Genes for Plant Abiotic Stress

Genes for Plant Abiotic Stress

Genes for Plant Abiotic Stress Editors Matthew A. Jenks and Andrew J. Wood © 2010 Blackwell Publishing ISBN: 978-0-813-81502-2

Genes for Plant Abiotic Stress

Editors MATTHEW A. JENKS Professor Horticulture and Landscape Architecture Center for Plant Environmental Stress Physiology Purdue University

ANDREW J. WOOD Professor Stress Physiology and Molecular Biology Department of Plant Biology Southern Illinois University

A John Wiley & Sons, Inc., Publication

Edition first published 2010 © 2010 Blackwell Publishing Blackwell Publishing was acquired by John Wiley & Sons in February 2007. Blackwell’s publishing program has been merged with Wiley’s global Scientific, Technical, and Medical business to form Wiley-Blackwell. Editorial Office 2121 State Avenue, Ames, Iowa 50014-8300, USA For details of our global editorial offices, for customer services, and for information about how to apply for permission to reuse the copyright material in this book, please see our website at www.wiley.com/wiley-blackwell. Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Blackwell Publishing, provided that the base fee is paid directly to the Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license by CCC, a separate system of payments has been arranged. The fee codes for users of the Transactional Reporting Service are ISBN-13: 978-0-8138-1502-2/2010. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. Library of Congress Cataloging-in-Publication Data Genes for plant abiotic stress / editors, Matthew A. Jenks, Andrew J. Wood. p. cm. Includes bibliographical references and index. ISBN 978-0-8138-1502-2 (hardback : alk. paper) 1. Crops–Effect of stress on. 2. Crop improvement. 3. Crops and climate. 4. Crops–Physiology. 5. Crops–Development. I. Jenks, Matthew A. II. Wood, Andrew J. SB112.5G46 2009 632′.1–dc22 2009031844 A catalog record for this book is available from the U.S. Library of Congress. Set in 10.5 on 12 pt Times by SNP Best-set Typesetter Ltd., Hong Kong Printed in the Singapore 1

2010

Contents

Contributors Preface Section 1 Chapter 1

Chapter 2

ix xiii

Genetic Determinants of Plant Adaptation under Water Stress Genetic Determinants of Stomatal Function SONG LI and SARAH M. ASSMANN

3 5

Introduction Arabidopsis as a Model System How Do Stomates Sense Drought Stress? Signaling Events inside Guard Cells in Response to Drought Cell Signaling Mutants with Altered Stomatal Responses Transcriptional Regulation in Stomatal Drought Response Summary References

5 7 7 11 15 22 24 25

Pathways and Genetic Determinants for Cell Wall–based Osmotic Stress Tolerance in the Arabidopsis thaliana Root System HISASHI KOIWA

35

Introduction Genes That Affect the Cell Wall and Plant Stress Tolerance Genes and Proteins in Cellulose Biosynthesis Pathways Involved in N-glycosylation and N-glycan Modifications Dolichol Biosynthesis Sugar-nucleotide Biosynthesis Assembly of Core Oligosaccharide Oligosaccharyltransferase Processing of Core Oligosaccharides in the ER Unfolded Protein Response and Osmotic Stress Signaling N-glycan Re-glycosylation and ER-associated Protein Degradation N-glycan Modification in the Golgi Apparatus Ascorbate as an Interface between the N-glycosylation Pathway and Oxidative Stress Response Biosynthesis of GPI Anchor Microtubules

35 35 36 38 38 39 40 40 42 42 44 44 46 46 47 v

vi

Chapter 3

CONTENTS

Conclusion References

48 49

Transcription and Signaling Factors in the Drought Response Regulatory Network MATTHEW GEISLER

55

Introduction Drought Stress Perception Systems Biology Approaches Transcriptomic Studies of Drought Stress The DREB/CBF Regulon ABA Signaling Reactive Oxygen Signaling Integration of Stress Regulatory Networks Assembling the Known Pathways and Expanding Using Gene Expression Networks’ Predicted Protein Interactions Acknowledgments References

Section 2 Chapter 4

Chapter 5

Genes for Crop Adaptation to Poor Soil Genetic Determinants of Salinity Tolerance in Crop Plants DARREN PLETT, BETTINA BERGER, and MARK TESTER

55 55 56 63 66 71 72 72 74 75 75

81 83

Introduction Salinity Tolerance Conclusion References

83 85 100 100

Unraveling the Mechanisms Underlying Aluminum-dependent Root Growth Inhibition PAUL B. LARSEN

113

Introduction Mechanisms of Aluminum Toxicity Aluminum Resistance Mechanisms Aluminum Tolerance Mechanisms Arabidopsis as a Model System for Aluminum Resistance, Tolerance, and Toxicity Aluminum-sensitive Arabidopsis Mutants The Role of ALS3 in A1 Tolerance ALS1 Encodes a Half-type ABC Transporter Required for Aluminum Tolerance Other Arabidopsis Factors Required for Aluminum Resistance/Tolerance Identification of Aluminum-tolerant Mutants in Arabidopsis The Nature of the alt1 Mutations Conclusions References

113 114 117 120 121 121 122 126 128 129 132 138 138

CONTENTS

Chapter 6

vii

Genetic Determinants of Phosphate Use Efficiency in Crops FULGENCIO ALATORRE-COBOS, DAMAR LÓPEZ-ARREDONDO, and LUIS HERRERA-ESTRELLA

143

Introduction Why Improve Crop Nutrition and the Relationship with World Food Security? Phosphorus and Crops: Phosphorus as an Essential Nutrient and Its Supply as a Key Component to Crop Yield Phosphorus and Plant Metabolism: Regulatory and Structural Functions Phosphate Starvation: Adaptations to Phosphate Starvation and Current Knowledge about Phosphate Sensing and Signaling Networks during Phosphate Stress Nutrient Use Efficiency Genetic Determinants for the Phosphate Acquisition Genetic Determinants for Pi Acquisition by Modulating Root System Architecture Genetic Determinants Involved with Phosphorus Utilization Efficiency Genetic Engineering to Improve the Phosphate Use Efficiency Conclusions References

143 143 144 145

146 150 150 153 155 156 158 158

Color Plate Section Chapter 7

Section 3 Chapter 8

Genes for Use in Improving Nitrate Use Efficiency in Crops DAVID A. LIGHTFOOT

167

Introduction The Two Forms of NUE: Regulation of Nitrogen Partitioning and Yield in Crops Mutants as Tools to Isolate Important Plant Genes Transcript Analysis Metanomic Tools for Extending Functional Genomics Transgenics Lacking A Priori Evidence for NUE Microbial Activity Nodule Effects and Mycorrhizal Effects Water Effects Conclusions References

167

Genes for Plant Tolerance to Temperature Extremes

169 169 174 174 175 176 178 178 178 179 183

Genes and Gene Regulation for Low-temperature Tolerance MANTAS SURVILA, PEKKA HEINO, and E. TAPIO PALVA

185

Introduction Protective Mechanisms Induced during Cold Acclimation Regulation of Gene Expression Cross Talk between Abiotic and Biotic Stress Responses

185 188 192 207

viii

Chapter 9

Section 4 Chapter 10

Chapter 11

Index

CONTENTS

Conclusions and Future Perspectives Acknowledgments References

207 209 209

Genetic Approaches toward Improving Heat Tolerance in Plants MAMATHA HANUMAPPA and HENRY T. NGUYEN

221

Introduction Thermotolerance High Temperature Impact and Plant Response to Heat Stress Mechanism of Heat Tolerance in Plants Genetic Approaches to Improve Heat Tolerance in Crops The Effect of Stress Combination Evolving Techniques Conclusion and Perspectives References

221 221 223 230 235 244 246 247 247

Integrating Plant Abiotic Stress Responses

261

Genetic Networks Underlying Plant Abiotic Stress Responses ARJUN KRISHNAN, MADANA M.R. AMBAVARAM, AMAL HARB, UTLWANG BATLANG, PETER E. WITTICH, and ANDY PEREIRA

263

Introduction Plant Responses to Environmental Stresses Transcriptome Analysis of Abiotic Stress Responses Gene Network of Universal Abiotic Stress Response Conclusions References

263 264 270 274 276 276

Discovering Genes for Abiotic Stress Tolerance in Crop Plants MICHAEL POPELKA, MITCHELL TUINSTRA, and CLIFFORD F. WEIL

281

Introduction Salt Stress Heat Stress Oxidative Stress Nutrient/Mineral Stress Plant Architecture and Morphology Evolutionary Conservation and Gene Discovery Conclusion References

281 286 287 288 289 290 291 292 292 303

Contributors

Fulgencio Alatorre-Cobos

Laboratorio Nacional de Genómica para la Biodiversidad, Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional Campus Guanajuato 36821 Irapuato, Guanajuato, México [email protected]

Madana M.R. Ambavaram Virginia Bioinformatics Institute Virginia Tech Washington Street Blacksburg, VA 24061 USA Sarah M. Assmann

Biology Department Penn State University 208 Mueller Laboratory University Park, PA 16802 USA

Utlwang Batlang

Virginia Bioinformatics Institute Virginia Tech Washington Street Blacksburg, VA 24061 USA

Bettina Berger

Australian Centre for Plant Functional Genomics and University of Adelaide, PMB1 Glen Osmond, SA Australia, 5064

Matthew Geisler

Southern Illinois University Department of Plant Biology 403 Life Science II Building 1125 Lincoln Drive Carbondale, IL 62901 USA ix

x

CONTRIBUTORS

Mamatha Hanumappa

Division of Plant Sciences and National Center for Soybean Biotechnology University of Missouri Columbia, MO 65211 USA

Amal Harb

Virginia Bioinformatics Institute Virginia Tech Washington Street Blacksburg, VA 24061 USA

Pekka Heino

Department of Biological and Environmental Sciences Division of Genetics University of Helsinki P.O. Box 56, FIN-00014 Helsinki Finland

Luis Herrera-Estrella

Laboratorio Nacional de Genómica para la Biodiversidad, Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional Campus Guanajuato 36821 Irapuato Guanajuato, México [email protected]

Hisashi Koiwa

Department of Horticultural Sciences 2133 Texas A&M University College Station, TX 77843-2133 USA

Arjun Krishnan

Virginia Bioinformatics Institute Virginia Tech Washington Street Blacksburg, VA 24061 USA

Paul B. Larsen

Department of Biochemistry University of California Riverside, CA 92521 USA 951-827-2026 [email protected]

CONTRIBUTORS

Song Li

Biology Department Penn State University 208 Mueller Laboratory University Park, PA 16802 USA

David A. Lightfoot

Department of Plant Soil and Agricultural Systems Southern Illinois University Carbondale, IL 62901 USA

Damar López-Arredondo

Laboratorio Nacional de Genómica para la Biodiversidad, Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional Campus Guanajuato 36821 Irapuato Guanajuato, México [email protected]

Henry T. Nguyen

Division of Plant Sciences and National Center for Soybean Biotechnology University of Missouri Columbia, MO 65211 USA

E. Tapio Palva

Department of Biological and Environmental Sciences Division of Genetics, University of Helsinki P.O. Box 56, FIN-00014 Helsinki Finland

Andy Pereira

Virginia Bioinformatics Institute Virginia Tech Washington Street Blacksburg, VA 24061 USA [email protected], 540-231-3784

Darren Plett

Australian Centre for Plant Functional Genomics and University of Adelaide, PMB1 Glen Osmond, SA Australia, 5064

xi

xii

CONTRIBUTORS

Michael Popelka

Agronomy Department Purdue University 915 West State Street West Lafayette, IN 47907 USA

Mantas Survila

Department of Biological and Environmental Sciences Division of Genetics, University of Helsinki P.O. Box 56, FIN-00014 Helsinki Finland

Mark Tester

Australian Centre for Plant Functional Genomics and University of Adelaide, PMB1 Glen Osmond, SA Australia, 5064

Mitchell Tuinstra

Agronomy Department Purdue University 915 West State Street West Lafayette, IN 47907 USA

Clifford F. Weil

Agronomy Department Purdue University 915 West State Street West Lafayette, IN 47907 USA

Peter E. Wittich

Virginia Bioinformatics Institute Virginia Tech Washington Street Blacksburg, VA 24061 USA

Preface

Conventional crop-breeding strategies have made limited progress in enhancing harvest indices in regions commonly beset by abiotic environmental stresses, such as those caused by drought, salinity, toxic metals, cold, heat, and nutrient-limiting soils. Worldwide, crop yield losses to abiotic stress range from minor to total, with actual losses being influenced by the timing, intensity, and duration of the stress. A major constraint to improving yield under abiotic stress is our limited understanding of the diverse genes and their alleles that underlie stress tolerance, as well as the difficulties faced by breeders and biotechnologists seeking to combine favorable alleles to create the desired stress-adapted high-yielding genotypes. Moreover, crop domestication has narrowed the genetic diversity for stress adaptation available within crops, and thus, limited options for traditional crop breeding. Consequently, a better understanding of gene function in plant-stress adaptation and the means to use these genes to enhance crop performance are needed if we are to realize the full potential of our efforts in crop improvement. Recent studies of gene function have revealed highly complex and surprisingly integrated genetic and metabolic networks for plant response to abiotic stress. These findings are revealing a new paradigm for effective crop improvement, one that adapts a systems-based approach that closely integrates new discoveries in fundamental biology with newly developed methods in plant breeding and biotechnology. This book integrates a broad cross-section of scientific knowledge and expertise around the key genetic determinants of plant abiotic stress adaptation, with gene function discussed in a way that bridges the physiological, biochemical, developmental, and molecular levels, and gives special consideration to the importance of signaling networks. New and creative approaches for manipulating these determinants for germplasm improvement are also discussed. Information presented in this book will be especially useful to agronomists and horticulturists, crop breeders, biotechnologists, and molecular geneticists, and serve as an important scholarly text for researchers, educators, and post-graduate students.

Matthew A. Jenks and Andrew J. Wood

xiii

Genes for Plant Abiotic Stress

Genes for Plant Abiotic Stress Editors Matthew A. Jenks and Andrew J. Wood © 2010 Blackwell Publishing ISBN: 978-0-813-81502-2

Section 1 Genetic Determinants of Plant Adaptation under Water Stress

Genes for Plant Abiotic Stress Editors Matthew A. Jenks and Andrew J. Wood © 2010 Blackwell Publishing ISBN: 978-0-813-81502-2

1

Genetic Determinants of Stomatal Function Song Li and Sarah M. Assmann

Introduction

Water is the major constituent of land plants, comprising 80–90% of the fresh weight of most herbaceous plants (Kramer and Boyer, 1995). As a small polar molecule with a high dielectric constant, water is a good general purpose solvent for many organic and inorganic ions. Since most mineral nutrients, photosynthetic products, and other biomolecules are charged molecules, water serves as the nutrient carrier and also the solvent for most biochemical reactions. Water has high heat content and high heat of vaporization, which also makes water an ideal substance for temperature regulation such as cooling through transpiration. Water is of particular importance to land plants in two other aspects. First, turgor pressure exerted against cell walls helps plants maintain their form and facilitates cell expansion and growth. Second, water is an essential reactant in photosynthesis, the principal mechanism of biomass accumulation for plants (Kramer and Boyer, 1995). Because water molecules are highly integrated into plant physiology, water-limiting conditions, normally defined as drought, are some of the major abiotic stresses that limit crop productivity (Bhatnagar-Mathur et al., 2008). In natural environments, drought stress is often accompanied by and complicated by a number of other abiotic stresses, such as high temperatures, cold or salinity. High temperatures increase the driving force for transpiration while cold and soil salinity limit soil water availability to roots. To prevent water loss, land plants have evolved vapor-resistant cuticles on their aerial surfaces and use stomatal apertures to regulate transpirational water loss and CO2 uptake (Jenks and Hasegawa, 2005; Kunst and Samuels, 2003). A stomatal complex consists of a pair of guard cells surrounding a microscopic pore called a stoma or stomate; in some species, stomatal complexes also include neighboring subsidiary cells (Willmer and Fricker, 1996). When ample water is present in the environment, stomatal pores can open in response to light to facilitate CO2 uptake and photosynthesis. When water is limited, stomata close to prevent further water loss and to maintain leaf water potential. Stomatal pores are regulated by swelling or shrinking of guard cells through changing cellular water content, which in turn is driven by changes in cellular concentrations of osmotically active solutes such as K+, Cl− and malate2− (MacRobbie, 1998). Guard cells respond to many different drought signals, including the best-characterized drought hormone, abscisic acid (ABA). Molecular details of drought and ABA response in stomata are better characterized in the model species Arabidopsis as compared to drought responses in other tissues and organs in Arabidopsis or in other species (Nilson and Assmann, 2007). This chapter describes recent advances in our understanding of genetic regulators of stomatal drought responses, with a focus on molecular mediators identified in Arabidopsis (see Table 1.1). We will also discuss recently identified genetic regulators of stomatal development in Arabidopsis. Other aspects of plant drought responses are reviewed elsewhere in this book and are not the focus of this chapter. Genes for Plant Abiotic Stress Editors Matthew A. Jenks and Andrew J. Wood © 2010 Blackwell Publishing ISBN: 978-0-813-81502-2

5

6

At5g67300 Human Gene At4g09570 At1g35670 At4g34000 At3g19290 At3g55530 At4g26070 At1g04120 At1g30270 At2g13540 At1g48270 At4g17615 At5g47100 At5g40280 At1g12110

MYB44 PI5P CPK4 CPK11 ABF3 ABF4 SDIR1 MEK1 AtMRP5 CIPK23 ABH1 GCR1 CBL1/CBL9 1 2

OE OE OE OE RN RN RN OE T-DNA Knockout T-DNA Knockout T-DNA Knockout T-DNA Knockout T-DNA Knockout

Mutant type

NT: Not tested in published results RN: Reduced number of open stomata WT: wild-type response OE: Overexpression 1. Fast neutron-generated mutant 2. γ-irradiation generated mutant 3. Better survival rate without rewatering

ERA1 AtCHL1

AGI locus identifier

Gene Name

Smaller Smaller

Smaller Smaller NT NT NT WT

Smaller WT NT NT

Hypersensitive WT

Hypersensitive Hypersensitive NT Hypersensitive NT NT NT WT Hyposensitive Hypersensitive Hypersensitive Hypersensitive Hypersensitive NT WT

NT Hyposensitive NT Hyposensitive NT NT NT NT NT Hypersensitive NT Hypersensitive NT

Inhibition of opening

NT Less

Less Less Less Less Less Less Less NT Less Less NT Less Less

Excised leaf water loss

Promotion of closure

Light

ABA

Leaf phenotype

Stomatal phenotype

Less Less

NT Less Less Less NT NT NT NT Less NT Less Less Less

Wilting

Whole plant phenotype

NT NT

Better NT Better Better Better Better Better Better 3 NT Better NT Better NT

Rewater survival rate

Table 1.1 Mutants with both stomatal phenotypes and increased drought tolerance. Among all genes mentioned in this chapter, not all have published results from whole plant drought response experiments. This table lists all published mutants for which both increased whole plant drought tolerance phenotypes and altered stomatal phenotypes have been reported. Among these 16 mutants, 8 are 35S driven overexpression mutants, 4 are T-DNA single gene knockouts, and 1 is a T-DNA double mutant. In response to light, 5 mutants have intrinsically smaller stomatal apertures, and 3 mutants have a smaller fraction of open stomata as compared to wild type. Nine mutants exhibit hypersensitivity in stomatal ABA responses. One mutant (MRP5) is insensitive for ABAinduced stomatal closure. All mutants tested for excised leaf water loss showed reduced water loss.

GENETIC DETERMINANTS OF STOMATAL FUNCTION

7

Arabidopsis as a Model System

Plant reactions to drought stress have been studied in many experimental species including crop species, trees, desiccation-resurrection plants, and many plant model species. In recent years, the model species Arabidopsis has become the most widely used system to dissect the genetic basis of plant drought tolerance (Nilson and Assmann, 2007). Arabidopsis has the advantage of a short life cycle and small stature. Also, Arabidopsis is easy to transform as compared to many other experimental and crop species. These features have allowed the creation and maintenance of large collections of mutants (Alonso et al., 2003), which are valuable tools for genetic analyses of stomatal drought response. The genome of Arabidopsis has been fully sequenced and is the best annotated plant genome to date (Arabidopsis Genome Initiative, 2000; Swarbreck et al., 2008). This unparalleled richness of genomic information underlies many research technologies. Because stomata can only be observed under a microscope, forward-genetic screens based on stomatal physiology traits have proven somewhat difficult. In fact, many genetic regulators of stomatal functions were first identified by forward genetic screens on more easily scored traits, such as ABA sensitivity in seed germination, rather than by scoring stomatal ABA responses. In reverse genetic approaches, gene functions are first predicted by sequence homology to known genes and then tested by phenotyping plants harboring mutations in the target gene. Alternatively, many cell physiological processes involved in stomatal drought responses were first characterized in species with large stomata by chemical inhibitor analyzes, and then the regulatory genes were identified in Arabidopsis by reverse genetic approaches (Negi et al., 2008; Vahisalu et al., 2008; Wang et al., 2001; Kwak et al., 2002). Further, the well-annotated Arabidopsis genome provides information regarding gene family sizes and sequence similarities between gene family members; hence double mutants can be generated based on the evolutionary relationships between family members to evaluate functional redundancy (Mori et al., 2006). A fully sequenced genome also helps in the application of microarray (Leonhardt et al., 2004) and newly developed sequencing-by-synthesis technologies (Cokus et al., 2008). Because naturally occurring Arabidopsis variants have different degrees of drought tolerance (Hausmann et al., 2005), both microarray and sequencing technologies provide opportunities to find novel genes and polymorphisms, which are correlated with drought tolerance traits (Nordborg and Weigel, 2008; Bouchabke et al., 2008). The rich information generated by novel technologies has already been used to identify new molecular mediators of drought tolerance and to correlate drought tolerance phenotypes with novel genetic loci at the whole plant level (Ossowski et al., 2008; Seki et al., 2002).

How Do Stomates Sense Drought Stress?

Drought causes decreases in soil water availability and, in some geographic regions, reduced air relative humidity, both of which are known to reduce stomatal conductance in multiple crop species (Kramer and Boyer, 1995). Given their location in aerial surfaces of plants, stomata cannot directly sense water-limiting conditions in the soil. Instead, stomata close in response to chemical signals such as ABA. ABA is known to be synthesized in roots and transported in xylem sap to leaves when roots sense drought in the soil (Davies and Zhang, 1991). Ample evidence suggests that stomata can also directly respond to the changes in leaf water status, and such responses may correlate with the accumulation of ABA (Comstock, 2002). In addition, stomatal humidity sensing is also connected to the ABA pathway by several recently published reports (Xie et al., 2006; Lake

8

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

Humidity

Drought

Cytokinins MeJA

ABA

Ethylene

Stomatal Movement

Stomatal Development

Figure 1.1 Stomatal drought response is mediated by plant hormones. Solid lines indicate regulation of stomatal opening and closure. Dashed lines indicate regulation of stomatal development. See text for details.

and Woodward, 2008). Because of the importance of ABA as a drought signal, a number of genetic regulators of ABA biosynthesis have been identified, and they will be discussed in this section. Cytokinins, ethylene, and jasmonates are known to be drought signals that act on guard cells, although none of these hormones has been found to override the dominant role of ABA in stomatal drought response (Lake et al., 2002; Acharya and Assmann, 2008). Molecular components mediating stomatal response to other hormones and interactions between ABA and other hormones during drought will also be discussed (see Figure 1.1).

ABA as a Drought Hormone

ABA is a 15 carbon (C15) molecule derived from the cis-xanthophyll (C40) carotenoid precursor zeaxanthin. The Arabidopsis gene ABA1 is a zeaxanthin epoxidase, which catalyzes the conversion

GENETIC DETERMINANTS OF STOMATAL FUNCTION

9

of zeaxanthin to violaxanthin (Xiong et al., 2002; Nambara and Marion-Poll, 2005). Both drought and ABA induce ABA1 gene expression. Violaxanthin is converted to neoxanthin and then 9′-cisneoxanthin and 9′-cis-violaxanthin, which are both cis-xanthophylls (C40). ABA4 was identified by map-based cloning as a positive regulator of neoxanthin synthase (North et al., 2007). Unlike ABA1, ABA4 is not induced by drought, but knockout of either of these two genes eliminates dehydration-induced ABA accumulation (North et al., 2007). Consistent with these results, both aba1 and aba4 mutants showed lower leaf temperatures than wild type after three days of drought stress (North et al., 2007). The ABA specific metabolic pathway starts with the cleavage of cis-xanthophylls into C15 xanthoxin and a C25 by-product catalyzed by 9-cis-epoxycarotenoid dioxygenases (NCED) (Nambara and Marion-Poll, 2005). In Arabidopsis, tissue specificity of ABA biosynthesis emerges at this step, since a family of nine NCED genes has been identified in the genome (Iuchi et al., 2001). Among these nine genes, five NCEDs have been studied by promoter-GUS assay (Tan et al., 2003). AtNCED2 is expressed in guard cells from senescing leaves, while AtNCED3 is expressed in guard cells in cotyledons, hypocotyls, and petioles. Both AtNCED2 and AtNCED3 are expressed in roots, implying that both of these genes play roles in root ABA biosynthetic processes. Surprisingly, none of the five AtNCED genes was found by reporter gene analysis to be expressed in guard cells from rosette leaves or cauline leaves. ABA2 and AAO3 are enzymes downstream of AtNCEDs and catalyze the transformation of xanthoxin to abscisic aldehyde and abscisic aldehyde to abscisic acid, respectively (Cheng et al., 2002b; Koiwai et al., 2004). AAO3 activity also requires another gene, ABA3, which encodes a MoCo sulfurase; and the homolog of this gene, flacca, was one of the first ABA biosynthetic genes found by genetic approaches in tomato, where its mutation results in a wilty phenotype (Sagi et al., 2002). Among all ABA biosynthesis genes in Arabidopsis, only NCED3 was strongly induced by drought in leaf vascular tissue (Iuchi et al., 2001; Endo et al., 2008). The expression of AAO3 was detected in guard cells and significantly induced in guard cells upon drought treatment (Koiwai et al., 2004), consistent with a previous report that guard cells can themselves synthesize ABA (Cornish and Zeevaart, 1986). ABA catabolism includes both conjugation and oxidation into inactive forms. The predominant pathway of ABA oxidation is thought to be 8′-hydroxylation. This process is catalyzed by a family of four CYP707A genes, which can be induced by seedling dehydration (Kushiro et al., 2004). CYP707A1 and CYP707A3, but not CYP707A2, were shown to be induced in guard cells and vascular tissue, respectively, by high humidity. A cyp707a1 cyp707a3 double mutant showed stronger defects in stomatal opening responses to high humidity than either cyp707a1 or cyp707a3 single mutants, suggesting that these two genes have synergistic functions (Okamoto et al., 2008). Because high air humidity could be the first sign of drought release due to precipitation, these results highlight the role of ABA oxidation in drought release responses of guard cells. ABA remobilization from conjugated forms seems to be involved in stomatal regulation. In Arabidopsis, ABA was shown to be released from a biologically inactivated, glucose conjugated form by a β-glucosidase, BG1 (Lee et al., 2006). Dehydration, and even moderate changes in air humidity, can rapidly activate BG1 by inducing BG1 protein polymerization. Under darkness, T-DNA insertional mutant bg1 showed defective stomatal closure, which could be rescued by exogenous ABA (Lee et al., 2006). The bg1 mutant loses more water in detached rosettes and is more sensitive to drought stress than wild type (Lee et al., 2006). Further experiments on the temporal expression patterns of genes involved in ABA biosynthesis and catabolism, their responses to different levels of drought stresses, their response to drought

10

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

release, and effects of ABA metabolism on stomatal movement will provide valuable insight regarding the interconnection of ABA metabolic pathways and stomatal drought responses.

Other Hormones in Stomatal Drought Responses

Besides ABA, cytokinins are also well-accepted root-to-shoot signaling hormones that participate in plant drought response (Schachtman and Goodger, 2008). In multiple species, drought stresses decrease cytokinin concentrations in xylem sap, while high concentrations of cytokinins antagonize ABA effects on stomatal closure and can induce stomatal opening (Wilkinson and Davies, 2002). In Arabidopsis, three cytokinin-receptor histidine kinases (CRHK) mutants, ahk2, ahk3, and cre1, are hypersensitive to ABA and osmotic stress and thus more tolerant to drought stresses (Tran et al., 2007). These CRHKs also showed functions antagonistic to AHK1, an osmotic-sensing receptor histidine kinase (Tran et al., 2007). Both of these experiments were carried out at the whole plant level; therefore, whether any of these cytokinin receptors directly function in guard cells remains unknown. Cytokinins inhibit ABA-induced stomatal closure in wild type but not in the ethylene insensitive mutant ein3-1. In the cytokinin over-producing mutant, amp1-1, ABA-induced stomatal closure is impaired but can be restored through inhibition of ethylene biosynthesis (Tanaka et al., 2006). Although these results suggest that cytokinins inhibit ABA-induced stomatal closure through ethylene biosynthesis, the possibility that cytokinin receptor kinases are also activated in guard cells cannot be ruled out. The role of ethylene in plant drought response is unclear. In different species, ethylene production differentially depends on the duration and intensity of drought (Acharya and Assmann, 2008). In Arabidopsis, ethylene inhibits ABA-induced stomatal closure (Tanaka et al., 2005), and also mediates an antagonistic effect of cytokinin on ABA-induced stomatal closure as described above (Tanaka et al., 2006). However, ethylene alone was shown to induce stomatal closure in Arabidopsis, and this process is mediated by H2O2 (Desikan et al., 2006). Ethylene receptor histidine kinase ETR1 and signaling proteins EIN2 and ARR2 are involved in ethylene-induced stomatal closure; and AtrbohF, a NADPH oxidase, seems to be an intermediate protein ABA and ethylene cross talk in guard cells (Desikan et al., 2006). Another histidine kinase, AHK5, is downstream of ethylene-induced reactive oxygen species (ROS) production but not ABA-induced stomatal closure (Desikan et al., 2008). In addition to regulating stomatal aperture, ethylene-treated plants or ethylene over-production mutants showed increased stomatal density in Arabidopsis (Serna and Fenoll, 1996). Jasmonic acid and methyl jasmonate (MeJA), collectively called jasmonates (JA), increase rapidly in response to drought (Creelman and Mullet, 1995). Unlike drought-induced ABA production, plant JA content drops after an initial peak, which suggests a transient role of JA in plant drought response (Creelman and Mullet, 1995, 1997; Deng et al., 2008). MeJA can induce stomatal closure in Arabidopsis, but such effects may depend on the plant growth conditions (Zhao et al., 2008). MeJA-induced stomatal closure is mediated by a number of common intracellular components of stomatal ABA responses, but is also mediated by several MeJA-specific components. In terms of common mediators, both MeJA and ABA induce stomatal closure by inducing ROS and nitric oxide (NO) production, which in turn activate membrane Ca2+ permeable channels, anion channels, and outward K+ channels in guard cells (Evans, 2003; Munemasa et al., 2007). Also, the induction of ROS in guard cells by either MeJA or ABA is dependent on protein phosphatase RCN1 and AtrbohD/F (Saito et al., 2008). However, MeJA but not ABA regulation of ROS, NO,

GENETIC DETERMINANTS OF STOMATAL FUNCTION

11

and ion channel activities is impaired in two JA insensitive mutants, jar1 and coi1 (Munemasa et al., 2007), while ABA but not JA-induced ROS and NO production is mediated by protein kinase OST1 (Suhita et al., 2004).

Signaling Events inside Guard Cells in Response to Drought

Drought stress is perceived by guard cells as a composite signal from multiple plant hormones but predominantly from ABA, which activates a wide range of intracellular messengers such as calcium, nitric oxide (NO), reactive oxygen species (ROS) phosphatidic acid (PA), and intracellular pH changes. A number of genetic regulators upstream of these intracellular messengers have been identified in Arabidopsis. In addition, many of these intracellular messengers can modulate membrane permeability through direct or indirect regulation of plasma membrane and vacuolar membrane ion channels, transporters, and pumps. Some of these intracellular messengers, such as calcium and proton concentrations, are themselves regulated by membrane channels and pumps. Membrane channels and transporters mediate the uptake and release of osmotically active solutes such as K+, Cl− and malate2− from guard cell cytosol and vacuoles, resulting in changes of cellular water content and guard cell volume, and eventually leading to changes in stomatal apertures (see Figure 1.2).

Ion Channels and Other Membrane Transport-related Proteins

Light activation of membrane H+ ATPases causes hyperpolarization of the plasma membrane, which is the driving force for ion flow into guard cells. Two dominant mutants ost2-1D and ost22D, which each harbor mutations in the guard cell–expressed H+ ATPase gene, AHA1, were found to have reduced leaf surface temperatures by a thermal imaging based screen. Stomata of ost2 mutants cannot close in response to ABA, due to constitutively active H+ ATPase (Merlot et al., 2007). This result suggests that downregulation of H+ ATPase activity is an important step in stomatal drought response (Goh et al., 1996). KAT1 is a well-characterized inward K+ channel subunit highly enriched in guard cells (Nakamura et al., 1995), which forms functional multi-protein complexes with KAT2 (Pilot et al., 2001) and potentially several other K+ channel proteins in guard cells (Szyroki et al., 2001; Ivashikina et al., 2005). At least five K+ channel proteins are expressed in guard cells (Szyroki et al., 2001), and knockout of KAT1 does not impair stomatal opening. However, plants that overexpress dominant negative alleles of KAT1 or KAT2 have reduced inward K+ current and reduced water loss (Kwak et al., 2001; Lebaudy et al., 2008), suggesting that K+ influx mediated by inward K+ channels is important for light-induced stomatal opening and plant drought response. ABA regulates inward K+ channel activity not only through secondary messengers, but also through vesicle trafficking to and from the cell membrane (Sutter et al., 2007). Two syntaxin proteins have been studied in detail in Arabidopsis. SYP121 was found to be directly involved in KAT1 membrane trafficking (Sutter et al., 2006), while SYP61 is enriched in guard cells, and is important for osmotic stress response (Zhu et al., 2002). In Arabidopsis, GORK is the major outward K+ channel gene expressed in guard cells. T-DNA knockout and dominant negative mutants of GORK showed impaired outwardly rectifying K+ channel activity (Hosy et al., 2003). Gork mutants also have increased water loss in both excised

Signaling

Protein Kinases

Enzymes

Proteins GPCRs

and Phosphatases InsP5-ptase

CIPK

CDPK

CBL

PP2C

PP2A

SnRK2

PTP

MAPK

Myrosinase HT G proteins PLD ROP G Proteins

LCBK SphK Rboh NR

ABA

Drought

LRR-RLKs

PLC

Membrane Transport

Secondary Messengers

TF and RNABinding Proteins

H+ATPase Ca2+in K+in K+out CHX

NRT

Ca2+

InsPs

phytoS1P S1P NO

PA

ROS

PIPs

bZIP bHLH Myb

MRP Anion Channels

isothiocynanates

CBP

Stomatal Drought Response Figure 1.2 Cellular mediators of stomatal drought response. The ABA-mediated drought stimulus acts on signaling proteins and membrane-transport–related proteins. Signaling proteins regulate membrane transport and intracellular enzymes. Although in mammalian systems, membrane-signaling proteins can directly activate intracellular protein kinases, this type of connection has not yet been found in guard cells. Changes in secondary messengers are regulated by enzymes that catalyze the formation of signal molecules or by membrane transport. Secondary messengers regulate many downstream signaling components. Protein kinases and phosphatases are also involved in stomatal drought response. Protein kinases and phosphatases can regulate membrane transporters, which is omitted from this figure for clarity. Transcription factors regulate both drought-induced gene expression and stomatal development. mRNA-binding proteins are involved in stomatal ABA and humidity responses. Ht G proteins: Heterotrimeric G proteins NR: Nitric reductases InsPs: Inositol phosphates CHX: Cation/H+ exchanger NRT: Nitrate transporter PIPs: Phosphatidylinositol phosphates MRP: ATP-binding cassette (ABC) transporters CBP: mRNA cap-binding proteins Rboh: NADPH oxidases

12

GENETIC DETERMINANTS OF STOMATAL FUNCTION

13

leaf and whole plant experiments (Hosy et al., 2003). Besides plasma membrane K+ channels, plants also have ion channels localized in internal membranes, including AtTPK1, which is a two-pore potassium channel mainly located in the tonoplast. Knockouts of AtTPK1 have reduced stomatal closure rate as compared to wild type, which is consistent with AtTPK1-mediated K+ release from vacuole to cytoplasm (Gobert et al., 2007). Besides K+ channels, anion channels are also important for stomatal movement in response to environmental stimuli. SLOW ANION CHANNEL-ASSOCIATED 1 (SLAC1) was recently cloned and proposed to encode a guard cell anion channel (Negi et al., 2008; Vahisalu et al., 2008). Knockouts of SLAC1 showed impaired stomatal responses to a range of environmental signals including ABA and humidity. Although knockouts of SLAC1 showed abolished anion channel activity in planta, heterologous expression of SLAC1 in yeast cannot complement a yeast malate uptake deficient mutant, and expression of SLAC1 in Xenopus oocytes does not result in anion channel activity (Negi et al., 2008; Vahisalu et al., 2008); one explanation is that additional components are required for SLAC1 anion channel activity. ATP-binding cassette proteins have been found to modulate guard cell anion channel activity and thus are potential partners for SLAC1 (Leonhardt et al., 1999). Two ATP-binding cassette (ABC) transporters, AtMRP4 and AtMRP5, have been studied in detail in Arabidopsis. Mutants of AtMRP4 and AtMRP5 have opposite phenotypes. In atmrp4 mutants, stomatal apertures are larger than wild type under both light and dark while stomatal ABA response is similar to wild type; therefore, plants lose more water under drought conditions (Klein et al., 2004). In contrast, atmrp5 mutants showed reduced water loss (Klein et al., 2003). Although ABA-induced stomatal closure is impaired in atmrp5 mutants, lightinduced stomatal opening is also reduced, which could be the main reason for the reduced water loss observed at the whole plant level (Klein et al., 2003). Besides the well-known K+ channels and anion channels, a number of other relevant membrane transporters have also been identified. For example, AtCHX20, a cation/H+ exchanger, was found to be a positive regulator of light-induced stomatal opening (Padmanaban et al., 2007). Knockouts of a nitrate transporter, AtCHL1/NRT1, implicated in NO3− uptake during stomatal opening, have enhanced drought tolerance (Guo et al., 2003). In addition, a PLEIOTROPIC DRUG RESISTANCE 3 transporter, AtPDR3 (Galbiati et al., 2008), and a sucrose transporter, AtSUC3 (Meyer et al., 2004), are both highly expressed in guard cells, although their roles in guard cell physiology remain unknown.

Calcium

ABA induces cytosolic Ca2+ increases through the activation of Ca2+ permeable channels in the plasma membrane and through the activation of Ca2+ release from internal stores. Elevated cytosolic Ca2+ ions can inhibit inward K+ channels and H+ pumps at the plasma membrane while activating anion channels (MacRobbie, 1998). Arabidopsis CYCLIC NUCLEOTIDE GATED CHANNEL2 (CNGC2/DND1) mediates cAMPinduced Ca2+ influxes through plasma membrane channels (Ali et al., 2007). Despite the importance of Ca2+ channels in stomatal ABA response, guard cell phenotypes have not yet been tested under ABA or drought treatment in the dnd1 mutant. Ca2+ release from internal stores may be partially mediated by a tonoplast channel, the two pore channel TPC1 (Furuichi et al., 2001; Peiter et al., 2005; but see Ranf et al., 2008). In addition, a thylakoid-localized calcium sensor (CAS1) may participate in Ca2+-induced Ca2+ release from chloroplasts (Han et al., 2003; Nomura et al., 2008). Both the cas1 mutant and the tpc1 mutant showed impaired extracellular Ca2+-induced stomatal

14

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

closure, but these two mutants did not have impaired stomatal ABA response (Han et al., 2003; Peiter et al., 2005). Little is known about the importance of Ca2+-induced closure under physiological conditions, thus the role of these two proteins in plant drought response is not clear (Ranf et al., 2008; Nomura et al., 2008).

ROS and NO

ABA activates plasma membrane Ca2+ channels by inducing cytosolic H2O2 production by the NADPH oxidases, AtrbohD, and AtrbohF in Arabidopsis guard cells (Kwak et al., 2003; Murata et al., 2001; Pei et al., 2000). AtrbohD/F-mediated ROS production is also involved in stomatal ethylene (Desikan et al., 2006) and jasmonate signaling (Munemasa et al., 2007). Other evidence also suggests that a small GTPase ROP2 and the phosphatidic acid (PA) pathway is involved in ABA-induced ROS production (Park et al., 2004). ROS also inhibit the enzymatic activities of other mediators of stomatal ABA responses, such as plasma membrane H+ ATPase activation (Zhang et al., 2004b), and protein phosphatases ABI1 and ABI2 (Meinhard and Grill, 2001). Further, an Arabidopsis glutathione peroxidase, AtGPX3, interacts with ABI1 and ABI2 and acts as a positive regulator of stomatal ROS responses (Miao et al., 2006). In Arabidopsis, ABA-induced NO production in guard cells is mainly mediated by NIA1 and NIA2 (Desikan et al., 2002), and also requires H2O2 production from the AtrbohD/F pathway (Bright et al., 2006). NO activates anion channels and inhibits inward K+ channels through cADPRand cGMP-dependent intracellular calcium release (Garcia-Mata et al., 2003; Sokolovski et al., 2005), whereas NO inhibition of outward K+ channels may be through direct protein modification (Sokolovski and Blatt, 2004).

