respectively, between the two points (Fiscus, 1975). Water uptake during growth maintains turgor pressure, which provides the driving force for cell expansion. The hydraulic conductivity (Lp ) of the water uptake pathway and the osmotic potential gradient (
) between the growing cell and the source of water regulate the ability of the tissue to supply water to growing cells and might therefore be related to cell expansion growth and to overall root growth. Some studies suggest that growth of plants is limited by the root ability to transport water to the shoot (Sa´nchez-Blanco et al., 1991; Alarco´n et al., 1994). This issue is controversial. Changes in hydraulic conductivity of water pathways were suggested to play a role in growth inhibition of root growth following salinization. NaCl stress was demonstrated to reduce the hydraulic conductivity and growth of bean, lupin, maize, tomato, and soybean roots (O’Leary, 1969; Munns and Passioura, 1984; Joly, 1989; Evlagon et al., 1990; Peyrano et al., 1997; Rodriguez et al., 1997) but not of barley, sunflower, or a different tomato cultivar (Shalhevet et al., 1976; Munns and Passioura, 1984). The conflicting reports may arise from differences in the size of the sampled root sections (large root systems vs. small root segments); problems associated with Lp measurements (see Chapters 39 by Nardini et al. and 38 by Sperry et al. in this volume), or inherent differences between roots of different cultivars or species, or different types of roots in the plant. In addition to possible damaging effects on shoot–water relations, an increased root hydraulic resistance under salinity was suggested to be of adaptive nature, restricting the loss of water from the root to the surrounding medium (Rodrigez et al., 1997). Parallel pathways—apoplastic, symplastic, and transmembrane—play a role during the passage of water through the root. Each of these pathways can contribute to overall root radial hydraulic resistance (Steudle and Peterson, 1998). Reduction of root hydraulic conductance under salt stress seems to involve changes in the symplastic transcellular pathways. Movement through water channels (aquaporins) is an important component in the regulation of water movement in the transcellular pathway (Steudle and Peterson, 1998). Reduced activity, or abundance, of water channels in the roots of NaCl-stressed plants decreased sap flow and osmotic pressure–dependent

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hydraulic conductance in pepper root systems (Carvajal et al., 1999). That the salinity reduced hydraulic conductivity of roots was not accompanied by changes in the content of ligninlike polymers, or in the activity of syringaldazine oxide (an enzyme associated with lignification of cell walls) (Peyrano et al., 1997) suggests as well that the NaCl-induced reduction in hydraulic conductance involves changes in the symplastic pathway. Measurements with cell pressure probe revealed that salinization reduced the root cells’ Lp by 30–66%. From the hydrostatic and osmotic relaxation of turgor, the hydraulic conductivity of cortical cells was also found to decrease significantly by salinization (Azaizeh and Steudle, 1991; Azaizeh et al., 1992).

VI.

ROOT CELL WALL PROPERTIES INVOLVED IN SALT SENSITIVITY AND TOLERANCE

Plant growth may be defined as an irreversible increase in size resulting from cell division and cell expansion. Cell expansion is thought to be controlled by cell wall mechanical properties and by cell turgor. Since root growth inhibition under salt stress is not necessarily caused by a decrease in turgor, information about effects of salinity on the cell wall is crucial for understanding growth inhibition and maintenance processes. A.

Mechanical Properties (Extensibility)

According to the combined growth equation of Lockhart (1965), cell expansion rate (ER) is a function of the wall extensibility coefficient (m), hydraulic conductivity (L), the osmotic potential gradient between the growing cell and the ambient water (
), and the yield threshold (the wall yield stress value; Y). ER ¼ ðm  L=m þ LÞð
  Y Þ Information accumulated so far suggest that inhibition of root growth by salinity is not caused by salinityinduced loss of the capacity to maintain
 or turgor, in the growing cells of the root. This was demonstrated for salt-sensitive maize roots (Neumann et al., 1994; Pritchard, 1994) and intact roots of the halopyte Aster trifolium (Zimmermann et al., 1992). Sudden decreases in turgor pressure following salinization are undoubtedly responsible for immediate, short-term inhibition of root growth following a rapid increase in root zone solute concentration. However, lowering of cell turgor does not appear to be the cause of long-

Root Growth Under Salinity Stress

term root growth inhibition. Long-term reduction of root elongation under salinization seems to involve hardening of cell walls of the expanding cells. The apparent yield threshold pressure (Y), is higher in salinized maize root tips than in nonsalinized ones (Pritchard et al., 1991; Neumann et al., 1994), and cell wall extensibility is lower under long-term exposure to stress (Neumann et al., 1994, Nonami et al., 1995). Changes in physical properties of cell walls are specific to elongation cells and are influenced more by sodium than by calcium salts (Nonami et al., 1995).

B.

Cell Wall Composition

Changes in the mechanical properties of cell walls following salinization must be caused ultimately by modification in wall structure at the molecular level. Salinity influences the biosynthesis of cell wall polymers, and wall metabolism. NaCl salinity was shown to delay cell wall thickening in cotton roots (Gerard and Hinjosa, 1973) and to inhibit the biosynthesis of polysaccharides (Zhong and La¨uchli, 1988, 1993b). The relative cell wall mass of NaCl-adapted tobacco cells in culture was also reduced by salinity to 50% of the nonsalinized, nonadapted mass (Iraki et al. 1989). In contrast, salinization increased synthesis of cell wall material in pea roots (Hasson-Porath and PoljakoffMayber, 1973; Solomon et al., 1987). Halophytes as well were reported to produce large amounts of cell wall material under saline conditions (Binet, 1985), and to have greater wall plasticity. The increased amount of cell wall material relative to that of the protoplasm was suggested to increase the Ca-binding capacity of the wall and thus maintain cell growth (Binet, 1985). Salinity-induced wall growth of xylem parenchyma cells in maize roots was also reported, and was suggested to allow the action of transfer cells (i.e., removal of Na from the xylem stream; Yeo et al., 1977). Growth regulators studies suggest that structural changes in cell wall polysaccharides underline the processes of cell wall loosening that lead to cell expansion (Taiz, 1984; Chapter 24 by Tanimoto in this volume). Salinization increased the total uronic acid content of cell wall of cotton root tips and decreased the cellulose content concomitantly to inhibition of growth (Zhong and La¨uchli, 1993b). Elevated concentrations of Ca reversed the salt inhibition of the biosynthesis of cellulotic material and reversed in part the reduction of growth. Salinization also reduced the biosynthesis of pectin and hemicellulose in cotton root cell walls in a

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process that does not respond to changes in external Ca2þ supply (Zhong and La¨uchli, 1993b). Infrared spectra of barley root cell walls also demonstrated a decrease in polysaccharide content (and carboxyl groups) following salinization. A decrease in water molecules, tightly bound to the cell wall matrix, was postulated to be a secondary effect, linked to a decreased polysaccharide content of the wall. Salt-induced Ca2þ deficiency may account for the decrease in polysaccharide content (Suhayda et al., 1994). Reduction in pectic polysaccharides and alterations in pectin structure are indeed observed under low-Ca2þ conditions (Konno et al., 1984). A salt-tolerant wild barley retained a higher relative content of cell wall polysaccharide than a salt-sensitive cultivar (Suhayda et al., 1994). It was postulated that the salt-tolerant wild barley has a higher pectic polysaccharide content in the cell wall, where polygalacturonan regions can crosslink by Ca2þ . Salinization increased the amount of polysaccharides of intermediate molecular size and decreased that of small-size molecules, indicating a possible inhibition of polysaccharide degradation (Zhong and La¨uchli, 1993b). This change was not observed with supplemental Ca2þ . An increase in cell wall protein and aromatic compounds following salinization was found for barley roots (Suhayda et al., 1994). Covalent bonding of wall residues is a mechanism by which cell wall extensibility may be controlled. Ferulic acid in pectin, and in the arabinoxylan fraction of hemicellulose, is covalently linked. Ferulic acid monomers, linked into diferulate, crosslink between polysaccharide chains in an oxidative reaction catalyzed by peroxidases. The glycoprotein extensin can also crosslink by the oxidative coupling of tyrosine residues, forming isodityrosine or trityrosine bridges between protein strands (Fry, 1986). The relative enrichment of proteins and aromatic compounds induced by salinization in barley root cell walls increases the potential for the oxidative coupling of proteins and phenolic entities. A portion of the root growth inhibition observed under salinity may therefore result from the increased crosslinkage of cell wall polymers (Suhayda et al., 1994). Salinity was also reported to induce deposition of -1,3-D-glucan, callose, in the walls of cortical root cells of sorghum. Such accumulation was suggested to hinder the expansion of root cells (Koryo, 1997). C.

Cell Wall Ultrastructure

Indirect evidence suggests that salt stress affects tissue and cell growth orientation (Section II). Alteration of

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cell growth anisotropy implies an effect of NaCl on the cytoskeleton. Anisotropic growth depends on anisotropic mechanical properties of the cell wall. In growing cells, the most prominent anisotropic wall elements are cellulose microfibrils. The ability of a cell to grow anisotropy seems to correlate with the presence of cellulose microfibrils aligned perpendicular to the axis of maximum growth rate (Green, 1980; Taiz, 1984; Brown, 1985). Salt stress has been observed to affect qualitatively the orientation of cell wall cellulose microfibrils in roots, leaves, and cells in culture (McCann et al., 1994; Koryo, 1997; Pareek et al., 1997). The effect of the stress on the dispersion of alignment among the microfibrils has not been assessed quantitatively. Therefore, the possible involvement of the observed stress-induced changes in cell wall ultrastructure in the processes of growth inhibition is unclear at this time. D.

Sorption Capacity of Cell Walls

Cation binding to plant cell walls has been implicated as a critical step in a wide range of physiological processes including wall extensibility and therefore cell elongation (Roelofsen, 1965; Peterson, 1974; Nakajima et al., 1981; Richter and Dainty, 1988). Interaction during sorption might also effect subsequent uptake into the cell by altering the availability of certain ions to the membrane (Mattson, 1948). The concentration of supplemental Ca2þ needed to ameliorate harmful effects of salt on root growth varies among species, as does the minimum concentration of Ca2þ needed to maintain growth (e.g., 0.2 mol m3 in Trifolium pratense and Agava spp. [Kinraide et al., 1985, Nobel and Berry, 1985] and 0.0075 mol m3 in peach [Edwards and Horton, 1979]). If such differences in responses to Ca2þ and Naþ /Ca2þ ratios do not arise from sorption affinities to the plasma membrane (see Section IV.C), binding affinities of Ca2þ to other media, such as the cell wall, should be further examined. Sorption specificity of the wall for different cations may be of importance for growth sensitivity under saline conditions. Ca2þ and Na+ were found to be sorbed on identical sites of barley root cell wall (Stassart et al., 1981). The selectivity for Ca2þ of cell walls of bean, a glycophyte, was higher than that of cell walls from the halophyte Cochearia anglica (Bigot and Binet, 1986) in accordance with their growth sensitivity, and while the cation exchange capacity of the halophyte decreased under salt stress, it tended to rise for the glycophyte. The selectivity of the wall for major cations was not affected by salinity.

Sorption of anions to cell walls, although low, may also change under saline conditions. Sorption of Cl to cell walls was shown to increase with external NaCl concentrations in the 0–20 mM NaCl range (Richter and Dainty, 1988). VII.

CONCLUDING REMARKS

Salt stress imposes inhibition of root elongation in most plant species. The complex response of root elongation to the stress can explain the sometimes conflicting concepts regarding mechanisms of root growth sensitivity and tolerance. Detailed spatial and temporal studies of growing root tissues’ and cells’ responses to a stress event is a required initial step toward resolving the mystery of adaptability of root responses to shortand long-term salt stress conditions. The most probable first site of salt effects is in cell walls of the elongation zone. Further plant responses might arise from changes imposed on the plasma membrane. Further development in microanalysis and noninterfering measurements and more quantitative understanding of cell membranes and cell wall responses will be useful for solving the enigma of root elongation inhibition under salt stress and its effect on whole-plant behavior. REFERENCES Alarco´n JJ, Sa´nchez-Blanco MJ, Boları´ n MC, Torrecillas A. 1994. Growth and osmotic adjustment of two tomato cultivars during and after saline stress. Plant Soil 166:75–82. Altman A. 1973. Passive and active components of chloride absorption in the bark of the and the wood of Citrus roots. Physiol Plant 29:163–168. Altman A, Mendel K. 1973. Characteristics of the uptake mechanisms of chloride ions in excised roots of woody plant (Citrus). Physiol Plant 29:157–162. Apse MP, Aharon GS, Snedden WA, Blumwald E. 1999. Salt tolerance conferred by overexpression of a vacuolar Naþ /Hþ antiport in Arabidopsis. Science 285:1256– 1258. Azaizeh H, Steudle H. 1991. Effects of salinity on water transport of excised maize (Zea mays L.) roots. Plant Physiol 97:1136–1145. Azaizeh H, Gunse B, Steudle E. 1992. Effects of NaCl and CaCl2 on water transport across root cells of maize (Zea mays L.) seedlings. Plant Physiol 99:886–894. Bar Y, Kafkafi U, Lahav E. 1987. Nitrate nutrition as a tool to reduce chloride toxicity in avocado. S Afr Avocado Growers Assoc Yearb 10:47–48. Bar Y, Apelbaum A, Kafkafi U, Goren R. 1997. Relationship between chloride and nitrate and its effect

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Root Growth Under Salinity Stress Yermiyahu U, Nir S, Ben Hayyim G, Kafkafi U, Scherer GFE, Kinraide TB. 1997. Surface properties of plasma membrane vesicles isolated from melon (Cucumus melo L.) root cells differing in salinity tolerance. Colloids and surfaces. Biointerfaces 14:237–249. Yu B, Gong H, Liu Y. 1998. Effects of calcium on lipid composition and function of plasma membrane and tonoplast vesicles isolated from roots of barley seedlings under salt stress. J Plant Nutr 21:1589–1600. Zenoff AM, Hilal M, Galo M, Moreno H. 1994. Changes in roots lipid composition and inhibition of the extrusion of protons during salt stress in two genotypes of soybean resistant or susceptible to stress. Varietal differences. Plant Cell Physiol 35:729–735. Zhang JS, Xie C, Li ZY, Chen Y. 1999. Expression of the plasma membrane Hþ -ATPase gene in response to salt stress in a rice salt-tolerant mutant and its original variety. Theor App Genet 99:1006–1001. Zhong H, La¨uchli A. 1988. Incorporation of [14C]glucose into cell wall polysaccharides of cotton roots: Effects of NaCl and CaCl2 . Plant Physiol 88:511–514. Zhong H, La¨uchli A. 1993a. Spatial and temporal aspects of growth in the primary root of cotton seedlings: effect of NaCl and CaCl2 . J Exp Bot 44:763–771.