Other Small Intracellular Molecules

ABA-induced cytosolic Ca2+ oscillation and stomatal closure are partially mediated by phospholipase C (PLC), which hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP2) into inositol-1,4,5trisphosphate (InsP3) (which induces Ca2+ release) and diacylglycerol (Staxen et al., 1999; Cousson, 2003). Inositol polyphosphate 5-phosphatase (InsP 5-ptase) catalyzes the hydrolysis of InsP3 and terminates the ABA induced InsP3 signaling (Perera et al., 2008). Transgenic plants with overexpression of InsP 5-ptase showed better whole plant drought tolerance and less water loss from excised leaves than wild type. Transgenic plants are hypersensitive to ABA-induced stomatal closure but insensitive to ABA inhibition of stomatal opening (Perera et al., 2008). These results suggest that, under this particular experimental condition, hypersensitivity to ABAinduced stomatal closure is more important for the observed whole plant drought tolerance phenotype. In a functional proteomic study, TGG1 was identified as one of the most abundant proteins in guard cells (Zhao et al., 2008). TGG1 encodes a myrosinase, which cleaves glucosinolates to produce toxic compounds such as isothiocyanates. T-DNA knockouts of tgg1 are hyposensitive to ABA-inhibited stomatal opening, consistent with a lack of ABA inhibition of inward K+ channels in guard cells of tgg1 mutants (Zhao et al., 2008). The glucosinolate-myrosinase system is well known for its function in plant biotic stress response (Barth and Jander, 2006). Because of the dual role of TGG1 in both biotic and abiotic stress responses, TGG1 is a good candidate for engineering pest and drought resistant crop plants.

GENETIC DETERMINANTS OF STOMATAL FUNCTION

15

Cell Signaling Mutants with Altered Stomatal Responses

Stomatal drought response involves not only ion channels and secondary messengers, but also a large number of cell-signaling proteins including protein kinases, protein phosphatases, G-proteins, and farnesyl transferase. Interestingly, many of these signaling proteins are also related to stomatal humidity response, which is also discussed in this section (see Figure 1.2).

Protein Kinases SnRK Protein Kinases Among many different types of protein kinases, members of the SNF1-

related protein kinase (SnRK) family are conserved mediators of ABA and osmotic stress responses in different plant species. Proteins from this family, including Vicia faba AAPK, barley PKABA, soybean SPK1/2, tobacco OSAK, rice SAPK, and those from Arabidopsis (Li et al., 2000; GomezCadenas et al., 2001; Monks et al., 2001; Mustilli et al., 2002; Kelner et al., 2004; Kobayashi et al., 2004) are activated by ABA, osmotic stress or both in various plant tissues. Among these genes, OST1 and AAPK are highly expressed in guard cells and play roles in stomatal function. AAPK was first identified as an ABA-activated protein kinase by in-gel kinase assay followed by mass spectrometry–based protein identification, while its ortholog OST1 was isolated in Arabidopsis by a thermal imaging-aided forward genetic screen. Originally, two mutant alleles of OST1, ost1-1 and ost1-2, were isolated by map-based cloning. Both mutants contain point mutations, and the mutant plants lack stomatal ABA responses but not stomatal light and CO2 responses (Mustilli et al., 2002). OST1 protein is activated by ABA in guard cells and roots in a calcium independent manner. This activation is blocked by the abi1-1 mutation but not by the abi2-1 mutation (Yoshida et al., 2006a). Also, ABA-induced ROS production in guard cells is interrupted by ost1 mutation, while direct application of ROS and calcium can cause stomatal closure in ost1 mutants. This evidence indicates that OST1 is upstream of ROS production and downstream of abi1-1 (Mustilli et al., 2002). Subsequent experiments showed that the ABI1 protein physically interacts with the C-terminal domain II of OST1, which is required for OST1 function in guard cell ABA responses. On the other hand, the OST1 C-terminal domain is not required for ABA response but is required for osmotic activation of OST1 kinase activity (Yoshida et al., 2006a). OST1 and several other SnRK2-like proteins phosphorylate AREB1, a transcription factor that binds to ABA-responsive elements (ABRE). These results support a critical role of the OST1 kinase in guard cell signaling. LRR Receptor Kinases Receptor-like kinases (RLK) are transmembrane kinases that function in

cell-to-cell communication and environmental signal perception in many eukaryotes (Dievart and Clark, 2004). The Arabidopsis genome contains more than 400 RLKs, with 216 Leucine-rich repeat (LRR)–containing receptor kinases representing the largest RLK family. In general, LRR receptor kinases have three domains: one LRR-containing extracellular domain, one intracellular serine/ threonine (ser/thr) protein kinase domain, and one single-pass transmembrane domain (Dievart and Clark, 2004). Arabidopsis RPK1 is an LRR receptor kinase that mediates ABA responses in plant germination, growth, and stomatal responses. The transcript level and protein level of RPK1 are both upregulated by ABA. In two independent mutant alleles of RPK1, ABA-induced stomatal closure was impaired (Osakabe et al., 2005). However, components downstream of RPK1 and the effects of rpk1 mutation on plants under drought conditions remain unknown.

16

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

The Arabidopsis ERECTA (ER) gene is a well-known LRR-receptor kinase that controls plant lateral organ size and flower development by regulating plant cell proliferation (Shpak et al., 2004). By quantitative trait loci (QTL) analysis, the Arabidopsis ERECTA gene was found to be a genetic regulator of transpirational efficiency (TE) (Masle et al., 2005), which is a measure of plant water use efficiency. All three erecta mutants used in the study showed increased stomatal conductance, partly due to the increased number of stomata on the leaf surface. Erecta mutants also have fewer mesophyll cells and reduced photosynthetic capacity as compared to plants with a functional ERECTA gene. Both increased stomatal conductance and decreased photosynthetic capacity negatively affect TE; consequently, erecta mutants have lower TE in both well-watered and drought conditions (Masle et al., 2005). Because the ABA sensitivities of erecta mutants have not yet been tested, to date, evidence suggests that ERECTA affects plant TE through multiple morphological changes. CDPK and CIPK Network Cytosolic Ca2+ is one of the key secondary messengers for multiple

cellular responses in guard cells. Ca2+-dependent protein kinases (CDPK) (Cheng et al., 2002), and calcineurin B–like (CBL) proteins (Cheong et al., 2007) are important intracellular calcium sensors, propagating the cytosolic Ca2+ signal to downstream targets such as membrane ion channels and transcription factors. The Arabidopsis genome encodes 34 CDPKs, each of which contains a kinase domain, an autoinhibitory domain and a calmodulin-like domain (Cheng et al., 2002). The calmodulin-like domain binds Ca2+ when the cytosolic free Ca2+ concentration is elevated, thus causing a conformational change that releases the autoinhibitory domain from the kinase domain and activates the CDPK. Recently, four Arabidopsis CDPKs were found to be related to stomatal functions. CPK3 and CPK6 were first identified by PCR from a guard cell–enriched cDNA library, and their expressions in guard cells were later confirmed by both RT-PCR and microarray analysis (Mori et al., 2006). In both single mutants, cpk3 and cpk6, and the double mutant cpk3cpk6, Ca2+ activation of slowanion channels was impaired. Furthermore, in cpk3cpk6 double mutants, ABA activation of S-type anion channel was strongly inhibited. In guard cells, one way of increasing cytosolic Ca2+ concentration is through activation of plasma membrane Ca2+ permeable-channels, which can be activated by ROS. In the cpk3cpk6 double mutant, ABA but not ROS activation of Ca2+ channels was inhibited. Finally, in both single mutants and the double mutant, ABA-induced stomatal closure was impaired (Mori et al., 2006). The functions of two other CDPKs, CPK4 and CPK11, have been characterized for various ABA responses in different organs and tissues (Zhu et al., 2007). For drought-related functions, the single mutants and double mutant of these two CDPKs are less sensitive to ABA-induced stomatal closure as compared to wild type, while overexpression of either CDPK conveys ABA hypersensitivity. In water-loss experiments, single and double mutants lost more water, while overexpression lines conserved more water than wild-type plants. Furthermore, in drought-rewater experiments, overexpression lines had higher survival rates than wild-type plants whereas all single and double mutants died under the same condition. Two ABA response transcription factors, ABF1 and ABF4 can be phosphorylated by these two CDPKs, which connects these CDPKs’ functions to transcriptional regulation (Zhu et al., 2007). Unlike the CDPKs, which can be directly activated by Ca2+, CBL proteins do not contain kinases domains and cannot directly phosphorylate downstream effectors. Instead, after Ca2+ binding at EF-hand domains, CBL proteins can bind and activate CBL-interacting protein kinases (CIPK). The Arabidopsis genome encodes 10 CBL proteins and 25 CIPKs (Kolukisaoglu et al., 2004), which form an interconnected network, with multiple CBLs interacting with the same

GENETIC DETERMINANTS OF STOMATAL FUNCTION

17

CIPK. T-DNA insertional mutants for all 25 CIPKs in Arabidopsis were screened for their phenotypes under drought conditions (Cheong et al., 2007). Among the CIPK mutants tested, the cipk23 mutant had the best survival rate (70%) while wild-type mutants had only a 20% survival rate under the same experimental condition. A number of drought- or ABA-inducible genes were induced to a similar level in wild-type and cipk23 mutants, and ABA content was not elevated in the cipk23 mutant. However, cipk23 mutant stomata are hypersensitive in both ABA-induced stomatal closure experiments and ABA-inhibited stomatal opening experiments. Earlier publications had shown that the upstream factors of CIPK23 are CBL1 and CBL9 (Kolukisaoglu et al., 2004), both of which can interact with CIPK23 and in turn, activate a K+ channel, AKT1, in roots (Xu et al., 2006). The double mutant, cbl1cbl9, but not cbl1 or cbl9 single mutants, conserved more water in water-loss experiments as compared to wild type (Cheong et al., 2007). Since CIPK23, CBL1, and CBL9 coexpress in guard cells (Cheong et al., 2007), these results suggest that CBL1 and CBL9 function synergistically upstream of CIPK23 in the regulation of stomatal ABA response and plant drought tolerance. In another study, CBL1 (called SCaBP5 in the publication) and CIPK15 (called PKS3) were shown to interact in yeast two hybrid experiments. RNA interference (RNAi) lines of cbl1 or cipk15 were hypersensitive to ABA in stomatal closure, and thus lost less water as compared to wild type (Guo et al., 2002). However, the effect of CBL1 RNAi lines, stronger than knockout lines, could be due to off target effects of RNAi. CIPK15 can interact with an APETALA2/EREBPtype transcription factor, AtERF7, which is a negative regulator in stomatal ABA responses (Song et al., 2005). MAP Kinases Cascade Mitogen-activated protein kinase (MAPK) cascades are conserved signaling modules, consisting of three types of protein kinases: MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). As indicated by their names, MAPKKKs phosphorylate MAPKKs and MAPKKs phosphorylate MAPKs, which propagate signals to downstream effectors. Plant MAPK cascades are known to be involved in multiple biotic and abiotic stress responses, hormonal responses, and plant development (Colcombet and Hirt, 2008). Recently, two members of Arabidopsis MAPK cascades, MPK3 (Gudesblat et al., 2007) and MEK1 (Xing et al., 2007), were found to be related to stomatal ABA responses. Antisense lines of MPK3 were found to be less sensitive to ABA inhibition of stomatal opening under normal conditions, while ABA promotion of stomatal closure was reduced in these lines if cytosolic pH was also clamped. Further experiments showed that stomata in these RNAi lines are less sensitive to H2O2-induced stomatal closure and inhibition of opening. However, ABA-induced H2O2 production was not affected. These results suggest that MPK3 is involved in cytosolic pH regulation and is downstream of H2O2 production (Gudesblat et al., 2007). Unlike MPK3, MEK1 was found to be upstream of H2O2 production in Arabidopsis. A mek1 T-DNA insertional mutant is less sensitive to ABA-induced closure and hypersensitive to drought stress, while overexpression lines of MEK1 are more resistant to drought treatment (Xing et al., 2007). Although MPK3 and MEK1 are related to stomatal ROS response, several other members of MAPK cascades have been found to be related to stomatal development and will be discussed in a later section.

Phosphatases

Plant protein phosphatases catalyze dephosphorylation reactions. Members from three categories of protein phosphatases, protein phosphatases 2C (PP2C), protein phosphatases 2A (PP2A), and

18

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

phosphotyrosine phosphatase (PTP) are involved in stomatal drought response (Farkas et al., 2007; Luan, 2003). PP2C Type 2C protein phosphatases are important modulators in stomatal ABA responses.

So far, four Arabidopsis PP2Cs—ABI1, ABI2, HAB1 and AtPP2CA—have been studied in detail. By forward genetic screen, dominant mutants abi1-1 and abi2-1 were both found to be ABA insensitive in germination assays (Koornneef et al., 1984). These loci were later found to encode two closely related PP2Cs, ABI1 and ABI2, whose phosphatase activities were diminished by point mutations at the same conserved amino acid in abi1-1 and abi2-1 (Leung et al., 1994; Meyer et al., 1994; Bertauche et al., 1996; Leung et al., 1997; Rodriguez et al., 1998a). Although phenotypes of these mutants suggest that both genes are positive regulators for various plant ABA response, experiments using recessive and loss of function mutants of ABI1 and ABI2 indicate that both genes are negative regulators of ABA signaling (Gosti et al., 1999; Merlot et al., 2001). Due to this difference in ABA sensitivities between different types of mutants, one should be cautious about the placement of these two proteins within the ABA signaling network. However, biochemical experiments using wild-type ABI1 and ABI2 showed that both proteins can be modulated by a number of signaling molecules downstream of ABA. For example, ABA causes increased cytosolic ROS, which inhibits the phosphatase activity of ABI1 and ABI2, while ABA also causes a cytosolic pH increase, which increases the phosphatase activity of ABI1 and ABI2 (Leube et al., 1998; Meinhard and Grill, 2001; Meinhard et al., 2002). In yeast, two hybrid experiments, ABI2, and, to a lesser extent, ABI1, bind to protein kinase CIPK15, which is a negative regulator of stomatal ABA response (Guo et al., 2002). Also, an important ABA-induced secondary messenger, phosphatidic acid (PA), interacts with ABI1 and represses ABI1 activity (Zhang et al., 2004). This process is related to ABA and G protein regulation of stomatal aperture and will be discussed in detail in a later section. Genetic analysis of abi1 and abi2 recessive mutants also showed that these two genes have synergistic functions in ABA-induced stomatal closure and hence plant drought response (Merlot et al., 2001). Interestingly, such synergistic interaction was also observed between ABI1 and another Arabidopsis PP2C, HAB1 (Saez et al., 2006). As a highly ABA-induced ABI1/2 homologue (Rodriguez et al., 1998), HAB1 is expressed ubiquitously in all plant tissues and is highly expressed in ABA target tissues, including guard cells, as shown by reporter gene (Saez et al., 2004) and microarray (Leonhardt et al., 2004) analyzes. Although stomata are hypersensitive to ABA promotion of closure in one hab1 knockout line (Leonhardt et al., 2004), another knockout line shows a wild-type response in water loss assays (Saez et al., 2004). Double mutants of both hab1 and one of the abi1-2 or abi1-3 knockout lines showed stronger stomatal ABA response and better drought tolerance than single recessive mutants of either gene or wild type (Saez et al., 2006). Such synergistic interactions between PP2C genes are likely due to their redundant yet not completely overlapping functions in plant ABA and drought response. The last PP2C protein discussed in this chapter is AtPP2CA, which interacts with K+ channels AKT2 and AKT3, and thus may be involved in regulation of plant K+ homeostasis (Vranova et al., 2001; Cherel et al., 2002). Atpp2ca knockout plants are hypersensitive to ABA promotion of stomatal closure, but perform similarly to wild-type plants in water-loss experiments. Overexpression of AtPP2CA causes stomatal ABA insensitivity and increased plant water loss (Kuhn et al., 2006). Interestingly, plants with overexpression of HAB1 also showed ABA insensitivity while knockout lines of the same gene were not significantly different from wild type. These results again suggest redundant functions between PP2C genes. Finally, a mutant with

GENETIC DETERMINANTS OF STOMATAL FUNCTION

19

reduced AtPP2CA phosphatase activity also has increased ABA accumulation in seeds (Yoshida et al., 2006b). Whether mutants of other PP2C genes also affect ABA accumulation is not known. PP2A RCN1 is a guard cell–enriched protein phosphatase 2A that is also expressed in other tissues including mesophyll cells, as revealed by promoter GUS analysis (Kwak et al., 2002). In guard cells, an rcn1 T-DNA knockout impairs two important intermediate ABA responses, cellular ROS increase and intracellular Ca2+ increase; loss of these responses in turn causes impaired anion channel and inward K+ channel responses to ABA. Thus, rcn1 mutant guard cells are less sensitive to ABA-induced stomatal closure (Kwak et al., 2002). MeJA-induced stomatal closure is also impaired in the rcn1 mutant; and MeJA no longer promotes ROS production, which suggests ABA and MeJA cross talk in guard cells is mediated by a RCN1-ROS pathway (Saito et al., 2008). PTP Experiments using a protein tyrosine phosphatase (PTP) inhibitor, phenylarsine oxide, supported a role of PTP in stomatal ABA response (MacRobbie, 2002). In a recent study, a T-DNA insertional mutant, phs1-3, which has reduced expression of tyrosine phosphatase PHS1, was found to have closed stomata under light when the measurement was taken immediately after harvest. When stomata of phs1-3 were opened using an opening medium under light, they showed hypersensitivity in ABA inhibition of light-induced stomatal opening (Quettier et al., 2006). Further investigation using knockout lines of PHS1 may provide more insight into the function of PTP in stomatal ABA response.

G Proteins

Several plant G-proteins, including members of the heterotrimeric G-protein complex and three small GTPases, have been found to mediate stomatal drought response. Heterotrimeric G-proteins are comprised of three subunits, Gα, Gβ, and Gγ, whereas proteins in the small GTPases family are monomeric proteins (Assmann, 2005). Heterotrimeric G Proteins Canonical heterotrimeric G-protein complexes are signaling mediators that transduce extracellular signals perceived by G-protein–coupled receptors (GPCR) to intracellular effectors such as phospholipases and ion channels. The GDP-bound Gα subunit, Gβ subunit, and Gγ subunit form a heterotrimer, which binds to cytosolic regions of the GPCR. Activation of the GPCR causes the exchange of GDP for GTP on the Gα subunit, and also the dissociation of GTP-bound Gα from the Gβγ dimer. Both the Gα-GTP and the Gβγ dimer can activate downstream effectors until the intrinsic GTPase activity of Gα hydrolyzes the GTP into GDP. Then, inactivated Gα recruits Gβγ back into the inactive heterotrimeric complex (Jones and Assmann, 2004; Assmann, 2005). The Arabidopsis genome encodes one canonical Gα subunit, GPA1, one Gβ subunit, AGB1 and two known Gγ subunits, AGG1 and AGG2 (Ma et al., 1990; Weiss et al., 1994; Mason and Botella, 2000). T-DNA insertional mutants of GPA1 and AGB1 are both insensitive to ABA inhibition of stomatal opening, but not ABA induction of stomatal closure (Wang et al., 2001; Fan et al., 2008). These phenotypes are consistent with the electrophysiological evidence that gpa1 and agb1 mutants are both insensitive to ABA inhibition of inward K+ channels and conditionally insensitive to ABA activation of anion channels (i.e., insensitive under strong cytosolic pH buffer). In addition, the gpa1 mutant has wider stomata in normal growth conditions and loses more water as compared to

20

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

wild-type plants. This evidence in total supports a role of GPA1 and AGB1 in stomatal drought response (Wang et al., 2001; Fan et al., 2008). However, the knockout mutants of two Gγ subunits, AGG1 and AGG2, do not show the same stomatal phenotype as gpa1 and agb1 in response to ABA (Trusov et al., 2008). Such an observation may suggest plant heterotrimeric G-proteins function differently from mammalian G proteins or that additional Gγ subunits exist. Sphingosine-1-phosphate (S1P) is one signaling molecule upstream of GPA1-mediated stomatal drought responses (Ng et al., 2001; Coursol et al., 2003; Worrall et al., 2008). Sphingosine kinase (SPHK) catalyzes S1P production, and SPHK activity is enhanced by ABA (Coursol et al., 2003). S1P inhibits stomatal opening and promotes stomatal closure by inhibiting inward K+ channels and activating outward anion channels in wild-type plants, whereas in the gpa1 mutant, S1P cannot regulate ion channels or stomatal apertures (Coursol et al., 2003). In addition to S1P, GCR1 is another upstream component of GPA1-mediated stomatal signaling. GCR1 has sequence homology to one class of mammalian GCPR and was shown to interact with GPA1 both in vitro and in vivo. Leaves from gcr1 mutants lose less water as compared to wild type. Also, gcr1 mutants showed better tolerance to drought treatment and higher survival rate after re-watering. In contrast to the gpa1 mutant, stomata of gcr1 mutant are hypersensitive to both ABA and S1P, which suggests that GCR1 is a negative regulator of G protein signaling in guard cells (Pandey and Assmann, 2004). Recently, several other candidate plant GPCRs have been cloned (Gookin et al., 2008). GGR2 had been proposed as an ABA receptor that interacts with GPA1 (Liu et al., 2007). However, the function of GCR2 is still under debate (Gao et al., 2007; Guo et al., 2008; Illingworth et al., 2008). Besides GCR2, two novel GPCR-Type G proteins, GTG1 and GTC2, have been identified and shown to bind to ABA (Pandey et al. 2009). The double mutant gtg1 / gtg2 is hyposensitive to ABA in assays of ABA-induced stomatal closure, but has a wild type response in ABA inhibition of stomatal opening. GTG1 and GTG2 each can interact with GPA1 in vitro and in vivo, which supports their roles in G protein signaling (Pandey et al., 2009). Phospholipase D (PLD) activity and the resultant product, phosphatidic acid (PA), are also related to GPA1 function in stomatal ABA response. In wild-type plants, ABA promotes PLD activity and PA production, which in turn promote stomatal closure and inhibit stomatal opening. Arabidopsis PLDα1 physically interacts with GPA1 and can stimulate the GTPase activity of GPA1. While GPA1 binding activates PLDα1 activity, adding GTP into the reaction negatively regulates GPA1-PLDα1 binding (Zhao et al., 2004). Stomatal ABA and PA responses were tested in two single mutants, pldα1 and gpa1, and double mutant pldα1gpa1 (Mishra et al., 2006). In these mutants, plants harboring gpa1 mutation are hyposensitive to ABA and PA inhibition of stomatal opening. Plants harboring pldα1 mutation are hyposensitive to ABA-induced stomatal closure, and this insensitivity can be rescued by exogenous PA treatment (Mishra et al., 2006). These results suggest PA is upstream of GPA1 in ABA inhibition of stomatal opening but not in ABA-induced stomatal closure. The observed stomatal phenotype of pldα1 knockout mutants can be partially explained by the interaction between PA and ABI1, because the latter is a negative regulator of stomatal ABA response. PA interacts with ABI1 and reduces the effect of ABI1 by both inhibition of phosphatase activity and by relocation of ABI1 from cytosol to cell membrane (Zhang et al., 2004). ABA induced a similar level of stomatal closure in wild type and abi1 knockout mutant, but not in an abi1 mutant expressing PA insensitive ABI1R73A protein. Also, ABA failed to induce stomatal closure in the pldα1 mutant, but this phenotype was reversed in the pldα1; abi1 double mutant. All the evidence above implies that the ABA-PLD-PA-ABI pathway is important for ABA-induced stomatal closure. However, both abi1 knockout and ABI1R73A mutants showed wild-type ABA response in opening experiments, suggesting that ABI1 is not related to the ABA inhibition of opening pathway (Mishra et al., 2006).

GENETIC DETERMINANTS OF STOMATAL FUNCTION

21

Small GTPase Two small GTPases, AtRAC1 (ROP6) and ROP10, have been found to be negative regulators of stomatal ABA responses. As a guard cell–enriched ROP GTPase, AtRAC1 mediates stomatal ABA responses through regulation of the guard cell actin cytoskeleton. In transgenic plants expressing dominant-positive (i.e., constitutively active) AtRAC1 (DP-RAC1), ABA-induced actin disruption as well as ABA-induced stomatal closure were impaired. These impaired phenotypes were also observed in the abi1-1 mutant, in which AtRAC1 cannot be activated by ABA. Furthermore, dominant negative AtRAC1 (DN-RAC1) transgenic plants showed stomatal closure even without ABA treatment, both in the wild-type background, and in an abi1-1 mutant background (Lemichez et al., 2001). However, as discussed above, current evidence is not sufficient to infer the relationship between AtRAC1 and ABI1 in the wild-type signaling network. Compared to AtRAC1, enrichment of ROP10 in guard cells is less pronounced, based on promoter GUS analysis. A null mutant of ROP10 is hypersensitive to ABA-induced stomatal closure and is more drought resistant than wild type. ROP10 protein localization at the guard cell membrane is weakly disrupted in an ABA-hypersensitive mutant, era1-2 (Zheng et al., 2002). Another small GTPase, ROP2, is related to the stomatal light response. Plants transformed with constitutively active ROP2 protein have large stomatal apertures under darkness and smaller stomatal apertures under light than wild type, while stomata of a dominant negative ROP2 mutant as well as an rop2 knockout mutant showed faster and larger opening under light. Further, thermal imaging showed that constitutively activated ROP2 (CA-ROP2) plants have higher leaf temperatures due to smaller stomatal apertures (Jeon et al., 2008). These results support a role of ROP2 in light responses of stomata; however, earlier results also suggest that ROP2 is involved in PA-induced ROS production in guard cells (Park et al., 2004). Because PA and ROS are positive regulators of ABA-induced stomatal closure, it can be postulated that ROP2 is also involved in stomatal ABA responses.

Farnesyl Transferase

ERA1 is the β-subunit of Arabidopsis farnesyl transferase (Cutler et al., 1996). In yeast and mammalian systems, farnesyl transferase catalyzes lipid modifications of signal transduction proteins to target them to the plasma membrane. Arabidopsis era1 mutants are hypersensitive to ABA-induced stomatal closure, partly due to reduced ABA activation of Ca2+ channels and anion channels in era1-2 guard cells (Pei et al., 1998; Allen et al., 2002). The era1-2 mutant has a reduced wilting phenotype under drought conditions but also has a reduced growth compared to wild type. The effect on crop drought tolerance of changing farnesyl transferase expression was tested in Brassica napus (Wang et al., 2005). The drought-inducible promoter used in this study provides reversible and conditional expression of an antisense ERA1 construct, which overcomes the pleiotropic effects of constitutive silencing of ERA1. Seed yield was increased in the transgenic plants under drought conditions, but was not affected under normal growth conditions (Wang et al., 2005). The conditional knockout strategy can be applied to crops using other regulators of stomatal ABA response to improve crop drought tolerance.

Genes Related to Humidity Sensing

Genetic regulators of plant humidity responses have begun to emerge in recent years. Early experiments showed that two Arabidopsis MAP kinases, AtMPK4 and AtMPK6, were not responsive at

22

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

the transcript level, but were activated by humidity and osmotic stresses (Ichimura et al., 2000). Later, a protein kinase SnRK2e (Yoshida et al., 2002), subsequently called OST1 (Xie et al., 2006), was not only activated by ABA but also by low humidity in plants. By a reverse genetic approach, a T-DNA insertional mutant of SnRK2e was isolated and young mutant plants showed a wilting phenotype under rapid low humidity treatment. In the same study, it was found that several other protein kinases were activated by low humidity or ABA. The identities of these other proteins remain elusive (Yoshida et al., 2002). OST1 was also identified, as was ABA2, from a forward genetic screen for humidity response mutants, using thermal imaging of leaf surface temperature on an EMS-mutagenized population (Xie et al., 2006). Another ABA catabolism gene, CYP707A1, is also induced at the transcript level by high humidity in guard cells in Arabidopsis (Okamoto et al., 2008). An mRNA cap binding protein mutant, abh1, also exhibits an altered response to humidity (Hugouvieux et al., 2002). Under low humidity growth conditions, abh1 mutants have smaller stomatal apertures as compared to wild-type plants, while subsequent electrophysiology experiments correlated that phenotype to enhanced slow anion channel activity and reduced inward K+ channel activity. The abh1 mutant is also hypersensitive to ABA-inhibited seed germination and has strong whole plant phenotypes (Hugouvieux et al., 2001). The Arabidopsis anion channel mutant, slac1, also has altered humidity responses (Vahisalu et al., 2008). Another experiment showed that LCBK, a sphingoid long-chain base (LCB) kinase, is slightly activated by humidity (Imai and Nishiura, 2005). As discussed above, sphingosine signaling has been found to be involved in stomatal ABA response (Coursol et al., 2003). All of these experiments imply a connection between ABA signaling and humidity responses; however, none of these results can rule out the possibility of a humidity specific pathway that is independent from ABA signaling (Assmann et al., 2000). Finally, a novel hat1 mutant was found to survive low humidity for 6 days while wild-type plants died in 24 hours. Hat1 maps to a 168kb region in chromosome 5, which contains 21 genes (Yan et al., 2006). Cloning of HAT1 may provide new insight into Arabidopsis humidity responses.

Transcriptional Regulation in Stomatal Drought Response

In addition to inducing fast and reversible responses such as stomatal closure, drought stress also causes dynamic changes in gene-expression patterns, which are considered to be slower than most of the above discussed signaling processes. Using microarray and sequencing technologies, thousands of gene-expression changes were found in response to drought or ABA at the whole plant level (Seki et al., 2002; Hoth et al., 2002) and in guard cells (Leonhardt et al., 2004). A protein phosphatase 2C gene, AtPP2C-HA, was identified based on the results from the guard cell microarray (Leonhardt et al., 2004). In this section, several transcriptional regulators involved in stomatal drought response will be discussed. Because drought and other environmental factors can also modulate stomatal size and distributions in developing leaves, recent advances in the stomatal development and patterning will also be discussed in this section.

Transcription Factors in Stomatal Drought Response

ABF2 (Kim et al., 2004), ABF3, and ABF4 (Kang et al., 2002) are basic leucine zipper (bZIP) proteins that can bind to ABREs of promoters. All three genes have strong expression in guard cells, suggesting functions in regulation of stomatal gene expression. Transgenic plants with overexpression of any of these three genes showed reduced water loss from excised leaves and increased

GENETIC DETERMINANTS OF STOMATAL FUNCTION

23

drought tolerance, which is likely due to reduced stomatal opening and changes in expression of genes such as ABI1, ABI2, KAT1, and KAT2 (Kang et al., 2002; Kim et al., 2004). A RING finger E3 ligase, SDIR1, is a putative upstream regulator of ABF3 and ABF4, because overexpression of ABF3 or ABF4 can partially rescue the ABA insensitive phenotype of an sdir1 knockout mutant (Zhang et al., 2007). SDIR1 is expressed in all tissues tested and significantly induced by drought in guard cells. Plants with SDIR1 overexpression are more tolerant to drought while sdir1 knockout plants are more sensitive to drought as compared to wild type. Under normal watering, SDIR1 overexpression lines also have smaller stomatal apertures and are hypersensitive to ABA-induced stomatal closure, while knockout lines have larger stomata and are less sensitive to ABA compared to wild type and sdir1 knockout lines (Zhang et al., 2007). Three R2R3-Myb transcription factors, AtMYB44, AtMYB60, and AtMYB61, are also related to stomatal function. All three Myb transcription factors are highly expressed in guard cells as shown by promoter reporter gene assays. However, each of these three genes acts on different aspects of stomatal responses. AtMYB44 overexpression lines have smaller stomatal apertures in normal conditions and faster closure rate under ABA treatment as compared to wild type, while knocking out AtMYB44 does not result in altered phenotypes (Jung et al., 2008). Unlike AtMYB44, AtMYB60 and AtMYB61 are not involved in stomatal ABA responses, but in light regulation of stomatal movements. T-DNA insertional mutant atmyb60 has smaller stomatal openings under light as compared to wild type, while a myb61 mutant showed defects in dark-induced stomatal closures (Newman et al., 2004; Cominelli et al., 2005). Drought stress responses were directly tested for AtMYB60 and AtMYB44. Both the atmyb60 mutant and AtMYB44 overexpression plants showed increased drought tolerance, which is consistent with their roles in stomatal regulation (Newman et al., 2004; Jung et al., 2008). ABO1 is homologous to a subunit in yeast and human elongator complex, which is a multiprotein complex functioning in transcript elongation. abo1-1 mutants lose water more slowly than wild-type plants, and are hypersensitive to ABA-induced stomatal closure. The abo1-1 mutation also affects stomatal development, because almost half of identified guard cell pairs form immature stomata with closed apertures (Chen et al., 2006).

Stomatal Development and Drought Responses

Stomata develop from leaf protodermal cells through multiple steps including stomatal spacing divisions, guard mother cell division, and guard cell maturation. Stomatal spacing determines stomatal distribution and density, while stomatal sizes are determined by guard cell maturation (Bergmann et al., 2004). Sizes and distribution of stomatal complexes on mature leaf surfaces are fixed, restricting acclimation to environmental changes to the changes of stomatal apertures. However, both the sizes and distribution of stomata as determined during leaf maturation determine the capacity of stomatal conductance (Spence et al., 1986). Many environmental signals can modify the density and sizes of stomata during leaf development. Under water-limiting conditions, cotton plants develop smaller and denser stomata in contrast to well-watered conditions (Cutler et al., 1977), and treatment of Tradescantia virginiana plants with ABA causes similar phenotype (Franks and Farquhar, 2001). Both the effects of CO2 and humidity on stomatal development are blocked in an ABA biosynthesis mutant, aba1, implying that both regulatory effects are mediated by ABA (Lake and Woodward, 2008). A number of genetic regulators of stomatal patterning have been discovered in recent years. A subtilisin-like serine protease, SDD1, and a secretory peptide (Berger and Altmann, 2000; Groll et al., 2002), EPIDERMAL PATTERNING FACTOR 1 (EPF1) (Hara et al., 2007), were hypoth-

24

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

esized to be involved in extracellular signaling during stomatal development. Transmembrane receptor-like protein TMM (Geisler et al., 2000), and transmembrane receptor-like kinases, ER, ERL1, and ERL2 (Shpak et al., 2005), are intermediate components between extracellular signals and intracellular signaling pathways of stomatal development. An MAPK cascade, including YODA, MKK4/MKK5, and MPK3/MPK6, acts downstream of membrane receptor kinases to regulate stomatal development (Bergmann et al., 2004; Wang et al., 2007). One member in this cascade, MPK3, is also a positive regulator of stomatal ABA and ROS responses (Gudesblat et al., 2007). Downstream of this MAPK cascade, a subfamily of basic helix-loop-helix (bHLH) transcription factors, SPCH, MUTE and FAMA, are involved in the control of stomatal lineage transitions (Ohashi-Ito and Bergmann, 2006; Pillitteri et al., 2007; MacAlister et al., 2007; Lampard et al., 2008). Another subfamily of bHLH transcription factors, ICE1 and SCRM2, form dimers with SPCH, MUTE, and FAMA, and thus control stomatal initiation, proliferation, and differentiation (Kanaoka et al., 2008). Interestingly, ICE1 was previously found to be important for plant cold tolerance (Chinnusamy et al., 2003). Finally, two genes, FLPS and Myb88, which are paralogous proteins of one subfamily of Myb transcription factors, also affect stomatal development (Lai et al., 2005). Because both ICE1 and MPK3 are also involved in plant response to environmental signals, these two genes are potential candidates for further analysis of how environmental factors modulate stomatal developmental programs (Casson et al., 2008).

Summary

Stomata are microscopic pores on leaf surfaces flanked by pairs of guard cells. Guard cells respond to a range of different environmental stimuli, such as light, humidity, temperature, and CO2 concentration, and also a number of internal stimuli mediated by plant hormones. The main role of stomata in plant drought response is to limit transpirational water loss, through reducing stomatal apertures in response to the stress hormone ABA. In Arabidopsis, many positive and negative regulators of guard cell drought responses have been identified by genetic and transgenic approaches. Mutants in these mediators sometimes showed improved plant drought tolerance, mainly due to increased guard cell ABA sensitivity (see Table 1.1) or increased ABA biosynthesis. However, as a general stress hormone, ABA also has other functions such as inhibiting seed germination and restricting plant growth. These traits, especially ABA inhibition of seed germination, are observed in many mutants that have hypersensitive stomatal ABA responses. ABA hypersensitive traits in organs other than guard cells could be detrimental to crop plants. Mutants with defects in lightinduced stomatal opening could also have better drought tolerance due to their intrinsically smaller stomatal apertures under light. However, the photosynthesis rate and total assimilation, although not measured in most published papers, is likely to be reduced in these mutants, because the reduced apertures can limit CO2 diffusion into the leaves. Therefore, more analyzes of transpiration efficiency are needed to assess the utility of these mutations for crop improvement. Some genes, for example Er family genes, simultaneously modify stomatal development and other aspects of leaf morphology, which both modulate plant water use efficiency. Genetic regulators with similar dual or multiple functions could be potential targets for further experimental analysis and for crop breeding. Arabidopsis heterotrimeric G proteins are good candidates, because G protein mutants, gpa1 and agb1, have both altered stomatal ABA responses, stomatal density changes, and leaf morphology changes. G protein mutants are hypersensitive to ABA inhibition of germination and root growth, but hyposensitive to ABA-inhibited stomatal opening (Pandey et al., 2006). Such opposite functions of G proteins imply that wild-type plants have longer root growth,

GENETIC DETERMINANTS OF STOMATAL FUNCTION

25

better germination rates, and better stomatal response as compared to mutants that lack G protein components. Thus, it can be postulated that G proteins are global positive regulators of plant growth. Despite the enormous progress made in understanding the genetic regulators of stomatal drought response in Arabidopsis, we are far from understanding the complex interactions between these regulators. To apply our knowledge in Arabidopsis in improving crop drought tolerance, one strategy would be generating overexpression or RNAi transgenic plants for all of the known regulators and then testing these plants under identical conditions, which is unfeasible for most other species. The alternative strategy would be to first synthesize our knowledge about the interconnection of these regulators into a network, and then use computational simulation to test the synthesized network until it can reproduce the observed interactions and responses in stomata (Li et al., 2006). We also need to keep in mind that those key regulators discovered in Arabidopsis, may have altered their function or gained functional redundancy in other species. As more fully sequenced plant genomes become available, comparative genomics can also help us to identify conserved genes that are universally involved in stomatal functions. Because drought is a complex stress interweaving with many other stresses, studies of cross talk are also of great importance for practical concerns such as crop breeding. During the last two decades, the Arabidopsis community has accumulated an unprecedented number of wellcharacterized gene-centric mutants involved in both drought response and other aspects of plant physiology. When studying stomatal drought responses, these well-characterized mutants are valuable biological tools for understanding cross talk between cellular processes under drought or between drought and other stresses.

References Acharya, B.R. and Assmann, S.M. (2008) Hormone interactions in stomatal function. Plant Mol Biol 64, 451–462. Ali, R., Ma, W., Lemtiri-Chlieh, F., Tsaltas, D., Leng, Q., von Bodman, S., and Berkowitz, G.A. (2007) Death don’t have no mercy and neither does calcium: Arabidopsis CYCLIC NUCLEOTIDE GATED CHANNEL2 and innate immunity. Plant Cell 19, 1081–1095. Allen, G.J., Murata, Y., Chu, S.P., Nafisi, M., and Schroeder, J.I. (2002) Hypersensitivity of abscisic acid-induced cytosolic calcium increases in the Arabidopsis farnesyltransferase mutant era1-2. Plant Cell 14, 1649–1662. Alonso, J.M., Stepanova, A.N., Leisse, T.J., Kim, C.J., Chen, H., Shinn, P., Stevenson, D.K., Zimmerman, J., Barajas, P., Cheuk, R., Gadrinab, C., Heller, C., Jeske, A., Koesema, E., Meyers, C.C., Parker, H., Prednis, L., Ansari, Y., Choy, N., Deen, H., Geralt, M., Hazari, N., Hom, E., Karnes, M., Mulholland, C., Ndubaku, R., Schmidt, I., Guzman, P., Aguilar-Henonin, L., Schmid, M., Weigel, D., Carter, D.E., Marchand, T., Risseeuw, E., Brogden, D., Zeko, A., Crosby, W.L., Berry, C.C., and Ecker, J.R. (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301, 653–657. Arabidopsis Genome Initiative. (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796–815. Assmann, S.M. (2005) G proteins go green: a plant G protein signaling FAQ sheet. Science 310, 71–73. Assmann, S.M., Snyder, J.A., and Lee, Y.R.J. (2000) ABA-deficient (aba1) and ABA-insensitive (abi1-1, abi2-1) mutants of Arabidopsis have a wild-type stomatal response to humidity. Plant Cell and Environment 23, 387–395. Barth, C. and Jander, G. (2006) Arabidopsis myrosinases TGG1 and TGG2 have redundant function in glucosinolate breakdown and insect defense. Plant J 46, 549–562. Berger, D. and Altmann, T. (2000) A subtilisin-like serine protease involved in the regulation of stomatal density and distribution in Arabidopsis thaliana. Genes Dev 14, 1119–1131. Bergmann, D.C., Lukowitz, W., and Somerville, C.R. (2004) Stomatal development and pattern controlled by a MAPKK kinase. Science 304, 1494–1497. Bertauche, N., Leung, J., and Giraudat, J. (1996) Protein phosphatase activity of abscisic acid insensitive 1 (ABI1) protein from Arabidopsis thaliana. Eur J Biochem 241, 193–200.