805 Zhong H, La¨uchli A. 1993b. Changes in cell wall composition and polymer size in primary roots of cotton seedlings under high salinity. J Exp Bot 44:773–778. Zhong H, La¨uchli A. 1994. Spatial distribution of solutes, K, Na, Ca and their deposition rates in the growth zone of primary cotton roots: effects of NaCl and CaCl2 . Planta 194:34–41. Zhang H, Zhou JM, Gguo Y, Chen SY. 1997. A physiological study on the salt-tolerant mutant of rice. Acta Physiol Sin 23:181–186. Zidan I, Azaizeh H, Neumann PM. 1990. Does salinity reduce growth of maize rot epidermal cells by inhibiting their capacity for cell wall acidification? Plant Physiol 93:7–11. Zidan I, Jacoby B, Ravina I, Neumann PM. 1991. Sodium does not compete with calcium in saturating plasma membrane sites regulating 22Na influx in salinized maize roots. Plant Physiol 96:331–334. Zimmermann U, Rygol J, Balling A, Klock G, Metzler A, Haase A. 1992. Radial turgor and osmotic pressure profiles in intact and excised roots of Aster trifolium. Pressure probe measurements and nuclear magnetic resonance imaging analysis. Plant Physiol 99:186–196.

45 High Soil Strength: Mechanical Forces at Play on Root Morphogenesis and in Root : Shoot Signaling Josette Masle Australian National University, Canberra, Australia

I.

INTRODUCTION

becomes limiting. As the severity of soil drying or compaction increases beyond a certain threshold, the effects of mechanical stress are compounded by those of water deficit or anaerobiosis, respectively. Mechanical stress is therefore an intrinsic component of drought and compaction, two of the most important causes of poor plant growth in the field. There have been a number of studies on root impedance in recent years providing us with a description of its phenotypic effects and with unambiguous evidence for a direct signaling pathway from roots to leaves. The mechanisms involved are little understood, however. Even less is known about the interactions between the signaling pathway of mechanical stress and that of other stresses that are often associated with it in the field. Most of the early work on root impedance focused on soil physics aspects and on the effects of soil mechanical properties on root growth. More recently, the attention has shifted to leaf responses and to root:shoot signaling mechanisms, i.e., implicitly to more systemic approaches. Several reviews published on root impedance over the two last decades reflect that shift (Greacen, 1986; Atwell, 1993; Bengough et al., 1997a; Masle, 1999). Here I limit myself to the most salient features of root impedance and their relevance to plant performance, with emphasis on the areas where progress has occurred recently or that offer exciting prospects.

Mechanical stress is inherent to root growth in nature. In order to push their way through soils, roots need to generate a force that can overcome the mechanical resistance of soil aggregates to displacement and deformation. This resistance, commonly referred to as soil strength, is influenced by a number of parameters; some are permanent characteristics of the soil, such as texture, while others, such as soil density and water content, vary with management and stochastic climatic variations, in rainfall and temperature especially. The drier or denser a soil is, the higher its strength (Taylor et al., 1969a). Depending on the length and intensity of soil-drying events and on the spatial heterogeneity in soil structure and compaction, mechanical impedance to root penetration may be a local, transient stress at the root tip or a chronic condition, relevant to a large fraction of the root system. It has long been recognized that roots can detect and respond to small changes in soil strength (Pfeffer, 1893; Barley, 1962). Only recent, however, is the recognition that subtle changes in mechanical forces on roots trigger a signaling cascade throughout the plant with profound distant physiological effects on leaves and stomata (Masle and Passioura, 1987). Upon soil drying or compaction, this cascade is activated before water, nutrient, or oxygen supply to roots 807

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MECHANICAL IMPEDANCE— MEASUREMENTS AND TERMINOLOGY

For quantitative analysis of plant responses to soil strength one needs to define appropriate parameters to describe soil mechanical properties. These parameters need to be measured reproducibly and be related to the actual mechanical forces exerted on roots and influencing their physiology. One of the most straightforward parameters is the resistance exerted by soil particles against displacement and deformation by the root tip. This resistance determines the pressure that the root tip needs to exert to push its way through the soil. When growing in soils, roots have to overcome axial and radial stresses as well as frictional forces (Greacen, 1986). Although the relative magnitude of these components varies depending on the granular and cohesive properties of the soil and to some extent on root shape and diameter, the axial component is generally dominant (Abdalla et al., 1969; Richards and Greacen, 1986; Bengough and Mullins, 1990). Root pressures are difficult to measure directly, even in laboratory conditions. Most often, ‘‘penetrometer resistances’’ are used as a substitute. They are measured as the pressure required for pushing a cylindrical metal probe through the soil. This probe consists of a shaft fitted with a conical tip, and the diameter of the shaft is usually slightly smaller than that of the cone in order to reduce soil–shaft friction (Bengough and Mullins, 1990; Bengough et al., 1997b). It is widely recognized that penetrometer pressures greatly overestimate actual root pressures, by factors of 2 to 8 (Eavis, 1967; Stolzy and Barley, 1968; Bengough et al., 1997b). This discrepancy is mostly due to an overestimation of friction and adhesion forces, and therefore varies depending on penetrometer design and also on soil texture and size of encountered aggregates (Cockroft et al., 1969; Bengough, 1992; Bengough et al., 1997b). A new generation of penetrometers has been produced, however, with a rotating probe, that allow realistic measurements of root pressures once soil–metal frictions are also measured and subtracted. In fact such penetrometers also minimize frictional resistance through a judicious choice of penetration speed and cone angle, dependent on root diameter and elongation rate (Bengough et al., 1997b). The problem remains, however, that metal probes and roots displace soil aggregates according to different spatial patterns (Greacen, 1986). Root growth induces axial and cylindrical compression of surrounding soil while

penetrometer probes create spherical displacement of soil aggregates. In conclusion, the actual forces exerted onto roots in soils are technically hard to measure. Provided penetrometers are used in a consistent manner and are appropriately designed, the variations in penetrometer resistances that are measured in a given soil with variation in water content and density are well correlated with variations in the overall resistance opposed to root penetration. III.

ROOT SENSITIVITY TO MECHANICAL STRESS—EVIDENCE FOR A DIRECT SIGNALING TO THE SHOOT

Plants grow poorly on compacted or dry soils and exhibit a range of physiological adaptations affecting morphogenesis, architecture, and nutrient and water use. These effects are generally referred to as ‘‘drought or compaction responses,’’ and their interpretation is confounded by the difficulty to separate the role of the several soil properties that are affected by soil drying or compaction, besides soil matric potential and soil aeration. One such property is soil mechanical strength. In fact, in a given soil, one can identify a range of both water content and density, in which modifications of plant growth and performance are driven by changes in soil mechanical strength and not by water deficit or anaerobiosis per se around the roots. This is demonstrated by results such as those of Taylor and Ratliff (1969a), who showed a negative correlation between root elongation rate and soil penetrometer resistance, regardless of whether changes in resistance were brought about by variations in soil water content or soil density. Not only can roots detect small changes in soil mechanical properties; they can also signal these changes, very quickly, to leaves. The first evidence for such a direct, long-range signaling was provided by Masle and Passioura (1987) in wheat. In these experiments, it was shown that, within a certain range, decreasing soil water content or increasing its density caused reductions in leaf expansion and transpiration rates which were uniquely related to the concurrent increases in soil strength. These effects were detected on very young plants, within a few days after germination. They persisted even when leaves were maintained continuously turgid through root pressurization (Masle, 1990) or when the soil was continuously aerated and supplied with plethoric amounts of nutrients (Masle and Passioura, 1987). Another strong argument for a direct signaling of root impedance to leaves is the fact that reduced

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expansive growth and transpiration rates occur even when a small fraction of the root system encounters mechanical impedance. This was unambiguously demonstrated in recent laboratory experiments using well-controlled spatial variations of soil strength either laterally through the soil profile (split-root systems; Hussain et al., 1999b) or with depth (multilayered systems; Masle, 1998; Hussain et al., 1999b). Experiments with soils, however, do not allow an in situ analysis of the mechanisms involved in the signaling of root impedance on time scales shorter than a day. They lack the time resolution necessary to clearly separate primary root–shoot signaling effects from secondary responses arising in the shoot as a consequence of physiological root adaptations to impedance or, conversely, to separate primary root responses from feedback effects from the shoot. This is because changes in soil mechanical properties at the root tip cannot easily be precisely timed or spatially controlled. Many experiments have thus been done using pressure cells where a hydrostatic pressure (commonly referred to as confining pressure) is applied onto roots growing in aeroponics or in a granular solid medium. Despite its drawbacks, such an approach partly overcomes the above problems. It has allowed to detect significant growth responses to a step change in confining pressure, within minutes (Russell and Goss, 1974; Goss, 1977; Bengough and MacKenzie, 1994; Young et al., 1997) to a few hours (e.g., Sarquis et al., 1991, 1992) of its occurrence, in both roots and shoots. In conclusion, combining the experimental systems, there is now overwhelming evidence for sensitive sensing mechanisms of soil mechanical forces by roots and a direct signaling of these forces locally and throughout the plant to the aerial parts. Supporting data have been obtained in a range of plant taxa (e.g. barley and wheat—Masle, 1992; tomato—Masle, 1990; Hussain et al., 1999a; Arabidopsis and citrus—Masle, unpublished data), demonstrating that it is a general phenomenon of broad ecological significance (Fig. 1). The magnitude of the threshold soil forces that can lead to significant physiological effects has been much debated. Measurements of soil penetrometer resistances suggest values on the order of 2 MPa. Experiments with pressure cells had led to conclude that confining pressures (i.e., the external hydrostatic pressures applied to the deformable wall(s) of the cell) as low as 0.01–0.02 MPa could induce significant reductions in root growth (e.g., Russell and Goss, 1974; Goss, 1977). It is now clear, however, that confining pressures grossly underestimate the actual pressures on the root walls. The few studies where

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resistance to root penetration in soil was determined directly (Eavis, 1967; Stolzy and Barley, 1968; Misra et al., 1986; Bengough and Mullins, 1991; Clark and Barraclough, 1999) have provided consistent values for the maximum axial pressure that a root can develop (0.9–1.3 MPa) and the pressures for which significant reductions in root elongation rates are observed (0.3–0.5 MPa). These are, however, values observed in short-term experiments. It has been suggested that the frequently observed thickening of the root behind the root tip allows in the long term relief of some of the mechanical stress at the root tip (Abdalla et al., 1969). Furthermore, there is evidence for soil

Figure 1 Schematic diagram illustrating the distant signaling of root impedance to leaf meristems and young expanding leaves (light gray) and to stomata in older and mature leaves. Rootborne signals affect the rate of development in the apical meristem, cell division, and cell expansion in the expanding leaves, and they induce stomatal closure. Stomatal conductance is usually proportionally more reduced than photosynthetic capacity, so transpiration efficiency is improved. Root signals are electrical and hormonal; ethylene, ABA, auxin, and most likely cytokinin signaling cascades are involved in mediating physiological effects in a complex network.

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temperature effects on the ability of roots to elongate at high soil strength (Greacen, 1986; Bengough et al., 1994) that need to be further explored.

IV.

BIOCHEMICAL AND DEVELOPMENTAL PLANT RESPONSES TO MECHANICAL IMPEDANCE

A.

Root Responses

Exposure to mechanical impedance induces a suite of physiological changes in roots, which at the macroscopic level have been extensively described. Within minutes (Goss, 1977; Moss et al., 1988; Sarquis et al., 1991; Bengough and MacKenzie, 1994) to hours (Eavis, 1967; Croser et al., 1999) of stress imposition on roots in the soil, root elongation rate is slowed down. The root cap generally becomes more rounded and the root diameter behind the meristem increases (Eavis, 1967; Atwell, 1990; Croser et al., 2000). The root meristem and elongation zone become shorter (Fig. 2). In Eavis’ experiments with pea radicles grown in soil, such effects were noticeable within < 24 h of treatment. They were accompanied by an increase in the number of lateral root primordia which, furthermore, were initiated closer to the root tip. The time scales on which reduction in root elongation rates are first detected following imposition of root impedance are too short for this initial reduction to be ascribed to changes in the number of elongating cells. This implies direct effects of impedance on the kinetics of cell elongation within the root tissue. There are no data on the cellular kinetics of expansion in the meristem, but the final size of meristematic cells appears to be little affected (Eavis, 1967). Impedance seems to mostly affect cells within the root elongation zone. Croser et al. (2000) recently showed a reduction in both the maximum elongation rate and in the duration of elongation of cortical cells in impeded pea roots. Interestingly, concurrently with reduced cell elongation they observed an increased radial enlargement. This appears to be a widespread response to root impedance, in a range of species (maize—Barley, 1965; pea—Eavis, 1967; barley—Wilson et al., 1977; Goss, 1977; eucalyptus—Misra, 1996). Assuming that water flux to the expanding cell is not limiting, and following Greacen and Oh (1972), the rate of cell elongation can be described by a modified form of the Lockhart equation: r ¼ mr ðP  s  Wc Þ

where mr is the extensibility of cell wall material, the quantity ðP  s Þ is the stress on cell walls, P is the turgor pressure within the cell, s is the soil pressure, Wc is the critical value that the stress on cell walls needs to exceed for elongation to occur. This equation suggests that the reduction in cell elongation under impedance may result from reduced cell turgor, from a stiffening of the cell wall, or from a direct effect of mechanical forces on wall rheology. Increases in cell osmotic pressure have consistently been measured in

Figure 2 Longitudinal sections of tomato roots grown in a silty loam soil of 1.1 MPa (a) and 4.5 (b) MPa penetrometer resistance and of Arabidopsis roots grown in a polymer of low (c) and high (d) strength, where compression forces were very small. The arrows in frames (c) and (d) indicate the distal end of the root cap (dashed arrow) and the distal end of the elongation zone (solid arrow). The horizontal bar in the corner of each frame corresponds to 50
m.

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impeded root tips (e.g., Greacen and Oh, 1972; Atwell and Newsome, 1990; Croser et al., 2000), leading to inference of reduction in cell turgor. In a few recent studies, however, cell turgor was actually measured using cell pressure probes, either in situ (Clark et al., 1996) or after removal of the root from its growth medium (Atwell and Newsome, 1990), during the recovery phase where root cells appear to keep the memory of conditions during impedance (Clark et al., 1996; Croser et al., 2000). Not surprisingly perhaps, given variations among studies in the type of impedance applied to roots and in the timing of the turgor measurements, the results are conflicting, showing either no change in turgor pressure (Barley, 1962; Atwell and Newsome, 1990; Croser et al., 2000) or an increase (Clark et al., 1996). More measurements are needed in a range of well-defined conditions to ascertain the extent that reduced turgor may contribute to reduced cell expansion. Meanwhile, there is mounting, although still indirect, evidence for direct effects of mechanical impedance on cell wall extensibility. This is the most likely explanation for the persistence of the reduced strain rates observed for several hours after release of impedance in pea roots (Clark et al., 1996; Croser et al., 2000) as well as in carrot and onion seedlings (Whalley et al., 1999). Moreover, the differential effects of root impedance on axial and radial expansion indicates a stiffening of cell walls in the axial direction but a loosening in the radial direction. In soil, cell growth anisotropy may be further influenced by the anisotropy of mechanical forces on cell walls within the root tissue. It is indeed striking that radial swelling of impeded roots seems to occur only if there is compressive stress around the elongation zone (Atwell, 1993; Fig. 2c,d). Moreover, most observations of radial cell enlargement refer to the outer cells of the cortex. Studies such as that of Wilson et al. (1977) indicate differential effects of root impedance on cell growth anisotropy from the outer to inner cortical cell files and the stele, whose diameter seems to be mostly unresponsive to impedance (cf. Atwell, 1990). On longer time scales of a day and beyond, the biophysical effects of root impedance are compounded by developmental responses. On such time scales whole-root elongation rate may be described as the product of cell flux and final cell length. While it is well established that impedance decreases both parameters, the mechanisms involved are not clear. Published comparisons of cell production rates are based on observations of mitotic figures or on calculations of mitotic indices or cell doubling times (Barley,

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1962; Eavis, 1967; Croser et al., 2000) that confound variations in the number of actively dividing meristemic cells and in the rate of cell cycling per se. The interpretation of these observations is further limited by the absence of information on the number of cortical cell files, which have been shown to increase in some studies (Wilson et al., 1977; Croser et al., 2000) but not in others (Atwell, 1988). Again, one may expect that the anisotropic soil forces exerted at the root tip and behind have differential effects on radial and tangential cell division. As for explanations of reduced mature cell length, they seem to lie in effects of impedance on both the maximum rate of cell elongation and the length of the elongation zone (Croser et al., 2000). As is obvious from this discussion, the cellular mechanisms underlying root growth responses to impedance, and their interrelationships, are poorly understood, even under near steady-state stress conditions. Most of the data concern mature cells and correspond to observations made at a given distance from root tips. Since impeded roots typically show a shorter meristem, some apparent anatomical differences compared to nonimpeded roots may simply be related to a more advanced differentiation of the tissue being sampled (i.e., in effect, to differences in cell biological age). Detailed kinematic analyses of cell division and elongation are required, distinguishing developmental effects related to position, age, and time (see Chapter 7 by Silk in this volume). Furthermore, as already indicated, these effects appear to depend on the type of mechanical stress: intensity, balance between axial and radial pressures on the root, and the part of the root where they are exerted (see Barley, 1962). B.

Leaf Responses

As mentioned earlier, direct physiological effects of root impedance on leaves and shoots have now been demonstrated in a number of studies where roots were either grown in soil (Masle and Passioura, 1987; Passioura, 1988; Passioura and Gardner, 1990; Masle, 1992; Hussain et al., 1999a,b) or in pressure cells (Goss, 1977; Young et al., 1997). Typically, two effects are detectable at the macroscopic scale: reduced leaf expansive growth and reduced stomatal conductance. Although the two effects are often observed concurrently, the stomatal response appears to lag behind the growth response (Masle, 1998; Young et al., 1997), as has also been observed under drought stress (Passioura and Gardner, 1988). It also involves partly independent signal transduction pathways (see below).

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The mechanisms underlying both responses have until recently received little attention. The decrease in leaf expansion growth observed by Young et al. (1997) within 10–20 min of imposition of a confining pressure is too fast to be the result of changes in developmental processes; if hydraulic effects can be excluded as the authors argue, that rapid decrease in tissue expansion may, as in roots, reflect changes in cell wall extensibility. Rapid changes in the expression of genes coding for enzymes that appear to be important for cell wall synthesis and mechanical properties have been demonstrated in leaves in response to a range of local mechanical stimuli (Braam and Davis, 1990; Antosiewicz et al., 1995; Braam et al., 1997). Direct measurements on leaves of impeded plants are needed with simultaneous measurements of elongation rate to ascertain whether similar kind of responses may be induced by distant mechanical stress. Interestingly, Lu and Newman (1998) did measure significant reductions of cell wall extensibility under mild root osmotic and water stress, in both barley and maize, but not in rice, indicating important genetic differences even among related species. No such data are available for mechanical stress. On time scales of hours to days, root impedance induces developmental responses in aerial meristems and young expanding leaves, which results in continued slower elongation rates and reduced adult leaf size. These responses include: delayed primordium initiation (Masle, unpublished); reduced subapical growth causing a reduction in the number of cellular files constituting the primordium and ultimately the blade, thereby reducing potential blade area; a shift in the composition of epidermal cellular files with an increase in the proportion of stomatal and associated files of trichome bearing cells (Beemster and Masle, 1996); and altered kinetics of cell partitioning and expansion of both meristemic and nonmeristemic cells (Beemster et al., 1996). These kinetics have been investigated in wheat in the studies just noted, using the theoretical framework first developed by Goodwin and Stepka (1945) and Erickson and Sax (1956) to analyze axial growth, where cellular elements within the growth zone are in effect treated as physical particles in a fluid. Such a framework allows the determination of the spatial and temporal patterns of cell partitioning and cell enlargement within meristems and expanding tissues and the quantitative examination of their role in whole-leaf expansion. In young leaves that had fully developed after mechanical impedance was first encountered by roots, impedance had no effect on cell production rates, so reduction in whole-leaf elon-

Masle

gation rate was directly proportional to the reduction in final cell length. In those leaves impedance caused no detectable changes in the rate of cell expansion whether in proliferative or nonproliferative cells nor in the duration of cell elongation. Mature cells were therefore shorter solely as a result of a reduced length upon entry into the elongation-only zone. Impedance modified the properties of meristemic cells, leading them to cycle faster and to divide at a smaller size, despite elongating as fast as in unimpeded plants. These accelerated cycling rates were offset by a reduction in the number of dividing cells—hence, the conserved cell flux. In contrast, in older leaves that were initiated prior to the encounter with root impedance, cell wall elongation rate was reduced throughout cell and leaf ontogeny; and although the duration of cell elongation was increased, the net result was a reduced final length. This reduction was compounded by reduced cell production rates due to slower cycling rates while meristem size was unaffected. There are thus several ways in which leaves adjust their overall elongation rate in response to the perception of high soil mechanical strength by the roots, and these depend on leaf position and on leaf developmental stage. Although rarely noted, leaf position effects on growth responses to environmental perturbations can be detected in a number of published reports (e.g., Masle, 2000). It is to be expected that, depending on when a stress is perceived during the ontogeny of an organ, various developmental processes may be affected. In the case of root impedance, however, a more complex developmental regulation is at play. This was demonstrated by a set of experiments where step changes in soil resistance to root penetration were imposed to the whole or part of the root system of young wheat seedlings, at various developmental stages (Masle, 1998). In every case, a step change in soil resistance caused changes in both leaf transpiration rates and leaf expansion rates. However, in every case these changes could only be detected after a significant time lag of several days. The growth of expanding emerged blades proceeded at the same rate as before the step change, and the duration of their elongation was unaffected. Only one interpretation could account for this as well as for the leaf position effects observed under steady-state conditions. The effects of root impedance on leaf expansive growth and adult leaf size must be set during a very early phase in the ontogeny of the wheat leaf, between initiation of the leaf primordium and formation of the intercalary meristem. It is fascinating that this ‘‘critical phase’’ much precedes the period where most of the

High Soil Strength

blade expansion occurs. Interestingly, while the events at the primordial and postprimordial stages need to be more widely tested, it has also emerged from studies of growth responses to other environmental perturbations in several species (Masle, 2000). When examining long-term responses to root impedance, time-dependent adaptive processes will complicate the detection of ontogenetic effects on responses to root impedance by causing apparent position or age effects. Thus, the morphological and biochemical adaptations that occur in impeded roots on scales of a few days, such as increased osmotic pressure, thickening of the cortex, and increased production of exudates and mucilage (e.g., Souty, 1987), all contribute to increase their ability to penetrate the soil. In effect, they concur to lower the level of mechanical impedance actually perceived by the roots and thus by the leaves. It is also likely that these adaptive root responses have a regulatory role in the signaling of root impedance to leaf meristems and stomata—for example, by causing changes in the composition of the xylem sap (Mulholland et al., 1999) and its pH (Wilkinson et al., 1998). Furthermore, while some adaptations may occur and be reversible within minutes to hours of stress release (e.g., osmotic pressure; Bengough et al., 1997a; Clark et al., 1996), others, such as changes in cell growth anisotropy or in cell wall extensibility, for example, cannot. Responses observed at one point in time therefore partly reflect past history, as has been shown in both roots and leaves during studies on the ‘‘recovery’’ from root impedance (Bengough and Young, 1993; Masle, 1998; Croser et al., 1999, 2000). Overall, this discussion emphasizes that physiological responses to a given soil resistance will vary depending on the organ ontogeny and, for roots, on where high resistance is encountered. Responses will also vary with time, through the effects of local and distant adaptive responses affecting the sensing and signaling of soil strength.

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involving several major hormones, with tissue- and process-specific regulation (see Chapter 28 by Bacon et al. in this volume). A.

Abscisic acid has been proposed as a putative signal for the regulation of stomatal conductance under root impedance. The reduction in stomatal conductance with increased resistance to root penetration, or its decrease upon release of stress, was correlated with an increase and a decrease, respectively, in xylem sap ABA concentration (Hartung et al., 1994; Mulholland et al., 1996; Hussain et al., 1999b; Hurley and Rowarth, 1999; Chapter 26 by Hose et al. in this volume). The absence of stomatal response to root impedance in ABA-deficient barley and tomato mutants is another argument in favor of a role for ABA (Hussain et al., 1999a, 2000). However, there has not been any study of root impedance where changes in stomatal conductances and levels of ABA in the xylem have been compared in intact transpiring plants, allowing the calculation of hormone delivery rates (Jokhan et al., 1996; Yong et al., 2000). Neither was the kinetics of changes in stomatal dynamics and ABA levels compared on fine time scales, thereby strengthening the case for a causal relationship. Moreover, recent studies with plants subjected to drought stress suggest that ABA conjugates and phaseic acid could be additional rootborne signaling compounds besides free ABA (Hansen and Do¨rfling, 1999) and that de novo ABA synthesis in leaves may be significant (Jeschke et al., 1997). Further studies of the temporal changes in the fluxes of ABA and ABA metabolites from impeded roots and in their compartmentation upon arrival in leaves are necessary to assess the role of ABA metabolism in the signaling of root impedance to stomata. B.

V.