26

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

Bhatnagar-Mathur, P., Vadez, V., and Sharma, K.K. (2008) Transgenic approaches for abiotic stress tolerance in plants: retrospect and prospects. Plant Cell Rep 27, 411–424. Bouchabke, O., Chang, F., Simon, M., Voisin, R., Pelletier, G., and Durand-Tardif, M. (2008) Natural variation in Arabidopsis thaliana as a tool for highlighting differential drought responses. PLoS ONE 3, e1705. Bright, J., Desikan, R., Hancock, J.T., Weir, I.S., and Neill, S.J. (2006) ABA-induced NO generation and stomatal closure in Arabidopsis are dependent on H2O2 synthesis. Plant J 45, 113–122. Casson, S. and Gray, J.E. (2008) Influence of environmental factors on stomatal development. New Phytol 178, 9–23. Chen, Z., Zhang, H., Jablonowski, D., Zhou, X., Ren, X., Hong, X., Schaffrath, R., Zhu, J.K., and Gong, Z. (2006) Mutations in ABO1/ELO2, a subunit of holo-Elongator, increase abscisic acid sensitivity and drought tolerance in Arabidopsis thaliana. Mol Cell Biol 26, 6902–6912. Cheng, S.H., Willmann, M.R., Chen, H.C., and Sheen, J. (2002a) Calcium signaling through protein kinases. The Arabidopsis calcium-dependent protein kinase gene family. Plant Physiol 129, 469–485. Cheng, W.H., Endo, A., Zhou, L., Penney, J., Chen, H.C., Arroyo, A., Leon, P., Nambara, E., Asami, T., Seo, M., Koshiba, T., and Sheen, J. (2002b) A unique short-chain dehydrogenase/reductase in Arabidopsis glucose signaling and abscisic acid biosynthesis and functions. Plant Cell 14, 2723–2743. Cheong, Y.H., Pandey, G.K., Grant, J.J., Batistic, O., Li, L., Kim, B.G., Lee, S.C., Kudla, J., and Luan, S. (2007) Two calcineurin B-like calcium sensors, interacting with protein kinase CIPK23, regulate leaf transpiration and root potassium uptake in Arabidopsis. Plant J 52, 223–239. Cherel, I., Michard, E., Platet, N., Mouline, K., Alcon, C., Sentenac, H., and Thibaud, J.B. (2002) Physical and functional interaction of the Arabidopsis K+ channel AKT2 and phosphatase AtPP2CA. Plant Cell 14, 1133–1146. Chinnusamy, V., Ohta, M., Kanrar, S., Lee, B.H., Hong, X., Agarwal, M., and Zhu, J.K. (2003) ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis. Genes Dev 17, 1043–1054. Cokus, S.J., Feng, S., Zhang, X., Chen, Z., Merriman, B., Haudenschild, C.D., Pradhan, S., Nelson, S.F., Pellegrini, M., and Jacobsen, S.E. (2008) Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452, 215–219. Colcombet, J. and Hirt, H. (2008) Arabidopsis MAPKs: a complex signalling network involved in multiple biological processes. Biochem J 413, 217–226. Cominelli, E., Galbiati, M., Vavasseur, A., Conti, L., Sala, T., Vuylsteke, M., Leonhardt, N., Dellaporta, S.L., and Tonelli, C. (2005) A guard-cell-specific MYB transcription factor regulates stomatal movements and plant drought tolerance. Curr Biol 15, 1196–1200. Comstock, J.P. (2002) Hydraulic and chemical signalling in the control of stomatal conductance and transpiration. J Exp Bot 53, 195–200. Cornish, K. and Zeevaart, J.A. (1986) Abscisic Acid Accumulation by in Situ and Isolated Guard Cells of Pisum sativum L. and Vicia faba L. in Relation to Water Stress. Plant Physiol 81, 1017–1021. Coursol, S., Fan, L.M., Le Stunff, H., Spiegel, S., Gilroy, S., and Assmann, S.M. (2003) Sphingolipid signalling in Arabidopsis guard cells involves heterotrimeric G proteins. Nature 423, 651–654. Cousson, A. (2003) Two potential Ca2+-mobilizing processes depend on the abscisic acid concentration and growth temperature in the Arabidopsis stomatal guard cell. J Plant Physiol 160, 493–501. Creelman, R.A. and Mullet, J.E. (1995) Jasmonic acid distribution and action in plants: regulation during development and response to biotic and abiotic stress. Proc Natl Acad Sci USA 92, 4114–4119. Creelman, R.A. and Mullet, J.E. (1997) Biosynthesis and Action of Jasmonates in Plants. Annu Rev Plant Physiol Plant Mol Biol 48, 355–381. Cutler, J.M., Rains, D.W., and Loomis, R.S. (1977) Importance of cell-size in water relations of plants. Physiol Plantarum 40, 255–260. Cutler, S., Ghassemian, M., Bonetta, D., Cooney, S., and McCourt, P. (1996) A protein farnesyl transferase involved in abscisic acid signal transduction in Arabidopsis. Science 273, 1239–1241. Davies, W.J. and Zhang, J.H. (1991) Root Signals and the Regulation of Growth and Development of Plants in Drying Soil. Annu Rev Plant Phys 42, 55–76. Deng, A., Tan, W., He, S., Liu, W., Nan, T., Li, Z., Wang, B., and Li, Q.X. (2008) Monoclonal antibody-based enzyme linked immunosorbent assay for the analysis of jasmonates in plants. J Integr Plant Biol 50, 1046–1052. Desikan, R., Griffiths, R., Hancock, J., and Neill, S. (2002) A new role for an old enzyme: nitrate reductase-mediated nitric oxide generation is required for abscisic acid-induced stomatal closure in Arabidopsis thaliana. Proc Natl Acad Sci USA 99, 16314–16318. Desikan, R., Last, K., Harrett-Williams, R., Tagliavia, C., Harter, K., Hooley, R., Hancock, J.T., and Neill, S.J. (2006) Ethylene-induced stomatal closure in Arabidopsis occurs via AtrbohF-mediated hydrogen peroxide synthesis. Plant J 47, 907–916.

GENETIC DETERMINANTS OF STOMATAL FUNCTION

27

Desikan, R., Horak, J., Chaban, C., Mira-Rodado, V., Witthoft, J., Elgass, K., Grefen, C., Cheung, M.K., Meixner, A.J., Hooley, R., Neill, S.J., Hancock, J.T., and Harter, K. (2008) The histidine kinase AHK5 integrates endogenous and environmental signals in Arabidopsis guard cells. PLoS ONE 3, e2491. Dievart, A. and Clark, S.E. (2004) LRR-containing receptors regulating plant development and defense. Development 131, 251–261. Endo, A., Sawada, Y., Takahashi, H., Okamoto, M., Ikegami, K., Koiwai, H., Seo, M., Toyomasu, T., Mitsuhashi, W., Shinozaki, K., Nakazono, M., Kamiya, Y., Koshiba, T., and Nambara, E. (2008) Drought induction of Arabidopsis 9-cis-epoxycarotenoid dioxygenase occurs in vascular parenchyma cells. Plant Physiol 147, 1984–1993. Evans, N.H. (2003) Modulation of guard cell plasma membrane potassium currents by methyl jasmonate. Plant Physiol 131, 8–11. Fan, L.M., Zhang, W., Chen, J.G., Taylor, J.P., Jones, A.M., and Assmann, S.M. (2008) Abscisic acid regulation of guard-cell K+ and anion channels in Gbeta- and RGS-deficient Arabidopsis lines. Proc Natl Acad Sci USA 105, 8476–8481. Farkas, I., Dombradi, V., Miskei, M., Szabados, L., and Koncz, C. (2007) Arabidopsis PPP family of serine/threonine phosphatases. Trends Plant Sci 12, 169–176. Franks, P.J. and Farquhar, G.D. (2001) The effect of exogenous abscisic acid on stomatal development, stomatal mechanics, and leaf gas exchange in Tradescantia virginiana. Plant Physiol 125, 935–942. Furuichi, T., Cunningham, K.W., and Muto, S. (2001) A putative two pore channel AtTPC1 mediates Ca2+ flux in Arabidopsis leaf cells. Plant Cell Physiol 42, 900–905. Galbiati, M., Simoni, L., Pavesi, G., Cominelli, E., Francia, P., Vavasseur, A., Nelson, T., Bevan, M., and Tonelli, C. (2008) Gene trap lines identify Arabidopsis genes expressed in stomatal guard cells. Plant J 53, 750–762. Gao, Y., Zeng, Q., Guo, J., Cheng, J., Ellis, B.E., and Chen, J.G. (2007) Genetic characterization reveals no role for the reported ABA receptor, GCR2, in ABA control of seed germination and early seedling development in Arabidopsis. Plant J 52, 1001–1013. Garcia-Mata, C., Gay, R., Sokolovski, S., Hills, A., Lamattina, L., and Blatt, M.R. (2003) Nitric oxide regulates K+ and Cl− channels in guard cells through a subset of abscisic acid-evoked signaling pathways. Proc Natl Acad Sci USA 100, 11116–11121. Geisler, M., Nadeau, J., and Sack, F.D. (2000) Oriented asymmetric divisions that generate the stomatal spacing pattern in arabidopsis are disrupted by the too many mouths mutation. Plant Cell 12, 2075–2086. Gobert, A., Isayenkov, S., Voelker, C., Czempinski, K., and Maathuis, F.J. (2007) The two-pore channel TPK1 gene encodes the vacuolar K+ conductance and plays a role in K+ homeostasis. Proc Natl Acad Sci USA 104, 10726–10731. Goh, C.H., Kinoshita, T., Oku, T., and Shimazaki, K. (1996) Inhibition of blue light-dependent H+ pumping by abscisic acid in Vicia guard-cell protoplasts. Plant Physiol 111, 433–440. Gomez-Cadenas, A., Zentella, R., Walker-Simmons, M.K., and Ho, T.H. (2001) Gibberellin/abscisic acid antagonism in barley aleurone cells: site of action of the protein kinase PKABA1 in relation to gibberellin signaling molecules. Plant Cell 13, 667–679. Gookin, T.E., Kim, J., and Assmann, S.M. (2008) Whole proteome identification of plant candidate G-protein coupled receptors in Arabidopsis, rice, and poplar: computational prediction and in-vivo protein coupling. Genome Biol 9, R120. Gosti, F., Beaudoin, N., Serizet, C., Webb, A.A., Vartanian, N., and Giraudat, J. (1999) ABI1 protein phosphatase 2C is a negative regulator of abscisic acid signaling. Plant Cell 11, 1897–1910. Groll, U.V.B., Dieter, B., and Altmann, T. (2002) The subtilisin-like serine protease SDD1 mediates cell-to-cell signaling during Arabidopsis stomatal development. Plant Cell 14, 1527–1539. Gudesblat, G.E., Iusem, N.D., and Morris, P.C. (2007) Guard cell-specific inhibition of Arabidopsis MPK3 expression causes abnormal stomatal responses to abscisic acid and hydrogen peroxide. New Phytol 173, 713–721. Guo, F.Q., Young, J., and Crawford, N.M. (2003) The nitrate transporter AtNRT1.1 (CHL1) functions in stomatal opening and contributes to drought susceptibility in Arabidopsis. Plant Cell 15, 107–117. Guo, J., Zeng, Q., Emami, M., Ellis, B.E., and Chen, J.G. (2008) The GCR2 gene family is not required for ABA control of seed germination and early seedling development in Arabidopsis. PLoS ONE 3, e2982. Guo, Y., Xiong, L., Song, C.P., Gong, D., Halfter, U., and Zhu, J.K. (2002) A calcium sensor and its interacting protein kinase are global regulators of abscisic acid signaling in Arabidopsis. Dev Cell 3, 233–244. Han, S., Tang, R., Anderson, L.K., Woerner, T.E., and Pei, Z.M. (2003) A cell surface receptor mediates extracellular Ca2+ sensing in guard cells. Nature 425, 196–200. Hara, K., Kajita, R., Torii, K.U., Bergmann, D.C., and Kakimoto, T. (2007) The secretory peptide gene EPF1 enforces the stomatal one-cell-spacing rule. Genes Dev 21, 1720–1725. Hausmann, N.J., Juenger, T.E., Sen, S., Stowe, K.A., Dawson, T.E., and Simms, E.L. (2005) Quantitative trait loci affecting delta13C and response to differential water availability in Arabidopsis thaliana. Evolution 59, 81–96.

28

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

Hosy, E., Vavasseur, A., Mouline, K., Dreyer, I., Gaymard, F., Poree, F., Boucherez, J., Lebaudy, A., Bouchez, D., Very, A.A., Simonneau, T., Thibaud, J.B., and Sentenac, H. (2003) The Arabidopsis outward K+ channel GORK is involved in regulation of stomatal movements and plant transpiration. Proc Natl Acad Sci USA 100, 5549–5554. Hoth, S., Morgante, M., Sanchez, J.P., Hanafey, M.K., Tingey, S.V., and Chua, N.H. (2002) Genome-wide gene expression profiling in Arabidopsis thaliana reveals new targets of abscisic acid and largely impaired gene regulation in the abi1-1 mutant. J Cell Sci 115, 4891–4900. Hugouvieux, V., Kwak, J.M., and Schroeder, J.I. (2001) An mRNA cap binding protein, ABH1, modulates early abscisic acid signal transduction in Arabidopsis. Cell 106, 477–487. Hugouvieux, V., Murata, Y., Young, J.J., Kwak, J.M., Mackesy, D.Z., and Schroeder, J.I. (2002) Localization, ion channel regulation, and genetic interactions during abscisic acid signaling of the nuclear mRNA cap-binding protein, ABH1. Plant Physiol 130, 1276–1287. Ichimura, K., Mizoguchi, T., Yoshida, R., Yuasa, T., and Shinozaki, K. (2000) Various abiotic stresses rapidly activate Arabidopsis MAP kinases ATMPK4 and ATMPK6. Plant J 24, 655–665. Illingworth, C.J., Parkes, K.E., Snell, C.R., Mullineaux, P.M., and Reynolds, C.A. (2008) Criteria for confirming sequence periodicity identified by Fourier transform analysis: application to GCR2, a candidate plant GPCR? Biophys Chem 133, 28–35. Imai, H. and Nishiura, H. (2005) Phosphorylation of sphingoid long-chain bases in Arabidopsis: functional characterization and expression of the first sphingoid long-chain base kinase gene in plants. Plant Cell Physiol 46, 375–380. Iuchi, S., Kobayashi, M., Taji, T., Naramoto, M., Seki, M., Kato, T., Tabata, S., Kakubari, Y., Yamaguchi-Shinozaki, K., and Shinozaki, K. (2001) Regulation of drought tolerance by gene manipulation of 9-cis-epoxycarotenoid dioxygenase, a key enzyme in abscisic acid biosynthesis in Arabidopsis. Plant J 27, 325–333. Ivashikina, N., Deeken, R., Fischer, S., Ache, P., and Hedrich, R. (2005) AKT2/3 subunits render guard cell K+ channels Ca2+ sensitive. J Gen Physiol 125, 483–492. Jenks, M.A. and Hasegawa, P.M. (2005) Plant abiotic stress. (Oxford, UK; Ames, Iowa: Blackwell Pub.). Jeon, B.W., Hwang, J.U., Hwang, Y., Song, W.Y., Fu, Y., Gu, Y., Bao, F., Cho, D., Kwak, J.M., Yang, Z., and Lee, Y. (2008) The Arabidopsis small G protein ROP2 is activated by light in guard cells and inhibits light-induced stomatal opening. Plant Cell 20, 75–87. Jones, A.M. and Assmann, S.M. (2004) Plants: the latest model system for G-protein research. EMBO Rep 5, 572–578. Jung, C., Seo, J.S., Han, S.W., Koo, Y.J., Kim, C.H., Song, S.I., Nahm, B.H., Choi, Y.D., and Cheong, J.J. (2008) Overexpression of AtMYB44 enhances stomatal closure to confer abiotic stress tolerance in transgenic Arabidopsis. Plant Physiol 146, 623–635. Kanaoka, M.M., Pillitteri, L.J., Fujii, H., Yoshida, Y., Bogenschutz, N.L., Takabayashi, J., Zhu, J.K., and Torii, K.U. (2008) SCREAM/ICE1 and SCREAM2 specify three cell-state transitional steps leading to Arabidopsis stomatal differentiation. Plant Cell 20, 1775–1785. Kang, J.Y., Choi, H.I., Im, M.Y., and Kim, S.Y. (2002) Arabidopsis basic leucine zipper proteins that mediate stress-responsive abscisic acid signaling. Plant Cell 14, 343–357. Kelner, A.P., Pekala, I., Kaczanowski, S., Muszynska, G., Hardie, D.G., and Dobrowolska, G. (2004) Biochemical characterization of the tobacco 42-kD protein kinase activated by osmotic stress. Plant Physiol 136, 3255–3265. Kim, S., Kang, J.Y., Cho, D.I., Park, J.H., and Kim, S.Y. (2004) ABF2, an ABRE-binding bZIP factor, is an essential component of glucose signaling and its overexpression affects multiple stress tolerance. Plant J 40, 75–87. Klein, M., Geisler, M., Suh, S.J., Kolukisaoglu, H.U., Azevedo, L., Plaza, S., Curtis, M.D., Richter, A., Weder, B., Schulz, B., and Martinoia, E. (2004) Disruption of AtMRP4, a guard cell plasma membrane ABCC-type ABC transporter, leads to deregulation of stomatal opening and increased drought susceptibility. Plant J 39, 219–236. Klein, M., Perfus-Barbeoch, L., Frelet, A., Gaedeke, N., Reinhardt, D., Mueller-Roeber, B., Martinoia, E., and Forestier, C. (2003) The plant multidrug resistance ABC transporter AtMRP5 is involved in guard cell hormonal signalling and water use. Plant J 33, 119–129. Kobayashi, Y.Y., Shuhei, Y., Minami, H., Kagaya, Y., and Hattori, T. (2004) Differential activation of the rice sucrose nonfermenting1-related protein kinase2 family by hyperosmotic stress and abscisic acid. Plant Cell 16, 1163–1177. Koiwai, H., Nakaminami, K., Seo, M., Mitsuhashi, W., Toyomasu, T., and Koshiba, T. (2004) Tissue-specific localization of an abscisic acid biosynthetic enzyme, AAO3, in Arabidopsis. Plant Physiol 134, 1697–1707. Kolukisaoglu, U., Weinl, S., Blazevic, D., Batistic, O., and Kudla, J. (2004) Calcium sensors and their interacting protein kinases: genomics of the Arabidopsis and rice CBL-CIPK signaling networks. Plant Physiol 134, 43–58. Koornneef, M., Reuling, G., and Karssen, C.M. (1984) The isolation and characterization of abscisic-acid insensitive mutants of Arabidopsis thaliana. Physiol Plantarum 61, 377–383. Kramer, P.J. and Boyer, J.S. (1995) Water relations of plants and soils. (San Diego: Academic Press).

GENETIC DETERMINANTS OF STOMATAL FUNCTION

29

Kuhn, J.M., Boisson-Dernier, A., Dizon, M.B., Maktabi, M.H., and Schroeder, J.I. (2006) The protein phosphatase AtPP2CA negatively regulates abscisic acid signal transduction in Arabidopsis, and effects of abh1 on AtPP2CA mRNA. Plant Physiol 140, 127–139. Kunst, L. and Samuels, A.L. (2003) Biosynthesis and secretion of plant cuticular wax. Prog Lipid Res 42, 51–80. Kushiro, T., Okamoto, M., Nakabayashi, K., Yamagishi, K., Kitamura, S., Asami, T., Hirai, N., Koshiba, T., Kamiya, Y., and Nambara, E. (2004) The Arabidopsis cytochrome P450 CYP707A encodes ABA 8′-hydroxylases: key enzymes in ABA catabolism. Embo J 23, 1647–1656. Kwak, J.M., Moon, J.H., Murata, Y., Kuchitsu, K., Leonhardt, N., DeLong, A., and Schroeder, J.I. (2002) Disruption of a guard cell-expressed protein phosphatase 2A regulatory subunit, RCN1, confers abscisic acid insensitivity in Arabidopsis. Plant Cell 14, 2849–2861. Kwak, J.M., Mori, I.C., Pei, Z.M., Leonhardt, N., Torres, M.A., Dangl, J.L., Bloom, R.E., Bodde, S., Jones, J.D., and Schroeder, J.I. (2003) NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis. Embo J 22, 2623–2633. Kwak, J.M., Murata, Y., Baizabal-Aguirre, V.M., Merrill, J., Wang, M., Kemper, A., Hawke, S.D., Tallman, G., and Schroeder, J.I. (2001) Dominant negative guard cell K+ channel mutants reduce inward-rectifying K+ currents and light-induced stomatal opening in arabidopsis. Plant Physiol 127, 473–485. Lai, L.B., Nadeau, J.A., Lucas, J., Lee, E.K., Nakagawa, T., Zhao, L., Geisler, M., and Sack, F.D. (2005) The Arabidopsis R2R3 MYB proteins FOUR LIPS and MYB88 restrict divisions late in the stomatal cell lineage. Plant Cell 17, 2754–2767. Lake, J.A. and Woodward, F.I. (2008) Response of stomatal numbers to CO2 and humidity: control by transpiration rate and abscisic acid. New Phytol 179, 397–404. Lake, J.A., Woodward, F.I., and Quick, W.P. (2002) Long-distance CO2 signalling in plants. J Exp Bot 53, 183–193. Lampard, G.R.M., MacAlister, C.A., and Bergmann, D.C. (2008) Arabidopsis stomatal initiation is controlled by MAPK-mediated regulation of the bHLH SPEECHLESS. Science 322, 1113–1116. Lebaudy, A., Vavasseur, A., Hosy, E., Dreyer, I., Leonhardt, N., Thibaud, J.B., Very, A.A., Simonneau, T., and Sentenac, H. (2008) Plant adaptation to fluctuating environment and biomass production are strongly dependent on guard cell potassium channels. Proc Natl Acad Sci USA 105, 5271–5276. Lee, K.H., Piao, H.L., Kim, H.Y., Choi, S.M., Jiang, F., Hartung, W., Hwang, I., Kwak, J.M., and Lee, I.J. (2006) Activation of glucosidase via stress-induced polymerization rapidly increases active pools of abscisic acid. Cell 126, 1109–1120. Lemichez, E., Wu, Y., Sanchez, J.P., Mettouchi, A., Mathur, J., and Chua, N.H. (2001) Inactivation of AtRac1 by abscisic acid is essential for stomatal closure. Genes Dev 15, 1808–1816. Leonhardt, N., Vavasseur, A., and Forestier, C. (1999) ATP binding cassette modulators control abscisic acid-regulated slow anion channels in guard cells. Plant Cell 11, 1141–1152. Leonhardt, N., Kwak, J.M., Robert, N., Waner, D., Leonhardt, G., and Schroeder, J.I. (2004) Microarray expression analyzes of Arabidopsis guard cells and isolation of a recessive abscisic acid hypersensitive protein phosphatase 2C mutant. Plant Cell 16, 596–615. Leube, M.P., Grill, E., and Amrhein, N. (1998) ABI1 of Arabidopsis is a protein serine/threonine phosphatase highly regulated by the proton and magnesium ion concentration. FEBS Lett 424, 100–104. Leung, J., Merlot, S., and Giraudat, J. (1997) The Arabidopsis ABSCISIC ACID-INSENSITIVE2 (ABI2) and ABI1 genes encode homologous protein phosphatases 2C involved in abscisic acid signal transduction. Plant Cell 9, 759–771. Leung, J., Bouvier-Durand, M., Morris, P.C., Guerrier, D., Chefdor, F., and Giraudat, J. (1994) Arabidopsis ABA response gene ABI1:features of a calcium-modulated protein phosphatase. Science 264, 1448–1452. Li, J.W., Wang, X.Q., Watson, M.B., and Assmann, S.M. (2000) Regulation of abscisic acid-induced stomatal closure and anion channels by guard cell AAPK kinase. Science 287, 300–303. Li, S., Assmann, S.M., and Albert, R. (2006) Predicting essential components of signal transduction networks: a dynamic model of guard cell abscisic acid signaling. PLoS Biol 4, e312. Liu, X., Yue, Y., Li, B., Nie, Y., Li, W., Wu, W.H., and Ma, L. (2007) A G protein-coupled receptor is a plasma membrane receptor for the plant hormone abscisic acid. Science 315, 1712–1716. Luan, S. (2003) Protein phosphatases in plants. Annu Rev Plant Biol 54, 63–92. Ma, H., Yanofsky, M.F., and Meyerowitz, E.M. (1990) Molecular cloning and characterization of GPA1, a G protein alpha subunit gene from Arabidopsis thaliana. Proc Natl Acad Sci USA 87, 3821–3825. MacAlister, C.A., Ohashi-Ito, K., and Bergmann, D.C. (2007) Transcription factor control of asymmetric cell divisions that establish the stomatal lineage. Nature 445, 537–540. MacRobbie, E.A. (1998) Signal transduction and ion channels in guard cells. Philos Trans R Soc Lond B Biol Sci 353, 1475–1488.

30

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

MacRobbie, E.A. (2002) Evidence for a role for protein tyrosine phosphatase in the control of ion release from the guard cell vacuole in stomatal closure. Proc Natl Acad Sci USA 99, 11963–11968. Masle, J., Gilmore, S.R., and Farquhar, G.D. (2005) The ERECTA gene regulates plant transpiration efficiency in Arabidopsis. Nature 436, 866–870. Mason, M.G., and Botella, J.R. (2000) Completing the heterotrimer: isolation and characterization of an Arabidopsis thaliana G protein gamma-subunit cDNA. Proc Natl Acad Sci USA 97, 14784–14788. Meinhard, M. and Grill, E. (2001) Hydrogen peroxide is a regulator of ABI1, a protein phosphatase 2C from Arabidopsis. FEBS Lett 508, 443–446. Meinhard, M., Rodriguez, P.L., and Grill, E. (2002) The sensitivity of ABI2 to hydrogen peroxide links the abscisic acid-response regulator to redox signalling. Planta 214, 775–782. Merlot, S., Gosti, F., Guerrier, D., Vavasseur, A., and Giraudat, J. (2001) The ABI1 and ABI2 protein phosphatases 2C act in a negative feedback regulatory loop of the abscisic acid signalling pathway. Plant J 25, 295–303. Merlot, S., Leonhardt, N., Fenzi, F., Valon, C., Costa, M., Piette, L., Vavasseur, A., Genty, B., Boivin, K., Muller, A., Giraudat, J., and Leung, J. (2007) Constitutive activation of a plasma membrane H+-ATPase prevents abscisic acid-mediated stomatal closure. Embo J 26, 3216–3226. Meyer, K., Leube, M.P., and Grill, E. (1994) A protein phosphatase 2C involved in ABA signal transduction in Arabidopsis thaliana. Science 264, 1452–1455. Meyer, S., Lauterbach, C., Niedermeier, M., Barth, I., Sjolund, R.D., and Sauer, N. (2004) Wounding enhances expression of AtSUC3, a sucrose transporter from Arabidopsis sieve elements and sink tissues. Plant Physiol 134, 684–693. Miao, Y., Lv, D., Wang, P., Wang, X.C., Chen, J., Miao, C., and Song, C.P. (2006) An Arabidopsis glutathione peroxidase functions as both a redox transducer and a scavenger in abscisic acid and drought stress responses. Plant Cell 18, 2749–2766. Mishra, G., Zhang, W., Deng, F., Zhao, J., and Wang, X. (2006) A bifurcating pathway directs abscisic acid effects on stomatal closure and opening in Arabidopsis. Science 312, 264–266. Monks, D.E.A., Aghoram, K., Courtney, P.D., DeWald, D.B., and Dewey, R.E. (2001) Hyperosmotic stress induces the rapid phosphorylation of a soybean phosphatidylinositol transfer protein homolog through activation of the protein kinases SPK1 and SPK2. Plant Cell 13, 1205–1219. Mori, I.C., Murata, Y., Yang, Y., Munemasa, S., Wang, Y.F., Andreoli, S., Tiriac, H., Alonso, J.M., Harper, J.F., Ecker, J.R., Kwak, J.M., and Schroeder, J.I. (2006) CDPKs CPK6 and CPK3 function in ABA regulation of guard cell S-type anion- and Ca2+-permeable channels and stomatal closure. PLoS Biol 4, e327. Munemasa, S., Oda, K., Watanabe-Sugimoto, M., Nakamura, Y., Shimoishi, Y., and Murata, Y. (2007) The coronatine-insensitive 1 mutation reveals the hormonal signaling interaction between abscisic acid and methyl jasmonate in Arabidopsis guard cells. Specific impairment of ion channel activation and second messenger production. Plant Physiol 143, 1398–1407. Murata, Y., Pei, Z.M., Mori, I.C., and Schroeder, J. (2001) Abscisic acid activation of plasma membrane Ca2+ channels in guard cells requires cytosolic NAD(P)H and is differentially disrupted upstream and downstream of reactive oxygen species production in abi1-1 and abi2-1 protein phosphatase 2C mutants. Plant Cell 13, 2513–2523. Mustilli, A.C., Merlot, S., Vavasseur, A., Fenzi, F., and Giraudat, J. (2002) Arabidopsis OST1 protein kinase mediates the regulation of stomatal aperture by abscisic acid and acts upstream of reactive oxygen species production. Plant Cell 14, 3089–3099. Nakamura, R.L., McKendree, W.L., Jr., Hirsch, R.E., Sedbrook, J.C., Gaber, R.F., and Sussman, M.R. (1995) Expression of an Arabidopsis potassium channel gene in guard cells. Plant Physiol 109, 371–374. Nambara, E. and Marion-Poll, A. (2005) Abscisic acid biosynthesis and catabolism. Annu Rev Plant Biol 56, 165–185. Negi, J., Matsuda, O., Nagasawa, T., Oba, Y., Takahashi, H., Kawai-Yamada, M., Uchimiya, H., Hashimoto, M., and Iba, K. (2008) CO2 regulator SLAC1 and its homologues are essential for anion homeostasis in plant cells. Nature 452, 483–486. Newman, L.J., Perazza, D.E., Juda, L., and Campbell, M.M. (2004) Involvement of the R2R3-MYB, AtMYB61, in the ectopic lignification and dark-photomorphogenic components of the det3 mutant phenotype. Plant J 37, 239–250. Ng, C.K., Carr, K., McAinsh, M.R., Powell, B., and Hetherington, A.M. (2001) Drought-induced guard cell signal transduction involves sphingosine-1-phosphate. Nature 410, 596–599. Nilson, S.E. and Assmann, S.M. (2007) The control of transpiration. Insights from Arabidopsis. Plant Physiol 143, 19–27. Nomura, H., Komori, T., Kobori, M., Nakahira, Y., and Shiina, T. (2008) Evidence for chloroplast control of external Ca2+-induced cytosolic Ca2+ transients and stomatal closure. Plant J 53, 988–998. Nordborg, M. and Weigel, D. (2008) Next-generation genetics in plants. Nature 456, 720–723. North, H.M., De Almeida, A., Boutin, J.P., Frey, A., To, A., Botran, L., Sotta, B., and Marion-Poll, A. (2007) The Arabidopsis ABA-deficient mutant aba4 demonstrates that the major route for stress-induced ABA accumulation is via neoxanthin isomers. Plant J 50, 810–824. Ohashi-Ito, K. and Bergmann, D.C. (2006) Arabidopsis FAMA controls the final proliferation/differentiation switch during stomatal development. Plant Cell 18, 2493–2505.

GENETIC DETERMINANTS OF STOMATAL FUNCTION

31

Okamoto, M., Tanaka, Y., Abrams, S.R., Kamiya, Y., Seki, M., and Nambara, E. (2008) High humidity induces ABA 8′hydroxylase in stomata and vasculature to regulate local and systemic ABA responses in Arabidopsis. Plant Physiol 149, 825–834. Osakabe, Y., Maruyama, K., Seki, M., Satou, M., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2005) Leucine-rich repeat receptor-like kinase1 is a key membrane-bound regulator of abscisic acid early signaling in Arabidopsis. Plant Cell 17, 1105–1119. Ossowski, S., Schneeberger, K., Clark, R.M., Lanz, C., Warthmann, N., and Weigel, D. (2008) Sequencing of natural strains of Arabidopsis thaliana with short reads. Genome Res 18, 2024–2033. Padmanaban, S., Chanroj, S., Kwak, J.M., Li, X., Ward, J.M., and Sze, H. (2007) Participation of endomembrane cation/H+ exchanger AtCHX20 in osmoregulation of guard cells. Plant Physiol 144, 82–93. Pandey, S. and Assmann, S.M. (2004) The Arabidopsis putative G protein-coupled receptor GCR1 interacts with the G protein α subunit GPA1 and regulates abscisic acid signaling. Plant Cell 16, 1616–1632. Pandey, S., Nelson, D.C., and Assmann, S.M. (2009) Two novel GPCR-type G proteins are abscisic acid receptors in Arabidopsis. Cell 136, 136–148. Pandey, S., Chen, J.G., Jones, A.M., and Assmann, S.M. (2006) G-protein complex mutants are hypersensitive to abscisic acid regulation of germination and postgermination development. Plant Physiol 141, 243–256. Park, J., Gu, Y., Lee, Y., and Yang, Z. (2004) Phosphatidic acid induces leaf cell death in Arabidopsis by activating the Rho-related small G protein GTPase-mediated pathway of reactive oxygen species generation. Plant Physiol 134, 129–136. Pei, Z.M., Ghassemian, M., Kwak, C.M., McCourt, P., and Schroeder, J.I. (1998) Role of farnesyltransferase in ABA regulation of guard cell anion channels and plant water loss. Science 282, 287–290. Pei, Z.M., Murata, Y., Benning, G., Thomine, S., Klusener, B., Allen, G.J., Grill, E., and Schroeder, J.I. (2000) Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells. Nature 406, 731–734. Peiter, E., Maathuis, F.J., Mills, L.N., Knight, H., Pelloux, J., Hetherington, A.M., and Sanders, D. (2005) The vacuolar Ca2+activated channel TPC1 regulates germination and stomatal movement. Nature 434, 404–408. Perera, I.Y., Hung, C.Y., Moore, C.D., Stevenson-Paulik, J., and Boss, W.F. (2008) Transgenic Arabidopsis plants expressing the type 1 inositol 5-phosphatase exhibit increased drought tolerance and altered abscisic acid signaling. Plant Cell 20, 2876–2893. Pillitteri, L.J., Sloan, D.B., Bogenschutz, N.L., and Torii, K.U. (2007) Termination of asymmetric cell division and differentiation of stomata. Nature 445, 501–505. Pilot, G., Lacombe, B., Gaymard, F., Cherel, I., Boucherez, J., Thibaud, J.B., and Sentenac, H. (2001) Guard cell inward K+ channel activity in arabidopsis involves expression of the twin channel subunits KAT1 and KAT2. J Biol Chem 276, 3215–3221. Quettier, A.L., Bertrand, C., Habricot, Y., Miginiac, E., Agnes, C., Jeannette, E., and Maldiney, R. (2006) The phs1-3 mutation in a putative dual-specificity protein tyrosine phosphatase gene provokes hypersensitive responses to abscisic acid in Arabidopsis thaliana. Plant J 47, 711–719. Ranf, S., Wunnenberg, P., Lee, J., Becker, D., Dunkel, M., Hedrich, R., Scheel, D., and Dietrich, P. (2008) Loss of the vacuolar cation channel, AtTPC1, does not impair Ca2+ signals induced by abiotic and biotic stresses. Plant J 53, 287–299. Rodriguez, P.L., Benning, G., and Grill, E. (1998a) ABI2, a second protein phosphatase 2C involved in abscisic acid signal transduction in Arabidopsis. FEBS Lett 421, 185–190. Rodriguez, P.L., Leube, M.P., and Grill, E. (1998b) Molecular cloning in Arabidopsis thaliana of a new protein phosphatase 2C (PP2C) with homology to ABI1 and ABI2. Plant Mol Biol 38, 879–883. Saez, A., Robert, N., Maktabi, M.H., Schroeder, J.I., Serrano, R., and Rodriguez, P.L. (2006) Enhancement of abscisic acid sensitivity and reduction of water consumption in Arabidopsis by combined inactivation of the protein phosphatases type 2C ABI1 and HAB1. Plant Physiol 141, 1389–1399. Saez, A., Apostolova, N., Gonzalez-Guzman, M., Gonzalez-Garcia, M.P., Nicolas, C., Lorenzo, O., and Rodriguez, P.L. (2004) Gain-of-function and loss-of-function phenotypes of the protein phosphatase 2C HAB1 reveal its role as a negative regulator of abscisic acid signalling. Plant J 37, 354–369. Sagi, M., Scazzocchio, C., and Fluhr, R. (2002) The absence of molybdenum cofactor sulfuration is the primary cause of the flacca phenotype in tomato plants. Plant J 31, 305–317. Saito, N., Munemasa, S., Nakamura, Y., Shimoishi, Y., Mori, I.C., and Murata, Y. (2008) Roles of RCN1, regulatory A subunit of protein phosphatase 2A, in methyl jasmonate signaling and signal crosstalk between methyl jasmonate and abscisic acid. Plant Cell Physiol 49, 1396–1401. Schachtman, D.P. and Goodger, J.Q. (2008) Chemical root to shoot signaling under drought. Trends Plant Sci 13, 281–287. Seki, M., Narusaka, M., Ishida, J., Nanjo, T., Fujita, M., Oono, Y., Kamiya, A., Nakajima, M., Enju, A., Sakurai, T., Satou, M., Akiyama, K., Taji, T., Yamaguchi-Shinozaki, K., Carninci, P., Kawai, J., Hayashizaki, Y., and Shinozaki, K. (2002)

32

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a fulllength cDNA microarray. Plant J 31, 279–292. Serna, L. and Fenoll, C. (1996) Ethylene induces stomata differentiation in Arabidopsis. Int J Dev Biol Suppl 1, 123S–124S. Shpak, E.D., Berthiaume, C.T., Hill, E.J., and Torii, K.U. (2004) Synergistic interaction of three ERECTA-family receptor-like kinases controls Arabidopsis organ growth and flower development by promoting cell proliferation. Development 131, 1491–1501. Shpak, E.D., Messmer, J., Pillitteri, L.J., Torii, K.U. (2005) Stomatal patterning and differentiation by synergistic interactions of receptor kinases. Science 309, 290–293. Sokolovski, S. and Blatt, M.R. (2004) Nitric oxide block of outward-rectifying K+ channels indicates direct control by protein nitrosylation in guard cells. Plant Physiol 136, 4275–4284. Sokolovski, S., Hills, A., Gay, R., Garcia-Mata, C., Lamattina, L., and Blatt, M.R. (2005) Protein phosphorylation is a prerequisite for intracellular Ca2+ release and ion channel control by nitric oxide and abscisic acid in guard cells. Plant J 43, 520–529. Song, C.P., Agarwal, M., Ohta, M., Guo, Y., Halfter, U., Wang, P., and Zhu, J.K. (2005) Role of an Arabidopsis AP2/EREBPtype transcriptional repressor in abscisic acid and drought stress responses. Plant Cell 17, 2384–2396. Spence, R.D., Wu, H., Sharpe, P.J.H., and Clark, K.G. (1986) Water-stress effects on guard-cell anatomy and the mechanical advantage of the epidermal-cells. Plant Cell and Environment 9, 197–202. Staxen, I., Pical, C., Montgomery, L.T., Gray, J.E., Hetherington, A.M., and McAinsh, M.R. (1999) Abscisic acid induces oscillations in guard-cell cytosolic free calcium that involve phosphoinositide-specific phospholipase C. Proc Natl Acad Sci USA 96, 1779–1784. Suhita, D., Raghavendra, A.S., Kwak, J.M., and Vavasseur, A. (2004) Cytoplasmic alkalization precedes reactive oxygen species production during methyl jasmonate- and abscisic acid-induced stomatal closure. Plant Physiol 134, 1536– 1545. Sutter, J.U., Campanoni, P., Tyrrell, M., and Blatt, M.R. (2006) Selective mobility and sensitivity to SNAREs is exhibited by the Arabidopsis KAT1 K+ channel at the plasma membrane. Plant Cell 18, 935–954. Sutter, J.U., Sieben, C., Hartel, A., Eisenach, C., Thiel, G., and Blatt, M.R. (2007) Abscisic acid triggers the endocytosis of the arabidopsis KAT1 K+ channel and its recycling to the plasma membrane. Curr Biol 17, 1396–1402. Swarbreck, D., Wilks, C., Lamesch, P., Berardini, T.Z., Garcia-Hernandez, M., Foerster, H., Li, D., Meyer, T., Muller, R., Ploetz, L., Radenbaugh, A., Singh, S., Swing, V., Tissier, C., Zhang, P., and Huala, E. (2008) The Arabidopsis Information Resource (TAIR): gene structure and function annotation. Nucleic Acids Res 36, D1009–D1014. Szyroki, A., Ivashikina, N., Dietrich, P., Roelfsema, M.R., Ache, P., Reintanz, B., Deeken, R., Godde, M., Felle, H., Steinmeyer, R., Palme, K., and Hedrich, R. (2001) KAT1 is not essential for stomatal opening. Proc Natl Acad Sci USA 98, 2917–2921. Tan, B.C., Joseph, L.M., Deng, W.T., Liu, L., Li, Q.B., Cline, K., and McCarty, D.R. (2003) Molecular characterization of the Arabidopsis 9-cis epoxycarotenoid dioxygenase gene family. Plant J 35, 44–56. Tanaka, Y., Sano, T., Tamaoki, M., Nakajima, N., Kondo, N., and Hasezawa, S. (2005) Ethylene inhibits abscisic acid-induced stomatal closure in Arabidopsis. Plant Physiol 138, 2337–2343. Tanaka, Y., Sano, T., Tamaoki, M., Nakajima, N., Kondo, N., and Hasezawa, S. (2006) Cytokinin and auxin inhibit abscisic acid-induced stomatal closure by enhancing ethylene production in Arabidopsis. J Exp Bot 57, 2259–2266. Tran, L.S., Urao, T., Qin, F., Maruyama, K., Kakimoto, T., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2007) Functional analysis of AHK1/ATHK1 and cytokinin receptor histidine kinases in response to abscisic acid, drought, and salt stress in Arabidopsis. Proc Natl Acad Sci USA 104, 20623–20628. Trusov, Y., Zhang, W., Assmann, S.M., and Botella, J.R. (2008) Gγ1 + Gγ2 not equal to Gβ: heterotrimeric G protein Gγ-deficient mutants do not recapitulate all phenotypes of Gβ-deficient mutants. Plant Physiol 147, 636–649. Vahisalu, T., Kollist, H., Wang, Y.F., Nishimura, N., Chan, W.Y., Valerio, G., Lamminmaki, A., Brosche, M., Moldau, H., Desikan, R., Schroeder, J.I., and Kangasjarvi, J. (2008) SLAC1 is required for plant guard cell S-type anion channel function in stomatal signalling. Nature 452, 487–491. Vranova, E., Tahtiharju, S., Sriprang, R., Willekens, H., Heino, P., Palva, E.T., Inze, D., and Van Camp, W. (2001) The AKT3 potassium channel protein interacts with the AtPP2CA protein phosphatase 2C. J Exp Bot 52, 181–182. Wang, H., Ngwenyama, N., Liu, Y., Walker, J.C., and Zhang, S. (2007) Stomatal development and patterning are regulated by environmentally responsive mitogen-activated protein kinases in Arabidopsis. Plant Cell 19, 63–73. Wang, X.Q., Ullah, H., Jones, A.M., and Assmann, S.M. (2001) G protein regulation of ion channels and abscisic acid signaling in Arabidopsis guard cells. Science 292, 2070–2072. Wang, Y., Ying, J., Kuzma, M., Chalifoux, M., Sample, A., McArthur, C., Uchacz, T., Sarvas, C., Wan, J., Dennis, D.T., McCourt, P., and Huang, Y. (2005) Molecular tailoring of farnesylation for plant drought tolerance and yield protection. Plant J 43, 413–424.