SIGNALING

The demonstration of a direct signaling of root impedance to leaves through nonhydraulic signals has generated intense interest in the compounds that may be involved. The majority of studies have focused on root–shoot signaling and attempted to identify the chemical signals responsible for stomatal and growth responses through the analysis of hormone levels in root and leaf tissues and more recently in xylem sap. The emerging picture is that of a multisignaling system

Stomatal Conductance

Developmental Effects

As for the regulation of developmental responses to root impedance, there is strong evidence for a pivotal role of ethylene. The growth phenotypes associated with root impedance and with ethylene exposure have a number of similarities: radial root swelling, reduced root and leaf elongation, leaf epinasty, and increased thickness (see also Chapter 27 by Hussain and Roberts in this volume). Increased ethylene evolution has long been known to occur in impeded roots. This was first reported by Goeschl et al. (1966) and Kays et al. (1974), investigating roots meeting an

814

impenetrable physical barrier. More recently, using a triaxial cell system, Sarquis et al. (1991, 1992) and He et al. (1996) found that step changes in confining root pressure stimulated ethylene production in roots and induced an increased accumulation of ACC (the immediate precursor of ethylene) in leaves as well as increased ACC synthase and ACC oxidase activities. However, other studies failed to detect such changes (Lachno et al., 1982; Moss et al., 1988) or detected them sometime after the growth response. Closer examination of these studies led me to suggest that the involvement of ethylene in the early-signaling steps of mechanical impedance depends on the type of impedance: localization on the root, duration, severity, and balance among axial, radial, and friction forces (Masle, 1999). This interpretation still awaits testing. However, the recent studies of Hussain et al. (1999a, 2000) with the tomato wild-type Alisa Craig and a genetically modified low-ethylene genotype, ACO1AS (Hamilton et al., 1990), give support to such a complex and subtle regulatory role of ethylene, at least in leaves. In these experiments, when impedance (high soil strength) was encountered by the whole root system, similar leaf growth reductions were observed in the wild-type and the ACO1AS genotype, despite a three- to fivefold difference in leaf ethylene evolution. In contrast, when impedance was confined to only a fraction of the roots using a split-root system, a significant reduction in leaf growth was still observed in the wild-type but not anymore in the ACO1AS plants, although the amounts of ethylene evolved in the two genotypes remained similar to those measured under ‘‘total impedance.’’ This difference in the response of the two genotypes could be inverted by artificially increasing ethylene production using etephon or, on the contrary, by limiting its perception using silver ions. Furthermore, under these conditions of partial impedance, ethylene evolution was negatively correlated with xylem sap ABA levels, suggesting an antagonistic relationship between the two hormones in regulating leaf growth. Such a relationship is reminiscent of that proposed by Spollen et al. (2000) for root growth under water stress, whereby increased ABA levels would allow growth maintenance by reducing ethylene production or decreasing its physiological effects. Recent molecular studies of Arabidopsis give undisputable evidence for important crosstalk between the ABA and the ethylene signaling cascades (Beaudoin et al., 2000; Ghassemian et al., 2000) that appears to occur at many levels and to be dependent on the tissue and process assayed. It seems that ABA may not affect

Masle

ethylene production directly as suggested by Hussain et al. (2000), but rather stimulate ethylene signaling— for example, through effects on its perception. It could also do so in both leaves and roots of impeded plants but through different mechanisms. The mechanisms involved in the growth inhibition induced by ethylene itself in impeded plants are open to debate. Ethylene inhibits polar transport of auxin in shoots and roots, and studies of Arabidopsis mutants in particular suggest that ethylene inhibition of root growth is largely mediated through internal auxin accumulation (e.g., Burg and Burg, 1967; Timpte et al., 1995; Luschnig et al., 1998). A number of mechanosensitive genes have been identified in a range of organisms, several of which are regulated by ethylene or auxin (Braam et al., 1997; Tatsuki and Mori, 1999; Arteca and Arteca, 1999) and involve calcium as a second messenger (e.g., Knight et al., 1991; Trewavas and Knight, 1994). There is evidence for significant increases of IAA levels in roots following mechanical stress (Lachno et al., 1982). It is not clear, however, whether such increases derive from increased production of ethylene, as suggested by Lachno and colleagues (1982), or are in fact causing them. Recent genetic studies show that ethylene enhances the activity of what seems to be an auxin response system capable of mediating differential growth (Harper et al., 2000). C.

An Elaborate Signaling Network

It is clear that the developmental responses to root impedance are regulated by several hormones interacting in elaborate networks. There is evidence for a role, in the first instance of ethylene, ABA, and auxin and for complex interactions between these hormone signaling cascades, dependent on organ, biological age, and level/type of mechanical stimulation. The fact that when the whole of the root system was impeded, similar growth reductions have been found in ABA or ethylene-deficient mutants and the isogenic wild-type plants (Hussain et al., 1999b, 2000), can be interpreted in two ways. It may imply that in certain impedance conditions, ethylene and ABA signaling are overriden by other, as yet unidentified but partly independent pathways. It may also reflect that over periods of a few days to a few weeks, severe impedance causes a much increased sensitivity to ABA and ethylene, so that even the low levels present in the mutants are beyond the thresholds required to induce maximum growth inhibition. Time-dependent changes in stomatal sensitivity to ABA have been shown under drought

High Soil Strength

stress (Davies et al., 1994). Further studies are required to test these two nonexclusive hypotheses and, more fundamentally, to dissect the signaling pathways involved in the growth and stomatal responses to root impedance. There is a total lack of kinetic data, especially during the early signaling steps. Available evidence is mostly correlative and fragmented, perhaps reflecting the technical and analytical difficulties involved in a quantitative and sensitive description of stress signaling cascades and associated growth processes, on compatible timescales.

VI.

ECOLOGICAL SIGNIFICANCE; CHALLENGES FOR THE FUTURE

Most experimental data on root impedance refer to steady-state conditions where the physical properties of the root surroundings were kept as constant and homogeneous as possible, or to simple step changes in impedance. In nature, however, soil mechanical properties, and hence root impedance, are highly variable characteristics, in both space and time. In these conditions one may expect plants to have evolved mechanisms giving them a relative tolerance to mechanical stress and allowing them to quickly adjust to its variations. Ideally, plants should be endowed with a very sensitive root–shoot communication system enabling roots and shoots to sense subtle changes in soil strength and to elicit a battery of conservative but quickly reversible responses for the best compromise at various time scales between growth maintenance and conservation of soil resources. Remarkably, this is to some extent the scenario emerging from the present knowledge of responses to mechanical stress. Electrical signals and changes in membrane potential that propagate quickly throughout the plant may act as first-line signals (see above and also Bose, 1913; Wildon et al., 1992), triggering rapid and short-lived responses in leaf meristems and stomata, which are appropriate to transient and mild stress or to repeated touch stimulus, such as occurs at the root tip in any solid medium. Under longer-lasting episodes of high impedance (scale of minutes to much longer), hormone signaling cascades are activated that modify gene expression and developmental patterns, resulting in morphological and functional adaptations in both roots and leaves (Fig. 3). These adaptations enhance the ability of roots to overcome soil resistance and enable the plant to grow more conservatively, especially with respect to water and nutrient consumption as shown by the increased

815

transpiration efficiency typical of impeded plants in many species (Masle and Farquhar, 1988; Masle, 1992). At macroscopic scales these adaptations show remarkable similarities with those induced by drought, and there seems to be a genetic association between the sensitivity of roots to mechanical impedance and to osmotic stress (e.g., Materechera et al., 1992). It may therefore be argued that the reduced expansive growth and transpiration rates induced by mechanical stress even under plentiful water, nutrients, and oxygen supply, are feed-forward responses to the increased likelihood that these resources will become scarce sooner as a result of poor root growth. Such feed-forward mechanisms would have an obvious adaptive value in environments prone to prolonged soil drying or to compaction. On the other hand, in the many cases where impedance affects only part of the root system, or is temporary, they may be undesirable, especially if there is no acclimation to mechanical stress that in the long term would decrease root sensitivity to impedance or accelerate growth recovery upon release of stress. This awaits analysis. Nonetheless, this discussion emphasizes that the agronomic significance of plant responses

Figure 3 Schematic diagram showing typical long-term developmental adaptations to root impedance: reduced root length; increased root mass and root:shoot ratios; reduced aerial growth; smaller, thicker leaves with increased N and C content per unit leaf area; reduced stomatal conductance.

816

to mechanical impedance needs to be assessed in relation to the duration, the intensity, the frequency, and the spatial distribution of mechanical stress. Depending on these parameters, different strategies will be appropriate to manipulate these responses to the advantage of the plant, whose aim may range from disabling the sensing and signaling mechanisms of root mechanical stress, to modulating them to various degrees, or even to reinforcing them so as to ensure completion of the life cycle on limited soil resources. Identifying these mechanisms and their interactions with the signal transduction pathways of related stresses—wounding, osmotic and drought stress in particular—is the challenge ahead. Basic questions still have to be answered such as: How do roots ‘‘measure’’ soil strength? Where is this monitoring system situated in the root? Further, how do plants decipher and integrate temporal and spatial variation in the mechanical forces encountered by different parts of the root system? What are the genetic determinants of the sensing and signaling of exogenous mechanical forces in roots? How does mechanical impedance influence the rhizosphere and the capacity of roots for water and nutrient uptake? It is known that the signaling of root impedance is strongly influenced by temperature and by the plant/ cell physiological context, its carbohydrate status in particular (Masle et al., 1990). The mechanisms involved are not understood. Genomic approaches hold great promise for addressing these questions and examining these interactions provided they are applied to physiologically well-characterized responses and well-defined, realistic impedance conditions. There is also much to be learned from combining these approaches with the analysis of natural genetic variation in response to impedance, which is significant both within and among species (Taylor and Ratliff, 1969a,b; Masle, 1992; Materechera et al., 1991; Whalley and Dexter, 1993).

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817 Greacen EL, Oh JS. 1972. Physics of root growth. Nature 235:24–25. Hamilton AJ, Lycett GW, Grierson D. 1990. Antisense gene that inhibits synthesis of the hormone ethylene in transgenic plants. Nature 346:284–287. Hansen H, Do¨rfling K. 1999. Changes of free and conjugated abscisic acid and phaseic acid in xylem sap of drought-stressed sunflower plants. J Exp Bot 50:1599– 1605. Harper RMH, Stowe-Evans EL, Luesse DR, Muto H, Tatematsu K, Watahiki MK, Yamamoto K, Liscum E. 2000. The NPH4 locus encodes the Auxin Response Factor ARF7, a conditional regulator of differential growth in aerial Arabidopsis tissue. Plant Cell 12:757–770. Hartung W, Zhang J, Davies WJ. 1994. Does abscisic acid play a stress physiological role in maize plants growing in heavily compacted soil? J Exp Bot 45:221–226. He C, Finlayson SA, Drew MC, Jordan WR, Morgan PW. 1996. Ethylene biosynthesis during aerenchyma formation in roots of maize subjected to mechanical impedance and hypoxia. Plant Physiol 112:1679–1685. Hurley MB, Rowarth JS. 1999. Resistance to root growth and changes in the concentrations of ABA within the root and xylem sap during root-restriction stress. J Exp Bot 50:799–804. Hussain A, Black CR, Taylor IB, Roberts JA. 1999a. Soil compaction. A role for ethylene in regulating leaf expansion and shoot growth in tomato. Plant Physiol 121:1227–1237. Hussain A, Black CR, Taylor IB, Mulholland BJ, Roberts JA. 1999b. Novel approaches for examining the effects of differential soil compaction on xylem sap abscisic acid concentration, stomatal conductance and growth in barley (Hordeum vulgare L.). Plant Cell Environ 22:1377–1388. Hussain A, Black CR, Taylor IB, Roberts JA. 2000. Does an antagonistic relationship between ABA and ethylene mediate shoot growth when tomato (Lycopersicon esculentum Mill.) plants encounter compacted soil? Plant Cell Environ 23:1217–1226. Jeschke WD, Peuke AD, Pate JS, Hartung W. 1997. Transport, synthesis and catabolism of abscisic acid (ABA) in intact plants of castor bean (Ricinus communis L.) under phosphate deficiency and moderate salinity. J Exp Bot 48:1737–1747. Jokhan AD, Else MA, Jackson MB. 1996. Delivery rates of abscissic acid in xylem sap of Ricinus communis L. plants subjected to part-drying of the soil. J Exp Bot 47:1595–1599. Kays SJ, Nicklow CW, Simons DH. 1974. Ethylene in relation to the response of roots to physical impedance. Plant Soil 40:565–571. Knight MR, Campbell AK, Smith SM, Trewavas AJ. 1991. Transgenic plant aequorin reports the effects of touch

818 and cold-shock and elicitors on cytoplasmic calcium. Nature 352:524–526. Lachno DR, Harrison-Murray RS, Audus LJ. 1982. The effects of mechanical impedance on the levels of ABA and IAA in root tips of Zea mays L. J Exp Bot 33:943–951. Lu Z, Newman PM. 1998. Water-stressed maize, barley and rice seedlings show species diversity in mechanisms of leaf growth inhibition. J Exp Bot 49:1945– 1952. Luschnig C, Gaxiola RA, Grisafi P, Fink GR. 1998. EIR1, a root-specific protein involved in auxin transport is required for gravitropism in Arabidopsis thaliana. Genes Dev 12:2175–2187. Masle J. 1990. Growth and stomatal behaviour: response to soil resistance to root penetration. In: Davies WJ, Jeffcoat B, eds. Importance of Root to Shoot Communication in the Responses to Environmental Stress. Bristol: British Society for Plant Growth Regulation, Monograph 21:95–113. Masle J. 1992. Genetic variation in the effects of root impedance on growth and transpiration rates of wheat and barley. Aust J Plant Physiol 19:109–125. Masle J. 1998. Growth and stomatal responses of wheat seedlings to spatial and temporal variations in soil strength of bi-layered soils. J Exp Bot 49:1245– 1251. Masle J. 1999. Root impedance: sensing, signalling and physiological effects. In: Lerner HR, ed. Plant Responses to Environmental Stresses. From Phytohormones to Genome Reorganization. New York; Marcel Dekker, pp 475–495. Masle J. 2000. The effects of elevated CO2 concentrations on cell division rates, growth patterns, and blade anatomy in young wheat plants are modulated by factors related to leaf position, vernalisation, and genotype. Plant Physiol 122:1399–1415. Masle J, Farquhar GD. 1988. Effects of soil strength on the relation of water-use efficiency and growth to carbon isotope discrimination in wheat seedlings. Plant Physiol 86:32–38. Masle J, Passioura JB. 1987. The Effect of soil strength on the growth of young wheat plants. Aust J Plant Physiol 14:643–656. Masle J, Farquhar GD, Gifford RM. 1990. Growth and carbon economy of wheat seedlings as affected by soil resistance to penetration and ambient partial pressure of CO2 . Aust J Plant Physiol 17:465–487. Materechera SA, Dexter AR, Alston AM. 1991. Penetration of very strong soils by seedling roots of different plant species. Plant Soil 135:31–41. Materechera SA, Dexter AR, Alston AM, Kirby JM. 1992. Growth of seedling roots in response to external osmotic stress by polyethylene glycol 20,000. Plant Soil 143:85–91.

Masle Misra RK. 1996. Maximum axial growth pressures of the lateral roots of pea and eucalypt. Plant Soil 188:161–170. Misra RK, Dexter AR, Alston AM. 1986. Maximum axial and radial growth pressures of plant roots. Plant Soil 95:315–326. Moss GI, Hall KC, Jackson MB. 1988. Ethylene and the responses of roots of maize (Zea mays L.) to physical impedance. New Phytol 109:303–311. Mulholland BJ, Black CR, Taylor IB, Roberts JA, Lenton JR. 1996. Effect of soil compaction on barley (Hordeum vulgare L.) growth. I. Possible role for ABA as a root sourced chemical signal. J Exp Bot 47:539–549. Mulholland BJ, Black CR, Taylor IB, Roberts JA. 1999. Influence of soil compaction on xylem sap composition in barley (Hordeum vulgare L.). J Plant Physiol 155:503–508. Passioura JB. 1988. Root signals control leaf expansion in wheat seedlings growing in drying soil. Aust J Plant Physiol 15:687–693. Passioura JB, Gardner PA. 1990. Control of leaf expansion in wheat seedlings growing in drying soil. Aust J Plant Physiol 17:149–157. Pfeffer W. 1893. Druck and Arbeitsleistung durch Wachsende Pflanzen. Abh. Sachs Akad Wiss Leipzig, Math Naturwiss Kl 33:235–474. Richards BG, Greacen EL. 1986. Mechanical stresses on an expanding cylindrical root analogue in granular media. Aust J Soil Res 24:393–404. Russell RS, Goss MJ. 1974. Physical aspects of soil fertility— the response of roots to mechanical impedance. Neth J Agric Sci 22:305–318. Sarquis JI, Jordan WR, Morgan PW. 1991. Ethylene evolution from maize (Zea mays L.) seedling roots shoots in response to mechanical impedance. Plant Physiol 96:1171–1177. Sarquis JI, Morgan PW, Jordan WR. 1992. Metabolism of 1aminocyclopropane-1-carboxylic acid in etiolated maize seedlings grown under mechanical impedance. Plant Physiol 98:1342–1348. Souty N. 1987. Mechanical behaviour of growing roots. I. Measurement of penetration force. Agronomie 7:623– 630. Spollen WG, LeNoble ME, Samuels TD, Bernstein N, Sharp RE. 2000. Abscisic acid accumulation maintains primary root elongation at low water potentials by restricting ethylene production. Plant Physiol 122:967–976. Stolzy LH, Barley KP. 1968. Mechanical resistance encountered by roots entering compact soils. Soil Sci 105:297– 301. Tatsuki M, Mori H. 1999. Rapid and transient expression of 1-aminocyclopropane-1-carboxylate synthase isogenes by touch and wound stimuli in tomato. Plant Cell Physiol 40:709–715.

High Soil Strength Taylor HM, Roberson GM, Parker JJ. 1965. Soil strengthroot penetration relations for medium- to coarse-textured soil material. Soil Sci 102:18–22. Taylor HM, Ratliff LF. 1969a. Root elongation rates of cotton and peanuts as a function of soil strength and soil water content. Soil Sci 108:113–119. Taylor HM, Ratliff LF. 1969b. Root growth pressures of cotton, peas, and peanuts. Agron J 61:398–402. Timpte C, Lincoln C, Pickett FB, Turner J, Estelle M. 1995. The AXR1 and AUX1 genes of Arabidopsis function in separate auxin response pathways. Plant J 8:561–569. Trewavas A, Knight M. 1994. Mechanical signalling, calcium and plant form. Plant Mol Biol 26:1329–1341. Whalley WR, Dexter AR. 1993. The maximum axial growth pressure of roots of spring and autumn cultivars of lupin. Plant Soil 157:313–318. Whalley WR, Finch-Savage WE, Cope RE, Rowse HR, Bird NRA. 1999. The response of carrot (Daucus carota L.) and onion (Allium cepa L.) seedlings to mechanical impedance and water stress at sub-optimal temperatures. Plant Cell Environ 22:229–242.

819 Wildon DC, Thain JF, Minchin PEH, Gubb I, Reilly A, Skipper Y, Doherty H, O’Donnell P, Bowles DJ. 1992. Electrical signalling and systemic proteinase inhibitor induction in the wounded plant. Nature 360:62– 65. Wilkinson S, Corlett JE, Oger L, Davies WJ. 1998. Effects of xylem pH on transpiration from wild-type and flacca tomato leaves. Plant Physiol 117:703–709. Wilson AJ, Robards AW, Goss MJ. 1977. Effects of mechanical impedance on root growth in barley, Hordeum vulgare L. II. Effects on cell development in seminal roots. J Exp Bot 28:1216–1227. Young IM, Montagu K, Conroy J, Bengough AG. 1997. Mechanical impedance of root growth directly reduces leaf elongation rates of cereals. New Phytol 135:613– 619. Yong JWH, Wong SC, Letham DS, Hocart CH, Farquhar GD. 2000. Effects of elevated [CO2] and nitrogen nutrition on cytokinins in the xylem sap and leaves of cotton. Plant Physiol 124:767–779.

46 Plant Roots Under Aluminum Stress: Toxicity and Tolerance Hideaki Matsumoto Okayama University, Okayama, Japan

I.

DISTRIBUTION OF ACID SOILS AND THEIR NATURE

Acid soils occupy 3.95 billion ha (30%) of the world’s ice-free land area (Baligar et al., 1998), comprising both the tropical and temperate belts. The distribution of acid soil in selected regions in the world is shown in Table 1. Depending on the degree of weathering and soil acidity, acid soil can be classified into 10 groups. Oxisols and Ultisols are the major acid soils in the tropical region and occupy 22% (846 m ha) and 18% (727 m ha), respectively, of the acid soil area in the world. Inceptisols including the sulfate soils of many tropical river deltas are very acidic owing to acid formed upon oxidation of sulfides. The level of acidification of acid soil generally reflects the degree of weathering and leaching it has experienced (Baligar et al., 1998). In addition to the natural factors that affect weathering, agricultural farming processes such as the excessive supply of inorganic fertilizers or removal of cations by harvest lower the pH. Furthermore, the acidity of soils is gradually increased owing to environmental pollution and acid rain. Acid soils are infertile because they lack the basic nutrients, such as Ca2þ , Mg2þ , and Kþ . Acid soils are characterized by high content of toxic elements such as Al, Mn, and Fe or deficiency of Ca2þ , Mg2þ , Kþ , N, and P. Most acid soils have low cation exchange capacity, leading to loss of essential minerals and to poor crop production.

II.

ALUMINUM TOXICITY IN ACID SOILS

A.

Occurrence and Chemistry of Al

Exchangeable Al and Mn are the major toxic elements in most acid soils. In most Oxisols and Ultisols, Al occupies 4–94% of the cation exchange sites (Baligar et al., 1998). Al is the most abundant metal in the earth’s crust. Aluminum exists in the soil in insoluble aluminosilicates or oxides. It has complicated chemical form and biological function. At pH < 5, Al3þ exists as the octahedral hexahydrate, AlðH2 OÞ3þ 6 , often abbreviated as Al3þ . As the solution becomes less acidic, AlðH2 OÞ3þ 6 undergoes successive deprotonations to yield AlðOHÞ2þ and AlðOHÞþ 2 . In neutral solution AlðOHÞ3 precipitates as gibbsite which redissolves in basic solutions owing to formation of tetrahedral AlðOHÞ 4 as aluminate anion. Time-dependent formation of polynuclear species may also take place (Martin, 1986). Since Al toxicity differs with the chemical form of Al, many studies have been done regarding this interaction, especially between Al3þ and mononuclear hydroxy-Al. Generally Al3þ is more phytotoxic than AlðOHÞ2þ or AlðOHÞþ 2 , but Alva et al. (1986) and Kinraide and Parker (1987) reported that dicotyledonous plants may be more sensitive to AlðOHÞ2þ and 3þ AlðOHÞþ 2 than to Al . The difference in behavior of Al species between monocots and dicots may be related to the fact that dicots have a much higher CEC in their

821

822

Matsumoto

Table 1 Extent of Acid Soils in the World and Selected Regionsa Region Distribution class 6

Acid land area (10 ha) Acid land area (%)c

Global

Central America

South America

Africa

Asiab

Australia/ New Zealand

North America

Europe

3,950 30

37 35

917 14

659 22

532 76

239 30

662 30

391 37

a

Von Uexkull and Mutert (1995). Excluding South and East Asia. c Ice-free land area of the globe. Source: Baligar et al. (1998). b

cell wall than monocots, but further studies are needed to elucidate the cause. In the soil solution Al3þ reacts not only with OH but also with phosphate, F , SO2 4 , silicate, and a large number of organic ligands. Under specific conditions of OH/Al ratio, total Al, and stirring rate, AlO4 Al12 ðOHÞ24 ðH2 OÞ7þ (Al13 polymer), which is 12 highly toxic, can be formed (Parker et al., 1989). Nevertheless, Al13 was not observed in soil solutions (Funakawa et al., 1993). Rhizotoxicity of the aluminate ion, AlðOHÞ 4 , which is formed at an alkaline pH was tested with wheat and red clover. Root elongation was inhibited to < 4% by 25
M AlðOHÞ 4 at pH 8 but not at pH 8.9, where elongation was unaffected. Thus, AlðOHÞ 4 is nontoxic, and the inhibition at a lower pH is attributable to the Al13 formed (Kinraide, 1990).

B.

Inhibition of Root Elongation by Al

Inhibition of root elongation is the first visible symptom of Al stress. In most plant species, root elongation is markedly inhibited by Al at the
mol level in a simple solution containing Ca2þ alone. Inhibition of root elongation of Al-sensitive maize occurred within 30 min of Al treatment (Llungany et al., 1995). However, inhibition of root elongation is reduced in the presence of other ions, because the interaction with other coexisting ions reduces the toxicity of Al3þ . Changes in the electric charge of the root surface by other ions, especially cations, affect the accessibility of Al3þ . Root elongation in Al-sensitive wheat cultivar, Scout 66, was apparently inhibited by a 3-h treatment with 5
M Al, but that of Atlas 66 was inhibited to the same degree only by a 10-fold higher concentration of Al (Fig. 1). The root apex (root cap, meristem, and elongation zone) accumulated more Al and plays a major role in the Al perception mechanism.

Indeed, only the apical 2–3 mm of maize and pea roots need to be exposed to Al for the inhibition of root elongation to take place (Delhaize and Ryan, 1995; Matsumoto et al., 1996). In near-isogenic wheat (Triticum aestivum) lines differing in Al tolerance, root apices of Al-sensitive genotypes were stained with hematoxylin after a short exposure to Al (10 min– h). Apices of Al-tolerant seedlings showed less intensive staining (Delhaize et al., 1993a). This indicates that inhibition of root elongation by Al varies among plant species or cultivars. Similar results were obtained with Al-tolerant wheat (Atlas 66) and Al-sensitive wheat (Scout 66) exposed to Al for 1 day (Sasaki et al., 1997b) (Fig. 2). These results suggest that (1) the differences in Al accumulation in the root apex are related to differences in Al sensitivity, (2) inhibition of root growth is related to the Al content in the root apex, and (3) tolerant cultivars possess a mechanism that excludes Al from root apices (Rinco´n and Gonzales, 1992; Samuels et al., 1997). 1.

Morphological Changes of Intact Roots and Root Cells Under Al Stress

Accumulation of coating materials on the epidermis of the apex and around the cap is commonly observed upon Al stress. Root meristem cells of canola (Brassica napus var. napus L. cv. barassa) plants, grown under control conditions, responded differently from the much larger control cap cells (Clune and Copeland, 1999). Under mild Al stress (20
M, 24 h), these cells expanded and increased in number, but under more severe treatment (80
M Al, 24 h) they diminished in size and number. Furthermore, the distinct boundary between cells in the root cap meristem and the elongation zone was no longer apparent, and the outer layer of cells in the root cap appeared to be only loosely attached. After only 4 h in 80
M Al, many ultrastructural changes were evident in periph-

Aluminum Stress

Figure 1 Time course for wheat root elongation of Atlas 66 and Scout 66. Seedlings were in the presence and absence of 5 (Scout 66) or 50 (Atlas 66)
M Al. Data are means (SE) of results from 10 roots. (From Sasaki, 1996.)

Figure 2 Hematoxylin-stained wheat roots of Atlas 66 and Scout 66. Seedlings were grown in the presence and absence of Al for 48 h. (From Sasaki et al., 1997b.)

823

eral root cells, including the appearance of numerous small vacuoles that occupied most of the cytoplasm. After 24 h, disorganization of the cellular contents of the peripheral root cap cells became obvious, and the plasma membrane was clearly separated from the cell wall. The cytoplasm was markedly reduced in volume and extensively vacuolated. Similarly, Al-induced vacuolations have been observed in several plant species (Ikeda and Tadano, 1993; Marienfeld et al., 1995). In Lemna minor, the vacuolation and numerous myelinlike whorls of membrane increased in the apical meristem of the root (Severi, 1991). Moreover, the vesicles produced by the Golgi apparatus were larger. However, whether vacuolation is related to the storage of Al remains to be determined. Al has a localized effect on auxin transport and unilateral application of Al inhibited root curvature. Inhibition of cell elongation in the elongation zone is the major outcome of the inhibition of root elongation. Shortening of the root elongation zone by Al is accompanied by an increase in the diameter and a decrease of the length of the cells in the second and third layers of the cortex of the elongation zone of Atlas 66 plants (Matsumoto, 2000; Sasaki et al., 1996) (Figs. 3, 4). The ratio of length to diameter of the cells in the control root was three to four times larger than that in the Al-treated roots, and cells in the second and third layers of the cortex were swollen laterally. The Al-induced inhibition of longitudinal cell expansion and cell swelling in the elongation zone might be related to the disorder of the cytoskeletal network. The orientation of the microtubules (MTs) is closely related to cell expansion. Longitudinally elongating cells have transversely oriented MTs. MTdisrupting agents promote lateral expansion but inhibit longitudinal expansion. Cortical MTs are known to be involved in the orientation of cellulose microfibrils. Indeed, the disappearance of the cortical MTs in elongating cells of wheat roots that was observed under Al stress (Sasaki et al., 1997a) might be responsible for these changes in cell growth. Moreover, the timedependent effect of Al on MTs stability was correlated with that on the reduction of root growth. The effect of Al on the behavior of structural proteins has been investigated intensively in recent years. The actin network plays an important role in the plant cell. Al induced a significant increase in the tension within the transvacuolar actin network in soybean cells (Grabski and Schindler, 1995). Al resulted in a reorganization of MTs in the inner cortex, but not outer cortex, and in the epidermis of the elongation zone of Zea mays (Blancaflor et al., 1998). They also

824

Matsumoto

Figure 3 Photomicrographs of longitudinal sections of wheat roots of Atlas 66. Roots were treated with or without 20
M Al for 24 h. Bar indicates 0.2 mm. (From Sasaki et al., 1996.)