GENETIC DETERMINANTS OF STOMATAL FUNCTION

33

Weiss, C.A., Garnaat, C.W., Mukai, K., Hu, Y., and Ma, H. (1994) Isolation of cDNAs encoding guanine nucleotide-binding protein beta-subunit homologues from maize (ZGB1) and Arabidopsis (AGB1). Proc Natl Acad Sci USA 91, 9554–9558. Wilkinson, S. and Davies, W.J. (2002) ABA-based chemical signalling: the co-ordination of responses to stress in plants. Plant Cell Environ 25, 195–210. Willmer, C.M. and Fricker, M. (1996) Stomata. (London; New York: Chapman & Hall). Worrall, D., Liang, Y.K., Alvarez, S., Holroyd, G.H., Spiegel, S., Panagopulos, M., Gray, J.E., and Hetherington, A.M. (2008) Involvement of sphingosine kinase in plant cell signalling. Plant J 56, 64–72. Xie, X., Wang, Y., Williamson, L., Holroyd, G.H., Tagliavia, C., Murchie, E., Theobald, J., Knight, M.R., Davies, W.J., Leyser, H.M., and Hetherington, A.M. (2006) The identification of genes involved in the stomatal response to reduced atmospheric relative humidity. Curr Biol 16, 882–887. Xing, Y., Jia, W., and Zhang, J. (2007) AtMEK1 mediates stress-induced gene expression of CAT1 catalase by triggering H2O2 production in Arabidopsis. J Exp Bot 58, 2969–2981. Xiong, L., Lee, H., Ishitani, M., and Zhu, J.K. (2002) Regulation of osmotic stress-responsive gene expression by the LOS6/ ABA1 locus in Arabidopsis. J Biol Chem 277, 8588–8596. Xu, J., Li, H.D., Chen, L.Q., Wang, Y., Liu, L.L., He, L., and Wu, W.H. (2006) A protein kinase, interacting with two calcineurin B-like proteins, regulates K+ transporter AKT1 in Arabidopsis. Cell 125, 1347–1360. Yan, C., Shen, H., Li, Q., and He, Z. (2006) A novel ABA-hypersensitive mutant in Arabidopsis defines a genetic locus that confers tolerance to xerothermic stress. Planta 224, 889–899. Yoshida, R., Hobo, T., Ichimura, K., Mizoguchi, T., Takahashi, F., Aronso, J., Ecker, J.R., and Shinozaki, K. (2002) ABAactivated SnRK2 protein kinase is required for dehydration stress signaling in Arabidopsis. Plant Cell Physiol 43, 1473–1483. Yoshida, R., Umezawa, T., Mizoguchi, T., Takahashi, S., Takahashi, F., and Shinozaki, K. (2006a) The regulatory domain of SRK2E/OST1/SnRK2.6 interacts with ABI1 and integrates abscisic acid (ABA) and osmotic stress signals controlling stomatal closure in Arabidopsis. J Biol Chem 281, 5310–5318. Yoshida, T., Nishimura, N., Kitahata, N., Kuromori, T., Ito, T., Asami, T., Shinozaki, K., and Hirayama, T. (2006b) ABAhypersensitive germination3 encodes a protein phosphatase 2C (AtPP2CA) that strongly regulates abscisic acid signaling during germination among Arabidopsis protein phosphatase 2Cs. Plant Physiol 140, 115–126. Zhang, W., Qin, C., Zhao, J., and Wang, X. (2004a) Phospholipase D alpha 1-derived phosphatidic acid interacts with ABI1 phosphatase 2C and regulates abscisic acid signaling. Proc Natl Acad Sci USA 101, 9508–9513. Zhang, X., Wang, H., Takemiya, A., Song, C.P., Kinoshita, T., and Shimazaki, K. (2004b) Inhibition of blue light-dependent H+ pumping by abscisic acid through hydrogen peroxide-induced dephosphorylation of the plasma membrane H+-ATPase in guard cell protoplasts. Plant Physiol 136, 4150–4158. Zhang, Y., Yang, C., Li, Y., Zheng, N., Chen, H., Zhao, Q., Gao, T., Guo, H., and Xie, Q. (2007) SDIR1 is a RING finger E3 ligase that positively regulates stress-responsive abscisic acid signaling in Arabidopsis. Plant Cell 19, 1912–1929. Zhao, J. and Wang, X. (2004) Arabidopsis phospholipase Dalpha1 interacts with the heterotrimeric G-protein alpha-subunit through a motif analogous to the DRY motif in G-protein-coupled receptors. J Biol Chem 279, 1794–1800. Zhao, Z., Zhang, W., Stanley, B.A., and Assmann, S.M. (2008) Functional proteomics of Arabidopsis thaliana guard cells uncovers new stomatal signaling pathways. Plant Cell 20, 3210–3226. Zheng, Z.L., Nafisi, M., Tam, A., Li, H., Crowell, D.N., Chary, S.N., Schroeder, J.I., Shen, J., and Yang, Z. (2002) Plasma membrane-associated ROP10 small GTPase is a specific negative regulator of abscisic acid responses in Arabidopsis. Plant Cell 14, 2787–2797. Zhu, J., Gong, Z., Zhang, C., Song, C.P., Damsz, B., Inan, G., Koiwa, H., Zhu, J.K., Hasegawa, P.M., and Bressan, R.A. (2002) OSM1/SYP61:a syntaxin protein in Arabidopsis controls abscisic acid-mediated and non-abscisic acid-mediated responses to abiotic stress. Plant Cell 14, 3009–3028. Zhu, S.Y., Yu, X.C., Wang, X.J., Zhao, R., Li, Y., Fan, R.C., Shang, Y., Du, S.Y., Wang, X.F., Wu, F.Q., Xu, Y.H., Zhang, X.Y., and Zhang, D.P. (2007) Two calcium-dependent protein kinases, CPK4 and CPK11, regulate abscisic acid signal transduction in Arabidopsis. Plant Cell 19, 3019–3036.

2

Pathways and Genetic Determinants for Cell Wall–Based Osmotic Stress Tolerance in the Arabidopsis thaliana Root System Hisashi Koiwa

Introduction

The growing tips of the plant roots are critical tissues for establishing a root network in the soil. The root tip contains the root apical meristem, where directional cell divisions continuously produce new cells (Benfey and Scheres, 2000). Subsequently, the elongation and differentiation of these cells produce ordered root cell profiles. The developmental program that regulates the plant root architecture has been studied intensively and involves layers of functions of multiple transcription factors (Nakajima and Benfey, 2002; Petricka and Benfey, 2008). When plants are exposed to environmental stress, the root tips are usually the first to encounter the new environment. Therefore, the cell divisions and differentiations at the root tips have greater ability to respond to environmental signals. Primary roots of wild-type Arabidopsis under salt stress reduce the growth rate by reducing the number of dividing cells in the meristems and producing smaller mature cells without changing duration of the cell cycle (West et al., 2004). In contrast, salt-sensitive mutant stt3a undergoes cell cycle arrest under the salt stress (Koiwa et al., 2003), indicating that sustaining cell cycle progression is an integral part of stress tolerance mechanism in plant roots. Furthermore, the developmental program that determines the identity of the cells determines the stress response outputs of individual root cells (Dinneny et al., 2008). Growth and differentiation of plant cells determines production and deposition of cell wall materials. At the same time, cell walls can influence the rate of cell division, elongation, and morphology of the cells. Because of this greater degree of coordination, growth, development, and cell wall biosynthesis of growing plant tissues are prone to environmental perturbations. Tobacco cultured cells exposed to salinity produce cell walls much weaker than those of unstressed cells (Iraki et al., 1989). This is accompanied by a decrease in crystalline cellulose in the primary cell wall and a decrease in hydroxyprolines in cell wall proteins, suggesting a cellulosic-extensin framework of cells are compromised during the salt stress. Few other studies indicate that osmotic stress tolerance of plants is directly affected by cell wall components (Amaya et al., 1999; Shi et al., 2003). Cell wall proteins undergo various post-translational modifications that directly influence the protein functions. This chapter will provide an overview of functions of cell wall proteins and their post-transcriptional modification pathways in stress tolerance. Genes That Affect the Cell Wall and Plant Stress Tolerance

The cell wall is composed from carbohydrate components and protein components. Carbohydrate components include crystalline cellulose microfibrils, hemicelluloses, xyloglucans, and pectin Genes for Plant Abiotic Stress Editors Matthew A. Jenks and Andrew J. Wood © 2010 Blackwell Publishing ISBN: 978-0-813-81502-2

35

36

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

matrix (O’Niell and York, 2003). Biosynthetic enzymes for carbohydrate polymers are often found on plasmamembranes or in the Golgi apparatus. Cell wall proteins such as extensins, glycine-rich proteins, hydroxyproline-rich glycoproteins, and arabinogalactan proteins are thought to be involved in structural frameworks, whereas enzymes such as expansins, xyloglucan endo-transglycosylase modulate the degree of cross-linking in the cell wall to allow cells to expand during growth and development. These functions provide an additional framework that complements the polysaccharide network and reinforces the assembly of wall materials. The first molecular genetic evidence that connects the cell wall and plant stress tolerance was provided when overexpression of cell wall peroxidase in tobacco plants improved the seed germination of transgenic plants under various osmotic stresses (Amaya et al., 1999). The authors proposed that increased linkage in cell walls could alter the pore size of the cell wall and increase the amount of water retained in the seed cell wall. Later, Shi and others reported that cell wall protein SOS5 was important for salt tolerance of Arabidopis (Shi et al., 2003). SOS5 encodes an arabinogalactan protein (AGP), an extensively glycosylated Hyp-rich proteoglycans. AGP family proteins are found in various cellular components including cell walls, plasma membranes, and vesicles (Majewska-Sawka and Nothnagel, 2000). Under salt stress, the sos5 mutant root tip swells and its growth arrests. SOS5 protein contains two arabinogalactan protein domains, two fasciclin-like domains, and C-terminal glycosylphosphatidylinositol anchor sequence. Since fasciclin domains are found in cell adhension proteins, the authors hypothesized that SOS5 is involved in cell-to-cell adhesion. The cell walls of sos5 are thinner and disorganized, implying that the normal cell wall structure is important for sustaining root growth under salt stress. Cellulose is a major component of cell walls. Forward genetic approaches identified a series of genes that are involved in cellulose biosynthesis (Somerville, 2006). Importance of cellulose biosynthesis in the plant stress tolerance was first indicated by the study of Arabidopsis leaf wilting 2 (lew2) mutants, which are enhanced for drought and osmotic stress tolerance (Chen et al., 2005). lew2 plants are tolerant to drought, sodium chloride (NaCl), and other osmotic stresses and accumulate more abscisic acid and compatible solutes that have been proposed to protect cells under osmotic stresses. Surprisingly, LEW2 is identical to the CesA8/IRX1 gene that encodes a subunit of cellulose synthase complex. In this instance, LEW2/CesA8/IRX1 functions in secondary cell wall formation in xylem and the mutant shows collapse of xylem structure (Taylor et al., 2000). Constant wilting phenotype and drought tolerance phenotype of lew2 have been attributed to the impeded water transport through xylem, which imposes constant water stress to the shoot tissue. This triggers biosynthesis of ABA and other stress response in the plants and thus preconditions the plants for more severe drought stress. More recently Kang and others showed enhanced salt sensitivity of rsw1 and rsw2, other cellulose biosynthesis mutants (Kang et al., 2008). These findings indicate that the cellulose biosynthesis pathway is a determinant of plant tolerance to osmotic stress.

Genes and Proteins in Cellulose Biosynthesis

Crystalline cellulose microfibrils are produced by cellulose synthase complex. The complex has been localized on the plasmamembranes of plant cells and has been observed as “rosettes” by the freeze fracture electron microscope technique. The cellulose synthase rosette structure consists of six globular units, each of which in turn consists of six subunits of cellulose synthases. It has been proposed that during cellulose synthesis, each subunit of rosette containing six subunits of cellulose synthase produces six strands of β-1,4-glycans that co-crystallize into a 36-chain microfibril/rosette (Herth, 1983; Somerville, 2006).

PATHWAYS AND GENETIC DETERMINANTS

37

A large number of cellulose synthase (CesA family) and cellulose synthase-like (CslA-CslG families) proteins are present in the Arabidopsis genome (Dhugga, 2001). CesA family proteins are shown to function in cellulose biosynthesis in primary and secondary wall formation. Mutant phenotype analysis, tissue specific expression profiles, and protein-protein interaction assays established that CesA1, 3, and 6 form a complex that function in the primary cell wall formation (Desprez et al., 2007; Persson et al., 2007; Wang et al., 2008). Null mutations in CesA1 and 6 cause lethality, whereas partial redundancy is observed among CesA6, 2, 5, and 9 (Desprez et al., 2007; Persson et al., 2007). Study of secondary wall biosynthesis revealed that CesA4, 7, and 8 are simultaneously required for the secondary wall biosynthesis (Taylor et al., 2000; Taylor et al., 2003). Inhibition of cellulose biosynthesis in the primary cell wall induces phenotypic abnormality similar to phenotype induced by salt stress. Arabidopsis roots treated with isoxaben or dichlorobenzonitrile cause radial swelling of the root tip (Arioli et al., 1998). Several Arabidopsis mutant plants selected based on aberrant swollen root morphology are defective in cellulose biosynthesis. For example, cellulose synthase mutations, namely, rsw1 (CesA1) (Arioli et al., 1998) and quill (Hauser et al., 1995) were identified in this approach. In addition, several other genes, which are not homologous to CesA family proteins, but shown to affect cellulose biosynthesis, were identified as the root morphology mutants. RSW2/LION’S TAIL is allelic to KORRIGAN1, and encodes β-1,4glucanase (Lane et al., 2001). RSW3 encodes α-glucosidase I in the endoplasmic reticulum involved in the processing of N-glycans of glycoproteins (Burn et al., 2002). COBRA encodes a glycosylphosphatidylinositol-anchored plasmamembrane protein (Schindelman et al., 2001; Roudier et al., 2005). KOBITO1/ELONGATION DEFECTIVE1/ABA INSENSITIVE 8 encodes a plasma membrane/cell wall protein of unknown function (Pagant et al., 2002; Lertpiriyapong and Sung, 2003; Brocard-Gifford et al., 2004). Mutations in these genes all resulted in decreased cellulose content in the mutant cell walls (Peng et al., 2000; Schindelman et al., 2001; Pagant et al., 2002). Similarity between root morphologies induced by salt stress and by cellulose deficiency prompted Kang and others (2008) to analyze their functional relationship. This study led to identification of RSW2/LIT/KOR1 as a functional link between cellulose biosynthesis, osmotic stress tolerance, and protein N-glycosylation (Kang et al., 2008). RSW2 belongs to the family of class II membrane proteins and contains a luminal catalytic domain with eight potential N-glycosylation sites (Nicol et al., 1998). N-glycosylation of Brassica RSW2 homolog is essential for its catalytic activity in vitro (Molhoj et al., 2001), and combining mutations in N-glycosylation or modification pathways with conditional rsw2-1 mutation induced strong constitutive growth defects in the double mutants (Kang et al., 2008). Further supporting evidence for this linkage became available with analysis of cobra mutants. COBRA protein has two kinds of modifications, N-glycosylation and GPI anchor. A severe mutant allele of cobra causes a cellulose deficiency. Combining N-glycosylation and modification mutations with mild conditional cobra mutation alleles causes constitutive growth defects similar to the results obtained with the rsw2-1 mutation (Koiwa et al., unpublished). Furthermore, a mutation in PEANUT/PIG-M, an endoplasmic reticulum-localized mannosyltransferase that is required for synthesis of the GPI-anchor resulted in a seedling lethal phenotype with a decrease of crystalline cellulose. These findings indicate close relations among the protein N-glycosylation, the GPI anchor addition, cellulose biosynthesis, and plant osmotic stress tolerance (Gillmor et al., 2005). Interestingly, the kobito1 mutant affected in cellulose biosynthesis and the aba-insensitive8 were allelic suggesting a direct connection between cell wall formation and ABA/ osmotic stress signaling (Pagant et al., 2002). In the following section, biosynthetic pathways that are required for these protein modifications are discussed.

38

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

Pathways Involved in N-glycosylation and N-glycan Modifications

In eukaryotes, secreted and membrane-bound proteins are post-transcriptionally modified by N-glycosylation. Proteins that enter the endoplasmic reticulum (ER) receive core oligosaccharides at conserved N-glycosylation motifs (N-X-S/T) (Helenius and Aebi, 2001). The core oligosaccharide has a structure of Glc3Man9GlcNac2, which is transferred from dolichol to amino group of proteins by oligosaccharyltransferase in the ER. Maturation of core oligosaccharides to highmannose and complex-type oligosaccharides requires a series of glucosidase and glycosyltransferases in the ER and in the Golgi apparatus.

Dolichol Biosynthesis

Core oligosaccharides are assembled on membrane dolichol-phosphate molecules. Dolichols are unsaturated polyisoprenoids that contain 15–23 units of isoprenoid units. The biosynthesis of dolichols mainly occurs in the ER. The initial step of dolichol biosynthesis is production of isopentenyl diphosphate (IPP) via the mevalonate pathway (Grabinska and Palamarczyk, 2002) (Figure 2.1). IPP is used by farnesyl diphosphate synthase, which conjugates three IPP to produce farnesyl diphosphate (FPP). The formation of longer chain polyprenyldiphosphate is catalyzed by cis-prenyltransferases. Cis-prenyltransferases catalyzes the sequential addition of IPP to FPP via electrophillic addition of carbocations (Cunillera et al., 2000; Oh et al., 2000). Data from yeasts and animals indicate that the resulting polyprenol is converted to dolichol by α-saturation reactions (Sagami et al., 1993; Szkopinska et al., 1996). Resulting dolichol is phosphorylated by dolichol kinase (Rymerson et al., 1992) to produce dolichyl phosphate that functions in protein N- and O-glycosylations and GPI-anchor biosynthesis.

Figure 2.1 Biosynthetic pathway of dolichols.

PATHWAYS AND GENETIC DETERMINANTS

39

Arabidopsis LEAF WILTING1 (LEW1) encodes an isoform of cis-prenyltransferases. Although T-DNA insertion in LEW1 caused lethality, a missense mutation of LEW1 gene resulted in a decrease of dolichol content by 85% and defects in protein N-glycosylation (Yang et al., 2008). Surprisingly, the lew1 mutant exhibited greater drought tolerance than wild type. Lew1 exhibited a greater hydraulic conductivity in the root system, which could be accounted for by enhanced aquaporin activity. In leaves, lew1 plants show increased electrolyte leakage, decreased turgor, and increased stomatal closure. Apparently, moderate stress at the cell membrane induced stomatal closure and drought tolerance in lew1 mutants.

Sugar-nucleotide Biosynthesis

Sugar substrates for assembly and modification of N-glycans are provided in forms of dricholphosphate-linked sugars and sugar nucleotides. The biosynthetic pathways for these substrates are summarized in Figure 2.2. In cytosol, UDP-glucose is produced from sucrose and/or Glucose

Figure 2.2 Integration of nucleotide-sugar substrates and ascorbate biosynthesis pathway. Steps in parentheses are implied by yeast pathway. Compounds used in protein glycosylations are boxed. ALG5, glucosylphosphodolichol synthase; SS, sucrose synthase; UGP, UDP-glucose pyrophosphorylase; UDP-Glc-DH, UDP-glucose dehydrogenase; UXS, UDP-xylose synthase; PGM, phosphoglucomutase; PGI, phosphoglucoisomerase; GFA1, glucosamine 6-P synthase; GNA1, glucosamine 6-P acetyltransferase; AGM1, phosphoacetyl-glucosamine mutase; UAP1, UDP-N-acetylglucosamine pyrophosphorylase; PMI, phosphomannoisomerase; PMM, phosphomannomutase; VTC1/CYT1, GDP-mannose pyrophosphorylase; GME, GDP-mannose epimerase; MUR1, GDP-mannose-4,6-dehydratase; GER1, GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase-4-reductase; DPM1, dolichol phosphate mannose synthase; VTC2, GDP-galactose phosphorylase; VTC4, galactose-1-P phosphatase; GalDH, galactose dehydrogenase; GLDH, galactono-1,4-lactone dehydrogenase.

40

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

1-phosphate, and is converted to UDP-glucuronic acid (UDP-GlcA) by UDP-glucose dehydrogenase (Tenhaken and Thulke, 1996). UDP-GlcA is then converted to UDP-xylose by UDP-GlcA decarboxylase (Pattathil et al., 2005). Biosynthesis of UDP-GlcNac has not been studied in plants, but it appears to be produced from fructose 6-P by a conserved four-step version of the Leloir pathway (Milewski et al., 2006). GDP-mannose and GDP-galactose also are produced from fructose 6-P as an intermediate of Wheeler-Smirnoff pathway of ascorbate biosynthesis (Wheeler et al., 1998). GDP-mannose also serves as a precursor for the production of GDP-L-fuc (Zablackis et al., 1996; Bonin and Reiter, 2000).

Assembly of Core Oligosaccharide

This process has not been characterized well in plants except for a few genes. However, the conserved pathway is well studied in yeasts (Figure 2.3). At the cytoplasmic side of the ER, assembly of core oligosaccharide starts with the transfer of GlcNAc1P from UDP-GlcNAc to dolichyl-P to form GlcNAc-PP-dolichol. This reaction is catalyzed by the enzyme UDP-GlcNAc:dolichol phosphate GlcNAc-1-P transferase (GPT), which is a target for an antibiotic tunicamycin. Overexpression of GPT increased tunicamycin tolerance of transgenic Arabidopsis (Koizumi et al., 1999). Subsequently, a series of reactions add another GlcNAc (by ALG13/ALG14 complex) and five mannose residues (by ALG1, ALG2, ALG11 mannosyltransferases) using UDP-GlcNAc and GDPMan as substrates and produce Man5-GlcNAc2-PP-Dol structure (Snider et al., 1980). The heptasaccharide-PP-Dol undergoes “flipping” across the ER membrane (Helenius and Aebi, 2002). Upon translocation of heptasaccharide-PP-Dol to the luminal side of the ER, the second phase of the core oligosaccharide starts. The oligosaccharide receives four more mannose and three glucose residues to form Glc3Man9GlcNAc2-PP-Dol (Lehle et al., 2006). In contrast to the cytoplasmic reactions, the reactions in ER use substrates attached to dolichol phosphates, such as Glc-P-Dol and Man-PDol. Dolichol-linked substrates are produced from UDP-Glc and GDP-Man by dolichyl phosphoglucose synthase (Heesen et al., 1994) and dolichyl phosphomannose synthase (Orlean et al., 1988). Similar to Man5-GlcNAc2-PP-Dol, Glc-P-Dol and Man-P-Dol are produced on the cytoplasmic side of the ER and must be flipped into the ER in order to serve as saccharide donors for the lumenoriented transferases (Rush et al., 1998). Three mannosyltransferases (ALG3, ALG9, ALG12) extend mannose branches whereas three glycosyltransferases (ALG6, ALG8, ALG10) cap the α-1,6 branch and complete the biosynthesis of the core oligosaccharides. Mutation in Arabidopsis ALG3 does not affect production of complex-N-glycans, but eradicates the production of high-mannosetype N-glycans (Henquet et al., 2008). The core oligosaccharide produced in the ER of alg3 has structure of Glc3Man5GlcNac2. Apparently, this structure is still transferred to nascent peptides; however, alg3 mutant activates unfolded protein response (see below).

Oligosaccharyltransferase

Oligosaccharyltransferase (OST) is a multisubunit enzyme that catalyzes en bloc transfer of core oligosaccharides to nascent peptides in the ER. OST consists of eight subunits in yeast (OST1, OST2, OST3/OST6, OST4, OST5, STT3, WBP1, SWP1) (Kelleher and Gilmore, 2006). Four genes encoding three subunits of OST have been reported in Arabidopsis thaliana. DAD1 was first reported as a suppressor of cell death phenotype of CHO cell dad1 mutant (Gallois et al., 1997). Later, DAD1 was found to be a homolog of yeast OST2 subunit. Arabidopsis STT3a and its paralog

PATHWAYS AND GENETIC DETERMINANTS

41

Figure 2.3 Biosynthetic pathway of core-N-glycan assembly in the cytosol and in the ER. Only GPT, ALG3, and OST subunit genes were characterized in plants. Other ALG gene products refer to corresponding proteins in Saccharomyces cerevisiae. GPT, UDP-GlcNAc:dolichol phosphate GlcNAc-1-P transferase.

STT3b were found by forward genetic screening for salt-sensitive root growth (Koiwa et al., 2003). Root growth of stt3a mutant was sensitive to various solutes, such as NaCl, potassium chloride (KCl), and mannitol. Stt3a mutation causes underglycosylation of glycoproteins and induces expression of ER chaperon, BiP. Mutation in STT3b by itself did not cause any phenotypic abnormality, however, stt3a stt3b double mutant was gamete lethal. This indicates that STT3a and STT3b redundantly exert essential cellular functions. Arabidopsis DGL1 is homologous to WBP1 in yeast (Lerouxel et al., 2005). A severe loss of function mutation in DGL1 causes lethality. Plants

42

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

homozygous for partial loss-of-function allele of DGL1 exhibit a decrease in protein N-glycosylation that is associated with defects in cell elongation and altered cell wall composition. Interestingly, overexpression of DAD1 and its paralog DAD2 in Arabidopsis can suppress program cell death induced by UV irradiation. The suppression was observed in the presence or absence of tunicamycin, indicating DAD1 has anti-PCD function independent of oligosaccharyltransferase activities (Danon et al., 2004).

Processing of Core Oligosaccharides in the ER

Figure 2.4 summarizes the processing and maturation processes of core oligosaccharides in the ER and in the Golgi. Upon translocation of the core oligosaccharide from dolichol-PP to a nascent peptide, the core oligosaccharide undergoes a series of trimmings by α-glucosidase I and II that remove glucose residues from the core oligosaccharide (Boisson et al., 2001; Burn et al., 2002; Gillmor et al., 2002). This process is closely associated with chaperon-assisted protein folding of the newly synthesized proteins, as the terminal glucose of the core oligosaccharide is recognized and bound by ER chaperons, calnexin and calreticulin (Crofts and Denecke, 1998). A successfully folded protein loses all the glucose residues from its N-glycans (Man9GlcNAc2) whereas N-glycans of misfolded proteins receive glucoses and re-enter the folding cycle. Ultimately, the N-glycans of correctly folded proteins are processed by ER-mannosidase I (ER manI). Trimming of N-glycans by ER ManI to Man8GlcNAc2 isomer B removes the glycoprotein from reglucosylation and calnexin binding cycles. ER ManI is concentrated together with the ERAD substrate in the pericentriolar ER-derived quality control compartment (ERQC). Proteins with Man8GlcNAc2 structure exit from the ER-quality control pathway, and the glycoproteins will be exported to the Golgi apparatus.

Unfolded Protein Response and Osmotic Stress Signaling

Overexpression or mutation of secreted proteins, mutations in the N-glycosylation enzymes, tunicamycin treatment, and changes in redox status in the ER can induce accumulation of unfolded and misfolded proteins in ER. The accumulation of unfolded proteins in the ER triggers the unfolded protein response (UPR). During the UPR, expressions of genes encoding proteins that promote protein foldings, such as calnexin, calreticulin, binding protein (BiP, ER isoform of HSP70) protein disulfide isomerase, are induced (Martinez and Chrispeels, 2003). In mammals, the activation of UPR is triggered by parallel signaling pathways (Kohno, 2007), but only the evolutionarily conserved IRE1 pathway has been characterized in Arabidopsis (Koizumi et al., 2001). IRE1 is an ER-localized transmembrane protein with luminal sensor domain and cytoplasmic kinase and RNAse domains. In the absence of unfolded proteins, the sensor domain binds to BiP and is inactive. Upon accumulation of unfolded protein, the BiP dissociates with the sensor domain and the sensor domain binds to unfolded proteins and induces dimerization and activation of IRE1 (Kimata et al., 2007). The signal-induced dimerization/oligomerization of IRE1 promotes transphosphorylation of the kinase domain and activation of nuclease domain. In yeasts, the activated nuclease domain catalyzes splicing of Hac1 transcription factor that activates transcription of UPR-target genes. Proper regulation of protein quality control via UPR is important for adaptation to water stress. Analysis of transgenic tobacco plants overexpressing and silenced for BiP demonstrated that BiP

Figure 2.4 Plant N-glycosylation pathway in the ER and in the Golgi apparatus. OST, oligosaccharyltransferase; GnTI, b1,2N-acetylglucosaminyltransferase I; GnTII, b1,2-N-acetylglucosaminyltransferase II.

43

44

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

is essential for normal plant growth and water-stress tolerance (Alvim et al., 2001). Under progressive drought stress, BiP-overexpressing plants maintain the shoot turgor and water content better than wild-type plants. Induction of endogenous BiP expression was observed in wild type during the course of water stress, suggesting that BiP has protective function during water stress. In transgenic potato plants expressing antisense-BiP, cDNA showed increased sensitivity to water deficit. Similarly, transgenic plants expressing antisense α-glucosidase II showed severe growth reduction and less tuber production in the field though the plants did not show the phenotype when grown in the greenhouse. This was accompanied by an increase in BiP expression. Cells of antisense plants show spontaneous plasmolysis, similar to the lew1 mutant, suggesting a similar constitutive stress response in these plants. Gene expression profiling of plants undergoing osmotic stress or UPR identified a group of transcripts that are synergistically regulated by these two stresses (Costa et al., 2008). This signaling pathway was termed the integrated pathway. Of the genes regulated by the integrated pathway are asparagine-rich protein (NRP) A and B, which can promote program cell death when overexpressed in soybean protoplasts. The symptoms induced by NRPs can be ameliorated by addition of zeatin, implying that both osmotic stress and ER-stress induce senescence-like PCD via common pathway.

N-glycan Re-glycosylation and ER-associated Protein Degradation

As described above, proteins that failed to fold in the calnexin chaperon system can re-enter another round of folding. The decision whether or not to enter another folding cycle is rendered, in yeasts and animals, by a folding sensor, UDP-glucose:glycoprotein glucosyltransferase, which recognizes unfolded structure of released polypeptides and adds back a glucose residue to N-glycans of unfolded proteins (Cannon and Helenius, 1999; Trombetta and Helenius, 2000). On the other hand, proteins that failed to fold in multiple rounds of folding cycle become a substrate of slow-acting ER-manI that removes single mannose residue from Man9GlcNAc2 and produces Man8GlcNAc2 isomer B (Jakob et al., 1998; Cabral et al., 2000). Unfolded protein with Man8GlcNAc2 isomer B structure is recognized by EDEM (ER degradation enhancing α-mannosidase–like protein) family proteins and targeted for translocation via the Sec61 translocation complex (Molinari et al., 2003; Oda et al., 2003; Wang and Hebert, 2003). Misfolded proteins are then deglycosylated by peptide: N-glycanases (Diepold et al., 2007) and degraded by the ubiquitin-proteasome system (Plemper and Wolf, 1999). This process is known as ER-associated degradation (ERAD) (Vembar and Brodsky, 2008).

N-glycan Modification in the Golgi Apparatus

Upon successful folding of polypeptides and trimming of single mannose residue from N-glycans, the glycoproteins are exported to the Golgi apparatus. Further modifications with glycosyltransferases and glycosylhydrolases in the Golgi apparatus produce mature complex N-glycans. In the cis-Golgi, Golgi α-mannosidase I (MANI) hydrolyzes the terminal α1,2-linked mannose residues (Mast and Moremen, 2006), and therefore converts the high-mannose-type oligosaccharide, Man8GlcNAc2, to Man5GlcNAc2. The Arabidopsis genome contains two redundant MANI genes, which are essential for the processing of Man8GlcNAc2 (Kajiura et al, unpublished

PATHWAYS AND GENETIC DETERMINANTS

45

data). N-acetylglucosaminyltransferase I (GnTI) then add single GlcNAc to Man5GlcNAc2 (Wenderoth and Schaewen, 2000). Resulting GlcNAcMan5GlcNAc2 is trimmed by Golgi αmannosidase II to produce GlcNAcMan3GlcNAc2 (Strasser et al., 2006); then single GlcNAc is added by N-acetylglucosaminyltransferase II (GnTII) to produce a common intermediate of eukaryotic complex N-glycan biosynthesis pathway, GlcNAc2Man3GlcNAc2 (Strasser et al., 1999). The unique structures of plant complex N-glycans are produced in the late N-glycan modification pathway, which employs plant-specific glycosyltransferases such as β1,2-xylosyltransferase (XYLT) (Strasser et al., 2000), two α1,3-fucosyltransferase (FUCT) (Bondili et al., 2006), β1,3galactosyltransferase (GALT) (Strasser et al., 2007) and α1,4-FUCT (Léonard et al., 2002). The functions of individual N-glycan modification enzymes are actively studied in Arabidopsis. The reactions by MANI and GnTI are prerequisite for the following modifications. The first Arabidopsis thaliana mutant lacking complex N-glycans (cgl1, complex glycan 1) was reported in 1993 (von Schaewen et al., 1993). CGL1 encode the Golgi GnTI, completely lack complex N-glycans, and accumulate oligomannosidic N-glycans, predominantly Man4GlcNAc2. Since then, several mutants and transgenic plants altered in N-glycan maturation in the Golgi apparatus have been reported. Arabidopsis hgl1 mutant lacking functional α-mannosidase II does not produce complex N-glycans, however, instead of accumulating high-mannose-type glycans, hgl1 plants accumulate hybrid type N-glycans. This indicates that mannose trimming from α-1,3mannose branch is not essential for subsequent modifications (Strasser et al., 2006). The following step is the parallel reactions by GnTII, XYLT, and FUCTa/FUCTb. Mutations in the genes encoding individual enzymes cause production of complex N-glycans lacking one or more residues (Strasser et al., 2004; Kang et al., 2008 von Schaewen, unpublished data). A triple mutant lacking XYLT, and FUCTa/FUCTb produces a complex N-glycans predominantly GlcNAc2Man3GlcNAc2. Arabidopsis GALT is expressed mainly in stem tissues where glycoproteins with antenna modified complex N-glycans accumulate. Complex N-glycans in galT-1 mutant plants lack Lewis a epitope structure. Since GALT reaction is a prerequisite for the FUCTc reaction to occur, galT-1 plants are not capable of generating any antenna modifications. Furthermore, a recent study indicates that manIamanIb double mutant, which cannot produce complex glycans, but accumulate the Man8GlcNAc2 form (Kajiura et al., unpublished data). The N-glycosylation in the ER and N-glycan modification in the Golgi apparatus is strongly connected with root growth under osmotic stress (Kang et al., 2008). Cgl1, hgl1, and fucTa fucTb xylT triple mutant were all salt sensitive like stt3a mutants. Unlike stt3a mutants, the mutations in the Golgi N-glycan modification enzymes did not activate the BiP promoter, confirming the prediction that these proteins function after the protein quality control step. In contrast, individual mutations in FUCTa/FUCTb, XYLT, GnTII, FUCTc did not cause salt sensitivity (Kang et al., 2008; Koiwa, unpublished results). This indicates that certain structures of complex N-glycan are required to sustain root growth under salt stress. Salt sensitivity of fucTa fucTbxylT triple mutant but not fucTa fucTb or xylT mutants indicates that the core β1,2-xylose and α1,3fucose redundantly function in salt tolerance. However, hgl1 mutant that produces hybrid N-glycans is salt sensitive in spite of the fact that the β1,2-xylose and α1,3-fucose modifications occur in this mutant (Strasser et al., 2006; Kang et al., 2008). Interestingly, gnTII mutant did not show salt sensitivity. GnTII does not modify α-1,3-mannose branch of N-glycans after trimming by αmannosidase II (HGL1) but has core β1,2-xylose and α1,3-fucose. Thus, gnTII is different from hgl1 only by the absence of α-1,3 and α-1,6-mannose extensions in the α-1,3-mannose branch. Further detailed analyzes are necessary to understand the function of fine N-glycan structures in salt tolerance.

46

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

Ascorbate as an Interface between the N-glycosylation Pathway and Oxidative Stress Response

Ascorbate is a major antioxidant in the ascorbate-glutathione cycle. Decrease of ascorbate in Arabidopsis results in sensitivity to various oxidative stresses including exposure to ozone, UV-B irradiation, and sulfur dioxide (Conklin et al., 1996). In plants, ascorbate is produced via WheelerSmirnoff pathway (Wheeler et al., 1998). The early part of this pathway is shared by the ascorbate biosynthesis pathway and protein glycosylation and cell wall biosynthesis pathways (Figure 2.2). The common intermediate is GDP-mannose produced from hexose-phosphate via mannose-1phosphate. Indeed, mutation in UDP-mannose pyrophosphorylase has been isolated as vtc1 mutant decreased in ascorbate as well as cyt1 mutant decreased in cellulose content (Conklin et al., 1996; Nickle and Meinke, 1998; Conklin et al., 1999; Lukowitz et al., 2001). Furthermore, compromising phosphomannomutase, which is a step upstream of VTC1 reaction, causes a decrease in ascorbate content in gene-silenced Nicotiana benthamiana (Qian et al., 2007), and protein underglycosylation and cell death in Arabidopsis pmm-12 mutant (Hoeberichts et al., 2008).

Biosynthesis of GPI Anchor

Glycosylphosphatidylinositol (GPI) anchors are eukaryotic mechanisms to attach proteins to the surface of the membranes (Paulick and Bertozzi, 2008). The GPI moiety of the anchor can be cleaved by phospholipase D, which allows regulated release of proteins from the cell surface. The core structure of GPI anchor (EtNP-6Manα1-2Manα1-6Manα1-4GlcNα1-6-inositol-phospholipid) is highly conserved among eukaryotes. Variety of side chain modifications exists for the mannosides and inositol moiety, resulting in the production of isoforms with microheterogeneity (Oxley and Bacic, 1999). GPI-anchor biosynthesis is poorly characterized in plants, however, the limited information available suggests that the pathway is similar to yeast and mammals (Lalanne et al., 2004; Gillmor et al., 2005). Figure 2.5 summarizes the biosynthesis pathway of the core GPIanchor structure in animals. The biosynthetic pathway of GPI starts on the cytosolic side of the ER with addition of N-acetylglucosamine to phosphatidylinositol (PI) by a GPI-Nacetylglucosaminyltransferase (GPI-GnT) complex. GPI-GnT is composed of six subunits, namely, PIG-A, PIG-C, PIG-H, GPI1, PIG-P, and DPM2. Mutations in Arabidopsis genes SETH1 and SETH2, encoding PIG-C and PIG-A homolog, respectively, affect pollen germination and tube growth, and cause very low male transmission of the mutated genes (Lalanne et al., 2004). Mutant pollen has normal cellulose and pectin staining profile, however, it was enhanced for the callose staining. The following step is catalyzed by GlcNAc-PI de-N-acetylase (PIG-L). The resulting GlcN-PI is flipped into the luminal side of the ER, and the acyl-group is added by inositol acyltransferase (PIG-W). In the ER, stepwise action of GPI-α-1,4-mannosyltransferase (PIG-M/PIG-X) (Ashida et al., 2005), phosphoethanolamine transferase (PIG-N), GPI-α-1,6-mannosyltransferase (PIG-V) (Kang et al., 2005), GPI-α-1,2-mannosyltransferase (PIG-B) (Takahashi et al., 1996), phosphoethanolamine transferase (PIG-F/PIG-O) (Inoue et al., 1993; Hong et al., 2000) completes the assembly of GPI precursor. Mutation in Arabidopsis PIG-M homolog results in peanut1 mutation that causes embryonic swelling, associated with cellulose deficiency (Gillmor et al., 2005). Other peanut mutants (pnt2-pnt5) may encode other enzymes in the same pathway. In addition, a homolog of PIG-F was identified in rice, and its expression profile has been characterized (Lee and Kang, 2008). The pre-assembled GPI is transferred en bloc to proteins that have the C-terminal GPI attachment signal sequence (CAAX) and single transmembrane domain. The transmembrane domain is pro-

PATHWAYS AND GENETIC DETERMINANTS

47

Figure 2.5 The biosynthetic pathway of the core GPI-anchor in the ER. The pathway is inferred by animal studies, and characterized plant homologs are indicated in the parenthesis.

teolytically cleaved, the C-terminus is attached to the GPI anchor by an amide bond formed between the carboxyl terminus, and phosphoethanolamine, which is attached to the third mannose of the anchor. This reaction is catalyzed by GPI transamidase complexes and consists of four subunits (GPI8, GAA1, PIG-S, PIG-T, and PIG-U) (Hong et al. 2000, 2003; Vainauskas and Menon, 2004). After attaching to protein, a GPI inositol-deacylase (PGAP1) removes the acyl group from the inositol moiety (Tanaka et al., 2004). To date, no plant GPI transamidase has been characterized.

Microtubules

The microtubule cytoskelton is an essential component of growth and morphology of the plant cells. The cortical microtubules form a structure under plasma membrane and guide patterning of

48

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

the cellulose microfibrils deposited to the cell wall. This in turn determines the expansion and morphology of the cells. Another linkage that relates cell wall function and salt tolerance was provided by recent studies of microtubules. Shoji and others (2006) reported that a helical growth phenotype and microtubule orientation of spiral1 can be suppressed by imposing salt stress or combining spr1 with either Na+/H+ antiporter mutant sos1 or with its regulatory protein mutant sos2 (Shoji et al., 2006). Spr1 is defective in microtubule organization and displays a right-handed helical growth phenotype. Furthermore, salt stress by itself alters the helical growth pattern of Arabidopsis roots. Based on this observation, Shoji and others proposed that cytoplasmic salt imbalance compromises cortical microtubule functions in which microtubule-localized SPR1 is specifically involved (Shoji et al., 2006). The significance of the above study in salt tolerance could be provided by the study of Wang and others (2007). In their study, dynamics of microtubule organization during the salt stress and effects of microtubule-altering drugs on salt tolerance were determined. They found that upon exposure to the salt stress, the cells undergo depolymerization and re-organization of the cortical microtubules. Inhibition of depolymerization by pacritaxel decreases the salt tolerance, but promoting depolymerization by oryzarin improved salt tolerance both in wild type and in the sos1 mutant. Interestingly, free cytoplasmic calcium concentration increased upon depolymerization of the microtubules, and the addition of calcium to the growth medium promoted the recovery of the cortical microtubule organization. In contrast, calcium deficiency inhibited the recovery of the microtubule organization. These results suggest calcium mediated reorganization of microtubule cytoskelton is essential for the salt tolerance (Wang et al., 2007). It has been known that orientation of microtubule and cellulose microfibrils correlates through the developmental stage. In the developing root, the cortical arrays in the meristem region are transversely oriented until the root hair emerges and alignment of cellulose microfibrils show similar direction to microtubules. Disruption of cortical microtubule arrays by genetic mutation or by oryzarin treatment causes radial swelling of the root cells that is accompanied with loss of alignment of microfibrils (Baskin et al., 2004). This suggests that microtubules guide the cellulose synthase complex on the cell surface; indeed, an elegant study using fluorescently tagged tubulin and CesA6 suggested that CesA6 travels on the microtubules (Paredez et al., 2006). Localization of KOR1 protein is also regulated by microtubules (Robert et al., 2005). Conversely, cellulose biosynthesis influences cortical array organization because a genetic screen to identify oryzarin hypersensitive mutants identified CesA6 and KOR1 (Paredez et al., 2008).

Conclusion

Recent advances in Arabidopsis genetic analysis demonstrated that formation of proper cell wall structure is essential for sustaining root growth under salt/osmotic stress. Multiple regulatory mechanisms for cell wall formation are also connected to the stress tolerance, such as protein N-glycosylation/modification of RSW2/KOR1 or COBRA that are involved in cellulose biosynthesis, and regulation of microtubule orientation by SOS1 Na+/H+ antiporter. Bidirectional regulation has been shown for microtubule cortical array formation and cellulose biosynthesis. Similarly, salt stress and SOS1 affect microtubule cortical array formation but establishment of salt tolerance requires microtubule cortical array rearrangement. Apparently, cell walls not only provide structural integrity during osmotic stress, but also likely function as a scaffold to mediate various signaling events juxtaposed to the plasma membranes that operate during the osmotic stress.

PATHWAYS AND GENETIC DETERMINANTS

49

References Alvim, F.C., Carolino, S.M., Cascardo, J.C., Nunes, C.C., Martinez, C.A., Otoni, W.C., and Fontes, E.P. (2001) Enhanced accumulation of BiP in transgenic plants confers tolerance to water stress. Plant Physiol 126: 1042–1054. Amaya, I., Botella, M.A., de la Calle, M., Medina, M.I., Heredia, A., Bressan, R.A., Hasegawa, P.M., Quesada, M.A., and Valpuesta, V. (1999) Improved germination under osmotic stress of tobacco plants overexpressing a cell wall peroxidase. FEBS Lett 457: 80–84. Arioli, T., Peng, L., Betzner, A.S., Burn, J., Wittke, W., Herth, W., Camilleri, C., Hofte, H., Plazinski, J., Birch, R., Cork, A., Glover, J., Redmond, J., and Williamson, R.E. (1998) Molecular analysis of cellulose biosynthesis in Arabidopsis. Science 279: 717–720. Ashida, H., Hong, Y., Murakami, Y., Shishioh, N., Sugimoto, N., Kim, Y.U., Maeda, Y., and Kinoshita, T. (2005) Mammalian PIG-X and yeast Pbnlp are the essential components of glycosylphosphatidylinositol-mannosyltransferase I. Mol Biol Cell 16: 1439–1448. Baskin, T.I., Beemster, G.T., Judy-March, J.E., and Marga, F. (2004) Disorganization of cortical microtubules stimulates tangential expansion and reduces the uniformity of cellulose microfibril alignment among cells in the root of Arabidopsis. Plant Physiol 135: 2279–2290. Benfey, P.N. and Scheres, B. (2000) Root development. Curr Biol 10: R813–815. Boisson, M., Gomord, V., Audran, C., Berger, N., Dubreucq, B., Granier, F., Lerouge, P., Faye, L., Caboche, M., and Lepiniec, L. (2001) Arabidopsis glucosidase I mutants reveal a critical role of N-glycan trimming in seed development. EMBO J 20: 1010–1019. Bondili, J.S., Castilho, A., Mach, L., Glossl, J., Steinkellner, H., Altmann, F., and Strasser, R. (2006) Molecular cloning and heterologous expression of beta1,2-xylosyltransferase and core alpha1,3-fucosyltransferase from maize. Phytochemistry 67: 2215–2224. Bonin, C.P. and Reiter, W.D. (2000) A bifunctional epimerase-reductase acts downstream of the MUR1 gene product and completes the de novo synthesis of GDP-L-fucose in Arabidopsis. Plant J 21: 445–454. Brocard-Gifford, I., Lynch, T.J., Garcia, M.E., Malhotra, B., and Finkelstein, R.R. (2004) The Arabidopsis thaliana ABSCISIC ACID-INSENSITIVE8 encodes a novel protein mediating abscisic acid and sugar responses essential for growth. Plant Cell 16: 406–421. Burn, J.E., Hurley, U.A., Birch, R.J., Arioli, T., Cork, A., and Williamson, R.E. (2002) The cellulose-deficient Arabidopsis mutant rsw3 is defective in a gene encoding a putative glucosidase II, an enzyme processing N-glycans during ER quality control. Plant J 32: 949–960. Cabral, C.M., Choudhury, P., Liu, Y., and Sifers, R.N. (2000) Processing by endoplasmic reticulum mannosidases partitions a secretion-impaired glycoprotein into distinct disposal pathways. J Biol Chem 275: 25015–25022. Cannon, K.S. and Helenius, A. (1999) Trimming and readdition of glucose to N-linked oligosaccharides determines calnexin association of a substrate glycoprotein in living cells. J Biol Chem 274: 7537–7544. Chen, Z., Hong, X., Zhang, H., Wang, Y., Li, X., Zhu, J.K., and Gong, Z. (2005) Disruption of the cellulose synthase gene, AtCesA8/IRX1, enhances drought and osmotic stress tolerance in Arabidopsis. Plant J 43: 273–283. Conklin, P.L., Norris, S.R., Wheeler, G.L., Williams, E.H., Smirnoff, N., and Last, R.L. (1999) Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis. Proc Natl Acad Sci USA 96: 4198–4203. Conklin, P.L., Williams, E.H., and Last, R.L. (1996) Environmental stress sensitivity of an ascorbic acid-deficient Arabidopsis mutant. Proc Natl Acad Sci USA 93: 9970–9974. Costa, M.D., Reis, P.A., Valente, M.A., Irsigler, A.S., Carvalho, C.M., Loureiro, M.E., Aragao, F.J., Boston, R.S., Fietto, L.G., and Fontes, E.P. (2008) A new branch of endoplasmic reticulum stress signaling and the osmotic signal converge on plantspecific asparagine-rich proteins to promote cell death. J Biol Chem 283: 20209–20219. Crofts, A.J. and Denecke, J. (1998) Calreticulin and calnexin in plants. Trends Plant Sci 3: 396–399. Cunillera, N., Arro, M., Fores, O., Manzano, D., and Ferrer, A. (2000). Characterization of dehydrodolichyl diphosphate synthase of Arabidopsis thaliana, a key enzyme in dolichol biosynthesis. FEBS Lett 477: 170–174. Danon, A., Rotari, V.I., Gordon, A., Mailhac, N., and Gallois, P. (2004) Ultraviolet-C overexposure induces programmed cell death in Arabidopsis, which is mediated by caspase-like activities and which can be suppressed by caspase inhibitors, p35 and Defender against Apoptotic Death. J Biol Chem 279: 779–787. Desprez, T., Juraniec, M., Crowell, E.F., Jouy, H., Pochylova, Z., Parcy, F., Hofte, H., Gonneau, M., and Vernhettes, S. (2007) Organization of cellulose synthase complexes involved in primary cell wall synthesis in Arabidopsis thaliana. Proc Natl Acad Sci USA 104: 15572–15577. Dhugga, K.S. (2001) Building the wall: genes and enzyme complexes for polysaccharide synthases. Curr Opin Plant Biol 4: 488–493.

50

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

Diepold, A., Li, G., Lennarz, W.J., Nurnberger, T., and Brunner, F. (2007) The Arabidopsis AtPNG1 gene encodes a peptide: N-glycanase. Plant J 52: 94–104. Dinneny, J.R., Long, T.A., Wang, J.Y., Jung, J.W., Mace, D., Pointer, S., Barron, C., Brady, S.M., Schiefelbein, J., and Benfey, P.N. (2008) Cell identity mediates the response of Arabidopsis roots to abiotic stress. Science 320: 942–945. Gallois, P., Makishima, T., Hecht, V., Despres, B., Laudie, M., Nishimoto, T., and Cooke, R. (1997) An Arabidopsis thaliana cDNA complementing a hamster apoptosis suppressor mutant. Plant J. 11: 1325–1331. Gillmor, C.S., Lukowitz, W., Brininstool, G., Sedbrook, J.C., Hamann, T., Poindexter, P., and Somerville, C. (2005) Glycosylphosphatidylinositol-anchored proteins are required for cell wall synthesis and morphogenesis in Arabidopsis. Plant Cell 17: 1128–1140. Gillmor, C.S., Poindexter, P., Lorieau, J., Palcic, M.M., and Somerville, C. (2002) Alpha-glucosidase I is required for cellulose biosynthesis and morphogenesis in Arabidopsis. J Cell Biol 156: 1003–1013. Grabinska, K. and Palamarczyk, G. (2002) Dolichol biosynthesis in the yeast Saccharomyces cerevisiae: an insight into the regulatory role of farnesyl diphosphate synthase. FEMS Yeast Res 2: 259–265. Hauser, M.T., Morikami, A., and Benfey, P.N. (1995) Conditional root expansion mutants of Arabidopsis. Development 121: 1237–1252. Heesen, S., Lehle, L., Weissmann, A., and Aebi, M. (1994) Isolation of the ALG5 locus encoding the UDP-glucose:dolichylphosphate glucosyltransferase from Saccharomyces cerevisiae. Eur J Biochem 224: 71–79. Helenius, A. and Aebi, M. (2001) Intracellular functions of N-linked glycans. Science 291: 2364–2369. Helenius, J. and Aebi, M. (2002) Transmembrane movement of dolichol linked carbohydrates during N-glycoprotein biosynthesis in the endoplasmic reticulum. Semin Cell Dev Biol 13: 171–178. Henquet, M., Lehle, L., Schreuder, M., Rouwendal, G., Molthoff, J., Helsper, J., van der Krol, S., and Bosch, D. (2008) Identification of the gene encoding the alpha1,3-mannosyltransferase (ALG3) in Arabidopsis and characterization of downstream n-glycan processing. Plant Cell 20: 1652–1664. Herth, W. (1983) Arrays of plasma-membrane rosettes involved in cellulose microfibril formation of spirogyra. Planta 159: 347–356. Hoeberichts, F.A., Vaeck, E., Kiddle, G., Coppens, E., van de Cotte, B., Adamantidis, A., Ormenese, S., Foyer, C.H., Zabeau, M., Inze, D., Perilleux, C., Van Breusegem, F., and Vuylsteke, M. (2008) A temperature-sensitive mutation in the Arabidopsis thaliana phosphomannomutase gene disrupts protein glycosylation and triggers cell death. J Biol Chem 283: 5708–5718. Hong, Y., Maeda, Y., Watanabe, R., Inoue, N., Ohishi, K., and Kinoshita, T. (2000) Requirement of PIG-F and PIG-O for transferring phosphoethanolamine to the third mannose in glycosylphosphatidylinositol. J Biol Chem 275: 20911–20919. Hong, Y., Ohishi, K., Kang, J.Y., Tanaka, S., Inoue, N., Nishimura, J., Maeda, Y., and Kinoshita, T. (2003) Human PIG-U and yeast Cdc9lp are the fifth subunit of GPI transamidase that attaches GPI-anchors to proteins. Mol Biol Cell 14: 1780–1789. Inoue, N., Kinoshita, T., Orii, T., and Takeda, J. (1993) Cloning of a human gene, PIG-F, a component of glycosylphosphatidylinositol anchor biosynthesis, by a novel expression cloning strategy. J Biol Chem 268: 6882–6885. Iraki, N.M., Bressan, R.A., Hasegawa, P.M., and Carpita, N.C. (1989) Alteration of the physical and chemical structure of the primary cell wall of growth-limited plant cells adapted to osmotic stress. Plant Physiol 91: 39–47. Jakob, C.A., Burda, P., Roth, J., and Aebi, M. (1998) Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomyces cerevisiae is determined by a specific oligosaccharide structure. J Cell Biol 142: 1223–1233. Kang, J.S., Frank, J., Kang, C.H., Kajiura, H., Vikram, M., Ueda, A., Kim, S., Bahk, J.D., Triplett, B., Fujiyama, K., Lee, S.Y., von Schaewen, A., and Koiwa, H. (2008) Salt tolerance of Arabidopsis thaliana requires maturation of N-glycosylated proteins in the Golgi apparatus. Proc Natl Acad Sci USA 105: 5933–5938. Kang, J.Y., Hong, Y., Ashida, H., Shishioh, N., Murakami, Y., Morita, Y.S., Maeda, Y., and Kinoshita, T. (2005) PIG-V involved in transferring the second mannose in glycosylphosphatidylinositol. J Biol Chem 280: 9489–9497. Kelleher, D.J. and Gilmore, R. (2006) An evolving view of the eukaryotic oligosaccharyltransferase. Glycobiology 16: 47R–62R. Kimata, Y., Ishiwata-Kimata, Y., Ito, T., Hirata, A., Suzuki, T., Oikawa, D., Takeuchi, M., and Kohno, K. (2007) Two regulatory steps of ER-stress sensor Ire1 involving its cluster formation and interaction with unfolded proteins. J Cell Biol 179: 75–86. Kohno, K. (2007) How transmembrane proteins sense endoplasmic reticulum stress. Antioxid Redox Signal 9: 2295–2303. Koiwa, H., Li, F., McCully, M.G., Mendoza, I., Koizumi, N., Manabe, Y., Nakagawa, Y., Zhu, J.-H., Rus, A., Pardo, J.M., Bressan, R.A., and Hasegawa, P.M. (2003) The STT3a subunit isoform of the Arabidopsis thaliana oligosaccharyltransferase controls adaptive responses to salt/osmotic stress. Plant Cell 15: 2273–2284. Koizumi, N., Martinez, I.M., Kimata, Y., Kohno, K., Sano, H., and Chrispeels, M.J. (2001) Molecular characterization of two Arabidopsis Ire1 homologs, endoplasmic reticulum-located transmembrane protein kinases. Plant Physiol 127: 949–962.

PATHWAYS AND GENETIC DETERMINANTS

51

Koizumi, N., Ujino, T., Sano, H., and Chrispeels, M.J. (1999) Overexpression of a gene that encodes the first enzyme in the biosynthesis of asparagine-linked glycans makes plants resistant to tunicamycin and obviates the tunicamycin-induced unfolded protein response. Plant Physiol 121: 353–361. Lalanne, E., Honys, D., Johnson, A., Borner, G.H., Lilley, K.S., Dupree, P., Grossniklaus, U., and Twell, D. (2004) SETH1 and SETH2, two components of the glycosylphosphatidylinositol anchor biosynthetic pathway, are required for pollen germination and tube growth in Arabidopsis. Plant Cell 16: 229–240. Lane, D.R., Wiedemeier, A., Peng, L., Hofte, H., Vernhettes, S., Desprez, T., Hocart, C.H., Birch, R.J., Baskin, T.I., Burn, J.E., Arioli, T., Betzner, A.S., and Williamson, R.E. (2001) Temperature-sensitive alleles of RSW2 link the KORRIGAN endo1,4-beta-glucanase to cellulose synthesis and cytokinesis in Arabidopsis. Plant Physiol 126: 278–288. Lee, D.H. and Kang, S.G. (2008) Characterization of phosphatidylinositol-glycan biosynthesis protein class F gene in rice. DNA Seq 19: 282–290. Lehle, L., Strahl, S., and Tanner, W. (2006) Protein glycosylation, conserved from yeast to man: a model organism helps elucidate congenital human diseases. Angew Chem Int Ed Engl 45: 6802–6818. Léonard, R., Costa, G., Darrambide, E., Lhemould, S., Fleurat-Lessard, P., Carlue, M., Gomord, V., Faye, L., and Maftan, A. (2002) The presence of Lewis a epitopes in Arabidopsis thaliana glycoconjugates depends on an active alpha4-fucosyltransferase gene. Glycobiology 12: 299–306. Lerouxel, O., Mouille, G., Andeme-Onzighi, C., Bruyant, M.P., Seveno, M., Loutelier-Bourhis, C., Driouich, A., Hofte, H., and Lerouge, P. (2005) Mutants in DEFECTIVE GLYCOSYLATION, an Arabidopsis homolog of an oligosaccharyltransferase complex subunit, show protein underglycosylation and defects in cell differentiation and growth. Plant J 42: 455–468. Lertpiriyapong, K. and Sung, Z.R. (2003) The elongation defective1 mutant of Arabidopsis is impaired in the gene encoding a serine-rich secreted protein. Plant Mol Biol 53: 581–595. Lukowitz, W., Nickle, T.C., Meinke, D.W., Last, R.L., Conklin, P.L., and Somerville, C.R. (2001) Arabidopsis cyt1 mutants are deficient in a mannose-1-phosphate guanylyltransferase and point to a requirement of N-linked glycosylation for cellulose biosynthesis. Proc Natl Acad Sci USA 98: 2262–2267. Majewsha-Sawka, A. and Nothnagel, E.A. (2000) The multiple roles of arabinogalactan proteins in plant development. Plant Physiol 122: 3–10. Martinez, I.M. and Chrispeels, M.J. (2003) Genomic analysis of the unfolded protein response in Arabidopsis shows its connection to important cellular processes. Plant Cell 15: 561–576. Mast, S.W. and Moremen, K.W. (2006) Family 47 alpha-mannosidases in N-glycan processing. Methods Enzymol 415: 31–46. Milewski, S., Gabriel, I., and Olchowy, J. (2006) Enzymes of UDP-GlcNAc biosynthesis in yeast. Yeast 23: 1–14. Molhoj, M., Ulvskov, P., and Dal Degan, F. (2001) Characterization of a functional soluble form of a Brassica napus membraneanchored endo-1,4-beta-glucanase heterologously expressed in Pichia pastoris. Plant Physiol 127: 674–684. Molinari, M., Calanca, V., Galli, C., Lucca, P., and Paganetti, P. (2003) Role of EDEM in the release of misfolded glycoproteins from the calnexin cycle. Science 299: 1397–1400. Nakajima, K. and Benfey, P.N. (2002) Signaling in and out: control of cell division and differentiation in the shoot and root. Plant Cell 14 Suppl: S265–276. Nickle, T.C. and Meinke, D.W. (1998) A cytokinesis-defective mutant of Arabidopsis (cyt1) characterized by embryonic lethality, incomplete cell walls, and excessive callose accumulation. Plant J 15: 321–332. Nicol, F., His, I., Jauneau, A., Vernhettes, S., Canut, H., and Hofte, H. (1998) A plasma membrane-bound putative endo-1,4-betaD-glucanase is required for normal wall assembly and cell elongation in Arabidopsis. Embo J 17: 5563–5576. O’Niell, M. and York, W.S. (2003) The composition and structure of plant primary cell walls. In KC Rose, Ed., The plant cell wall, Vol 8. Blackwell Publishing, Oxford. Oda, Y., Hosokawa, N., Wada, I., and Nagata, K. (2003) EDEM as an acceptor of terminally misfolded glycoproteins released from calnexin. Science 299: 1394–1397. Oh, S.K., Han, K.H., Ryu, S.B., and Kang, H. (2000) Molecular cloning, expression, and functional analysis of a cis-prenyltransferase from Arabidopsis thaliana. Implications in rubber biosynthesis. J Biol Chem 275: 18482–18488. Orlean, P., Albright, C., and Robbins, P.W. (1988) Cloning and sequencing of the yeast gene for dolichol phosphate mannose synthase, an essential protein. J Biol Chem 263: 17499–17507. Oxley, D. and Bacic, A. (1999) Structure of the glycosylphosphatidylinositol anchor of an arabinogalactan protein from Pyrus communis suspension-cultured cells. Proc Natl Acad Sci USA 96: 14246–14251. Pagant, S., Bichet, A., Sugimoto, K., Lerouxel, O., Desprez, T., McCann, M., Lerouge, P., Vernhettes, S., and Hofte, H. (2002) KOBITO1 encodes a novel plasma membrane protein necessary for normal synthesis of cellulose during cell expansion in Arabidopsis. Plant Cell 14: 2001–2013. Paredez, A.R., Persson, S., Ehrhardt, D.W., and Somerville, C.R. (2008) Genetic evidence that cellulose synthase activity influences microtubule cortical array organization. Plant Physiol 147: 1723–1734.

52

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

Paredez, A.R., Somerville, C.R., and Ehrhardt, D.W. (2006) Visualization of cellulose synthase demonstrates functional association with microtubules. Science 312: 1491–1495. Pattathil, S., Harper, A.D., and Bar-Peled, M. (2005) Biosynthesis of UDP-xylose: characterization of membrane-bound AtUxs2. Planta 221: 538–548. Paulick, M.G. and Bertozzi, C.R. (2008) The glycosylphosphatidylinositol anchor: a complex membrane-anchoring structure for proteins. Biochemistry 47: 6991–7000. Peng, L., Hocart, C.H., Redmond, J.W., and Williamson, R.E. (2000) Fractionation of carbohydrates in Arabidopsis root cell walls shows that three radial swelling loci are specifically involved in cellulose production. Planta 211: 406–414. Persson, S., Paredez, A., Carroll, A., Palsdottir, H., Doblin, M., Poindexter, P., Khitrov, N., Auer, M., and Somerville, C.R. (2007) Genetic evidence for three unique components in primary cell-wall cellulose synthase complexes in Arabidopsis. Proc Natl Acad Sci USA 104: 15566–15571. Petricka, J.J. and Benfey, P.N. (2008) Root layers: complex regulation of developmental patterning. Curr Opin Genet Dev 18: 354–361. Plemper, R.K. and Wolf, D.H. (1999) Retrograde protein translocation: ERADication of secretory proteins in health and disease. Trends Biochem Sci 24: 266–270. Qian, W., Yu, C., Qin, H., Liu, X., Zhang, A., Johansen, I.E., and Wang, D. (2007) Molecular and functional analysis of phosphomannomutase (PMM) from higher plants and genetic evidence for the involvement of PMM in ascorbic acid biosynthesis in Arabidopsis and Nicotiana benthamiana. Plant J 49: 399–413. Robert, S., Bichet, A., Grandjean, O., Kierzkowski, D., Satiat-Jeunemaitre, B., Pelletier, S., Hauser, M.T., Hofte, H., and Vernhettes, S. (2005) An Arabidopsis endo-1,4-beta-D-glucanase involved in cellulose synthesis undergoes regulated intracellular cycling. Plant Cell 17: 3378–3389. Roudier, F., Fernandez, A.G., Fujita, M., Himmelspach, R., Borner, G.H., Schindelman, G., Song, S., Baskin, T.I., Dupree, P., Wasteneys, G.O., and Benfey, P.N. (2005) COBRA, an Arabidopsis extracellular glycosyl-phosphatidyl inositol-anchored protein, specifically controls highly anisotropic expansion through its involvement in cellulose microfibril orientation. Plant Cell 17: 1749–1763. Rush, J.S., van Leyen, K., Ouerfelli, O., Wolucka, B., and Waechter, C.J. (1998) Transbilayer movement of Glc-P-dolichol and its function as a glucosyl donor: protein-mediated transport of a water-soluble analog into sealed ER vesicles from pig brain. Glycobiology 8: 1195–1205. Rymerson, R.T., Carroll, K.K., and Rip, J.W. (1992) Dolichol and polyprenol kinase activities in microsomes from etiolated rye seedlings. Biochem Cell Biol 70: 455–459. Sagami, H., Kurisaki, A., and Ogura, K. (1993) Formation of dolichol from dehydrodolichol is catalyzed by NADPH-dependent reductase localized in microsomes of rat liver. J Biol Chem 268: 10109–10113. Schindelman, G., Morikami, A., Jung, J., Baskin, T.I., Carpita, N.C., Derbyshire, P., McCann, M.C., and Benfey, P.N. (2001) COBRA encodes a putative GPI-anchored protein, which is polarly localized and necessary for oriented cell expansion in Arabidopsis. Genes Dev 15: 1115–1127. Shi, H., Kim, Y., Guo, Y., Stevenson, B., and Zhu, J.K. (2003) The Arabidopsis SOS5 locus encodes a putative cell surface adhesion protein and is required for normal cell expansion. Plant Cell 15: 19–32. Shoji, T., Suzuki, K., Abe, T., Kaneko, Y., Shi, H., Zhu, J.K., Rus, A., Hasegawa, P.M., and Hashimoto, T. (2006) Salt stress affects cortical microtubule organization and helical growth in Arabidopsis. Plant Cell Physiol 47: 1158–1168. Snider, M.D., Sultzman, L.A., and Robbins, P.W. (1980) Transmembrane location of oligosaccharide-lipid synthesis in microsomal vesicles. Cell 21: 385–392. Somerville, C. (2006) Cellulose Synthesis in Higher Plants. Annual Review of Cell and Developmental Biology 22: 53–78. Strasser, R., Altmann, F., Mach, L., Glossl, J., and Steinkellner, H. (2004) Generation of Arabidopsis thaliana plants with complex N-glycans lacking beta1,2-linked xylose and core alpha1,3-linked fucose. FEBS Lett 561: 132–136. Strasser, R, Bondili, J.S., Vavra, U., Schoberer, J., Svoboda, B., Glossl, J., Leonard, R., Stadlmann, J., Altmann, F., Steinkellner, H., and Mach, L. (2007) A unique (beta) 1,3-galactosyltransferase is indispensable for the biosynthesis of N-glycans containing Lewis a structures in Arabidopsis thaliana. Plant cell 19: 2278–2292. Strasser, R., Mucha, J., Mach, L., Altmann, F., Wilson, I.B., Glossl, J., and Steinkellner, H. (2000) Molecular cloning and functional expression of betal,2-xylosyltransferase cDNA from Arabidopsis thaliana. FEBS Lett 472: 105–108. Strasser, R., Schoberer, J., Jin, C., Glossl, J., Mach, L., and Steinkellner, H. (2006) Molecular cloning and characterization of Arabidopsis thaliana Golgi alpha-mannosidase II, a key enzyme in the formation of complex N-glycans in plants. Plant J 45: 789–803. Strasser, R., Steinkellner, H., Boren, M., Altmann, F., Mach, L., Glossl, J., and Mucha, J. (1999) Molecular cloning of cDNA encoding N-acetylglucosaminyltransferase II from Arabidopsis thaliana. Glycoconj J 16: 787–791. Szkopinska, A., Karst, F., and Palamarczyk, G. (1996) Products of S cerevisiae cis-prenyltransferase activity in vitro. Biochimie 78: 111–116.

PATHWAYS AND GENETIC DETERMINANTS

53

Takahashi, M., Inoue, N., Ohishi, K., Maeda, Y., Nakamura, N., Endo, Y., Fujita, T., Takeda, J., and Kinoshita, T. (1996) PIG-B, a membrane protein of the endoplasmic reticulum with a large lumenal domain, is involved in transferring the third mannose of the GPI anchor. Embo J 15: 4254–4261. Tanaka, S., Maeda, Y., Tashima, Y., and Kinoshita, T. (2004) Inositol deacylation of glycosylphosphatidylinositol-anchored proteins is mediated by mammalian PGAP1 and yeast Bstlp. J Biol Chem 279: 14256 –14263. Taylor, N.G., Howells, R.M., Huttly, A.K., Vickers, K., and Turner, S.R. (2003) Interactions among three distinct CesA proteins essential for cellulose synthesis. Proc Natl Acad Sci USA 100: 1450 –1455. Taylor, N.G., Laurie, S., and Turner, S.R. (2000) Multiple cellulose synthase catalytic subunits are required for cellulose synthesis in Arabidopsis. Plant Cell 12: 2529–2540. Tenhaken, R. and Thulke, O. (1996) Cloning of an enzyme that synthesizes a key nucleotide-sugar precursor of hemicellulose biosynthesis from soybean: UDP-glucose dehydrogenase. Plant Physiol 112: 1127–1134. Trombetta, E.S. and Helenius, A. (2000) Conformational requirements for glycoprotein reglucosylation in the endoplasmic reticulum. J Cell Biol 148: 1123–1129. Vainauskas, S. and Menon, A.K. (2004) A conserved praline in the last transmembrane segment of Gaal is required for glycosylphosphatidylinositol (GPI) recognition by GPI transamidase. J Biol Chem 279: 6540–6545. Vembar, S.S. and Brodsky, J.L. (2008) One step at a time: endoplasmic reticulum-associated degradation. Nat Rev Mol Cell Biol. von Schaewen, A., Sturm, A., O’Neill, J., and Chrispeels, M. (1993) Isolation of a mutant Arabidopsis plant that lacks N-acetyl glucosaminyl transferase I and is unable to synthesize Golgi-modified complex N-linked glycans. Plant Physiol 102: 1109–1118. Wang, C., Li, J., and Yuan, M. (2007) Salt tolerance requires cortical microtubule reorganization in Arabidopsis. Plant Cell Physiol 48: 1534–1547. Wang, J., Elliott, J.E., and Williamson, R.E. (2008) Features of the primary wall CESA complex in wild type and cellulosedeficient mutants of Arabidopsis thaliana. J Exp Bot 59: 2627–2637. Wang, T. and Hebert, D.N. (2003) EDEM an ER quality control receptor. Nat Struct Biol 10: 319–321. Wenderoth, I. and von Schaewen, A. (2000) Isolation and characterization of plant N-acetyl glucosaminyltransferase I (GntI) cDNA sequences. Functional analysis in the Arabidopsis cgl mutant and in antisense plants. Plant Physiol 123: 1097–1108. West, G., Inze, D., and Beemster, G.T. (2004) Cell cycle modulation in the response of the primary root of Arabidopsis to salt stress. Plant Physiol 135: 1050–1058. Wheeler, G.L., Jones, M.A., and Smirnoff, N. (1998) The biosynthetic pathway of vitamin C in higher plants. Nature 393: 365–369. Yang, L., Tang, R., Zhu, J., Liu, H., Mueller-Roeber, B., Xia, H., and Zhang, H. (2008) Enhancement of stress tolerance in transgenic tobacco plants constitutively expressing AtIpk2beta, an inositol polyphosphate 6-/3-kinase from Arabidopsis thaliana. Plant Mol Biol 66: 329–343. Zablackis, E., York, W.S., Pauly, M., Hantus, S., Reiter, W.D., Chapple, C.C., Albersheim, P., and Darvill, A. (1996) Substitution of L-fucose by L-galactose in cell walls of Arabidopsis mur1. Science 272: 1808–1810.

3

Transcription and Signaling Factors in the Drought Response Regulatory Network Matthew Geisler

Introduction

Drought, even if only one day mild water stress, affects the expression of more than 1,000 genes in Arabidopsis and rice. Some of these expression changes are the unplanned consequences of drought, as the loss of water leads to damage or malformation of proteins and membranes, which in turn impacts metabolism, cell division, and other cellular processes. Other changes to gene expression are part of a highly regulated survival strategy that confers at least some drought tolerance, avoidance, or resistance. Here we separate tolerance as the ability to continue biological processes during water deficit; resistance as the ability to survive drought such as by shifting to a dormant state; and avoidance as an escape mechanism either by alleviating the drought symptoms, or by giving up on the somatic tissue, speeding up reproduction and surviving as seeds. Plants may possess one or more of such strategies, and the regulatory system is thus faced with a decision as to which survival strategy to employ. This decision is probably based on the severity of the drought, but also could involve other factors, such as the current developmental stage, the level of metabolic reserves, and also the ability of the plant to predict the nature of the stress it faces using memory (physiological or genetic) and environmental indicators other than the drought stress itself (i.e., light level, temperature, time of year, etc). Although most of the components of drought signaling and response seem to be conserved across land plants, the wiring of these decision-making circuits, the weights assigned to each input value, and the threshold values for arriving at different decision states are probably quite variable. Thus, each species may have a unique policy for dealing with drought stress. Drought Stress Perception

A number of different pathways for the sensing and signaling of drought stress have been explored. These involve signaling molecules such as reactive oxygen species (ROS), calcium, the sugar trehalose, and the hormones abscisic acid and methyl jasmonate (Bartels and Sunkar 2005; Kaur and Gupta, 2005; Huang et al., 2008; Wasilewska et al., 2008; Paul 2008; Sakamoto et al., 2008; Ge et al., 2008). A number of signaling proteins have also been identified, including members of many known transcription factor families (i.e., AP2, basic leucine zipper [bZIP], ZAT, WRKY; Uno et al., 2000; Kim et al., 2004; Amoutzias et al., 2006; Li et al., 2008; Schutze et al., 2008; Jiang and Deyholos, 2009) and protein-protein interactors (i.e., map kinases, calcium-binding proteins; Bartels and Sunkar 2005; Menges et al., 2008). Despite identification of many of the signaling components and downstream regulators, attempts to discover the drought sensor itself have only recently been successful. Genes for Plant Abiotic Stress Editors Matthew A. Jenks and Andrew J. Wood © 2010 Blackwell Publishing ISBN: 978-0-813-81502-2

55

56

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

Although it is possible that there are many ways in which plants sense drought, at least one of the drought sensors appears to be a histidine kinase (AtHK1), part of a two component signaling complex also found in other organisms as a membrane-bound sensory system (Urao et al., 1999). Overexpression of AtHK1 increases drought resistance, while knockout mutants are less tolerant (Wohlbach et al., 2008). The expression of the AtHK1 gene is osmotically regulated and abundant in roots. The orthologous gene in yeast (Sln1) also appears to encode an osmotic stress sensor. A transcriptomic study of knockout and overexpression lines showed that AtHK1 is a regulator of NCED4, a gene controlling a critical step in ABA biosynthesis, and ABI2, involved in ABA regulation, linking AtHK1 to ABA. Other ABA biosynthetic genes and ABA responsive genes are also affected by overexpression or loss of AtHK1. Meta-analysis of expression patterns of more than 400 stress microarrays showed co-expression of AtHK1 with several ARR (Arabidopsis response regulator) genes, which interact with the (pseudo response regulator) PRR genes central to the circadian clock (Harrisingh and Nitabach, 2008). This is not surprising because ABA responses are clock gated, and co-expression of the drought sensor with the circadian clock may explain some of this gating, especially if the expression or output of the receptor was gated by or under the direct control of the circadian clock rather than the other way around (co-expression does not inform us of the direction of causality). Early stages of the drought signaling and tolerance in Arabidopsis use many of the same genes found in the desiccation tolerance strategy of seeds (Santos-Mendoza et al. 2008). If all seeds require a desiccation-tolerant dormant phase, this would mean all seed plants have the potential genes for drought tolerance. Differences between drought tolerant and drought sensitive seed plants would be in the vegetative regulation of these pre-existing genes. The AtHK1 receptor is involved in the activation of ABA biosynthetic genes in late seed development (Wohlbach et al., 2008). ABA treatment of seedlings, other abiotic stresses, including cold, osmotic, salt, ozone, and biotic stresses like infection by bacterial or fungal pathogens, induce global gene expression patterns that are highly correlated to drought stress, especially early in stress response (see Table 3.1). This indicates that many of the genes involved in ABA response and these other stresses are the same. It is likely that a single complex network of decision-making regulators chooses a survival strategy by integrating the inputs from multiple stress sensors. This circuit is also connected to the circadian clock and likely receives inputs by determining the level of metabolic reserves (sucrose, starch) through trehalose signaling (Kamin et al., 2007; Iordachescu and Imai, 2008; Ge et al., 2008; Paul et al., 2008), and current stage of development for example through ABA and auxin signals via ABI3, ABI5, LEC, and FUS genes (Santos-Mendoza et al., 2008). As we will delve into the regulatory circuitry discovered, we must necessarily include intersections with other networks that directly integrate with drought, and perhaps begin detailing this single decision-making network.

Systems Biology Approaches

The application of bioinformatics analysis to whole genome and post-genome high throughput data has made possible a top-down view of biological systems (see Yuan et al., 2008 for overview). This is a comprehensive view of all genetic and metabolic components simultaneously as the plant is perturbed by stresses. It suffers from false positive discoveries (incorrect identification of a component or interaction) just as bottom up approaches suffer from false negatives (missing or leaving out major components). The systems biology approach to drought-stress regulation begins by first identifying all components involved. The easiest way to do this would seem to be identifying all genes that are drought induced or suppressed (Figure 3.1, bottom left). This assumes (wrongly) that

Table 3.1 Correlation of drought stress transcriptome with other stresses. For color detail, please see color plate section.

57

58

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

Individual microarray experiment

Individual microarray experiment

Normalization

Microarray matrix

PCC

Global similarity

Individual microarray experiment

SA HC

Regulated gene list

NV

Co-expressed network

SOR

Regulatory motifs

Co-expressed genes

Figure 3.1 A typical top-down transcriptomic meta-analysis workflow. Individual microarray experiments undergo statistical analysis (SA) to determine which genes have had significant changes to expression between experiment and control groups. In order to compare microarrays, they are normalized and globally scaled so that the genomic average expression is set to an arbitrary value (usually 100 units). The microarray matrix is the assembly where each column is an individual scaled microarray dataset (there can be up to thousands of such datasets), and each row is one gene. The contents of each element in the matrix (X, Y position) are either the normalized scaled signal, or the log (base 2) of the ratio of experiment over control (known as the m-value). Columns can be run through pairwise linear regression using the Pearson correlation coefficient (PCC) to determine the similarity of global expression pattern (all genes) between experiments. Hierarchical clustering (HC) can be used on rows to determine which genes have similar patterns of expression. Network visualization (NV) of co-expressed genes usually employs a distance cutoff to decide if an edge (co-expression) should be drawn between two nodes (genes), and then presents the entire genome as a network.

any gene thus regulated must be part of the survival strategy. Thousands of genes are affected by drought, and there are different cohorts of genes induced or suppressed at different times after the onset of water deficit. Some of these genes must be part of the drought regulatory network (Shinozaki et al., 2003) and others part of the survival strategy, so it is likely this gene list is enriched for such genes; however, identifying candidate genes from the background of genes changing expression indirectly or as a result of dysfunction is a challenge. A more focused strategy groups genes by expression pattern across an expression matrix, either a time course or an array of different stresses and tissues to produce a gene co-expression network (Figure 3.1 lower right). These co-expressed genes thus have similar responses to abiotic stimuli, and are assumed to be part of the same regulon (group of co-regulated genes), and thus may have the same transcription factor or factors controlling them. Co-expression maps have been generated for Arabidopsis, and are becoming a tool for systems biology approaches, with the caution that genes with co-related expression are not necessarily working for the same cause. A co-expression map was employed to study co-regulated components of the drought sensor ATKH1 (Wohlbach et al., 2008). This was done by assembling a meta-database of some 444 different microarray experiments on which ATHK1 gene was present. The expression

TRANSCRIPTION AND SIGNALING FACTORS IN THE DROUGHT RESPONSE REGULATORY NETWORK

59

values (M-values is the log base 2 of the expression ratio of treated versus control) were globally scaled and normalized to allow comparison as a matrix of 444 arrays by 22,800 genes. Hierarchical clustering of this matrix identifies small co-expressed clades (groups) surrounding ATHK1, with the presumption that any gene that is tightly regulated with the drought sensor must either be drought regulated (i.e., downstream) or else regulating the sensor itself (i.e., upstream). This tightly regulated cluster included several response regulator genes (ARR4, 5, 6, 8, and 9), an unknown gene (At4g37080), and an 18S ribosomal RNA. The co-expression of ARRs, known to interact with the circadian clock (Harrisingh and Nitabach 2008; Más 2008) leads to the assumption that ATHK1 is then connected to the clock, and thus drought regulation is connected to this sensor. This type of transcriptomic meta-analysis study (as outlined in Figure 3.1) is increasingly common, especially for regulatory systems and metabolic pathways. An individual microarray experiment can be used to generate lists of induced and suppressed genes. After normalization, microarrays can be merged from all available sources into a single matrix. Currently there are now more than 2,000 different published array datasets for Arabidopsis, and more than 100 for rice either at TAIR (www. arabidopsis.org), NASC (www.arabidopsis.info), or gene expression omnibus (GEO; http://www. ncbi.nlm.nih.gov/geo/). Linear regression is employed to compare experiments, and yields a ranking of similarity of the global expression pattern of genes for different treatments. Thus, different treatments, mutants, and tissues can be evaluated and compared on a global level (most or all genes in the genome). Pearson correlation (or Spearman’s correlation) coefficient is then used as a metric for hierarchical clustering of genes, generating tables of co-expressed groups of genes. Genes that are co-regulated are assumed to be part of the same biological process or pathway. The strength of co-expression becomes the edge value to tie genes (nodes) into a network model of gene regulation. Co-expressed genes can also be mined for statistically over-represented sequences in promoters using algorithms such as Gibbs sampling (Thijs et al. 2001) and hidden Markov models (Bailey and Gribskov 1998). These efforts can identify cis-regulatory elements, but often discover non-regulatory motifs such as microsattelites. See Figure 3.2. An examination of all regulatory clusters of Arabidopsis has revealed a higher-level organization for nuclear and plastid regulons, and identified some 998 clusters of co-regulated genes overall (Mentzen and Wurtele, 2008). Maps of co-regulated genes using different matrixes of microarray-based expression data and different metrics (measurements of similarity of expression pattern) have been generated by several groups (Rhee et al., 2006; Kilian et al., 2007; Horan et al., 2008; Less and Galili, 2008; Matsui et al., 2008). These so far have been presented as unbiased tools available to researchers of regulatory systems to make candidate lists of potential pathway members. All of these tools so far come with the caveat that the lists of genes they generate are based on inference, and not direct experimental evidence, so caution is urged when drawing conclusions from such datasets. A complementary approach is to create a map of protein-protein interactions using high throughput yeast-2-hybrid, co-precipitation, or other methods to capture physical interactions between proteins. This effort is an attempt to capture some of the regulatory steps that do not rely on changes to the mRNA level of the regulators. Currently there is no large-scale dataset available for plants, however a predicted interactome has been constructed based on protein orthology (Geisler-Lee et al., 2007). The assumption is that if two proteins interact in yeast and their orthologs interact in Drosophila, these are conserved interactions, and the orthologs in plants probably also interact. As it turns out, and this is true in all eukaryotes studied so far, all genes in a genome are part of the same interacting pathway! There is a single interconnected web of interacting proteins, not discreet smaller pathways each belonging to a biological function. We find transcription factors, metabolic enzymes, cytoskeletal proteins, DNA replication machinery, and everything else to be part of the same pathway (e.g., compare Figure 3.3 to Figure 3.4A). Interesting, but this resulting map is not

Figure 3.2 The DREB/CBF circuit anchored with cis-regulatory elements. This view of the CBF portion of cold/drought response is centered on 4 members of DREB A-1 subfamily. Genes are shown as proteins (colored ovals) and promoters (black lines) with cis-regulatory elements (colored small rectangles). Regulators and elements are color matched. The ICE protein (inducer of CBF; blue oval) binds to the ICEr3 and ICEr4 elements (blue rectangles) in ZAT12, NAC72, HOS9, and CBFs 1-3. HOS9 binds to the HOS9r1 element (yellow ovals and rectangles), possibly acting as a repressor by displacing ICE since the elements are adjacent on the promoter. CBFs and DREB2 all bind to the DRE element (green) to induce downstream genes (light green) and the growth regulator ZAT10 (pink). CBF2 suppresses CBF1 and 3, but probably does not bind to the DRE element itself. ZAT10, which is also induced by ROS and other abiotic stresses, has 2 ICE elements, and suppresses the activity of DREbinding genes through an unknown mechanism. NAC72 is both upstream and downstream of CBFs, having both DRE and ICE elements. It binds to NAC72r1 element in CBF2, 3 and HOS9 to suppress expression. DREB2 is activated by drought, CBF4 is activated by increased ABA and interacts with ABRE binding factors (ABF). GA oxidases possess DRE elements and are activated by CBF1 to reduce GA levels, which leads to DELLA mediated dwarfism. For color detail, please see color plate section.