Figure 4 Effects of Al on the lengths and diameters of wheat root cells in the second layer from surface in Atlas 66. Roots were treated with or without 20
M Al for 24 h (a) or 48 h (b). Data are means (SE) of results from five or six samples. (From Sasaki et al., 1996.)

Aluminum Stress

found that the auxin-induced reorientation and coldinduced depolymerization of MTs in the outer cortex were blocked by Al, suggesting that Al increased the stability of MTs in these cells. The changes of behavior of MTs against Al stress may depend on the growth phase of the cells (Sivaguru et al., 1999). This stability effect of Al in the outer cortex coincided with growth inhibition. Aluminate [AlðOHÞ 4 ] at pH 10.0 induced the bending of roots of salt-tolerant grass (Thinopyrum bessarabium A. Lo¨ve). The roots under aluminate treatment displayed a number of morphological and structural malformations (Eleftheriou et al., 1993). The root cap decreased in size, and both the calyptrogen and the meristemic region occupied a smaller area. Amyloplasts in the root cap cells were hardly distinguishable and showed less evidence of sedimentation. The swollen cells of wheat roots were characterized by the drastic accumulation of lignin on their cell walls under Al stress (Sasaki et al., 1996). The decrease of cell viability of the elongation zone of wheat roots were also observed after 3 h of exposure to Al which coincided with the time required for the inhibition of the root elongation as well as for lignin deposition (Sasaki et al., 1997b). It is still unknown whether lignin deposition is involved in the mechanism of Al toxicity. The morphological changes in roots were characterized by the cracks on the root surface (Sasaki, 1996) (Fig. 5). Cracking might be caused by the outward pressure of the cells in the second and third layers of the cortex of wheat roots. The distal part of the elongation zone of maize roots, where cells are undergoing a preparatory phase for rapid elongation, is the primary target of Al influence (Sivaguru and Horst, 1998). To understand the primary event of Al toxicity, we must know how the cells in the specific zones, i.e., elongation and/or transition zone of the root, accumulate Al and how their elongation is inhibited by ultrastructural alterations. Why do such events cause death of the cell? In other words, are the matured root cells resistant to Al toxicity after their elongation has ended? 2.

Inhibition of Cell Division

Cell division in root meristems of several plants is inhibited by Al (Clarkson, 1965; Morimura et al., 1978). However, cell division accounts for only 1–2% of the overall root elongation. Furthermore, cell cycle in plants takes about 1 day. However, the primal phenomenon of Al toxicity is the inhibition of root elongation that occurs within hour(s) of Al treatment.

825

Figure 5 Photographs with scanning electron microscope of wheat root surface of Atlas 66. Roots were treated without and with 50
M Al for 4 h in the presence of 0.1 mM CaCl2 . (From Sasaki, 1996.)

Thus, attention has been largely paid to the inhibition of root cell elongation as the primary site of Al toxicity. On the other hand, the lethal consequence of Al toxicity might be inhibition of cell division (Matsumoto, 2000). Large amounts of Al accumulate in the developing lateral roots of pea roots where cells are actively dividing (Matsumoto et al., 1976a). Al was detected in nuclei of root hair cells by staining and by chemical determination of Al in purified nuclei prepared from Al-treated pea roots. Furthermore, Al accumulation in the nuclei of soybean root tips was detected with Al-sensitive stain lumogallion and confocal laser scanning microscopy (Silva et al., 2000). The nuclei isolated from pea roots treated with 1 mM AlCl3 at pH 5.5 for 1 day were fractionated; 73% of the total Al in nuclei was recovered in the chromatin fraction, and 94% of Al in chromatin was recovered in DNA (Matsumoto et al., 1977b). Expression of genetic information of DNA is regulated by structural changes of DNA and chromatin. Unwinding of double strands of DNA is a prerequisite

826

for expression of genetic information, but separation of double strands of DNA was interrupted by Al (Fig. 6) (Matsumoto, 1991). Furthermore, the structural change of chromatin in pea roots treated with Al in vivo implied that Al induced the condensation and/or aggregation of chromatin (Matsumoto, 1988). These results suggested that the template activity of DNA and/or chromatin of pea roots for RNA synthesis is repressed by Al. This is caused by Al-induced structural alteration of DNA and chromatin through the association of the negative charge of phosphates of DNA and positively charged Al3þ (Morimura and Matsumoto, 1978). Plant cells require dynamic cytoskeleton-based networks for various cell activities (e.g., differentiation and division). With suspension of tobacco cells, Sivaguru et al. (1999) found that the

Figure 6 Proposed mechanism of inhibition of the transcription of RNA by Al. (A) Normal transcription of RNA on a sense strand of DNA template in the absence of Al. A short section of the double strands must open and, thus, only the sense strand can act as the template. (B) Transcription is inhibited in the presence of Al. Sections of the double strands (indicated by two arrows) are captured by Al polymers with various structures shown as ‘Al ¼ Al ¼ Al’, through the strong electrostatic interaction between phosphate groups with a negative charge and the large positive charge of the Al polymer. Thus, separation of the strands is blocked and limited synthesis of RNA results. In addition, the Al polymer with its large positive charge on one helix, shown as nAlmþ where n and m are highly variable and depend on coexisting factors, can associate with the other helix and cause aggregation of chromatin fibers. (From Matsumoto, 1991.)

Matsumoto

actively dividing log-phase cells were characterized with faint and larger phragmoplasts and unusually enlarged daughter nuclei after 6 h of Al treatment. After a 24-h treatment, no phragmoplasts and spindle MTs (SMT) from cells having metaphase plate chromosomes were observed. The disintegration of SMT and disorganization of phragmoplasts caused by Al might block cell division directly at the metaphase. As mentioned before, inhibition of cell division will be the cause for the complete inhibition of root elongation by Al and subsequent death. Therefore, elucidation of the detailed mechanism of cell division inhibition by Al will be required in order to understand the mechanism of Al toxicity (Matsumoto, 2000). C.

Site of Al Toxicity

1.

Apoplast

Although there is disagreement with regard to the site of Al toxicity, namely, symplastic or apoplastic, many investigators have stated that 30–90% of the absorbed Al is localized in the apoplast (cf. Tice et al., 1992; Rengel, 1996). Two possible interpretations has been given to the role of CEC in connection with Al accumulation in the apoplast. On the one hand, high CEC will be associated with large quantities of Al accumulated in the apoplast. On the other hand, high CEC may prevent Al from entering the symplast, where it exerts its lethal effect. However, a clear relationship between root CEC and Al sensitivity and/or tolerance was not found across a wide range of plant genotypes (Grauer, 1992). As to the binding site of Al in the apoplast, pectin carboxyl was suggested as a plausible candidate although almost no evidence has been found to show the binding of Al to pectin in vivo (Matsumoto et al., 1977a; Horst, 1995). Although Al is bound by the negative charge of pectin, the binding capacity of pectin varies with the plant species, and the pectin content is extremely different between monocots and dicots. Even in the same species, the pectin content of the roots differs with the position on the root or with the chemical modification of pectin, such as methylation or demethylation changes with the physiological activity of the cell. A Ca-pectate membrane was used as a model system. There was a rapid reaction between Al and Ca pectate, but there was no difference in Al remaining in solution even after 16 min. Only a slight decrease was observed after 24 h. The solution containing 29
M Al and 1 mM Ca reduced the flow through the Ca pectate membrane by > 80% compared to the

Aluminum Stress

solution containing 1 mM Ca only. These results suggest that an important effect of toxic Al is a reduction in water movement into roots (Blamey et al., 1993). Interactions of Al with other cell-wall components, such as enzymes, extensin and xyloglucan may affect the functional integrity of cell walls. In the roots of cotton seedlings, Al impaired the sucrose utilization for cell wall formation (Huck, 1972). Al also induced the production of cell-wall components of squash seedlings, especially of hemicellulose, (Van et al., 1994). Al stress increased the level of covalently bound cell wall proteins in pea roots (Pisum sativum cv. Alaska). In vitro and in vivo Al binding experiments have suggested that extensin has the highest capacity to bind Al among cell wall proteins (Kenjebaeva et al., 2001). 2.

Plasma Membrane

The plasma membrane is one of the first targets of Al (Haug, 1984; Matsumoto, 2000). Al binds readily to the plasma membrane because of its high content of phosphate such as phospholipids and the negative charge of the membrane surface. Al3þ has a 560-fold higher affinity for the phosphatidyl choline surface than Ca2þ (Akeson et al., 1989). Structural and functional changes of the plasma membranes are induced by Al binding, although the evidence of Al binding to the plasma membrane in vivo is limited (Matsumoto et al., 1992). Al-induced changes in membrane behavior of intact root cortex cells of Quercus rubra root showed that Al altered the activation energy required to transport water (þ32%), urea (þ9%), and monoethyl urea (7%) across cell membranes as measured by the plasmometric method (Zhao et al., 1987). Al increased the lipid partiality of the plasma membrane at > 9 C but decreased it at temperatures < 7 C. These changes in membrane behavior are explainable if Al reduces membrane lipid fluidity and kink frequency and increases packing density and the occurrence of straight lipid chains (Chen et al., 1991). It can be concluded that Al3þ (1) increased membrane permeability to the nonelectrolytes, (2) decreased the membrane partiality for lipid permeators, and (3) decreased membrane permeability to water caused by increased activation energy. It is thus implied that Al3þ alters the architecture of membrane lipids (Vierstra and Haug, 1978). Furthermore, Al3þ inhibited the influx of cations and enhanced the influx of anions. This was caused by the Al-induced formation of positively charged layer at membrane surface influencing ion movement to the binding sites of the transport proteins. A posi-

827

tively charged layer would retard the movement of cations to the plasma membrane. This is explained by the charge of the membrane surface potential, Zeta potential, through the binding of Al. It was also argued from the relationship between Al tolerance and surface negativity of plasma membranes (Wagatsuma et al., 1995). Al reduced the negative charge associated with phospholipids, i.e., depolarization of Zeta potential, and proteins by binding to these charged groups or shielding the surface charges. Alteration of Kþ efflux and Hþ influx by Al also affect the Zeta potential as well as the potential difference (PD) across plasma membrane (Sasaki et al., 1994a). A more direct effect of Al3þ is its binding to transport proteins and impairs their function. For instance, Al3þ blocks inward-rectifying Kþ channels in root hairs of wheat (Triticum aestivum) and VDAC, channel-forming protein located in the outer mitochondrial membranes (Dill et al., 1987). Recently, a special interaction was found between depolarization of Zeta potential and decrease of Hþ -ATPase of plasma membrane of squash roots treated with Al. The interaction was typically observed at root tips, 0–5 mm portion of the root (Ahn et al., 2001). However, the varietal sensitivity to Al3þ is not based on the difference in cell surface electrical potential (Kinraide et al., 1992), and inhibition of root growth by Al is not caused by the reduction in current or H+ influx at the root apex (Ryan et al., 1992). On the other hand, a different membrane potential depolarization of root cap cells preceded Al tolerance in snapbean (Phaseolus vulgaris L.) (Olivetti et al., 1995). The Al-tolerant cultivar Dade depolarized rapidly upon exposure to Al, but the Alsensitive cultivar Romano was only slightly depolarized. This might be related to the fact that Al reduces the Kþ efflux channel conductance in the tolerant Dade root cap cells, but does not affect it in the sensitive cultivar Romano. Further research is needed to understand the interrelationship between Al toxicity and/or tolerance and electrophysiology. One of the biochemical changes of the plasma membrane is the Al-dependent lipid peroxidation in the root tip of soybean (Glycine max). A close relationship existed between lipid peroxidation and inhibition of root elongation induced by Al and/or Fe toxicity and/or Ca deficiency (Cakmak and Horst, 1991). Enhanced lipid peroxidation by oxygen free radicals can be a consequence of primary effects of Al on membrane structure. The tolerance mechanism against Al toxicity in terms of lipid peroxidation was proposed for tobacco suspension cells (Yamamoto et al., 1998). Lipid composition can be a determinant of the varietal

828

difference for Al sensitivity. The largest change of the lipid composition of the microsomal fraction in the root is sterylglucosides upon Al treatment between Al-resistant (PT741) and Al-sensitive (Katepwa) cultivar of Triticum aestivum (Zhang et al., 1997). 3. Calcium Ca2þ is an essential element for root growth and much work was devoted to Al toxicity in terms of Ca2þ . Alinduced changes in cell physiology, occurring in the cytoplasm and at the plasma membrane, might be caused by the disorder of Ca2þ homeostasis. Al inhibited Ca2þ influx into the root apex of Al-sensitive wheat cultivar Scout 66, but had a much smaller effect on the Al-tolerant cultivar Atlas 66. In the absence of Al3þ both cultivars maintained similar rates of net Ca2þ uptake (Huang et al., 1992). A similar inhibitory effect of Al was observed not only on Ca2þ uptake but also on its translocation from the apical region. Furthermore, Ca2þ transport activity due to the membrane potential was inhibited by externally added Al to isolated plasma membrane vesicles. But there was no difference in the inhibition effect between tolerant and sensitive wheat cultivars (Jacob and Northcote, 1985; Sasaki et al., 1994b; Huang et al., 1996). Such results imply that Al blocks the Ca2þ channels on the plasma membrane. The observed difference in Al uptake between intact roots and membrane vesicles might be due to different chelation of Al with the different efflux level of organic acids from the roots triggered by Al. The antagonistic effect between Ca2þ and Al toxicity is well known. The question is how Al inhibits the physiological functions of Ca2þ . Replacement of functional Ca2þ from the membranes and from the cell walls in the root might be one reason although the evidence for such displacement in vivo is limited (Kinraide et al., 1992). Competition between Ca2þ and Al3þ is thought to either weaken the cell walls, by reducing the number of Ca crosslinks or by replacing Ca2þ crosslinks with stronger Al3þ ones, making them too rigid for growth. Displacement of apoplasmic Ca2þ by Al is in part due to the competition for ligands, such as pectin carboxyl radical. It can also be caused by reduction of the negative potential difference, on the surface of plasma membrane. In isolated cell walls equilibrated with 50
M Ca2þ at pH 4.4, 100
M Al displaced > 80% of the bound Ca2þ with half-time of 25 min (Reid et al., 1995). Ca2þ was not displaced by Al in wheat (Ryan et al., 1997a), but the signal initiated or disrupted by excess Al inhibited the growth in the meris-