60

Figure 3.3 Assembly of osmosensor, ABA and DREB/CBF regulatory circuits. Individual interactions identified in this chapter were assembled into an excel file, identifying the locus identifier for each gene, and downloading attributes from the Arabidopsis information resource (TAIR), the subcellular localization database for Arabidopsis (SUBA), and input into the network visualization tool cytoscape 2.6.1 (www.cytoscape.org) using the automated hierarchical layout. Shapes represent genes (triangles as hormones), colors are localizations (blue = nucleus; red = endomembrane; green = chloroplast, tan = cytostole, pink = unknown. Edges represent interactions (arrowhead = induces or activates, bar = suppresses). For color detail, please see color plate section.

61

Figure 3.4 Systems level view of drought/cold stress network. Genes described in this chapter were identified in the predicted interactome of Arabidopsis (Geisler-Lee et al. 2007), and all interaction between, and were captured with first neighbors using cytoscape. This predicted and neighbor ’s network was then merged with known interactions from Figure 3.3 to produce a single network of 323 genes and 1,638 interactions. Genes are identified by shape (octagon = known transcription factor or DNA binding; hexagon = protein, metabolite, or hormone binding; parallogram = structural or transporter; triangle = nucleotide binding; circle = everything else or unknown) and color (blue = nucleus, light blue = mitochondria; green = chloroplast; tan = cytosol; red = endomembrane; pink = unknown). The whole network is shown (A) without gene labels, and two smaller portions show interactions near the CBF/DREB regulatory circuit (B) and the osmosensing ATHK1, ARR phosphor-relay genes and PRR genes of the circadian clock (C). For color detail, please see color plate section.

62

TRANSCRIPTION AND SIGNALING FACTORS IN THE DROUGHT RESPONSE REGULATORY NETWORK

63

itself very useful in trying to identify the connections related to drought stress regulation or any other discreet process. The best that systems biologists can do to subdivide this map into manageable portions is to identify the first neighbors (with direct interactions) of “bait” genes that we know are part of the system we are interested in (e.g., Figure 3.4B, 3.4C). Bioinformatic approaches can also attempt to identify molecular function and potentially subcellular localization of genes by discovering domains or family alignments. These are patterns within the coding sequence that are either of known molecular function (in the case of domains), or are correlated with a known function but lack direct evidence. In this way, genes are assigned a function without any direct evidence, and grouped into families. For example, the MYB transcription factor family is populated by 128 genes in Arabidopsis, all classified as transcription factors although experimental evidence exists for less than half of the members (based on TAIR 8.0 classification at www.arabidopsis.org). The remaining genes are assumed to be transcription factors as well because they have a MYB domain. Other gene families are curated with much less support. Networks in systems biology often use domain or functional assignment by alignment rather than by evidence, typically using the classifications found in the gene ontology (GO) database. These GO codes are often used as a post-hoc test of network validity in protein-protein interaction and co-expression networks. Ontology can also be used to create a network, but these are probably even more prone to false positives than co-expression. Even noisier are the bibliographic or text-mining networks that look for the co-occurrence of search terms (proteins, metabolites, signaling molecules) in the abstracts of all scientific literature and web pages (Rihn et al. 2003). This is usually a tool for curators to identify papers of interest, but lately has been proposed as a crude network reconstruction method if one wants to quickly evaluate the information contained in tens of thousands of papers without having to read each one.

Transcriptomic Studies of Drought Stress

Numerous transcriptomic studies have been carried out by subjecting Arabidopsis and other model plant species to various abiotic stresses, and then capturing a snapshot of the mRNA levels of each gene in the genome. (For recent drought transcriptome papers, see Matsui et al., 2008, Huang et al., 2008, Degenkolbe et al., 2009.) The best of these so far from a systems standpoint was carried out by the Arabidopsis Genome Expression project (AtGen Express; see http://www.arabidopsis. org/portals/expression), which conducted a series of abiotic stress time courses with root and shoot tissues harvested separately at six different time points (0.5, 1, 3, 6, 12, and 24 hours) (Kilian et al., 2007). These series, conducted in magenta boxes on agar media, included a 4 °C cold shift, mechanical wounding of leaves, lid-off dehydration, shift to media with either 150 milliMole (mM) NaCl, 300 mM mannitol, or 10 mM of one of the plant hormones. RNA extracted from these samples was then hybridized to the ATH1 DNA microarray to get a comprehensive picture of gene mRNA levels during the first day of stress. Using global scaling normalization, it is possible to directly compare the results of these experiments (see Denby and Gehring, 2005 for example). The genes induced by abiotic stress are numerous. Up to 25% of the genome is differentially regulated by any given stress. The first changes to the mRNA expression level were rapid, occurring within the first hour. Surprisingly a cohort of 59 genes including 21 transcription factors was induced no matter what stress was applied. These universal stress genes include known stress regulators ZAT10 and ERF5 as well as members of the MYB, AP2, and C3H2 zinc finger families, proteins involved in calcium ion signaling, and other signaling genes (Kilian et al., 2007; Khandelwal et al., 2008). This observation suggested that the first response to all abiotic stresses is universal, like a person

64

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

might say “ahhh” if suddenly wounded, burnt, frozen or shocked, in simple recognition that a stress has been encountered. In longer exposures to cold or drought (6−24 hours), the fraction of genes that respond to all stresses equally diminishes, and more stress specific genes are expressed. The function assignment of most genes in Arabidopsis is still hypothetical, predicted based on homology of sequence or domain motif or frequently completely unknown. There are clear patterns to expression however. Genes are grouped by the similarity of expression patterns across several abiotic stresses using hierarchical or k-means statistical clustering methods (Horan et al., 2008). In this way, 206 new genes were identified specific to a particular stress, and 104 new genes were found responsive to multiple stresses. This top-down annotation by co-regulation may help expand the list of genes that need to be considered. This list is already daunting, with more than 877 annotated stress responsive genes and 210 novel ones found by Horan and others (2008). There is a clear separation of the timing of response, with early-induced and late-induced transcription factor gene groups. Roots and shoots have the same stress expression profile for many differentially regulated genes; however, there are clear-cut groups of root-only and shoot-only responding genes. Each of these transcriptomic profiles can be thought of as the mixture of response and reaction to drought. Mathematically, a result set is a series of numbers corresponding to each gene, and can thus be directly compared to other transcriptomic profiles via Pearson correlation (PCC) or Spearman’s rank correlation (SRC) to estimate the similarity of responses. A matrix table of transcriptomes (Table 3.1) gives us a map of which stress, tissue, and time point is most similar to another. Hierarchical clustering these treatments, a dendrogram shows which stresses are most related, that is, those having the most similar patterns of gene expression. Comparing a drought time-course to other stresses, it is clear that time is the most important factor in the pattern of whole genome expression in response to stress (blue boxes in Table 3.1). Just looking at correlations within drought stress (Table 3.1 top brown rows), expression is most similar at nearby time points (compare shoot at 0.5 hours and shoot at 1.0 hours), and roots and shoot expression patterns coalign to the same time points, indicating that neither is “out of phase” (i.e., by delays caused by signal propagation). Comparing drought stress to salt stress (Table 3.1 yellow rows), there is strong correlation (PCC > 0.4) between genome expression along the same time points. That is, whatever drought stress is regulating at 1 hour, salt stress for 1 hour regulates many of the same genes the same way. This is true for cold and osmotic (blue rows) but has an especially strong correlation with wounding stress (green rows). Indeed the global expression pattern of wounding stress more closely resembles drought than any other stress, but only along the time course. Salt, cold, and osmotic stresses across all time points more broadly correlate to drought stress for 0.5 to 1 hour (notice the faint blue vertical rectangular boxes indicating high correlation across all time points in salt versus 0.5 and 1 hour in drought). Drought stress at the 0.5- to 1-hour time point also correlates (more weakly) with ozone gassing and ABA application (Table 3.1 pink rows), indicating that genes induced early in drought stress are similar to ABA- and ROS-induced genes. Methyl jasmonate correlates with drought at 3 hours, after the peak correlating time points for ABA application indicating that jasmonate-induced genes activate later in the drought response than ABAinduced genes. Pathogen infestation, by either bacteria or fungi at 24 hours post inoculation, correlate weakly with all time points in drought stress (Table 3.1, faint blue horizontal rectangles in the purple rows). This would mean that genes turned on after a day of infection are similar to genes expressed throughout drought stress, many of these being WRKY type transcription factors. The time course profile of a stress response might indicate more than just the progress of signal propagation. There are internal timekeeping mechanisms centered on the cell cycle and circadian clock. Cell cycle is tied directly to proliferation, growth and production of new organs, and organ size. Mutations reducing cell cycle result in plants with smaller and misshapen organs and abnor-

TRANSCRIPTION AND SIGNALING FACTORS IN THE DROUGHT RESPONSE REGULATORY NETWORK

65

malities in development. Severe stresses often result in similar phenotypes, and may either directly affect cell cycle, or represent the indirect effect of stress pathology, that is, the stress reduces photosynthetic and metabolic efficiency, diverts energy toward stress abatement, and results in fewer resources for growth, thus reducing cell division rather indirectly. The circadian clock is a vital part of plant short-term memory, allowing it to turn on and off genes in anticipation of abiotic events, including light, temperature, water availability, and potentially the activity cycle of herbivores and pathogens (Gardener et al., 2006). Plants with intact circadian clocks are healthier and survive better than those with mutations in clock genes (Green et al., 2002; Dodd et al. 2005). Approximately one-third of all Arabidopsis genes are influenced by the circadian clock (Covington et al. 2008), and in particular the response to ABA and MeJA hormones is highly dependent on the position of the circadian clock (Mizuno and Yamashino, 2008). The clock itself is a free-running cascade of 5 PSEUDO-RESPONSE REGULATOR (PRR) genes, which each reciprocally activate the next gene in the cascade and inhibit the previous gene (McClung 2006; Más 2008). The PRR genes encode the central pacemaker of the clock, and it is entrained daily through a phytochrome-based light signal proteins PhyA and B and PIF3 (phytochrome interacting factor) interacting with TOC1 (Más 2008). Martin-Tryon and Harmer (2008) identified XAP5 CIRCADIAN TIMEKEEPER (XCT), which also connects the light signal to the circadian clock. Mutants of XCT have altered circadian rhythm, and show different photomorphogenic phenotypes depending on light color. When plants are subjected to a cold temperature shift, the clock stops, clock-type genes and clock-regulated genes exhibit constitutive expression (Ibañez C et al. 2008; Bieniawska et al. 2008). Thus, circadian rhythm is a major factor in the regulatory pattern of cold-induced genes. Given the high degree of overlap between cold and drought stress (Table 3.1), and that ABA response is clock-gated, it is very likely that drought stress also is directly tied to the circadian clock. This may go a long way in explaining the timeresponse correlation between abiotic stresses. The controls in the Killian and others (2007) experiments were plants grown in ideal conditions and harvested at the same time as the treated plants. If the clock has been stopped by cold stress, then the differences in gene expression are a combination of stress response, and the diverging clock positions. If this is causing the correlation of stresses across the time course, then it is likely that all abiotic stresses interfere with the circadian clock, because this correlation appears systemically throughout the dataset no matter what stress was applied. Covington and others (2008) determined that a third of all expressed genes are influenced by the circadian rhythm in Arabidopsis. Sorting by hormone, ABA-responsive and methyl jasmonate (MeJA)-responsive genes were found to oscillate diurnally, but not ethylene, brassinolide, auxin, or cytokinin responsive genes (Mizuno Yamashino, 2008). Thus, ABA- and JA-mediated pathways should both be strongly altered by changes to circadian rhythm. We saw this pattern as well in the global correlation of transcriptomes (Table 3.1). Why only these hormones are tied to the clock is unknown, and as responses to ABA and JA depend on time of day, extra care must be taken in experimental design to produce repeatable results. Since drought is strongly regulated by ABA, experiments following the time course and application of drought stress must consider the time of day the stress was applied. If the processes being observed are programmed responses to drought, or to clock phase, this likely will have different outcomes depending on when the experiment is begun. Two controls for each drought experiment are thus necessary, an untreated control and a zero time point (taken at the moment just before the stress is applied). The untreated control will continue to cycle through the circadian rhythm. If drought alters the phase, the two clocks will get increasingly “out of sync,” and differences between drought and control will be due to clock cycle. The zero time control is a snapshot of the “un-droughted” plant at the same clock phase as the

66

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

treatment (initially). If the clock stops in drought-treated plants, this control will stay in sync with the drought stress, but not the untreated control. Between these two controls, it will be possible to separate clock-independent drought stress regulation from circadian clock components.

The DREB/CBF Regulon

One of the most clearly understood abiotic stress regulatory networks from both the cis-regulatory and transcription factor side is mediated through the dehydration-responsive element (DRE) and several layers of interconnecting transcription factors. Much of our understanding of this circuit has been built from the bottom-up by linking the results of promoter binding assays and mutant analysis (Riera et al., 2005; Nakashima et al., 2006; Yamaguchi-Shinozaki and Shinozaki, 2006). Studies from the top down using DNA microarrays have largely supported the established components and wiring of the CBF regulon and have added new putative components. Cold, salt, and drought stress all cause rapid (1–2 hour) expression of related AP2 type transcription factors called DREB1A-D (drought element binding) or CBF1-4 (C-repeat binding) and DREB2 (Agarwal et al., 2006). The AP2-ERBP family of transcription factors (146 members in Arabidopsis) is responsible for a diverse range of biological functions including flower organ identity (APETELA2, for which the family is named), ethylene, JA, and cytokinin response (ERF, JRF, CRF), and still has many members of unknown roles (Riechmann and Meyerowitz, 1998; Pierik et al., 2007; Nakano, 2006). The CRF (cytokinin response) type AP2 transcription factor is regulated by a histidine kinase two-component system, similar to that identified as the drought stress receptor (Rashotte, et al. 2006). A subgroup with a single AP2 repeat includes DREB (drought response), ERF, TINY (tiny root hairs), and ABI4 (abscisic acid insensitive) genes. Within this subgroup DDF1 and 2 (DWARF AND DELAYED FLOWERING), regulators of both gibberellins and salt tolerance are part of DREB subfamily A-1, which includes all four CBFs. Over-expression of either DDF gene results in reduced gibberellins, dwarfism, and increased salt tolerance. Overexpression of CBF1-4 (DREB1A-D) results in increased cold, salt, and drought tolerance through increases in proline and cytosolic sugars and expression of drought resistance genes such as LEA, late embryogenesis abundant (Gilmour et al., 2004; Kasuga et al., 1999, Huang et al., 2008; Hundertmark and Hincha, 2008; Gutha and Reddy, 2008). Overexpression of CBF genes also results in GA loss and dwarfism, similar to that of DDF genes, indicating that all subfamily members may compete for the same binding sites, and have significantly overlapping regulons. DREB2 belongs to a related subfamily (A-2) of eight genes, including two that are drought induced (DREB2A and B) and are regulators of drought tolerance genes. The cis-regulatory element DRE or LTRE is the promoter target for CBF, DREB2 (and likely DDF) transcription factors. It is specifically recognized by CBF proteins, but 1-base mismatches of the primary motif sequence are not bound in vitro. Cold and drought regulated genes are enriched for DRE elements in their promoter in Arabidopsis, rice, and the moss Physcomitrella patens (Maruyama et al., 2004; Liu et al., 2007). DREB/CBF homologs have been found in virtually all plant species indicating that this pathway is potentially common to all land plants. Using microarray data in the reverse correlation, all genes with DRE elements were first obtained based on pattern search, then that group of DRE-containing genes was assayed on several microarrays for changes in group-expression patterns using statistical analysis. DRE-containing genes are not only enriched for cold induction, but also for drought, wounding, osmotic, heat, and salt stresses, as well as the hormone ABA, sfr6 (sensitive to freezing), and cbf2 mutants, during seed

TRANSCRIPTION AND SIGNALING FACTORS IN THE DROUGHT RESPONSE REGULATORY NETWORK

67

imbibitions and strangely, in atrichoblast cells (Geisler et al., 2006; Table 3.2). The SRF genes (there are seven) all protect plants from freezing either as regulators or as cryoprotectors (i.e., SFR2; Fourrier et al., 2008). SRF6 acts post-translationally on CBF proteins or downstream as overexpression of CBF1 and does not induce the DRE regulon in sfr6-1 mutants (Knight et al., 2009). However, cold stress still does induce the DRE regulon in sfr6-1 mutants (see Table 3.2) indicating that alternative transcription factors such as CBF4 and DREB2 are unaffected. SFR6 is linked to the circadian clock through the presence of an evening element, in the promoter (Knight et al., 2008). The expression pattern for DRE-regulon (DRE containing genes), known as a cis-regulatory fingerprint (Table 3.2) describes the collective behavior for the element itself, without knowledge or bias as to the binding transcription factor. It is likely that all DREB subfamily A-1 members will bind to DRE, but so will other transcription factors, each with different binding strengths, connecting that element/gene to different pathways. By calculating the expression pattern from the perspective of the cis-regulatory element, we get a complementary view of gene regulation. This will help flesh out regulatory pathways and identify the interconnections of different signals. Using the same strategy, the allowable sequence variants were determined by a bioinformatic process called in silicio mutagenesis, in which all genes with a naturally occurring variant sequence (one base mismatch) were identified in Arabidopsis, and this variant subgroup was analyzed for enrichment of induction or suppression by cold (Geisler et al., 2006). Allowable sequence variants determined by this method agreed in in vitro binding results, in that GCCGAC and ACCGAC sequences both showed cold induction, while TCCGAC and CCCGAC did not. The orientation of the element to the gene, however, did not make a difference. The DRE element can be located up to 400 bp upstream, in the 5′UTR, or in the first few introns and still show correlation to cold induction. The correlation disappears any further upstream. The ABRE element by contrast, can be located up to 1,200 bp upstream and still show significant correlation to ABA induction, but it cannot be located in the UTR or introns (Geisler et al., unpublished results). Using this bioinformatic method, correlating the presence, orientation, position, and naturally occurring variations in sequence, a great deal can be learned about the function, mechanism, and responses of a cis-regulatory element, and a large number of false-positives can be removed (elements that occur in the wrong position and are non-regulatory). The comprehensive identification of downstream targets of CBF genes will be greatly facilitated by the analysis of DRE cis-regulatory elements. CBF genes show some interactions amongst themselves (Figure 3.2). CBF2 is a negative regulator of its paralogs, knockout of CBF2 increases expression of CBF1 and 3, but CBF2 may not bind members of the same downstream regulon (CBF1 and 3 have no DRE element). CBF2 is expressed later than CBF1 and 3 and may act to control the timing of the cold/drought response (Novillo et al., 2007). CBF3 was thought to suppress the activity of CBF2 (due to increased expression in an ice1 mutant; Chinnusamy et al., 2007); however, individual knockouts of CBF1 and 3 do not show this (Novillo et al., 2007). CBF2 is thus likely directly regulated by ICE1. CBF1 and 3 regulons significantly overlap, with some genes requiring both transcription factors for expression, but do not affect the regulation of other CBF genes. Knockouts of either CBF1 or 3 result in reduced cold and drought resistance, indicating that they do not act redundantly and that both genes are required for resistance. CBF4, a drought and ABA-induced member, is not affected by knockout or overexpression of CBF2. Upstream of the CBF gene is the ICE (Inducer of CBF) gene, a c-MYC-like basic helix-loop-helix that is phosphorylated in cold temperatures (but with no change in transcription), and binds to the ICE box (ICEr3/ICEr4) found in CBF1, 2, and 3 (Zarka et al., 2003; Benedict et al., 2006). Other ICE elements (ICEr1 and 2) overrepresented in the ICE regulon were subsequently shown not to be bioactive (Benedict et al, 2006). Light and circadian

Table 3.2 Regulatory fingerprint of drought responsive element (DRE). Experimental design

Overall p-value

Induced genes

Ind. enrich

Ind. p-value

Supp. genes

Supp. enrich

Supp. p-value

Cold stress 24 hrs shoot Cold stress 12 hrs shoot Cold stress 24 hrs root Cold stress 12 hrs root Freezing 3 hrs Cold stress 24 hrs on soil Freezing 3 hrs cold acclimated 35S:CBF2 Wounding of shoot 6 hrs sfr3 mutant 24 hrs cold stress dormant seed of cvi ecotype sfr6 mutant 24 hrs cold stress seed imbibed in light vs dark cvi ecotype fad2 mutant in cold 3 hrs dry seed vs imbibed cvi ecotype prolonged imbibition vs dry cvi ecotype sfr6 mutant 24 hrs cold wounding 3 hrs Salt stress 3 hrs shoot 3 μm ABA 24 hrs 30 °C heat shock wounding 12 hrs fad3/7/8 mutant 3 hrs cold stress Atrichoblast cells vs whole root Salt stress 24 hrs shoot 30 um ABA 24 hrs gpa1 mutant w/ABA Drought stress 0.5 hr Osmotic stress 3 hrs Drought stress 1 hr Whole root vs lateral root cap Osmotic stress 6 hrs shoot Greenhouse vs growth chamber

2.82E-34 4.12E-26 1.66E-25 6.23E-20 3.81E-18 1.64E-17 1.71E-15 1.77E-14 2.56E-12 2.02E-11 1.42E-10 1.68E-10 2.79E-10 5.20E-10 6.42E-10 6.42E-10 2.46E-09 1.44E-08 1.56E-08 1.74E-08 6.42E-08 1.22E-07 1.96E-07 2.36E-07 4.44E-07 5.11E-07 5.84E-07 6.45E-07 7.41E-07 1.79E-06 1.96E-06 2.15E-06 2.39E-06

206 158 159 126 123 133 114 105 109 131 204 132 149 72 193 171 205 80 113 172 109 107 80 166 128 183 108 111 132 105 149 151 87

2.15 2.17 2.11 2.15 2.10 1.98 2.00 2.04 1.89 1.73 1.52 1.66 1.64 2.02 1.50 0.79 1.47 1.90 1.69 0.82 1.68 1.65 1.75 1.48 1.54 0.86 1.63 1.55 1.49 1.52 0.91 1.44 1.49

3.21E-33 9.93E-26 5.77E-24 5.72E-20 1.62E-18 4.50E-17 4.04E-15 9.08E-15 2.34E-12 2.61E-11 4.62E-11 5.25E-10 6.55E-11 3.96E-10 7.25E-10 0.00037 9.19E-10 1.96E-09 3.49E-09 0.00290 8.66E-09 4.51E-08 1.54E-07 3.60E-08 2.12E-07 0.026 8.62E-08 8.98E-07 8.60E-07 5.95E-06 0.20 1.03E-06 9.72E-05

91 72 87 62 56 58 36 34 50 67 170 65 112 31 171 193 55 49 56 209 93 39 21 114 55 192 80 14 51 11 191 87 21

0.61 0.65 0.62 0.69 0.72 0.65 0.61 0.68 0.70 0.71 0.81 0.68 0.83 0.68 0.79 1.5 0.74 0.91 0.82 1.43 0.94 0.75 0.64 0.89 0.70 1.41 0.96 0.54 0.71 0.46 1.40 0.80 0.48

2.99E-07 0.0001 1.22E-06 0.002 0.011 0.0006 0.0020 0.02 0.008 0.003 0.002 0.001 0.03 0.02 0.0003 7.25E-10 0.02 0.50 0.12 7.50E-09 0.53 0.074 0.03 0.195 0.041 1.13E-07 0.763 0.018 0.0124 0.0076 3.00E-07 0.0297 0.00044

The regulatory fingerprint for extended DRE ([A/G/T][A/G]CCGACN[A/T]) in the 500-bp upstream promoter region. There are 2,381 genes with the extended DRE element in the 500-bp upstream of the transcription start site, the region in which DRE is statistically enriched and demonstrated to be functional. Looking at each transcriptome (microarray experiment), of these DRE genes, 851 are present on microarrays. The number of induced, suppressed, and neutral genes with DRE is compared to expected values based on the genome as a whole. The enrichment is the ratio of observed versus expected genes. An overall p-value is determined from a chi-squared test (2df) between observed and expected numbers of genes, and specific p-values (1df) were calculated for induced/not induced and suppressed/not suppressed genes. Treatments with a significant p-value are thus enriched (enrichment >1), or depleted (enrichment <1) for differentially regulated genes. In many cases, there is significant enrichment for induction and significant depletion of suppression (such as Row 1) indicating a one-directional regulation of the DRE element. Not all genes with a DRE element are drought/cold/osmotic stress induced, as other elements in their promoter bind to different transcription factors that may squelch or override CBF or DREB2 inputs.

68

TRANSCRIPTION AND SIGNALING FACTORS IN THE DROUGHT RESPONSE REGULATORY NETWORK

69

rhythm gating may take place through interactions between ICE and phytochromes PhyA and PhyB based on mutants and regulon overlap (Kim et al., 2004; Benedict et al., 2006). ICE phosphorylation has been shown to induce expression of CBF3 (Chinnusamy et al., 2007; Nakashima and Yamaguchi-Shinozaki, 2006). ICE boxes also appear in the promoters of transcription factors NAC072 and ZAT12. The ICE cis-regulon (all genes containing H[A/C]CACGT or in the upstream promoter) responds significantly to cold, drought, ABA, and salt stress. This indicates that steps downstream of ICE signaling are dependent on ABA, which is not surprising because the ICE regulon is highly integrated with other stresses. Time course microarray data show that genes with the ICE box in their promoter become significantly cold induced 1 hour after treatment, while genes with the DRE element are not significantly cold regulated until 6 hours after treatment (Benedict et al., 2006). This establishes the temporal separation of ICE box and DRE cis-regulons, and confirms the placement of ICE upstream of CBF transcription factors. ZAT12 is a repressor of CBF, but importantly is involved in reactive oxygen (ROS), heat, and high light stress regulation (Vogel et al., 2005; Davletova et al., 2005a, 2005b). The regulation of ZAT12 by ICE closely links excess light and cold stress perception pathways. This is important because cold temperature greatly exacerbates stress on photosynthetic machinery by high light levels. HOS9 also acts upstream of CBFs. It contains an ICE box and a NAC072 target. The HOS9 regulon significantly overlaps ICE, and is enriched for the cis-regulatory element [A/C]CGCGT. This element is correlated with cold at 3 hours post-treatment (temporally 2 hours later than the ICE cis-regulon, but prior to the CBF cis-regulon), and drought stress induction after 0.5 hours, temporally much closer to stress perception. HOS9 cis-regulon is only weakly correlated with ABA, and not at all to salt stress. ICE and HOS9 elements are quite similar in sequence, differing only by 1 nucleotide, and occurring adjacent to one another in HOS9 and CBF2 promoters. One possible interacting mechanism is that both transcription factors compete for the same sites with different efficiencies. HOS9 is also autoregulatory, with both HOS9r1 and ICE box elements present in the HOS9 promoter. ICE also activates NAC072, which is in turn a suppressor of CBF2, CBF3, and HOS9. NAC072 contains ICE box and DRE elements, so it is difficult to assign it as upstream or downstream, but rather the wiring seems a little more complex. As ICE directly activates both NAC072 and CBF3, there is an initial induction of CBF3, followed by a later suppression via NAC072. This network arrangement is known as an incoherent feed forward loop, and is one of the most overrepresented motifs in biological networks (Dekel et al., 2005). CBF2 is induced by ICE, but suppresses CBF1 and 3, creating a second incoherent FFL, possibly as a damping or noise filter to prevent regulatory runaway. This design is typical of time-dependent regulation, such as the negative feedback loops found in circadian clock circuits. Downstream of the ICE/NAC072/CBF machinery are genes with DRE elements, such as RD29A, one of the dehydration protecting LEA (late embryogenesis abundant) genes. Many LEA genes have both ABRE and DRE elements, thus fall under regulation by both cold and ABA (Narusaka et al. 2003). DRE elements are found in other regulators such as ZAT10/STZ. ZAT10 is a suppressing regulator of photosynthetic and primary metabolic genes, and induction of ZAT10 by the upstream drought/cold sensor circuitry can act to slow plant growth. Gain or loss of ZAT10 leads to increased drought tolerance (Mittler et al., 2006). CBF1 overexpression also reduces the level of gibberellins by inducing expression of GA 2-oxidase genes, which in turn increases the amount of DELLA proteins (Achard et al., 2006). GA causes the proteolytic degradation of DELLA. GA is thus linked to cold-induced dwarfism and late flowering, as mutants lacking GAI (gibberellins insensitive) and REPRESSOR OF GA1-3, both DELLA proteins, do not show CBF1-induced dwarfism (Willige et al., 2008). Upstream of the CBF/DREBs is also a signaling cascade beginning with PI-4 phosphate and PI4P5 kinase. IP3 and calcium signaling causes the

70

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

drought stress activation of DREB2 (Gupta and Kaur, 2005; Perera et al., 2008). DREB2 overexpression does not improve drought tolerance, although it is drought induced (Nakashima and Yamaguchi-Shinozaki, 2006). DREB2 is negatively regulated through a domain in the central portion of the protein, and overexpression of a constitutively active DREB2 does improve drought tolerance through the activation of dehydration inducible genes, including several that are not regulated by the CBF/DREB1 pathway. This indicates that DREB2 is part of a parallel drought-sensing pathway that intersects with CBFs by competing for binding to the DRE element. CBF4 is not part of the ICE-induced pathway, is not known to interact with any of the components described thus far but is ABA regulated. CBF4 thus begins to activate the DRE element cis-regulon at a much later time point (12 to −24 hours), possibly to augment CBF1-3 activity in a more prolonged water deficit. Microarray studies have been extensively used in these studies and complement forward and reverse genetics approaches. Microarrays have been made for overexpression and knockout of ICE, ZAT, CBF, as well as cold and drought time courses (Lee et al., 2005; Maruyama et al., 2004; Davletova et al., 2005; Killian et al., 2007). A regulon in the broadest sense is the list of all of the genes downstream of a regulator, induced or suppressed (in comparison to wild type) by the knockout and overexpression of the transcription factor. The simplest gene lists are those induced by the overexpression and suppressed in the knockout (or vice versa), but some genes appear to be induced or suppressed by both, or else inexplicably neutral (no change) in one mutant, but regulatory in the other. This latter case indicates a more complex regulatory logic such as partial redundancy or the fact that the gene in question is only indirectly related to the regulator. In the narrow sense, a regulon consists of only genes immediately interacting with that regulator. A cis-regulon is an attempt to predict the narrow sense regulon by creating a subset of the induced and suppressed genes that contain a known or predicted cis-regulatory element, preferably one that occurs within the operational window (location within the promoter where the element is bioactive). Both broad and narrow sense regulons can be compared to determine degree of overlap; for example, if a regulator is truly downstream and epistatic to another regulator, its narrow regulon should be entirely contained within the broad regulon of the higher regulator. A complete picture of the DREB/CBF section of the drought-sensing network can therefore be reconstructed from both the top down and bottom up. Sensors (Figure 3.3; top row) perceive drought and cold stress perhaps independently. There is likely more than one sensor for each stress, possibly different mechanisms of sensing, as this would make the system more robust and less sensitive to noise. Robustness is also created by the negative regulators (CBF2, ZAT12, NAC072) working antagonistically with the input signals from ICE and HOS9. A temporal order is observed (first ICE, then HOS, finally CBFs) in both transcription factors and their cis-regulons, which may be explained in part by the incoherent feed forward loop wiring. ABA is clearly involved somewhere in this circuitry, as drought and cold stress, and regulons of ice, cbf, and zat12 mutants significantly overlap with ABA-induced transcriptomes. In the downstream signaling pathways and wiring, we can establish connections on the regulator side (looking at downstream targets of each regulator) and also by looking at the promoter side (establishing the regulatory fingerprint of the cis-regulatory element). A confident connection can then be established by both the presence of functionally annotated CREs and co-activated or co-expressed transcription factors. There are also many unknown nodes in the system that have not been identified in bottom-up analysis. From the top down, there are still hundreds or perhaps thousands of drought-induced genes (nodes) and interactions (edges) discovered by high throughput experiments, including some key regulators and steps among the noise, but are not yet integrated into our models.

TRANSCRIPTION AND SIGNALING FACTORS IN THE DROUGHT RESPONSE REGULATORY NETWORK

71

ABA Signaling

Abscisic acid (ABA) is a hormone whose synthesis, transport, and perception form essential links in many stress and developmental pathways (see Wasilewska et al., 2008 for review). Although it is often thought of as a drought/osmotic stress hormone, or a general stress hormone, it is prevalent in so many processes that it is more likely an integration step for multiple pathways. The level of ABA in any given cell is the summary of the outputs from different biotic and abiotic stress pathways, as well as developmental programs. How competent a cell is to respond to ABA, dependent on expression and activity of ABA receptors and signaling machinery, is also regulated and part of the integral survival strategy. ABA is synthesized from abscisic aldehyde by the ABA3 and AAO enzymes, which is synthesized from xanthoxin by ABA2 (Nambara and Marion-Poll, 2005). This pathway branches off from β-carotene synthesis in which ABA1 turns zeaxanthin into violaxanthin, and then ABA4 or NCED convert to neoxanthin. ABA is transported as an active hormone, or an inactive hormone bound to glucose, which can be collected into inactive pools and subsequently released rapidly by β-glucosidase (AtBGL1; AT1G52400) during a stress response (Lee et al., 2006). When a plant is not stressed, ABA can be normally found in a tissue-specific distribution, with concentrations high enough to drive ABA-specific promoters carrying reporter genes in guard cells, collumella and quiescent center in the root, veins, and the shoot apical meristem suggesting a role in development and tissue organization of the vegetative plant. ABA is also induced during seed development, specifically during desiccation and dormancy stages. To what degree changes in ABA pattern during development and stress perception are due to transport or local production is still somewhat unknown, although biosynthetic genes are normally expressed. The identity of the ABA receptor is somewhat more elusive, and it is likely that there are multiple pathways for ABA perception, and that ABA signaling may be essential to early development, in such a way that a knockout mutant is embryo lethal. The ABAP1 protein isolated from barley is thought to be an ABA receptor, however the Arabidopsis homolog FLOWERING LOCUS C (FLC; AT5G10140) does not have a significant phenotype when knocked out, possibly due to a redundancy. An auto-regulatory splicing step in FLC mRNA processing FLC protein interacts with FY (AT5G13480) in an ABA-dependent manner to correctly splice FLC transcript. Other candidates such as GUN5 (genomes uncoupled, a chloroplast localized magnesium chelatase) may have parallel roles in ABA sensing and connect ABA to different pathways downstream, such as in chlorophyll pigment biosynthesis (Wasilewska et al., 2008). The cis-regulatory element ABRE is bound by a subfamily of bZIP transcription factors known as ABREBs or ABFs (see Kim, 2006 for analysis of the family). Variants of the basic core sequence (ACGT) may bind to different bZIP family members, including the G-box (CCACGTGG), ABRE (ACGTGTC; Hattori et al., 2002), and TGA type ROS-regulated bZIPs binding to AS-1 element (TGACGTCA; Garretón et al., 2002). Each of these variations of flanking nucleotides may confer favorable binding to specific bZIP family members, though a certain amount of cross binding is expected (Suzuki et al., 2005). This choice of dozens of transcription factors, each binding to numerous variant cis-regulatory elements, perhaps each with a different strength, makes a multiplexing switchbox called a regulatory dense overlapping region (DOR) in network parlance and is typical of decision making circuits with multiple inputs (Alon, 2008). ABRE elements function if the motif is present anywhere in the first 1,200 bp upstream of the transcription start site (Geisler et al., 2006), but do not confer ABA induction if found in the UTR or introns, unlike DRE (Geisler, unpublished data). There are 2,501 Arabidopsis genes bearing the ABRE element in the first 1,000 bp upstream, and they as a group are more likely (compared to the rest of the genome) to respond to a large number of biotic and abiotic stresses, most notably ABA, osmotic stress, and drought (Geisler et al., 2006). These genes

72

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

are also suppressed by glucose, during seed imbibitions and in roots, induced in flowers, and during seed set and show strong circadian influence. It seems that ABA is involved in too many processes to be considered part of a drought-response or even a general stress response. The hormone is likely an integrator of many different inputs, each increasing or decreasing the intracellular ABA level, which acts as an additive function and short-term memory.

Reactive Oxygen Signaling

Hydrogen peroxide is an active signaling molecule in most biotic and abiotic plant stresses, and is also a secondary messenger in animals and fungi (Quan et al., 2008). Free oxygen radicals are normally produced by both chloroplast and mitochondria as by-products of metabolism, but can be generated deliberately by the plant cell through calcium signaling (Vandenbroucke et al., 2008). ROS begins as singlet oxygen produced in mitochondria and in chloroplasts. Knockout of mitochondrial alternate oxidase (AOX1a) leads to severe drought sensitivity, presumably due to increase in ROS generation (Giraud et al., 2008). Singlet oxygen is converted rapidly to superoxide, which itself is dangerously unstable and reactive. Superoxides from these and other reactions are converted to hydrogen peroxide, a slightly more stable form by superoxide dismutase. Hydrogen peroxide is long lived enough to travel to adjacent cells and act as signal, but is also highly reactive and damaging. Peroxide is an evolutionarily conserved signal, found in all kingdoms (Vandenbroucke et al., 2008). It is scavenged by a variety of antioxidant pathways, including ascorbate and glutathione peroxidase, and catalase. These act as detoxifying agents to prevent cellular damage, but also serve to attenuate and modulate the ROS signal. Although microarray-based studies show only a R = 0.12 peak correlation between drought stress and hydrogen peroxide treatment, a number of known key drought network genes are common to both datasets. Hydrogen peroxide activates DREB2A, ZAT12, and two drought responding NAC-type transcription factor genes (At5g63790 and At1g77450). Drought and salt stresses produce an increase in intracellular ROS. It is unclear whether this increase in ROS is part of the signaling, part of the pathology of drought stress, or both. Plants overexpressing superoxide dismutase (SOD), catalase, and glutathione S-transferase (GST) are resistant to salt stress. How extracellular ROS is sensed is currently unknown but is predicted to be a membrane-based receptor or alternatively by the detoxifying enzymes themselves. The signaling is more clearly understood, using a map kinase cascade MAPK3 and MAPK6, ANP1, OXI1. These MAPK components are drought induced (Xiong et al., 1999; Menges et al., 2008). Intracellular ROS causes changes in the redox state of the cellular glutathione, and LSD1, NPR1 bZIP transcription factors (Foyer and Noctor, 2005). ROS signaling feeds into and ties together much of the biotic and abiotic stress responses.

Integration of Stress Regulatory Networks

The simplest form of regulation is that of an auto-regulating enzyme, which is itself the sensor, the regulator, and the adaptive response. It is theoretically possible for all proteins involved in a stress response to be of this type, acting independently and in parallel to produce an adaptive response. However, this is not likely to be the case especially in eukaryotic cells. Separation of these three functions into a linear regulatory pathway has been the paradigm for models of stress gene regulation in which a membrane sensor or receptor protein triggers a regulatory protein, which

TRANSCRIPTION AND SIGNALING FACTORS IN THE DROUGHT RESPONSE REGULATORY NETWORK

73

then activates one or many downstream adaptive response genes (i.e., see Mahajan and Tuteja, 2005). More recently, linear pathways have been shown to have a shared node, which is a gene or protein that technically belongs to two or more pathways. One such example is the WRKY30 transcription factor, a regulator responding to drought stress, cold stress, pathogen attack, wounding, and several hormone pathways probably via reactive oxygen species signaling. The WRKY family of transcription factors is greatly expanded in land plants, and may be regulator nodes unique to plant abiotic stress adaptation (Zhang and Wang, 2005). In rice, overexpression of WRKY13, encoding a regulator of bacterial and fungal pathogen resistance, suppressed salt and cold stress, and showed interactions with developmental pathways and secondary metabolism (Qiu et al., 2008). These nodes provide cross talk between pathways, and more importantly function as integrators, which sum up the total signal coming from a variety of different sensors or connected pathways. Because post-genomic strategies have been employed to comprehensively identify all the components of regulatory pathways in plants, it is becoming increasingly obvious that such integrators are common. Interconnections between ethylene, jasmonic acid, salicylic acid, light- and pathogen-sensing signaling pathways have become sufficiently complex that a Boolean road map or wiring diagram (Devoto et al., 2005) is now essential when discussing them. Rather than speaking of separate linear pathways with the occasional cross talk, the new paradigm is that regulation is more deeply and perhaps holistically interconnected, and it is perhaps not surprising that it should be. When a plant experiences drought stress, a number of physiological and metabolic changes occur. Some of these changes are part of the pathology (damage) caused by the stress. Other changes are adaptive responses by the plant in order to ensure survival either of the plant itself (the soma), or that of the germ line through speeding up of reproduction and seed set. In order to gauge the level or type of response, the plant must be able to perceive the degree of all stresses impacting its survival, and also the current metabolic status and availability of stored reserves, and perhaps even include memory or some predictive capacity. A complex interacting network of regulatory proteins and transcription factors likely performs this survival strategy calculation. The core components of this network have been identified from the bottom up, through forward and reverse genetics and identification of potential cis-regulatory elements through in vitro binding and promoter reporter genes. Several preliminary models of the abiotic stress network have been made by identifying key genes that have an effect on drought tolerance when mutated, and through discovery of droughtinduced genes. More recently, these efforts have been paired with top-down approaches to seek and simultaneously identify all components of the drought response network by using global gene expression analysis, such as transcriptomic, proteomic and more recently interactomic datasets. An unfortunately large number of genes significantly changes expression upon drought stress. The genes change with time after stress, and are dependent on tissue and developmental stage. Mixed into these lists of drought regulatory and response genes are likely those indirectly drought induced, not part of the regulatory network, but rather are part of the pathology. Inducing this latter category of genes transgenically will more likely re-create the pathological symptoms of drought rather than act as a remedy. Although nobody knows how big the drought regulatory network is, it is probably large and highly integrated with other networks for stress, metabolism, and development. Systems biology is the field that addresses such large and complex networks and attempts to return from molecular and genetic roots back to a comprehensive and useful understanding of organismal physiology. Given the high degree of overlap and interaction, it is likely that a systems biology approach that joins together many areas of plant biology beyond drought stress will succeed in building a clearer picture of drought stress regulation.

74

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

Assembling the Known Pathways and Expanding Using Gene Expression Networks’ Predicted Protein Interactions

Systems biology is all about assembling pathways and linking them together into a network so that the emergent properties (i.e., phenotype) can be predicted based on analysis of perturbations to that network. In this way, a working network view can be used to predict the consequences of a null mutant or overexpression of one component (gene or interaction). Unfortunately, it seems that systems are not isolated from one another. A protein interaction map of yeast links the entire genome into a single network, rather than separate networks for separate functions. There is even no modularity in the yeast network, no clearly visible clumps or modules of interacting genes that share a common biological role. The structure of the yeast interactome has been described as like stratus clouds (the thin wispy ones at high altitude) rather than cumulus ones (the lumpy white rain clouds Batada et al., 2006). Preliminary analysis of the predicted Arabidopsis interactome by Geisler-Lee and others (2007) has come to much the same conclusion. So a truly comprehensive integrated view of drought stress is by necessity also a view of the entire plant genome. To make sense of things, however, we humans must create smaller windows through which to see and understand regulatory patterns. As a first approximation, we limit our view to the genes mentioned thus far in this chapter. If we consider only ATHK1, the ARR genes downstream and biosynthetic genes of ABA, then add the known components of the DREB/CBF pathway, and a few genes involved in GA oxidation, DELLAs, and GA response, we get a network view pictured in Figure 3.3. We can roughly block out areas of ABA biosynthesis, drought perception, signal transduction through the phosphorelay (ARR genes). From ABA we connect to the DRE/DREB network via CBF4 and the ABRE elements in late embryogenesis abundant (LEA) genes represented here by RD29A. This view, although easy to follow, is a gross simplification and leaves out other connections, such as light and ROS regulation of ZAT12 and NAC72, as well as many other known and predicted interactions of these components. A more comprehensive view, but still incomplete, is constructed by adding all predicted and known interactions using the Geisler-Lee and other (2007) dataset, which combines all known protein-protein and some known protein-promoter interactions mined from the literature with about 70,000 predicted interactions based on orthology from other species. Predicting protein interactions is tricky; the primary dataset is high throughput (HTP) yeast 2 hybrid (1/3), co-immunoprecipitation (1/3), and other techniques such as FRET, reconstituted complexes, synthetic lethality, phenotype enhancement or rescue, and so on. The primary dataset thus is only as good as the techniques, and can contain false positive results, so a confidence value is built based on the number of times the interaction is replicated and the number of different experimental techniques that detected it. A predicted interactome is built by first identifying all orthologs between the organism of interest and the reference genome (with the HTP data). Where orthologs in the reference organism interact, that interaction is mapped to the organism of interest. A confidence value is also built, and this time includes the number of reference species in addition to replications and experiments. If we use this dataset to discover all interactions known and predicted, between the genes in Figure 3.3, and add to that all first neighbors (all genes interacting with genes in Figure 3.3), we get a much larger pathway map (Figure 3.4). This expanded pathway now includes 323 genes, including GI and all PRR (circadian clock genes), MEK1 and MPK6 map kinases, ethylene pathway genes EIN1 and CTR (Figure 3.4C), HOS1, and several unknown BHLH and WD40 genes (Figure 3.4B). There are more than 1,600 known and predicted interactions between these genes, much more than can reasonably be followed by a human eye. Superimposing a gene expression network (GEN) from Ma and coworkers (2008) produces much the same type of map. These interactions are probably full of false positives, and that can be controlled somewhat

TRANSCRIPTION AND SIGNALING FACTORS IN THE DROUGHT RESPONSE REGULATORY NETWORK

75

by restricting our view to increasingly high confidence value edges, at the cost of dropping some real interactions. How then do we proceed? Should every interaction be verified experimentally? Should we begin to make predictions based on the map at hand? Is drought stress regulation much simpler, and have we needlessly complicated it by this kind of global analysis? What if drought stress really is this complicated? How do we process the knowledge generated here, study the system and make useful predictions, or design useful mutants that have improved drought tolerance or resistance? The programs that generate these data can also be employed to study it. Mapping of regulatory topology, analysis, and modeling of complex systems are beginning to emerge as common tools in the field of systems biology (Albert, 2007; Palsson, 2006; Alon, 2008; Yuan et al., 2008). This is a mathematical and computer science exercise, but draws data from wet-bench as well as high throughput (chip/column) research. Gene regulation in the near future will face potential bifurcation into these two (theoretical, experimental) fields.

Acknowledgments

The author wishes to thank Southern Illinois University Carbondale for financial support in some of the work described and Dr. Jane Geisler-Lee for her assistance in preparation of this chapter.

References Achard, P., Gong, F., Cheminant, S., Alioua, M., Hedden, P., and Genschik, P. (2008) The cold-inducible CBF1 factor-dependent signaling pathway modulates the accumulation of the growth-repressing DELLA proteins via its effect on gibberellin metabolism. Plant Cell 20:2117–29. Agarwal, P.K., Agarwal, P., Reddy, M.K., and Sopory, S.K. (2006) Role of DREB transcription factors in abiotic and biotic stress tolerance in plants. Plant Cell Rep 25:1263–74. Albert, R. (2007) Network inference, analysis, and modeling in systems biology. Plant Cell 19:3327–38. Alon, U. (2008) An introduction to systems biology: design principles of biological circuits. Chapman & Hall/CRC Mathematical and Computational Biology. Amoutzias, G.D., Bornberg-Bauer, E., Oliver, S.G., and Robertson, D.L. (2006) Reduction/oxidation-phosphorylation control of DNA binding in the bZIP dimerization network. BMC Genomics 7:107. Bailey, T. and Gribskov, B. (1998) Combining evidence using p-values: application to sequence homology searches. Bioinformatics 14:48–54. Bartels, D. and Sunkar, R. (2005) Drought and salt tolerance in plants. Critical Reviews in Plant Sciences 24:23–58. Batada, N.N., Reguly, T., Breitkreutz, A., Boucher, L., Breitkreutz, B.J., Hurst, L.D., and Tyers, M. (2006) Stratus not altocumulus: a new view of the yeast protein interaction network. PLoS Biol 4:e317. Benedict, C., Geisler, M., Trygg, J., Huner, N., and Hurry, V. (2006) Consensus by democracy. Using meta-analyzes of microarray and genomic data to model the cold acclimation signaling pathway in Arabidopsis. Plant Physiol 141:1219–32. Bieniawska, Z., Espinoza, C., Schlereth, A., Sulpice, R., Hincha, D.K., and Hannah, M.A. (2008) Disruption of the Arabidopsis circadian clock is responsible for extensive variation in the cold-responsive transcriptome. Plant Physiol 147:263–79. Chinnusamy, V., Zhu, J., and Zhu, J.K. (2007) Cold stress regulation of gene expression in plants. Trends Plant Sci 12:444–51. Covington, M.F., Maloof, J.N., Straume, M., Kay, S.A., and Harmer, S.L. (2008) Global transcriptome analysis reveals circadian regulation of key pathways in plant growth and development. Genome Biol 9:R130. Davletova, S., Schlauch, K., Coutu, J., and Mittler R. (2005a) The zinc-finger protein Zat12 plays a central role in reactive oxygen and abiotic stress signaling in Arabidopsis. Plant Physiol 139:847–56. Davletova, S., Rizhsky, L., Liang, H., Shengqiang, Z., Oliver, D.J., Coutu, J., Shulaev, V., Schlauch, K., and Mittler, R. (2005b) Cytosolic ascorbate peroxidase 1 is a central component of the reactive oxygen gene network of Arabidopsis. Plant Cell 17:268–81. Degenkolbe, T., Do, P.T., Zuther, E., Repsilber, D., Walther, D., Hincha, D.K., and Köhl, K.I. (2009) Plant Mol Biol 69:133–53.

76

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

Dekel, E., Mangan, S., and Alon, U. (2005) Environmental selection of the feed-forward loop circuit in gene-regulation networks. Phys Biol 2:81–8. Denby, K. and Gehring, C. (2005) Engineering drought and salinity tolerance in plants: lessons from genome-wide expression profiling in Arabidopsis. Trends Biotechnol 23:547–52. Devoto, A., Ellis, C., Magusin, A., Chang, H.S., Chilcott, C., Zhu, T., and Turner, J.G. (2005) Expression profiling reveals COI1 to be a key regulator of genes involved in wound- and methyl jasmonate-induced secondary metabolism, defence, and hormone interactions. Plant Mol Biol 58:497–513. Dodd, A.N., Salathia, N., Hall, A., Kével, E., Tóth, R., Nagy, F., Hibberd, J.M., Millar, A.J., and Webb, A.A. (2005) Plant circadian clocks increase photosynthesis, growth, survival, and competitive advantage. Science 309:630–3. Fourrier, N., Bédard, J., Lopez-Juez, E., Barbrook, A., Bowyer, J., Jarvis, P., Warren, G., and Thorlby, G. (2008) A role for SENSITIVE TO FREEZING2 in protecting chloroplasts against freeze-induced damage in Arabidopsis. Plant J 55:734–45. Foyer, C.H. and Noctor, G. (2005) Redox homeostasis and antioxidant signaling: a metabolic interface between stress perception and physiological responses. Plant Cell 17:1866–75. Gardner, M.J., Hubbard, K.E., Hotta, C.T., Dodd, A.N., and Webb, A.A.R. (2006) How plants tell the time. Biochem J 397:15–24. Garreton, V., Carpinelli, J., Jordana, X., and Holuigue, L. (2002) The as-1 promoter element is an oxidative stress-responsive element and salicylic acid activates it via oxidative species. Plant Physiol 130:1516–26. Ge, L.F., Chao, D.Y., Shi, M., Zhu, M.Z., Gao, J.P., and Lin, H.X. (2008) Overexpression of the trehalose-6-phosphate phosphatase gene OsTPP1 confers stress tolerance in rice and results in the activation of stress responsive genes. Planta 228:191–201. Geisler, M., Kleczkowski, L.A., and Karpinski, S.A. (2006) A universal algorithm for genome-wide in silicio identification of biologically significant gene promoter putative cis-regulatory-elements; identification of new elements for reactive oxygen species and sucrose signaling in Arabidopsis. Plant Journal 45(3):384–98. Geisler-Lee, J., O’Toole, N., Ammar, R., Provart, N.J., Millar, A.H., and Geisler, M. (2007) A predicted interactome for Arabidopsis thaliana. Plant Physiology 145:317–29. Gilmour, S.J., Fowler, S.G., and Thomashow, M.F. (2004) Arabidopsis transcriptional activators CBF1, CBF2, and CBF3 have matching functional activities. Plant Mol Biol 54:767–81. Giraud, E., Ho, L.H., Clifton, R., Carroll, A., Estavillo, G., Tan, Y.F., Howell, K.A., Ivanova, A., Pogson, B.J., Millar, A.H., and Whelan, J. (2008) The absence of ALTERNATIVE OXIDASE1a in Arabidopsis results in acute sensitivity to combined light and drought stress. Plant Physiol 147:595–610. Green, R.M., Tingay, S., Wang, Z., and Tobin, E.M. (2002) Circadian rhythms confer a higher level of fitness to Arabidopsis plants. Plant Physiol 129:576–84. Gupta, A.K. and Kaur, N. (2005) Sugar signalling and gene expression in relation to carbohydrate metabolism under abiotic stresses in plants. J Biosci 30:761–76. Gutha, L.R. and Reddy, A.R. (2008) Rice DREB1B promoter shows distinct stress-specific responses, and the overexpression of cDNA in tobacco confers improved abiotic and biotic stress tolerance. Plant Mol Biol 68:533–55. Harrisingh, M.C. and Nitabach, M.N. (2008) Integrating circadian timekeeping with cellular physiology. Science 320:870–80. Hattori, T., Totsuka, M., Hobo, T., Kagaya, Y., and Yamamoto-Toyoda, A. (2002) Experimentally determined sequence requirement of ACGT-containing abscisic acid response element. Plant Cell Physiol 43:136–40. Horan, K., Jang, C., Bailey-Serres, J., Mittler, R., Shelton, C., Harper, J.F., Zhu, J.K., Cushman, J.C., Gollery, M., and Girke, T. (2008) Annotating genes of known and unknown function by large-scale coexpression analysis. Plant Physiol 147:41–57. Huang, D., Wu, W., Abrams, S.R., and Cutler, A.J. (2008) The relationship of drought-related gene expression in Arabidopsis thaliana to hormonal and environmental factors. Journal of Experimental Botany 59:2991–3007. Hundertmark, M. and Hincha, D.K. (2008) LEA (Late Embryogenesis Abundant) proteins and their encoding genes in Arabidopsis thaliana. BMC Genomics 9:118. Ibañez, C., Ramos, A., Acebo, P., Contreras, A., Casado, R., Allona, I., and Aragoncillo, C. (2008) Overall alteration of circadian clock gene expression in the chestnut cold response. PLoS ONE 3:e3567. Iordachescu, M. and Imai, R. (2008) Trehalose biosynthesis in response to abiotic stresses. J Integr Plant Biol 50:1223–9. Jiang, Y. and Deyholos, M.K. (2009) Functional characterization of Arabidopsis NaCl-inducible WRKY25 and WRKY33 transcription factors in abiotic stresses. Plant Mol Biol 69:91–105. Karim, S., Aronsson, H., Ericson, H., Pirhonen, M., Leyman, B., Welin, B., Mäntylä, E., Palva, E.T., Van Dijck, P., and Holmström, K.O. (2007) Improved drought tolerance without undesired side effects in transgenic plants producing trehalose. Plant Mol Biol 64:371–86. Kasuga, M., Liu, Q., Miura, S., Yamaguchi-Shinozaki, K., and Shinozaki, K. (1999) Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat Biotechnol 17:287–91. Kaur, N. and Gupta, A.K. (2005) Signal transduction pathways under abiotic stresses in plants. Current Science 88:1771–80.

TRANSCRIPTION AND SIGNALING FACTORS IN THE DROUGHT RESPONSE REGULATORY NETWORK

77

Khandelwal, A., Elvitigala, T., Ghosh, B., Ralph, S., and Quatrano, R.S. (2008) Arabidopsis transcriptome reveals control circuits regulating redox homeostasis and the role of an AP2 transcription factor. Plant Physiology 148:2050–8. Kilian, J., Whitehead, D., Horak, J., Weinl, S., Batistic, O., D’Angelo, C., Bornberg-Bauer, E., Kudla, J., and Harter, K. (2007) The AtGenExpress global stress expression data set: Protocols, evaluation and exemplary data analysis of UV-B light, drought and cold stress responses. Plant J 50:347–63. Kim, S. (2006) The role of ABF family bZIP class transcription factors in stress response. Physiologia Plantarum 126(4):519–27(9). Kim, S., Kang, J.Y., Cho, D.I., Park, J.H., and Kim, S.Y. (2004) ABF2, an ABRE-binding bZIP factor, is an essential component of glucose signaling and its overexpression affects multiple stress tolerance. Plant Journal 40:75–87. Knight, H., Thomson, A.J., and McWatters, H.G. (2008a) SENSITIVE TO FREEZING 6 integrates cellular and environmental inputs to the plant circadian clock. Plant Physiol 148(1):293–303. Knight, H., Mugford Nee Garton, S.G., Ulker, B., Gao, D., Thorlby, G., and Knight, M.R. (2009) Identification of SFR6, a key component in cold acclimation acting post-translationally on CBF function. Plant journal advanced publication DOI: 10.1111/j.1365-313X.2008.03763.x. Lee, B.H., Henderson, D.A., and Zhu, J.K. (2005) The Arabidopsis cold-responsive transcriptome and its regulation by ICE1. Plant Cell 17:3155–75. Lee, K.H., Piao, H.L., Kim, H.Y., Choi, S.M., Jiang, F., Hartung, W., Hwang, I., Kwak, J.M., Lee, I.J., and Hwang, I. (2006) Activation of glucosidase via stress-induced polymerization rapidly increases active pools of abscisic acid. Cell 126:1109–20. Less, H. and Galili, G. (2008) Principal transcriptional programs regulating plant amino acid metabolism in response to abiotic stresses. Plant Physiol 147:316–30. Li, W.X., Oono, Y., Zhu, J., He, X.J., Wu, J.M., Iida, K., Lu, X.Y., Cui, X., Jin, H., and Zhu, J.K. (2008) The Arabidopsis NFYA5 transcription factor is regulated transcriptionally and posttranscriptionally to promote drought resistance. Plant Cell 20:2238–51. Liu, N., Zhong, N.Q., Wang, G.L., Li, L.J., Liu, L.X., He, Y.K., and Xia, G.X. (2007) Cloning and functional characterization of PpDBF1 gene encoding a DRE-binding transcription factor from Physcomitrella patens. Planta 226:827–38. Ma, S.S. and Bohnert, H.J. (2008) Gene networks for the integration and understanding of gene expression characteristics. Weed Sci 56:314–21. Mahajan, S. and Tuteja, N. (2005) Cold, salinity and drought stresses: an overview. Arch Biochem Biophys 444:139–58. Martin-Tryon, E.L. and Harmer, E.L. (2008) XAP5 CIRCADIAN TIMEKEEPER coordinates light signals for proper timing of photomorphogenesis and the circadian clock in Arabidopsis. Plant Cell 20:1244–59. Maruyama, K., Sakuma, Y., Kasuga, M., Ito, Y., Seki, M., Goda, H., Shimada, Y., Yoshida, S., Shinozaki, K., and YamaguchiShinozaki, K. (2004) Identification of cold-inducible downstream genes of the Arabidopsis DREB1A/CBF3 transcriptional factor using two microarray systems. Plant J 38:982–93. Más, P. (2008) Circadian clock function in Arabidopsis thaliana: time beyond transcription. Trends Cell Biol 18:273–81. Matsui, A., Ishida, J., Morosawa, T., Mochizuki, Y., Kaminuma, E., Endo, T.A., Okamoto, M., Nambara, E., Nakajima, M., Kawashima, M., Satou, M., Kim, J.M., Kobayashi, N., Toyoda, T., Shinozaki, K., and Seki, M. (2008) Arabidopsis transcriptome analysis under drought, cold, high-salinity and ABA treatment conditions using a tiling array. Plant Cell Physiol 49:1135–49. McClung, C.R. (2006) Plant circadian rhythms. Plant Cell 18:792–803. Menges, M., Dóczi, R., Ökrész, L., Morandini, P., Mizzi, L., Soloviev, M., Murray, J.A.H., and Böogre, L. (2008) Comprehensive gene expression atlas for the Arabidopsis MAP kinase signalling pathways. New Phytologist 179:643–62. Mentzen, W.I. and Wurtele, E.S. (2008) Regulon organization of Arabidopsis. BMC Plant Biology 8:99. Mittler, R., Kim, Y.S., Song, L., Coutu, J., Coutu, A., Ciftci, S., Lee, H., Stevenson, B., and Zhu, J.-K. (2006) Gain- and lossof-function mutations in Zat10 enhance the tolerance of plants to abiotic stress FEBS letters 580(28–29):6537–42. Mizuno, T. and Yamashino, T. (2008) Comparative transcriptome of diurnally oscillating genes and hormone-responsive genes in Arabidopsis thaliana: insight into circadian clock-controlled daily responses to common ambient stresses in plants. Plant Cell Physiol 49:481–7. Nakano, T., Suzuki, K., Fujimura, T., and Shinshi, H. (2006) Genome-wide analysis of the ERF gene family in Arabidopsis and rice. Plant Physiol 140:411–32. Nakashima, K. and Yamaguchi-Shinozaki, K. (2006) Regulons involved in osmotic stress-responsive and cold stress-responsive gene expression in plants. Physiologia Plantarum 126(1):62–71. Nakashima, K., Fujita, Y., Katsura, K., Maruyama, K., Narusaka, Y., Seki, M., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2006) Transcriptional regulation of ABI3- and ABA-responsive genes including RD29B and RD29A in seeds, germinating embryos, and seedlings of Arabidopsis. Plant Mol Biol 60:51–68.

78

GENETIC DETERMINANTS OF PLANT ADAPTATION UNDER WATER STRESS

Nambara, E. and Marion-Poll, A. (2005) Abscisic acid biosynthesis and catabolism. Annu Rev Plant Biol 56:165–85. Narusaka, Y., Nakashima, K., Shinwari, Z.K., Sakuma, Y., Furihata, T., Abe, H., Narusaka, M., Shinozaki, K., and YamaguchiShinozaki, K. (2003) Interaction between two cis-acting elements, ABRE and DRE, in ABA-dependent expression of Arabidopsis rd29A gene in response to dehydration and high-salinity stresses. Plant J 34:137–48. Novillo, F., Medina, J., and Salinas, J. (2007) Arabidopsis CBF1 and CBF3 have a different function than CBF2 in cold acclimation and define different gene classes in the CBF regulon. Proc Natl Acad Sci USA 104:21002–7. Palsson, B. (2006) Introduction to systems biology. Properties of reconstructed networks. Cambridge University Press. Paul, M.J. (2008) Trehalose 6-phosphate: a signal of sucrose status. Biochem J 412:e1–2. Paul, M.J., Primavesi, L.F., Jhurreea, D., and Zhang, Y. (2008) Trehalose metabolism and signaling. Annu Rev Plant Biol 59:417–41. Perera, I.Y., Hung, C.Y., Moore, C.D., Stevenson-Paulik, J., and Boss, W.F. (2008) Transgenic Arabidopsis plants expressing the type 1 inositol 5-phosphatase exhibit increased drought tolerance and altered abscisic acid signaling. Plant Cell 20:2876–93. Pierik, R., Sasidharan, R., and Voesenek, L.A.C.J. (2007) Growth control by ethylene: adjusting phenotypes to the environment. J Plant Growth Regul 26:188–200. Qiu, D., Xiao, J., Xie, W., Liu, H., Li, X., Xiong, L., and Wang, S. (2008) Rice gene network inferred from expression profiling of plants overexpressing OsWRKY13, a positive regulator of disease resistance. Molecular Plant 1:538–51. Quan, L.J., Zhang, B., Shi, W.W., and Li, H.Y. (2008) Hydrogen peroxide in plants: a versatile molecule of the reactive oxygen species network. J Integr Plant Biol 50:2–18. Rashotte, A.M., Mason, M.G., Hutchison, C.E., Ferreira, F.J., Schaller, G.E., and Kieber, J.J. (2006) A subset of Arabidopsis AP2 transcription factors mediates cytokinin responses in concert with a two-component pathway. PNAS 103(29):11081–5. Rhee, S.Y., Dickerson, J., and Xu, D. (2006) Bioinformatics and its applications in plant biology. Annu Rev Plant Biol 57:335–60. Riechmann, J.L. and Meyerowitz, E.M. (1998) The AP2/EREBP family of plant transcription factors. Biol Chem 379:633–46. Riera, M., Valon, C., Fenzi, F., Giraudat, J., and Leung, J. (2005) The genetics of adaptive responses to drought stress: abscisic acid-dependent and abscisic acid-independent signalling components. Physiologia Plantarum 123:111–9. Rihn, B.H., Vidal, S., Nemurat, C., Vachenc, S., Mohr, S., Mazur, F., Houdry, P., Grandjean, F., Visvikis, S., and Ducloy, J. (2003) From transcriptomics to bibliomics. Medical Science Monitor 9:MT89–95. Sakamoto, H., Matsuda, O., and Iba, K. (2008) ITN1, a novel gene encoding an ankyrin-repeat protein that affects the ABAmediated production of reactive oxygen species and is involved in salt-stress tolerance in Arabidopsis thaliana. Plant Journal 56:411–22. Santos-Mendoza, M., Dubreucq, B., Baud, S., Parcy, F., Caboche, M., and Lepiniec, L. (2008) Deciphering gene regulatory networks that control seed development and maturation in Arabidopsis. Plant Journal 54:608–20. Schutze, K., Harter, K., and Chaban, C. (2008) Post-translational regulation of plant bZIP factors. Trends in Plant Science 13:247–55. Shinozaki, K., Yamaguchi-Shinozaki, K., and Seki, M. (2003) Regulatory network of gene expression in the drought and cold stress responses. Curr Opin Plant Biol 6:410–7. Suzuki, M., Ketterling, M., and McCarty, D. (2005) Quantitative statistical analysis of cis-regulatory sequences in ABA/VP1- and CBF/DREB1-regulated genes of Arabidopsis. Plant Physiol 139:437–47. Thijs, G., Lescot, M., Marchal, K., Rombauts, S., De Moor, B., Rouzé, P., and Moreau, Y. (2001) A higher order background model improves the detection of regulatory elements by Gibbs Sampling. Bioinformatics 17(12):1113–22. Uno, Y., Furihata, T., Abe, H., Yoshida, R., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2000) Arabidopsis basic leucine zipper transcription factors involved in an abscisic acid-dependent signal transduction pathway under drought and high-salinity conditions. Proc Natl Acad Sci USA 97:11632–7. Urao, T., Yakubov, B., Satoh, R., Yamaguchi-Shinozaki, K., Seki, M., Hirayama, T., and Shinozaki, K. (1999) A transmembrane hybrid-type histidine kinase in Arabidopsis functions as an osmosensor. Plant Cell 11:1743–54. Vandenbroucke, K., Robbens, S., Vandepoele, K., Inzé, D., Van de Peer, Y., and Van Breusegem, F. (2008) Hydrogen PeroxideInduced Gene Expression across Kingdoms: A Comparative Analysis. Mol Biol Evol 25:507–16. Vogel, J.T., Zarka, D.G., Van Buskirk, H.A., Fowler, S.G., and Thomashow, M.F. (2005) Roles of the CBF2 and ZAT12 transcription factors in configuring the low temperature transcriptome of Arabidopsis. Plant J 41:195–211. Wasilewska, A., Vlad, F., Sirichandra, C., Redko, Y., Jammes, F., Valon, C., dit Frey, N.F., and Leung, J. (2008) An Update on Abscisic Acid Signaling in Plants and More … Molecular Plant 1:198–217. Willige, B.C., Ghosh, S., Nill, C., Zourelidou, M., Dohmann, E.M., Maier, A., and Schwechheimer, C. (2008) The DELLA domain of GA INSENSITIVE mediates the interaction with the GA INSENSITIVE DWARF1A gibberellin receptor of Arabidopsis. Plant Cell 19:1209–20.

TRANSCRIPTION AND SIGNALING FACTORS IN THE DROUGHT RESPONSE REGULATORY NETWORK

79

Wohlbach, D.J., Quirino, B.F., and Sussman, M.R. (2008) Analysis of the Arabidopsis histidine kinase ATHK1 reveals a connection between vegetative osmotic stress sensing and seed maturation. Plant Cell 20:1101–17. Xiong, L., Ishitani, M., and Zhu, J.K. (1999) Interaction of osmotic stress, ABA and low temperature in the regulation of stress gene expression in Arabidopsis thaliana. Plant Physiol 119:205–11. Yamaguchi-Shinozaki, K. and Shinozaki, K. (2006) Transcriptional Regulatory Networks in Cellular Responses and Tolerance to Dehydration and Cold Stresses. Annu Rev Plant Biol 57:781–803. Yuan, J.S., Galbraith, D.W., Dai, S.Y., Griffin, P., Stewart, Jr., C.N. (2008) Plant systems biology comes of age. Trends Plant Sci 13:165–71. Zarka, D.G., Vogel, J.T., Cook, D., and Thomashow, M.F. (2003) Cold induction of Arabidopsis CBF genes involves multiple ICE (inducer of CBF expression) promoter elements and a cold-regulatory circuit that is desensitized by low temperature. Plant Physiol 133:910–8. Zhang, Y. and Wang, L. (2005) The WRKY transcription factor superfamily: its origin in eukaryotes and expansion in plants. BMC Evol Biol 5:1.

Section 2 Genes for Crop Adaptation to Poor Soil

Genes for Plant Abiotic Stress Editors Matthew A. Jenks and Andrew J. Wood © 2010 Blackwell Publishing ISBN: 978-0-813-81502-2

4

Genetic Determinants of Salinity Tolerance in Crop Plants Darren Plett, Bettina Berger, and Mark Tester

Introduction

Soil salinity is a significant issue worldwide. It is estimated by salinity-monitoring organizations, such as the United States Salinity Laboratory (USSL) and the United Nations (UN) Food and Agriculture Organization (FAO), that 1 billion of the 13 billion hectares (ha) of land worldwide are salt affected, including ∼30% of all irrigated land (Rengasamy, 2006). This estimate includes ∼200 million ha in the Americas, portions of southern and eastern Europe, 120 million ha in the Middle East, 80 million ha in Africa, 35 million ha in Asia, and more than 6 million ha in Australia. Soil salinity is an especially acute problem in Australia with projections that 17 million ha will be affected by 2050, much of which is in the western wheat belt (Rengasamy, 2006). One-third of the world’s food comes from irrigated land (Munns, 2002), thus salinization of agricultural soils is a critical issue.

Definitions

Saline soils are defined as loose and sandy soils with significant amounts of water-soluble salts (e.g., sodium, chloride, calcium, magnesium, and sulphate) and have an electrical conductivity (EC) greater than 4 deciSiemens per meter (dS/m), an exchangeable sodium percentage (ESP) less than 15%, and a pH of less than 8.5 (Rengasamy, 2002). Sodic soils are generally dense and clogged, with low soluble salt content, an EC less than 4 dS/m, an ESP of greater than 15%, and a pH higher than 8.5. These soils have high concentrations of insoluble sodium carbonate and bicarbonate, which cause a crust over the soil surface (Rengasamy, 2002). Saline-sodic soils are common in arid and semi-arid regions and are intermediate types of saline soils with an EC greater than 4 dS/m, ESP greater than 15%, and pH less than 8.5 (Rengasamy, 2002).

Causes

Two main types of salinity exist as a result of both natural processes and human intervention. Primary salinity is a result of evolutionary processes such as evaporation and underground water movements that bring salt to the surface. Seawater influences and climatic changes are also primary causes of salinity (Rengasamy, 2006). Genes for Plant Abiotic Stress Editors Matthew A. Jenks and Andrew J. Wood © 2010 Blackwell Publishing ISBN: 978-0-813-81502-2

83

84

GENES FOR CROP ADAPTATION TO POOR SOIL

Secondary salinity has been caused by human intervention through irrigation, destruction of critical watersheds, and clearing of natural, deep-rooted vegetation for agricultural, industrial, and urban development, thereby altering the dynamic equilibrium of water and salt circulation. Some salinity problems are aggravated by application of certain chemical fertilizers and soil amendments (Rengasamy, 2006).

Effects on Plant Health

Salinity stress in plants is a result of ionic and osmotic components, whose effects can be difficult to distinguish in a salt-stressed plant (Munns and Tester, 2008). One common feature of saltstressed plants is an inhibition of root growth, which appears to be related more to the osmotic potential of the external solution than to the Na+ content of the plant itself (Munns et al., 2000; Munns, 2002). The same inhibition of leaf elongation by leaf Na+ content has been observed in several grass species (Cramer, 2003). A potentially useful method to separate the effects of the ionic and osmotic components of salt stress is to compare the effects of lithium chloride (LiCl) to those of NaCl on plant growth (Tester and Davenport, 2003). LiCl is toxic at one-tenth the concentration of NaCl and shares many of the transport pathways; thus, comparing the effects on plants of solutions equal in molarity and/or that are different in concentration but which have the same inhibition on growth would help to separate the components of the salt stress. Ionic toxicity of Na+ is largely related to its competition with K+ for binding sites crucial to metabolic processes, which Na+ cannot fulfill correctly (Bhandal and Malik, 1988). Thus, maintaining a low Na+: K+ ratio is critical to salinity tolerance, and may even be more critical to salt tolerance than the absolute Na+ level itself, at least at some Na+ concentrations (Dubcovsky et al., 1996; Maathuis and Amtmann, 1999). A significant component of salt stress is related to the build-up of Na+ (and Cl−) in the apoplastic spaces in leaf tissue from the evaporating water of the xylem stream. With elevated Na+ levels in the apoplastic space, water is drawn out of leaf cells osmotically, causing dehydration (Flowers et al., 1991). Shoots accumulate more Na+ than do roots, thus it can appear that they are more sensitive to osmotic and ionic Na+ stress than roots. Elevated Na+ levels also impede efficient uptake of other nutrients through nutrient transporters, which can result in nutrient deficiency (Silberbush and Ben-Asher, 2001; Hu and Schmidhalter, 2005). An obvious result of decreases in plant vigour due to the osmotic and ionic stresses as well as the nutrient deficiencies imposed on plants by salinity stress is a significant penalty in yield (Quarrie and Mahmood, 1993; Katerji et al., 2003). The negative effects of shoot Cl− accumulation on plant health are also significant in species such as grapevine (White and Broadley, 2001; Storey et al., 2003), but will not be discussed because the effects of Na+ are thought to be more significant in the majority of crop species.

Management-based Solutions

Control of water movement on land is important to reduce the salts present in the soil and to reduce further accumulation of soil salts, especially those resulting

Water and Vegetation Management

GENETIC DETERMINANTS OF SALINITY TOLERANCE IN CROP PLANTS

85

from irrigation (Qadir and Oster, 2004). Alternatively, trees and other vegetation may be used to change the level of the water table and saline layer of soil, improve soil structure and even to remove salt from the soil by improving drainage (Qadir and Oster, 2004). Soil Reclamation Several techniques exist for reclamation of saline soils. Physical manipulation

by removing saline topsoil or deep ploughing to bury saline soil can be useful in some instances. Leaching of the salt in the topsoil may, in some cases, flush sufficient amounts of salt out of the topsoil to reclaim it for agricultural use. Improvement of drainage to prevent further salt accumulation also helps remove the salt present in the soil. The addition of calcium and organic amendments has also been useful to combat the effects of salt on plants as well as to improve the structure of the soil (Rengasamy, 2006). Use of Salt Tolerant or Halophytic Crops Halophytes and other salt-tolerant species can help in remediation of saline soils. Salinity tolerant perennials have deep root penetration to improve soil structure, and some extreme halophytes can accumulate significant levels of sodium in their aboveground tissues, potentially removing sodium from the land (Malcolm et al., 2003). However, the cost of removing the aboveground biomass would be a drawback to this method on all but high value land (Tester and Davenport, 2003). Nevertheless, in most agricultural systems, mild to moderate soil salinity exists, which means salt-tolerant crops need to be grown. Wide ranges in salinity tolerance exist among crop species from relatively salt-tolerant species like barley to salt-sensitive species such as rice or wheat. Considering the importance of these crops to many countries’ economies and food supplies, it is necessary to keep growing these crops. Thus, improving their salinity tolerance is important.

Salinity Tolerance

Salinity tolerance in plants is derived from the contributions of three components—exclusion of sodium from the shoot, tissue tolerance of the accumulated sodium, and tolerance to the osmotic stress imposed by elevated external sodium concentrations (Munns and Tester, 2008). The contributions of the three mechanisms of salinity tolerance have recently been quantified using an image capture technique (Rajendran et al., 2009). Surprisingly, there appeared to be no preferred mechanism for tolerance among the Triticum monococcum germplasm used in the study. This indicates the importance of the continued study of particularly the tissue and osmotic tolerance mechanism for salinity tolerance, which have not received as much attention as the sodiumexclusion mechanism, but appear to be equally crucial to providing salinity tolerance to plants (Genc et al., 2007). These three components are completely interconnected and are regulated by signaling pathways, which are only beginning to be elucidated. Because so little is known about these other components, most of this review is focused on processes related directly to Na+ transport and its relevance to Na+ exclusion and tissue tolerance. Similarly, there is more known about the genetic determinants of sodium exclusion, but there is relatively less known about the genetic determinants of osmotic tolerance. This chapter will discuss the genetic determinants of salinity tolerance that are known and will discuss several physiological phenomena that have no link to genetic determinants. However, it is hoped that genetic approaches in the coming years will reveal more about osmotic tolerance.

86

GENES FOR CROP ADAPTATION TO POOR SOIL

Sodium Exclusion

There are several salinity responses in plants that require the coordination of several cell types, each responding to salinity in a different manner. These responses are largely centered on controlling Na+ uptake by the roots and its subsequent distribution within the plant tissue. The genetic determinants associated with several of these responses are summarized in Figure 4.1 (single cell level) and Figure 4.2 (whole plant level). Generally, salinity tolerance is related to the minimization of Na+ reaching the shoot tissue. Two important species where this relationship does not necessarily hold are the relatively salt-tolerant barley and cotton, which tolerate higher Na+ levels within shoot tissue than does a salt-sensitive species, like wheat. The model plant species, Arabidopsis, also appears to have a poor correlation between salinity tolerance and sodium accumulation, which calls into question its usefulness as a species to study salinity tolerance (Møller and Tester, 2007). (This does not preclude its utility for studying the molecular basis of component processes, but makes it difficult to then relate these to salinity tolerance of the Arabidopsis plants in a way that is directly relevant to salinity tolerance of cereals.) Another, perhaps equally important factor, is maintaining a low ratio of Na+ to K+ reaching the shoot tissues as Na+ is largely toxic due to its competition for K+ binding sites in cells (Gorham et al., 1990, Dubcovsky et al., 1996, Maathuis and Amtmann, 1999, Cuin et al., 2003). Increased K+ efflux from Na+-stressed root cells creates added difficulty for plants to maintain a low Na+ to K+ ratio in the shoot, thus, K+ efflux from roots may provide a suitable measure of salinity tolerance (Chen et al., 2005, Chen et al., 2007, Cuin et al., 2008). Despite this, the actual cytosolic Na+ to K+ ratio in barley roots does not seem to properly explain salinity stress reduction of growth (Kronzucker et al., 2006). The rate of unidirectional Na+ influx is significantly higher than the net rate of influx, and this indicates that significant rates of efflux must exist (Jacoby and Hanson, 1985, Davenport et al., 1997). The fact that unidirectional influx is so high in glycophytes is related to the nonselectiveness of most of the Na+ transporters (Demidchik et al., 2002), which appear likely to function in the uptake of other cations like Ca2+ and NH +4 (White, 1996, White and Davenport, 2002). Also, the rate of net uptake of Na+ in glycophytes appears to be regulated more by the efflux of Na+ by Na+selective Na+/H+ antiporters than it is by control of influx (Qiu et al., 2002). Net influx is not determined exclusively by the difference between unidirectional influx and efflux, rather internal controls of Na+ level appear to be also involved (Tester and Davenport, 2003). Root Na+ concentration differs much less significantly than does shoot Na+ concentration among a variety of species and salt-tolerance levels (e.g., rice and Phragmites communis) (Matsushita and Matoh, 1991). Root cells apparently detect internal Na+ concentrations and control Na+ transporters appropriately, which shows a nonlinear relationship between root Na+ concentration and external Na+ levels (Munns, 2002). As well, shoot Na+ concentration is higher than root Na+ concentration due to the influx from roots and minor efflux levels, while the root may efflux Na+ to the external media or to shoot tissues. Thus, alterations affecting the net uptake of Na+ by the root may actually alter the shoot Na+ levels more significantly. Further evidence indicates that inhibiting unidirectional Na+ influx with Ca2+ decreased shoot Na+ concentration more significantly than root Na+ concentration (Reid and Smith, 2000). Another factor controlling Na+ uptake appears to be external K+ supply as K+ starved rice plants were shown to activate Na+ uptake mechanisms including OsHKT2;1 (Horie et al., 2007). Influx to the Root Concentration and voltage favor passive entry for Na+ from the soil into the root

cortical cytoplasm (Cheeseman, 1982). However, net accumulation of Na+ within the cell is due to

87

Na+

Na+

Na+

OsHKT2;1

Na+ H+

Na+

Na+

SOS1

Na+

SOS2

H+

NHX1

H+

AVP1

2 Pi

SOS3

Vacuole

PPi

H+

PPi 2 Pi

[Ca2+]cyt

ATP

ADP + Pi

AVP1 ?