Matsumoto

tem of Allium cepa (Schofield et al., 1998). Al also impaired Ca-mediated plant defense responses to low pH conditions (Plieth et al., 1999). The disruption of Ca2þ metabolism by Al was debated. In wheat seedlings, root growth can be severely inhibited by Al3þ concentrations that do not affect Ca2þ uptake, while the addition of other ameliorating cations—e.g., 30 mM Naþ , 3 mM Mg2þ , or 50
M Tris (ethylenediamine) cobalt (III), depressed Ca2þ uptake (Ryan et al., 1994). Phytotoxic action of Al in the root hairs of Arabidopsis is not due to blockage of Ca2þ -permeable channels required for Ca2þ influx into the cytoplasm (Jones et al., 1998). On the other hand, Zhang and Rengel (1999) found that the increase of cytoplasmic free Ca2þ ions ([Ca2þ c ) in root apical cells was higher in Al-sensitive wheat (ES8) than in Al-tolerant wheat (ET8). The Al-related increase in [Ca2þ c was correlated with inhibition of root growth and was reversible upon removing the ambient AlCl3 . At present, we are far from elucidating the mechanism of Al/Ca interaction. The question whether Al increases or decreases cytoplasmic Ca2þ and how these alterations in Ca2þ level are related to Al toxicity syndrome in plant roots should be solved. Technical advancement for the detection of free Ca2þ in the cytosol of plant cells is also needed. 4.

Callose

Callose (-1,3-D-glucane) synthesis is very sensitive to Al stress and has been considered to be a reliable marker for Al toxicity. Callose formation was induced by Al as low as 5
M and as early as 10 min after Al addition to a suspension of cultured cells of soybean (Horst, 1995). Callose formation is most intensive in the root apex and confined only to the outer cortical cell layers of soybean seedlings (Wissemeier et al., 1992). The callose concentration in the 10- to 30-mm root tip of cowpea was inversely related to the root elongation rate when the roots were subjected to Al concentration > 10
M. Furthermore, a negative correlation was found between the relative callose concentration and relative root elongation rates of three soybean genotypes differing in Al sensitivity. Therefore, Al-induced callose formation in root tips might be used as a selection criterion for Al sensitivity. Callose is synthesized by -1,3-glucane synthetase on the plasma membrane and activated by Ca2þ . It is still unknown where the Ca2þ required for callose synthesis comes from. Ca2þ is released from the exchange sites of the cell wall and plasma membranes. Thus, an increase

Aluminum Stress

in the free Ca2þ concentration and triggering callose synthesis cannot be excluded. Ca2þ may not be the only signal for callose formation, and alteration in the plasma membrane architecture might also be important for callose synthesis (Jacob and Northcote, 1985). As callose is released into the apoplast after its synthesis on the plasma membrane, cell walls of root cells of Al-treated plants most likely contain callose depositions. What is the inhibitory function of callose under Al stress? Callose can be considered as a sealing system in plants. Callose is localized in the wall around the plasmodesmata, which appear to be structurally subdivided. Thus, the constricting force upon callose synthesis would be transmitted to the plasmodesmal core (Turner et al., 1994). This will inhibit the transport of cellular compounds through plasmodesmata under Al stress. A large increase of callose accumulation at the plasma membrane under Al stress was found in Al-sensitive wheat roots. Such an increase inhibited the cell-to-cell trafficking of molecules through the plasmodesmata, resulting in the inhibition of root elongation. Furthermore, these events are markedly repressed in the presence of 2-deoxyD-glucose, which is a callose synthase inhibitor (Sivaguru et al., 2000). However, in an Al-tolerant Arabidopsis mutant, no direct relationship between Al uptake and callose formation was established (Larsen et al., 1996). D.

Signal Transduction and Al Signal

Plants respond to Al stress quickly. Inhibition of root elongation is observed within less than an hour, and homeostasis of cytoplasmic free Ca2þ is broken instantly after Al addition. Special attention has been paid to phosphoinositide-associated signal transduction. AlCl3 and Al-citrate inhibited phospholipase C (PLC) of the microsomal membrane in a dose-dependent manner in wheat roots. I50 was observed at 15–20
M Al (Jones and Kochian, 1995). Binding of Al to microsomes and liposomes was found to be lipid dependent, with the signal transduction element PIP2 having the highest affinity for Al with an Al:lipid stoichiometry of 1:1. These results suggest an Al effect on the signal transduction pathway that is associated with the mechanism of Al toxicity. How is the Al signal recognized by receptor and how is it transported into the cytoplasm at the root apices? Bennet and Breen (1991) proposed that the Al signal is perceived in the root cap of Zea mays. Matsumoto et al. (1996) speculated that the transduc-

829

tion of Al signal in barley roots is related to an increase of ABA. ABA induces both the ATP- and PPi-dependent Hþ pump activity of the tonoplast (Matsumoto et al., 1996). Contrary to ABA, transport of exogenously applied [3H]indole-3-acetic acid to the meristemic zone was significantly inhibited by Al in maize roots. The signaling pathway in the root apex mediating the Al signal may be responsible for the genotypic difference in Al resistance (Kollmeier et al., 2000). The biochemical mechanism in terms of the transduction of Al signal is poorly understood. Protein phosphorylation may be involved because of the strong association of Al with phosphate.

E.

General Metabolism Affected by Al

The effect of Al on excised root apices and isolated mitochondria of wheat was investigated. O2 uptake by excised roots was reduced by 23% and 35% after 12- and 24-h treatment with 75
M Al. Mitochondria isolated from Al-treated roots had reduced oxidative capacity with supply of electrons to complexes I and II. It was found that initially Al affected electron flow through complexes I and II, and after longer exposure interacted with other sites in the mitochondria (de Lima and Copeland, 1994). Al tolerance of Phaseolus vulgaris (cv. Dade) was an inducible trait. In this cultivar, the resumption of root elongation during recovery from Al treatment was accompanied by increased rates of respiration. Respiration rates slowly declined over the 72-h treatment of Al-sensitive Romano. When partitioned into growth and maintenance expenditures, a larger proportion of root respiration of Al-treated Dade plants was allocated to maintenance processes, potentially reflecting diversion of energy to metabolic pathways that offset the adverse effects of Al toxicity (Cumming et al., 1992). Carbon metabolism is also affected by Al. Al stress increased alcohol dehydrogenase activity in wheat (Triticum aestivum cv. Vulcan) roots. Sucrose synthase and lactate dehydrogenase were also increased in Altreated roots, suggesting that the early effect of Al on wheat roots may be a shift from aerobic to anaerobic metabolism. The first two enzymes in the pentose phosphate pathway (G-6-PDH and 6-PGDH) decreased in Al-sensitive wheat cv. Grana. However, these two enzymes first increased, but then decreased in Al-tolerant rye (Sla´ski et al., 1996). These results suggest that the mechanism of Al resistance involves the regulation of the pentose pathway.

830

Matsumoto

III.

ALUMINUM TOLERANCE MECHANISM

A.

Al Tolerance on Genetic Basis

The bimodal distribution of phenotypes corresponding to 3:1 segregation ratio for Al tolerance/sensitivity in populations derived from crosses between tolerant and sensitive cultivars revealed the presence of single major genes for Al tolerance (Gain, 1998). In addition to major genes conferring major differences in Al tolerance, there is also some evidence that minor genes or modifier genes may play a role in modulating the effect of major Al tolerance genes. Other studies have suggested that genetic factors located on the long arm of chromosome 2D prevent accumulation of Al in root apical meristems of the BH1146 euploid wheat (Aniol, 1995). However, other genetic factors are also located on these chromosome segments that control Al detoxification in the root tips of Al-tolerant lines. The D genome of wheat may determine the tolerance to acid soil and consequently contribute to the increased adaptation of hexaploid wheats during their evolution. Atlas 66 is a well-known Al-tolerant cultivar of wheat. However, not all the genes for tolerance to Al in Atlas 66 are located on the D genome chromosome (Berzonsky, 1992). Furthermore, Al tolerance in wheat is a dominant trait and the majority of observed variability could be explained by hypotheses of two or three gene pairs, each gene affecting the same character with complete dominance of each gene pair. Al tolerance in the ditelosomic line of Chinese Spring wheat cultivar revealed that genes controlling this character are located on the short arm of chromosome 5A and the long arm of chromosomes 2D and 4D (Delhaize et al., 1993a). Conservation of the Al tolerance gene by various species was investigated. RFLP markers for a major wheat Al tolerance gene AltBH were found on the long arm of chromosome 4D, while in rye, the Al tolerance gene was located on chromosome 4, which harbors chromosome segments homologous to regions of wheat chromosome 4D. In barley, the Al tolerance gene Alp is almost certainly orthologous to the wheat AltBH gene due to the fact that the relative positions of Alp and AltHB with respect to a common set of molecular markers are virtually identical in both genomes (Berzonsky, 1992). In spite of efforts made so far, the genetics of Al tolerance is little known for any single species. As to the physiological functions of Al tolerance genes, Delhaize et al. (1993b) found that Al tolerance, controlled by the Alt gene in wheat, appeared to be dominant across a range of Al concentrations

based on identical Al tolerance in heterozygotes and Alt 1 homozygotes. This gene controls the excretion of malate upon Al stress. B.

Genetic Basis of Al Tolerance

Application of lime to acid soils to increase the soil pH is one strategy for alleviating Al toxicity. However, this technique is problematic from the economical and environmental points of view. Another strategy is to use Al-tolerant crops. Breeding may depend on classical techniques and/or transgenic plants to which Al tolerance genes are being introduced. Snowden et al. (1995) isolated several cDNA (wali 1–7) whose transcript accumulates in wheat under Al treatment. A cDNA library constructed from the mRNA of Al-treated roots of Al-sensitive wheat (cv. Victory). It was screened with a degenerate oligonucleotide probe derived from a partial amino acid sequence of the Al-induced protein TAl-18. Out of seven clones that initially hybridized with the probe, one encoding a novel 1,3--glucanase mRNA was upregulated in Altreated roots, with highest expression after 12 h. A second cDNA showed similarity to genes encoding cytoskeletal fimbinlike protein. Unfortunately, a transgenic protein enriched with those genes was not constructed (Cruz-Ortega et al., 1997). Al ions bind to phospholipids, and the plasma membrane is a primary barrier to the entry of Al into the cells (Matsumoto, 1988; Kochian, 1995). Thus, a change in lipid composition of the plasma membrane could improve the resistance of the cell by excluding Al (Delhaize et al., 1999). Cloned wheat cDNA (TaPSS1) that codes for phosphatidyl serine synthase (PSS) was tested. Overexpression of PSS increased Al resistance in yeast. However, a high level of TaPSSI expression in Arabidopsis and tobacco led to the appearance of necrotic lesions on leaves, which may have resulted from the excessive accumulation of PS (Delhaize et al., 1999). An Arabidopsis blue-copper-binding protein gene, a tobacco glutathione S-transferase gene, a tobacco peroxidase gene, and a tobacco GDP dissociation inhibitor gene conferred a certain degree of resistance to Al (Ezaki et al., 2000). These lines also showed increased resistance to oxidative stress, suggesting a link between Al stress and oxidative stress in plants. One successful approach was the generation of transgenic tobacco and papaya with the citrate synthase (CS) gene from Pseudomonas aeruginosa with the 35S promoter of cauliflower mosaic virus introduced using a Ti plasmid derived from a transfor-

Aluminum Stress

mation system. The idea was that organic acids serve as chelating agents and may prevent Al toxicity (Delhaize and Ryan, 1995; Fuente et al., 1997). Hematoxylin staining was employed to examine whether the increased Al tolerance of the CSb lines is due to the inhibition of Al uptake by the root tip. Following exposure to Al CSb lines showed a considerably lighter staining than the control. Apparently, the expression of a citrate synthase in the cytoplasm increases the concentration of citrate, which led to a higher rate of its efflux; the higher synthesis and excretion of citrate confers Al tolerance.

C.

Organic Acids as Al-Chelating Substance

Since higher plants cannot move away from the acid soil, they have developed ways to reduce this edaphic stress. An effective strategy to reduce the stress is to chelate the Al3þ in the rhizosphere and by that reduce its toxicity. Exclusion of malate from wheat (Delhaize et al., 1993b); citrate from maize, Cassia tora, and rye (Miyasaka et al., 1991; Zheng et al., 1998; Li et al., 2000b); and oxalic acid from taro and buckwheat (Ma and Miyasaka, 1998; Ma et al., 1997b) have been reported. Some plants exclude both malate and citrate under Al stress. Malate and oxalate are excreted instantly under Al stress, but citrate is excreted only after a lag phase. These results suggest that stored malate and oxalate are excreted while citrate is synthesized by a gene-regulated system. The organic acids were classified into three groups of Al detoxifiers: (1) strong (citrate, oxalic, tartaric); (2) moderate (malic, malonic, salicylic); and (3) weak (succinic, lactic, formic, acetic phthalic) (Hue et al., 1986). The following facts support the role of excreted organic acids as detoxifiers; During the first 20 h of Al exposure, the root growth rate of both tolerant and sensitive maize varieties was severely inhibited. However, after this period, root growth was resumed in the tolerant plants, but remained severely inhibited in the Al-sensitive one. A dose-dependent citrate and malate exudation was observed from tolerant but not from sensitive roots (Jorge and Arruda, 1997; Yang et al., 2000). The root of the Al-resistant snapbean released 70 times more citrate in the presence of Al as in its absence, and the amount of citrate excreted was 10 times as much as that of Al-sensitive cultivars (Miyasaka et al., 1991). Similar results regarding malate exclusion were obtained with Al-tolerant and Al-sensitive isogenic wheat lines.

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In > 36 lines of wheat cultivars differing in Al resistance that were screened, Al-stimulated malate release was correlated with Al resistance (Ryan et al., 1995). It was shown that addition of organic acids to the solution ameliorated Al toxicity in the root of Al-sensitive varieties and reduced dramatically the loss of viability of root cells (Li, 2000). This may be explained by the fact that complexes of Al with di- and tricarboxylic acids were not transported through root cell membranes (Ma et al., 1998). Excretion of organic acids is Al3þ specific and is not induced by other trivalent cations (Ma et al., 1997b). The loss of a certain chromosome arm resulted in a decrease in Al resistance in ditelosomic wheat lines and decreased rates of root apical malate release concomitant with decreased Al exclusion (Kochian, 1998). Exposure to Al induced depolarization of the membrane potential (Em) in Al-tolerant wheat cultivars but not in an Al-sensitive cultivars. Depolarization was specific to Al. Al-induced depolarization of root cap cell membrane potentials is probably linked to malate release (Papernick and Kochian, 1997). Al3þ triggers the opening of the putative malatepermeable channel. Several antagonists of anion channels inhibited the Al-stimulated efflux of malate. The anion channel antagonist niflumate inhibited the current in whole-cell measurements by 83% at 100
M Al. Patch clamp recordings revealed a multistate channel with single-channel conductance of between 27 and 66 ps. This is a good candidate to be the transport system facilitating Al-induced malate release (Ryan et al., 1997b). K-252a, a potent inhibitor of protein phosphorylation, reduced dramatically the excretion of malate from Al-treated wheat, suggesting the involvement of protein phosphorylation for the regulation of malate excretion under Al stress (Osawa and Matsumoto, 2001). Transgenic introduction of the bacterial cytosolic citrate synthetase gene into tobacco and papaya resulted in Al tolerance (Fuente et al., 1997). Excretion of organic acids from the root apex where Al injury is located seems to be a reasonable strategy for the effective use of carbon and energy by the plant (Delhaize and Ryan, 1995). Such experimental evidence indicates that the mechanism of Al exclusion that depends on the chelation of Al3þ with excreted organic acids is an effective strategy. However, it is unclear whether the quantities of organic acids released are adequate to explain the insensitivity to Al of the more tolerant genotypes. Consumption of the excreted organic acids by soil bacteria should also be considered. The question why different plant species excrete

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different organic acids in different ways upon Al stress also remains to be solved. The efflux of organic acids is probably switched on or off by the Al3þ stress, because continuous excretion of organic acids from roots would consume carbon and energy in extraordinary amounts. The precise mechanism remains to be elucidated, and a more integrative, multifaceted model of tolerance is needed (Parker and Pedler, 1998). The role of intracellular organic acids on Al tolerance was investigated using the Al-tolerant plant Hydrangea macrophylla. Leaves of hydrangea may contain up to 15.66 mmol Al kg1 fresh weight. About 77% of the total Al exists in the cell sap (Ma et al., 1997a). The ligand in the Al complex of hydrangea leaves was citric acid with a molecular ratio of 1 Al to 1 citrate. The purified Al:citrate complex from hydrangea leaves did not inhibit the root elongation of maize and did not decrease the cell viability although both parameters were strongly inhibited by the same concentration of AlCl3 . This means that Al in the form of Al:citrate is not toxic. Also, buckwheat contains large amounts of Al compared to other crop plants but can grow normally under Al stress. This suggests that buckwheat can detoxify Al. The measurement of 27 Al-NMR revealed that the Al in buckwheat leaves and roots existed as an Al:oxalate (1:3) complex. The Al:oxalate (1:3) complex is the least toxic complex compared to other complexes. Buckwheat contains a large amount of oxalate regardless of Al stress and can excrete oxalate immediately after the plant is exposed to Al. Even if some Al is incorporated into the plant tissues, it is immediately chelated there. D.

Protein Expression in Roots Under Al Stress

Wheat genotypes differing in Al tolerance were compared for qualitative and quantitative differences of their proteins. However, no conclusive evidence for upregulation of a certain protein that confer Al tolerance was reported (Delhaize et al., 1991; Ownby and Hruschka, 1991). Most of the changes in protein expression associated with Al stress probably result from the effects of Al on cell metabolism. Another approach was to look for proteins that are characterized by their binding capacity to Al. Appearance of the 23-kDa peptide in root exudates cosegregated with the Al-resistant phenotype in F2 populations and had a significant Al-binding capacity (Basu et al., 1999). Similarly, a 51-kDa membrane-bound protein accumulated in the root tip of Al-tolerant wheat (PT741) under Al stress. The specific induction of the 51-kDa

band in PT741 suggested a potential role of these proteins in mediating resistance to Al, associated with the tonoplast. Antibodies raised against tonoplast H+ATPase and H+-PPiase did not crossreact with 51kDa protein, although Al stress induced these enzymes in the barley roots (Matsumoto et al., 1996; Taylor et al., 1997). E.

Mucilage

The root apices of most plant species are covered by mucilaginous substances that are excreted from root cap cells (see also Chapter 3 by Sievers et al. in this volume). The meristem and cap region where Al toxicity is dominant are coated with mucilage that ranges in thickness from 50
m to 1 mm. Mucilage consists mainly of polysaccharides > 2  106 daltons. Abundant sugars are glucose, galactose, and arabinose. Uronic acids are smaller in amount but characteristic of the mucilage. Mucilage has various protective functions against toxic metals in the soil and has a high Al-binding capacity. Fifty percent of the total Al of root apices of cowpea was associated with mucilage (Horst et al., 1982). Al bound to mucilage of wheat roots accounted for
25–35% of the Al remaining after desorption by citric acid. The Al in rhizosphere is bound to mucilage that blocks the entry of Al into the root. When the mucilage was periodically removed from the root tips of cowpea with a brush, inhibition of root elongation was increased. Apparently, binding of Al to mucilage is a mechanism of Al tolerance. A good correlation exists between mucilage volume and Al tolerance. Organic acids released into a mucilage droplet would diffuse slowly; thus, the mucilage droplet would form a region of high concentration of organic acids where Al is captured before reaching the root surface. However, a protective role of the mucilage against Al injury is difficult to reconcile with the lack of evidence that mucilage excretion affected by Al. On the contrary, disappearance of mucilage is one of the first visible symptoms of Al toxicity (Puthota et al., 1991). The role of mucilage in the protection of roots against Al toxicity depends on the amount of mucilage excreted and how strongly Al bounds to mucilage. Mucilage from maize roots is strongly bound to Al but failed to prevent Al-induced inhibition of root elongation (Li et al., 2000a). Approximately 50% of the total Al of the root apices was located in the mucilage of cowpea, while only 9–22% of Al in maize root was bound to mucilage. The binding is decreased by the lower content of uronic acids (3%) in maize muci-

Aluminum Stress

lage as compared to 11.5% in cow pea mucilage (Li et al., 2000a). It will be necessary to determine the Albinding sugar component in mucilage as well as total amount of mucilage and kinetic data of synthesis and excretion of mucilage in order to understand the role of mucilage in Al resistance. F.

pH in the Rhizosphere

Solubility of Al depends strongly on pH. Thus, maintenance of a high solution pH may reduce the solubility and toxicity of Al. An increase in the pH of dilute nutrient solution from 4.5 to 4.6 caused a 26% decline in soluble Al concentration (Blamey et al., 1983). This suggests that even a slight pH change can affect the toxicity of Al as well as tolerance. However, measurement of pH should be done carefully because the change of pH near the root surface is important. For this purpose, a vibrating microelectrode was used to measure pH at a radial distance of 20 and 50
m from the surface of the root tip of a wild-type and of an Al-tolerant Arabidopsis (Degenhardt et al., 1998). The Al-tolerant Arabidopsis mutant alr-104 showed a clear increase of pH at the rhizosphere in the presence of Al, but the wild type did not. Al exposure of alr-104 induced a twofold increase in net Hþ influx localized at the root tip. The increased flux raised the root surface pH of alr-104 by 0.15 unit, suggesting that Al resistance in alr-104 is mediated by pH change in the rhizosphere. Difference in Al resistance between wild type and alr-104 disappeared when roots were grown in pHbuffered medium. It is interesting that no difference in root Hþ fluxes between wild type and alr-104 was detected in the absence of Al.

IV.

BENEFICIAL EFFECT OF ALUMINUM ON PLANT GROWTH

Plants that contain > 1000 ppm Al are called Al accumulators. Among the 259 plant families, 37 Al accumulator species were found and most of them are arborescent cryptogams (Chenery and Sporne, 1976). Al accumulators can grow in acid soils, and growth of some of them is even promoted. Tea (Camellia sinensis) may contain as much as 30,000 ppm Al in old leaves but only 600 ppm in young leaves. The specific locations of Al in epidermis of old leaves and in the cell lumen of bean and barley root was demonstrated by the staining of Al with aluminon and by x-ray (EMX) microanalysis (Waisel et al., 1970; Matsumoto et al., 1976b). The secondary cell wall of epidermal cells of

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old leaves is thicker and Al may have accumulated in the cell wall during thickening. The formation of new roots was greatly accelerated after 1 month of Al treatment and thereafter the growth of tops was positively affected (Matsumoto et al., 1976b). Tea is a sensitive plant for phosphate nutrition and markedly inhibited by the excess of phosphate. Konishi et al. (1985) showed that maximum growth of tea occurred with coexistence of Al with phosphate. The reduced growth of tea at 0.8 mM phosphate was dramatically stimulated by the presence of 1.6 mM Al with new root formation. Al plays a regulatory role in the effective absorption and utilization of phosphate. Another strategy of tea plants against Al toxicity is binding of most of Al to catechin, phenolics, and organic acids (Nagata et al., 1992). The mechanism of Al tolerance is generally carried out by internal detoxification or excretion of chelators, but the mechanism of beneficial effect of Al on the growth has not been clearly demonstrated.

V.

CONCLUDING REMARKS

Inhibition of root elongation caused by Al toxicity is one of the most deleterious factors for plant growth in acid soils. Al3þ concentrations as low as 1
mol at pH 4.5–5.0 inhibit root elongation within 1 h. Absorbed Al is localized at the root apex, where it inhibits cell functions. It is therefore important to know the effect of Al on the processes of cell elongation and cell division at the root apex. There are several unsolved problems underlying the mechanism of Al toxicity. The receptor of the Al signal on root cell membrane and how the signal is transmitted remain unknown. Does the signal work only in the apoplast? If so, there must be a signal transduction system through the plasma membrane into the symplast. Structural proteins like tubulin and actin are candidates for participation in that system. Another possibility is that Al itself is active in the symplast. In this case, we must know the mechanism of Al transport through the plasma membrane. The role of organic acids, both intracellular and extracellular, in the mechanism of Al tolerance has been clarified markedly during the last decade. Al-tolerant plants accumulate less Al than sensitive ones, and formation of chelaters reduces Al toxicity. The major organic acids are citric, malic, and oxalic acids. Why do different plant species excrete different organic acids by the same Al signal? Is there any other Al-chelating compounds of plant origin other than organic acids? Is

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the amount of excreted organic acids sufficient for Al chelation in the rhizosphere in acid soil? (See also Chapter 36 by Neumann and Ro¨mheld in this volume.) Much progress has been made in the molecular aspects of Al binding to oxalic acid and excretion mechanism of malic acid. However, an important problem still to be solved is the regulatory mechanism of the synthesis and excretion of organic acids upon the Al signal. The knowledge of the cell responses to the short-term effects of Al is expected to help us to understand the whole-plant responses and would lead to the improvement of crop production under long-term effects of Al. ACKNOWLEDGMENT The author wishes to thank Mrs. S. Rikiishi for her careful preparation of the manuscript. REFERENCES Ahn SJ, Sivaguru M, Osawa H, Chung GC, Matsumoto H. 2001. Aluminum inhibits the Hþ -ATPase activity by permanently altering the plasma membrane surface potentials in squash roots. Plant Physiol (in press). Akeson MA, Munns DN, Burau RG. 1989. Adsorption of Al3þ to phosphatidylcholine vesicles. Biochim Biophys Acta 986:33–40. Alva AK, Edwords DG, Asher CJ, Blamey FP. 1986. Relationships between root length of soybean and calculated activities of aluminum monomers in nutrient solutions. Soil Sci Soc Am J 50:959–962. Aniol AM. 1995. Physiological aspects of aluminium tolerance associated with the long arm of chromosome 2D of the wheat (Triticum aestivum L.) genome. Theor Appl Genet 91:510–516. Baligar VC, Beaver WV, Ahlrichs JL. 1998. Nature and distribution of acid soils in the world. In: Schaffert RE, ed. Proceedings of a Workshop to Develop a Strategy for Collaborative Research and Dissemination of Technology in Sustainable Crop Production in Acid Savannas and other Problem Soils of the World. Purdue University, pp 1–12. Basu U, Good AG, Aung T, Slaski JJ, Basu A, Briggs KG, Taylor GJ. 1999. A 23-kDa, root exudates polypeptide co-segregates with aluminum resistance in Triticum aestivum. Physiol Plant 106:53–61. Bennet RJ, Breen CM. 1991. The aluminium signal: New dimensions to mechanisms of aluminium tolerance. In: Wright RJ, Baligar VC, Murrmann RP eds. Plant–Soil Interactions at Low pH. Dordrecht, Netherlands: Kluwer Academic Publishers, pp 703– 716.

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