V-ATPase

H+

Figure 4.1 The ion transporters, channels, and pumps, which have been characterized as being involved in Na+ exclusion from the shoot. The various proteins are localized on the putative membrane that they are associated with, and all proteins have been localized in a single cell to simply show their putative function in the movement of Na+. Responsible for influx of Na+ into cells are cyclic-nucleotide gated channels (CNGC), glutamate receptors (GLR), nonselective cation channels (NSCC), and HKT transporters (AtHKT1;1, OsHKT1;4, OsHKT1;5 and OsHKT2;1). Responsible for efflux of Na+ from cells is the Na+/H+ antiporter (SOS1), which interacts with the serine/threonine protein kinase (SOS2), which interacts with the calcium binding protein (SOS3), which is activated by [Ca2+]cyt. Vacuolar storage of Na+ is mediated by a vacuolar Na+/H+ antiporter (NHX1) and the proton gradient is provided by the vacuolar H+-pyrophosphatase (AVP1) and the vacuolar H+-ATPase (V-ATPase). AVP1 has also been associated with acidifying the apoplastic space. For color detail, please see color plate section.

NSCCs

GLRs

CNGCs

Cytoplasm

Apoplast

OsHKT1;4 OsHKT1;5 AtHKT1;1

MX

XP

SOS1 ?

AtHKT1;1

OsHKT1;4

OsHKT2;1

NSCCs SOS1 ? GLRs

AtHKT1;1

OsHKT1;5

CNGCs

SOS1 ?

EN

Soil

PR

XP

MX

CO EP

Figure 4.2 Whole plant localization of several Na+ transporters or channels involved in Na+ transport in the shoot (top) or root (bottom). Depicted are proteins putatively involved in Na+ influx from soil to the epidermis: OsHKT2;1, nonselective cation channels (NSCC), glutamate receptors (GLR), and cyclic-nucleotide gated channels (CNGC). Transporters responsible for Na+ retrieval from the metaxylem to the xylem parenchyma in the root include HKT transporters (AtHKT1;1 and OsHKT1;5) and a Na+/H+ antiporter (SOS1). Transporters responsible for Na+ retrieval from the metaxylem to the xylem parenchyma of the shoot include the HKT genes (AtHKT1;1 and OsHKT1;4) and a Na+/H+ antiporter (SOS1). Cell types depicted include epidermis (EP), cortex (CO), endodermis (EN), pericycle (PR), xylem parenchyma (XP), and metaxylem (MX). Also depicted is the apoplastic influx pathway for Na+ (green arrow). For color detail, please see color plate section.

88

GENETIC DETERMINANTS OF SALINITY TOLERANCE IN CROP PLANTS

89

the difference between passive influx and active efflux. Thus, maximizing the ability of the plant to exclude Na+ from entering root cells is important to salinity tolerance (Schubert and Läuchli, 1990). Some discrepancy can be observed in estimates of the rate of Na+ influx into root cells. Much of this may be explained by the method of measurement used in the experiment. When short time courses are used (<3 minutes), estimates in the range of 0.5–2.0 μmol g−1 FW min−1 with 50 milliMole (mM) external Na+ have been found in wheat (Davenport, 1998), rice (Plett, 2008) and Arabidopsis (Essah et al., 2003). Lower estimates published prior to the studies cited above (Zidan et al., 1991, Elphick et al., 2001) probably did not take into account the rate of efflux that becomes significant after about 3 minutes of exposure of the cell to Na+ (Tester and Davenport, 2003). It is apparent that the rate of unidirectional Na+ influx in halophytes may be lower than those of glycophytes. The halophytic dicotyledon, Spergularia marina was found to have a low rate of influx (0.24 μmol g−1 FW min−1 at 100 mM external Na+), significantly lower than those of glycophytes (Cheeseman et al., 1985). The halophytic relative of Arabidopsis, Thellungiella halophila, had significantly lower influx than Arabidopsis (Wang et al., 2006). Similar measurements of halophytic monocotyledons have produced similar results with influx in Triglochin maritima being estimated at 0.065 and 0.21 μmol g−1 FW min−1 at 100 mM external Na+ (Jefferies, 1973) and 0.13 μmol g−1 FW min−1 at 74 mM Na+ in Eleocharis uniglumis (Shepherd and Bowling, 1979). Ca2+-Sensitive Influx Addition of up to 10 mM Ca2+ to the external solution will (generally) reduce the toxic effects of Na+ through a complex set of effects (Cramer, 2002, El-Hamdaoui et al., 2003). This effect is, at least in part, due to the inhibition of unidirectional Na+ influx by Ca2+. (Also of note is the stimulation of K+ influx by Ca2+). Interestingly, Ca2+ also reduces K+ efflux stimulated by high Na+ levels (Cramer et al., 1985, Shabala et al., 2006). These effects have been interpreted in relation to the SOS signaling pathway (Liu and Zhu, 1998). In this model, elevated Na+ stimulates a rise in cytosolic Ca2+, which activates SOS3 leading to a change in expression and activity of Na+ and K+ transporters. Since sos3 mutants produce a mutant form of SOS3, they are less sensitive to Ca2+ and thus require higher levels of external Ca2+ for normal root elongation (Horie et al., 2006). However, in wheat, external Ca2+ directly inhibits unidirectional influx of Na+, which suggests that Ca2+ may not necessarily be involved in a signaling pathway (Davenport and Tester, 2000). It is possible that the means of Ca2+-sensitive Na+ influx is non-selective cation channels (Amtmann and Sanders, 1999; Tyerman and Skerrett, 1999; White, 1999; Davenport and Tester, 2000; Demidchik et al., 2002; Demidchik and Maathuis, 2007). There are many candidates for these channels including the cyclic nucleotide-gated channels (CNGC) (Leng et al., 2002; Gobert et al., 2006) and glutamate-activated channels (GLR) (Cheffings, 2001; Lacombe et al., 2001; Demidchik et al., 2004; Qi et al., 2006). The family of 20 glutamate receptors is proving a difficult group of ion channels to understand. Recent evidence indicates that there may be three classes of receptors that are activated by different group of amino acids (Stephens et al., 2008). An additional complication is the potential that the genes form heteromers, as a recent study was unable to identify tissue-specific expression patterns for the genes with the exception of GLR3.7, which appears to be expressed in all tissues (Roy et al., 2008). Interestingly, the evidence for (Maathuis and Sanders, 2001; Demidchik and Tester, 2002; Essah et al., 2003; Demidchik et al., 2004) or against (Leng et al., 2002) these channels being the nonselective cation channels seems to depend on whether the experiments are performed in plants or in heterologous systems. Another important gene to mention in this discussion is LCT1 from wheat, which, when expressed in yeast, leads to an increase in cation influx and hypersensitivity to Na+ (Schachtman et al., 1997;

90

GENES FOR CROP ADAPTATION TO POOR SOIL

Clemens et al., 1998; Amtmann et al., 2001). Addition of external Ca2+ reduced Na+ influx and sensitivity, but the cation profile influxed by LCT1 resembled the profile of endogenous ion transport in yeast, suggesting that LCT1 may be stimulating the ion transporters already present (Amtmann et al., 2001). Ca2+-insensitive Influx It is possible that Ca2+-insensitive influx of Na+ is partially due to a component of the Na+ influx through nonselective cation channels, as the inhibition of Na+ by Ca2+ is partial (Davenport and Tester, 2000). There are several other potential transporters mediating this influx, including those encoded by the HKT, KUP, and HAK gene families (Platten et al., 2006; Grabov, 2007). TaHKT2;1 is a Ca2+-insensitive transporter from wheat (Schachtman and Schroeder, 1994; Tyerman and Skerrett, 1999), which acts as a high affinity Na+/K+ symporter in Xenopus oocytes or yeast and in high Na+ concentrations catalyzes low affinity Na+ uniport (Rubio et al., 1995). A screen to identify gene knockouts in Arabidopsis complementary to sos3 revealed several individuals with reduced AtHKT1;1 activity, thus it was suggested that the gene is involved in Na+ influx (Rus et al., 2001). It was also suggested that AtHKT1;1 is under control of SOS3 and may simply be suppressed under elevated Na+ conditions in wild-type plants (Zhu, 2002). Further studies showed the athkt1;1 mutant has no change in Na+ influx from wild-type plants, suggesting another function for AtHKT1;1 (Berthomieu et al., 2003; Davenport et al., 2007). However, there are nine HKT genes in rice (Platten et al., 2006) and other cereals (Huang et al., 2008), and OsHKT2;1 has been shown to catalyze high affinity Na+ uptake into roots under K+-starvation conditions as it appears Na+ can partially replace the function of K+ (Horie et al., 2007). Interestingly, similar highaffinity Na+ uptake was observed in K+-starved barley roots, but when heterologously expressed in yeast, HvHKT2;1 was shown to mediate Na+ (or K+) uniport, Na+-K+ symport, or a mix of both, depending on the construct from which the transporter was expressed (Haro et al., 2005; Banuelos et al., 2008). Bypass Flow Apoplastic leakage may be a significant Na+ entry point for some salt-sensitive plants such as rice, where external Ca2+ has little effect on Na+ uptake and salinity tolerance (Yeo and Flowers, 1985; Yeo et al., 1987). Using an apoplastic dye, it was observed that rice plants with high shoot Na+ accumulation had high apoplastic water flow (Yadav et al., 1996; Yeo, 1999), indicating leaks in endodermis at root branch points, root apices, or simply through permeable endodermis. This pathway also varies between species, with bypass flow in rice being 10 times greater than that of wheat (Garcia et al., 1997). Variation in influx also apparently exists between salt-tolerant and -sensitive rice lines, perhaps indicating a difference in bypass flow rates within the species (Malagoli et al., 2008). This information meshes well with classical observations that the Casparian band is two to three times wider in halophytes than nonhalophytes (Poljakoff-Mayber, 1975; Peng et al., 2004), and salinization of cotton enhances its formation of the Casparian band and exodermis (Reinhardt and Rost, 1995). Apoplastic leakage is reduced by addition of silicon to the growth media, apparently because silicon deposits decrease the gaps in the endodermal and exodermal layer of the rice root (Gong et al., 2006). A relatively unknown cell type, the phi cell, has recently been suggested to be a physical barrier to apoplastic sodium movement from the cortex to the stele of Brassica plants, thereby preventing higher transfer to the xylem stream and shoot of plants possessing this cell type (Fernandez-Garcia et al., 2009). Efflux to Soil Maximizing efflux of Na+ from the root may be just as important to improving salin-

ity tolerance as minimizing influx. Efflux may occur through a Na+/H+ antiporter (Blumwald et al.,

GENETIC DETERMINANTS OF SALINITY TOLERANCE IN CROP PLANTS

91

2000), but evidence for this transporter can be difficult to find (Mennen et al., 1990). Though unlikely, this transporter has been proposed to operate in influx and efflux depending on Na+ concentration of the growth medium, while stoichiometry of the Na+/H+ exchange also depends on the energy costs of the plasma membrane ATPase extrusion of H+ versus the actual cost of moving a Na+ ion out of the root cell (Briskin and Reynolds-Niesman, 1991; Briskin et al., 1995). Addition of Na+ elevated the transcription of H+-translocating ATPases in rice and tobacco roots (Niu et al., 1996; Zhang et al., 1999), but these results have yet to be linked to the increased requirement of the Na+/H+ antiporter for H+-extrusion. The bryophyte Physcomitrella patens is receiving much attention because it is tolerant to several stresses, and is potentially a source of genes to improve stress tolerance in higher plants (Nishiyama et al., 2003; Frank et al., 2005; Saavedra et al., 2006; Cuming et al., 2007). It has two Na+-ATPases, PpENA1 and PpENA2, and heterologous expression in yeast indicated that PpENA1 was functional as it improved the growth of yeast deficient in ScENA1 when grown on high Na+ media (and high K+ media) (Benito and Rodriguez-Navarro, 2003). Expression of PpENA1 is significantly upregulated by Na+ stress (and to a lesser extent by osmotic stress), but is unaffected by other stresses (Lunde et al., 2007). The wild-type line maintained a higher K+/Na+ ratio and plants were 40% larger than an ena1 knockout line at moderate Na+ concentrations (100 mM), but the difference disappears at high Na+ concentrations (Lunde et al., 2007). Controlled expression of these ATPases in plants may improve Na+ extrusion from root cells. Influx to Xylem An important concept in salt tolerance is that specific cell types are required to

perform different functions to minimize the transfer of Na+ to the shoot tissues. In order to minimize Na+ transfer to the shoot, the outer cortical cells of the root need to maximize Na+ efflux to or minimize influx from the external media, while the inner stelar cells of the root need to maximize Na+ influx from or minimize efflux to the xylem (Tester and Leigh, 2001). For example, the salinity tolerance of a salt-tolerant wheat line and an amphiploid cross between wheat and the salt-tolerant wheat grass, Lophopyrum elongatum, was related to the minimization of Na+ entry to the xylem from the root cortex (Gorham et al., 1990; Santa-María and Epstein, 2001). Cryo-scanning electron microscopy and x-ray microanalysis was used to show that Na+ is removed by the epidermal and cortical cells of durum wheat roots before it reaches the transpirational stream (Läuchli et al., 2008). The pericycle and xylem parenchyma also appeared to prevent Na+ movement to the xylem stream, while the endodermis did not. The mechanisms that control xylem Na+ loading and the energetics of Na+ transfer into the xylem are poorly understood. The loading of Na+ to the xylem may be an active process, despite the counter-intuitiveness of this idea. Estimates of xylem sap Na+ concentrations range from 1 to 10 mM using excised plants (Munns, 1985; Shi et al., 2002) and spittlebugs (Watson et al., 2001), whereas estimates of root cytoplasmic Na+ concentrations have been made at 10–30 mM using x-ray microanalysis (Koyro and Stelzer, 1988) or ion-sensitive electrodes (Carden, 1999). An estimate of 100 mV negative inside xylem parenchyma cells relative to the xylem has been made (DeBoer, 1999; Wegner et al., 1999); the energy difference is mainly a result of the potential difference across the plasma membrane. This situation would create active Na+ transport into the xylem, detrimental to the plant, but supported by the report that the SOS1 Na+/H+ antiporter is preferentially expressed in the xylem symplast boundary in roots and sos1 mutants accumulate less shoot Na+ than wild type (Shi et al., 2002). However, if stelar cytosolic Na+ levels were closer to 100 mM (Harvey 1985) and xylem Na+ was closer to 2 mM (Munns, 1985), passive leakage of Na+ to the xylem would be favored. Alternatively, Na+ loading could be active at low external Na+ concentrations and passive at high external Na+ concentrations (Shi et al., 2002).

92

GENES FOR CROP ADAPTATION TO POOR SOIL

Xylem loading is controlled by ABA, in the case of K+ and Cl− (Roberts, 1998; Gilliham, 2002). However, control of Na+ loading by ABA is much less studied. ABA does stimulate H+ extrusion to the xylem (Clarkson and Hanson, 1986), which could stimulate the Na+/H+-mediated transport of Na+ to the xylem. Other candidates for the control of Na+ loading to the xylem include the plasma membrane + H -translocating ATPases, one of which, when knocked out, increases salt sensitivity and shoot Na+ concentrations (Vitart et al., 2001). Inositol stimulates Na+ transfer to the shoot in Mesembryanthemum crystallinum, possibly as a means of lowering osmotic potential in shoots during drought stress (Nelson et al., 1999). Retrieval from Xylem Removal of Na+ prior to its arrival in the shoot has been proposed to occur

in the mature root (Kramer, 1983), mesocotyl (Drew and Läuchli, 1987), base of the shoot (Matsushita and Matoh, 1992), mature extended shoot (Blom-Zandstra et al., 1998), or internodal tissues (Wolf et al., 1991). One possibility is that a Na+-permeable, inwardly rectifying channel in the xylem parenchyma cells moves the Na+ out of the xylem into the cytosol (Wegner and Raschke, 1994). However, the suggestion that the Na+/H+ antiporter functions in reverse in this capacity under high Na+ (Lacan and Durand, 1996) is unlikely to be thermodynamically possible (Tester and Davenport, 2003). Based on the observation that sos1 mutants have lower shoot Na+ concentrations than wild type at modest salinity and higher shoot Na+ concentration at high salinity (100 mM), it was proposed that SOS1 could act as a Na+-scavenging mechanism at the root xylem-symplast interface (Shi et al., 2002). It has also been suggested that AtHKT1;1 may have variable activity at different solute levels (Rubio et al., 1995). However, the likelihood of Arabidopsis plants surviving extended experimental conditions of active transpiration and 100 mM Na+, seriously damage the validity of this idea (Møller and Tester, 2007). Further data suggest that AtHKT1;1 is responsible for Na+ retrieval from the xylem, which would be the reason the athkt1;1 mutant has elevated shoot Na+ levels (Davenport et al., 2007). A recent flurry of information on the function of the HKT gene family (Platten et al., 2006) has shown that members of the family are indeed responsible for Na+ retrieval from the xylem and reducing transfer to the shoot tissue. AtHKT1;1 has been shown to be localized to the stele (Sunarpi et al., 2005), and it was shown that athkt1;1 knockout lines had increased levels of Na+ in the xylem sap (Sunarpi et al., 2005). Unidirectional flux measurements using 22Na+ in athkt1;1 knockouts showed AtHKT1;1 functions in root accumulation of Na+ and retrieval of Na+ from the xylem, but is not involved in root influx or recirculation in the phloem (Davenport et al., 2007). Somewhat counter-intuitively, overexpression of AtHKT1;1 using its native promoter resulted in no change in shoot and root Na+ accumulation, but did result in a decrease in Na+ tolerance (Rus et al., 2004). Large variation in Na+ accumulation has been observed among Arabidopsis ecotypes, and some of this variation was explained by a deletion found in the AtHKT1;1 promoter region in two independent ecotypes, both of which overaccumulate Na+ in the shoot tissue (Rus et al., 2006). Interestingly, this deletion also resulted in increased Na+ tolerance, again indicating there is a complex relationship between Na+ accumulation and Na+ tolerance in Arabidopsis (Møller and Tester, 2007). Rice has a similar mechanism of xylem retrieval of Na+. Genetic analysis revealed an important + K -homeostasis QTL called SKC1 (Lin et al., 2004). The SKC1 gene was cloned and found to be OsHKT1;5 (Ren et al., 2005). Heterologous expression revealed it was a Na+ transporter, and whole plant analysis indicated it functions in the root xylem parenchyma to retrieve Na+ from the xylem stream thereby reducing Na+ accumulation in the shoot (Ren et al., 2005).

GENETIC DETERMINANTS OF SALINITY TOLERANCE IN CROP PLANTS

93

Flux analysis of a salt-tolerant durum wheat landrace called line 149 revealed that two individual traits that provide sodium exclusion to the line are decreased Na+ transfer to the shoot and increased Na+ retrieval to the leaf sheath tissue (Davenport et al., 2005). Two previously mapped QTLs, Nax1 and Nax2, linked to salinity tolerance in the line, were found to control the two transport traits (James et al., 2006). The Nax2 locus coincided with a sodium transporter related to OsHKT1;5 in rice, and this gene was shown to be responsible for removal of Na+ from the xylem in the roots (Byrt et al., 2007). Anoxia in maize roots seems to increase the transfer of Na+ to shoots at modest salinity, while it has an inhibitory effect at higher Na+ concentrations (Drew and Dikumwin, 1985). Also, transfer cells may be involved in Na+ removal from the xylem as subjection of maize and bean to modest salinity has been observed to stimulate transfer cell induction (Kramer et al., 1977; Yeo et al., 1977). Recirculation in Phloem Despite the widely held idea that movement of Na+ from the shoot through

the phloem to the roots is negligible, studies in lupin (Munns et al., 1988), Trifolium alexandrium (Winter, 1982), sweet pepper (Blom-Zandstra et al., 1998), and maize (Lohaus et al., 2000) indicate significant recirculation may be occurring. As well, this process has been linked to salinity tolerance in the salt-tolerant species Lycopersicon pennellii (Perez-Alfocea et al., 2000) and Phragmites communis (Matsushita and Matoh, 1991), when compared to their close salt-sensitive relatives. Also, sas1 (sodium accumulation in shoots) mutants accumulate two to seven times the Na+ in the shoot as wild-type Arabidopsis (while maintaining similar root Na+ levels), which may indicate a role in control of vascular loading and unloading for SAS1 (Nublat et al., 2001). A link has also been drawn between the sas2 mutant and an inability to recirculate Na+ in the phloem (Berthomieu et al., 2003). The sas2 locus was found to correspond to the AtHKT1;1 gene, and the sas mutants were found to have lower phloem Na+ concentration, higher shoot Na+ concentration, lower root Na+ concentration and increased Na+ sensitivity. This led to the conclusion that AtHKT1;1 mediates Na+ loading into the phloem in shoot tissue and unloading in root tissue and that the recirculation of Na+ from the shoot plays a critical role in salinity tolerance (Berthomieu et al., 2003). Subsequently however, the role of AtHKT1;1 has been shown to be in xylem retrieval and not in phloem recirculation (Davenport et al., 2007), thus transporters functioning in this regard have yet to be identified. Shoot Storage Low levels of Na+ are often observed in young leaves and are sometimes attributed

to their low rates of transpiration and short existence (Munns, 1993), but also to protection processes (Jeschke, 1984; Sibole et al., 2003; Wei et al., 2003). Movement of Na+ could occur through phloem and xylem elements towards older “sacrificial leaves” (Wolf et al., 1991) as has been described for other solutes (Pate et al., 1979). Using autoradiography, P has been observed to move throughout the plant from a source leaf, while Na+ did not enter the new leaves and roots (Marschner, 1995). Preferential Na+ accumulation has been observed in leaf epidermal cells, possibly due to activity of nonselective cation channels (Karley et al., 2000). Similar observations have been made in bundle sheath cells, which may be a mechanism to avoid Na+ accumulation in more photosynthetically important cells (Stelzer, 1981; Karley et al., 2000). Two putative sodium transport genes related to OsHKT1;4, were found to be in the Nax1 chromosomal region, and these transporters were responsible for retaining Na+ in the sheath tissue (Huang et al., 2006). Given that HKT genes have been shown to function in xylem retrieval in several species, it seems likely that homologs of this gene will be identified as being important to Na+ tolerance in other cereal species, like rice and barley.

94

GENES FOR CROP ADAPTATION TO POOR SOIL

Discontinuous distribution of Na+ within the leaf blades of rice has been observed using radioactive tracers (Yeo and Flowers, 1982). Sodium accumulated in the older leaves before the younger ones and to a decreased concentration in salinity-tolerant varieties. Time of exposure and difference in growth rates between the lines could not explain the difference in Na+ distribution between the leaves. This has important ramifications for experimental sampling of leaf blade tissue in rice meaning valuable comparisons between cultivars may only be made when precisely the same leaf blade is harvested from each plant. Salt Glands Salt glands are found in many halophytes. They function by moving salt into apoplastic

space where it accumulates and is pushed out of the leaf by bulk flow of water due to the negative osmotic potential created by the Na+ accumulation. This process is generally limited to salt marshes where water is not a limiting factor, but grasses have also been observed to have bicellular glands that secrete salt (Amarasinghe and Watson, 1989; McWhorter et al., 1995). A wild relative of rice, Porteresia coarctata, can grow on 25% sea water and has salt-secreting microhairs (Flowers et al., 1990). Recently it was shown that a mutant of the common ice plant without epidermal bladder cells (EBC) was more salt sensitive than the wild type since the EBCs play an important role as water reservoirs and in Na+ sequestration (Agarie et al., 2007). Some halophytes get around the problem of using bulk flow to push out accumulated Na+ by using “salt hairs,” which accumulate salt and water then die, thereby reducing transpirational losses. Alternatively, hydathodes, which release water by guttation during low transpiration periods, may be able to be adapted into salt glands through transformation of specific cell types with Na+ transporting genes.

Tissue Tolerance

Sodium exclusion mechanisms are not perfect and can also become overwhelmed in high sodium growth conditions. Plants continue to grow despite the sodium reaching the shoot, indicating mechanisms exist to tolerate potentially toxic levels of sodium in the leaf tissues. Vacuolar Storage Maintenance of low concentrations of Na+ within the cytoplasm of cells is of

utmost importance to the survival of plants in saline environments. The simplest method for plants to achieve this is the sequestration of Na+ within vacuoles. Na+ that enters the roots and is transported to the leaves must be compartmentalized in the vacuoles in order to avoid build-up of levels of Na+, which are toxic to proteins in the cytoplasm. Central to this process is the vacuolar Na+/H+ antiporter (NHX), which moves Na+ into the vacuole in exchange for H+ (Blumwald et al., 2000) and may be regulated by the SOS signaling pathway (Qiu et al., 2004). The original H+ gradient is created by both vacuolar H+-ATPase and H+-pyrophosphatase proteins (Gaxiola et al., 2001). This strategy also appears to work in roots as elevated levels of Na+ increase the activity of the Na+/H+ antiporter in the roots of barley (Garbarino and DuPont, 1989), tomato (Wilson and Shannon, 1995), sunflower (Ballesteros et al., 1997), maize (Zörb et al., 2005), Medicago (Zahran et al., 2007), and cotton (Wu et al., 2004), but not in salt-sensitive rice (Fukuda et al., 1998). There are eight NHX gene family members in Arabidopsis (Yokoi et al., 2002) of which only 1, 7, and 8 have functional assignments. NHX7 is also known as SOS1, and NHX8 has been shown to be a Li+/H+ antiporter (An et al., 2007), although the biological relevance of Li+ transport seems obscure. Several reports indicate that constitutive overexpression of the vacuolar transporters increases the salt tolerance of a variety species. Constitutive overexpression of the Arabidopsis vacuolar Na+/

GENETIC DETERMINANTS OF SALINITY TOLERANCE IN CROP PLANTS

95

H+ antiporter, AtNHX1, appears to increase salinity tolerance significantly in yeast (Aharon et al., 2003), Arabidopsis (Apse et al., 1999), tomato (Zhang and Blumwald, 2001), Brassica napus (Zhang et al., 2001), and cotton (He et al., 2005). Similarly, constitutive overexpression of various cereal homologues has been reported to improve the salinity tolerance of Arabidopsis (Brini et al., 2007), rice (Fukuda et al., 2004b; Zhao et al., 2006b), wheat (Xue et al., 2004), and barley (Fukuda et al., 2004a). The overexpression of NHX1 does not alter its role in the regulation of cytoplasmic and vacuolar pH (Fukada-Tanaka et al., 2000; Viehveger et al., 2002), and its cation selectivity is regulated by a luminal C-terminus (Yamaguchi et al., 2003). The overexpression of NHX1 in Arabidopsis leads to a small increase in shoot Na+ accumulation (Apse et al., 1999), which possibly allowed the cells to maintain a favorable osmotic balance, yet maintain low cytoplasmic Na+ levels with the Na+ sequestered to the vacuole. The nhx1 mutant had much lower Na+/H+ and K+/H+ exchange capabilities in isolated vacuoles, fewer large epidermal cells, and less overall leaf area, indicating NHX1 also plays a developmental role (Apse et al., 2003). Overexpression and knockout of the NHX1 gene in Arabidopsis has been shown to significantly and differentially alter the expression of a large number of genes in the plant’s salt stress response, indicating Arabidopsis is able to respond to change in one Na+ transporter by regulating other genes (Sottosanto et al., 2004; Sottosanto et al., 2007). The overexpression of AVP1, the vacuolar H+-pyrophosphatase, increases salinity tolerance and Na+ accumulation in Arabidopsis (Gaxiola et al., 2001). Activity of the Na+/H+ antiporter must have increased as a result of the AVP1 overexpression as the vacuolar Na+ levels of the transformants were higher than those of wild-type plants. This was shown in barley, where expression of the vacuolar H+-pyrophosphatase, HVP1, and the vacuolar Na+/H+ antiporter, NHX1, were similarly upregulated by salt stress (Fukuda et al., 2004a), and are similarly regulated by ABA, auxin, and gibberellin (Fukuda and Tanaka, 2006). NHX and AVP genes expressed simultaneously were found to increase salinity tolerance beyond that provided by expression of the genes individually in Arabidopsis (Brini et al., 2007) and rice (Zhao et al., 2006a). The overexpression of AVP1 also appears to increase growth rates of plants due to an interaction with PIN1, the auxin transporter, which increases auxin transport and leads to more robust plants that are better able to survive stress situations (Li et al., 2005; Yang et al., 2007). Na+ transport across the tonoplast is bidirectional and dynamic and efflux of Na+ from vacuoles is thought to occur through nonselective cation channels, which are highly permeable to other cations as well (Demidchik et al., 2002). Their activity is presumed to be quite low since they are highly permeable to cations (Na+, Ca2+), and since it appears there are no major differences in the properties of vacuolar channels of salt-sensitive and -tolerant species (Maathuis and Prins, 1990). Tolerance to Cytoplasmic Na+ Cytoplasmic reactions have been shown to be quite tolerant of

elevated levels of Na+ (100 mM) in the presence of osmoprotectants in in vitro studies (Cheeseman, 1988; Shomer-Ilan et al., 1991). The cytoplasmic enzymes of some salt-tolerant plants have been shown in vitro to be tolerant of elevated Na+ levels, likely due to increased ability to substitute Na+ for enzyme functions normally requiring K+ (Flowers and Dalmond, 1992). Additionally, the structure of some enzymes in halophytic bacteria provides tolerance to very high Na+ (Dym et al., 1995). However, if these enzymes were to be engineered into plants, the metabolic efficiency of the plant may drop significantly under nonsaline conditions (possibly avoided by stress-inducible induction of osmoprotectants) (Su and Wu, 2004; Urano et al., 2004). As well, the studies above must be complemented by in vivo studies to prove the validity of using this approach to produce salt-tolerant plants.

96

GENES FOR CROP ADAPTATION TO POOR SOIL

Response to Damage and Maintenance of Growth Salt stress increases the synthesis of osmotins

and dehydrins, which have similar properties to chaperones and seem to be responsible for maintenance of protein structure under elevated Na+ concentrations (Ingram and Bartels, 1996; Campbell and Close, 1997). Care needs to be taken with these studies because osmotic shock from sudden experimental changes may be responsible for the increase in these protective proteins. Constitutive overexpression of a late embryogenesis abundant (LEA) protein from barley increased salt tolerance in rice (Xu et al., 1996), while overexpression of a heat shock protein from a halotolerant bacterium in tobacco increased salt tolerance (Sugino et al., 1999). However, the lack of osmotic controls in these experiments makes the results more difficult to interpret (Flowers, 2004). As well, extreme care needs to be taken when salinity tolerance is claimed by authors without any quantification of growth parameters. Growth penalties associated with overexpression of a transgene have been hidden by use of such parameters as “relative root growth” (Flowers, 2004). Glycinebetaine (Chen and Murata, 2002), putrescine (Galston and Sawhnet, 1990), spermine (Mansour, 2000; Capell et al., 2004; Urano et al., 2004), and tyramine (Lefevre et al. 2001) have all been reported to increase in response to salt stress and to be involved in protective functions such as reducing lipid peroxidation and protecting mitochondrial electron transport reactions (Chen and Murata, 2002). The scavenging of reactive oxygen species (ROS) by these compounds is a favored hypothesis to explain their protective action (Zhu, 2001; Xiong et al., 2002), but they may also reduce the efflux of K+ from roots associated with salinity stress and ROS production (Cuin and Shabala, 2007). Another, more recent suggestion is that molecules like polyamines actually block NaCl-induced K+ efflux by non-selective cation channels (NSCC), thereby improving the ionic balance of the plant under salinity stress (Shabala et al., 2007). Interestingly, this process may also operate in reverse with new evidence indicating that salinity stress causes the release of spermidine into the apoplast where it is catabolized into H2O2 and results in upregulation of various stress-related genes and even programmed cell death (Moschou et al., 2008). The accumulation of ROS during salinity stress has been linked to various signaling and stress response pathways. The long cytoplasmic tail of SOS1 was found to interact with RCD1, a regulator of oxidative stress responses, thus indicating SOS1 is regulated, in part, by oxidative stress (Katiyar-Agarwal et al., 2006). Increase of ROS also appears to stabilize SOS1 transcripts (Chung et al., 2008). The accumulation of ROS also affects the accumulation of DELLA proteins, which promote plant growth; and salinity stress appears to affect the balance between ROS, DELLA proteins, and gibberellic acid (GA), which promotes plant growth (Achard et al., 2008). Another potentially related line of evidence indicates that the reduction in growth of salinity-stressed plants appears to be correlated with a decrease in endogenous GA within the plant; the molecular basis of this phenomena was recently identified as the salt-responsive transcription factor, DDF1, and the gibberellin-deactivating gene, GA2ox7 (Magome et al., 2008). The maintenance of plant growth under salinity stress has also been linked to the ability of the plant to maintain cell wall development, and in particular the N-glycosylation process, with mutants of this process being particularly sensitive to salt stress (Kang et al., 2008).

Osmotic Tolerance Synthesis of Osmoprotectants With the compartmentation of Na+ within the vacuole, and/or the

osmotic stress caused by high external salt concentrations, must come the increase of solutes in the cytoplasm to avoid dehydration of the cytoplasm. These solutes must not inhibit biochemical cel-

GENETIC DETERMINANTS OF SALINITY TOLERANCE IN CROP PLANTS

97

lular processes, and must potentially even protect them from inorganic ion damage (Shomer-Ilan et al., 1991). They are often soluble, neutral, or zwitterionic secondary metabolites such as glycinebetaine (Sulpice et al., 2003) and mannitol or primary metabolites such as proline (Ábrahám et al., 2003; Hien et al., 2003), trehalose (Garg et al., 2002; Jang et al., 2003), and sucrose (Hu et al., 2000). Overexpression of genes encoding compatible solutes (or osmoprotectants) generally result in osmotically insignificant levels of the particular metabolite (Chen and Murata, 2002), indicating that the role of such solutes in increasing salt tolerance may be to protect protein structures or scavenging of reactive oxygen species (Skopelitis et al., 2006). Somewhat problematically, most osmoprotectant transgenic studies have occurred in tobacco, a plant that is more sensitive to the osmotic component of NaCl than the ionic component (Murthy and Tester, 1996). A more appropriate species for these studies may be maize, in which a clear relationship between naturally varying levels of osmoprotectant and salt tolerance exists (Saneoka et al., 1995). Related to this is the striking number of salinity tolerance studies using the salt-sensitive Arabidopsis as the model plant; but a closely related salt-tolerant plant, Thellungiella halophila, may be a more appropriate model for discovering salinity-tolerance mechanisms (Taji et al., 2003; Volkov et al., 2003; Wang et al., 2004; Gong et al., 2005; Volkov and Amtmann, 2006). For instance, one study suggests that Thellungiella halophila may be more salt tolerant than Arabidopsis due to more efficient production of osmoprotectants (Kant et al., 2006). Stomatal Closure The salinity tolerance of Aster tripolium has been attributed to its ability to close stomata in response to leaf apoplastic Na+, whereas salt sensitivity was attributed to an inhibition of stomatal closure by Na+ in A. amellus (Robinson et al., 1997). However, it seems that both glycophytes and halophytes tend to have reduced stomatal conductance in elevated Na+ conditions (Ball, 1988; James et al., 2002). Even though the low productivity of crops in saline conditions is largely due to the low rates of transpiration imposed by osmotic stress, improving stomatal control may improve crop-salinity tolerance. This must be coupled, of course, with the ability to maintain a sufficient photosynthetic rate to sustain growth. The potential variability in osmotic stress tolerance was recently examined by measuring stomatal conductance before toxic levels of Na+ built up in the leaves of durum wheat (James et al., 2008). This analysis revealed that growth under the osmotic component of salinity stress was highly correlated to stomatal conductance and that there was a 2- to 3-fold difference in stomatal conductance among the 50 durum lines examined, indicating there is potential in breeding for this trait.

Genomics and Signaling

The three components of salinity tolerance do not exist in isolation. It is likely that all three operate simultaneously and interact to a greater or lesser extent to provide a plant with its overall salinity tolerance. This requires several signaling pathways and components operating simultaneously to finely tune the salinity stress response of a plant. The components of these pathways are becoming known due to the availability of vast amounts of genetic information from the suite of genomicscale technologies that have become available in the past decade. Genomics An incredible amount of data is being developed through the use of microarrays, comparing the expression of genes under salt-stress conditions to the expression under normal growth conditions (Bohnert et al., 2001; Kawasaki et al., 2001; Seki et al., 2001; Chen et al., 2002; Ozturk et al., 2002; Walia et al., 2005; Walia et al., 2006). Some of the data indicates that ∼8% of all genes

98

GENES FOR CROP ADAPTATION TO POOR SOIL

are transcriptionally altered by salt stress in Arabidopsis, and about 70% of the genes affected are distinct from those altered by drought stress (Bohnert et al., 2001). Similar studies in rice (Rabbani et al., 2003) and in maize indicated a figure of around 15% of genes being regulated by salt stress (Wang et al., 2003). Additionally, the gene expression is dependent on tissue type, developmental stage, and the extent of the stress treatment (Bohnert et al., 2001) as well as being largely ABAindependent (Wei et al., 2000). However, the reconciliation of these experiments with the results of previous physiologically based data needs to occur. Some microarray data suggest that the early responses are more important to salt-tolerance responses of plants (Bohnert et al., 2001), while older physiologically based data indicated the initial response to salt stress was largely unrelated to the long-term tolerance of the plant to salinity stress (Munns, 1993; Munns, 2002). Additionally, levels of Na+ used in most microarray experiments to induce salt stress, were likely to be high enough to induce death in Arabidopsis (Kilian et al., 2007) or rice (Kawasaki et al., 2001), and it has been suggested that future experiments should be undertaken at more relevant levels to increase the validity of the claims (Munns, 2002). More appropriate stress levels have been used in several more recent experiments performed in rice (Walia et al., 2005) and barley (Walia et al., 2006) in collaboration with the U.S. Salinity Laboratory. An Arabidopsis study examined the effects of either an 80-mM Na+ treatment, K+ starvation, or Ca2+ starvation on gene expression in roots and found significant overlap in genes being regulated by the three stresses (Maathuis et al., 2003). However, the interactions among the three ions in many cellular functions are extensive and complex, making interpretation very difficult. Recently, a “reverse functional genomic” technique was used to discover salinity tolerance genes/ alleles. A Thellungiella halophila cDNA library was overexpressed in Arabidopsis, and several previously uncharacterized genes encoding small unidentified proteins were identified by the increased salinity tolerance of their respective transformants (Du et al., 2008). Signaling Pathways The onset of salinity stress in plants activates pathways, which involve a

receptor, which perceives the stress, alterations in protein activity, changes in gene transcription via signaling intermediates, and phosphoprotein cascades (Leung and Giraudat, 1998; Hasegawa et al., 2000; Schroeder et al., 2001; Xiong et al., 2002; Zhu, 2002). Recently, the small ubiquitinlike modifier (SUMO) has been identified as being crucial to the modification of salt-signaling molecules (Conti et al., 2008). However, as with the microarray data, the interpretation of the data in many studies is difficult due to the high levels of Na+ used to elicit the stress response (Munns, 2002). One of the first responses to a sudden increase in Na+ is a significant rise in Ca in the cytoplasm of the cell (Knight et al., 1997; Kader et al., 2007). Although sudden increases in Na+ are not of immediate agricultural relevance, they provide a useful tool to reveal processes that are likely to be relevant to longer-term responses to salinization. Several components of the Ca2+ signal transduction pathway and Ca2+ transporters are induced by Na+ stress (Wimmers et al., 1992; Allen and Sanders, 1994; Hirayama et al., 1995). The nature of the Ca2+ signaling response was examined using Arabidopsis plants expressing the fluorescent [Ca2+]cyt reporter molecule, aequorin, and revealed highly specific root responses to various NaCl stresses (Tracy et al., 2008). The calcium regulated calcinuerin B-like protein/serine threonine protein kinase (CBL-CIPK) signaling pathway is central to signaling in plants. The Arabidopsis genome contains 10 CBLs and 25 CIPKs, while rice has 10 CBLs and 30 CIPKs, indicating the large signaling network potential that exists for this pathway (Kolukisaoglu et al., 2004). CBL1, CBL9, and CIPK23 regulate the K+ transporter, AKT1, which is central to K+ nutrition (Xu et al., 2006). Similarly, SOS2 Cytosolic Calcium Activity 2+

GENETIC DETERMINANTS OF SALINITY TOLERANCE IN CROP PLANTS

99

(CIPK24) and SOS3 (CBL4) regulate SOS1, a Na+ transporter crucial to the Na+ response in Arabidopsis. Protein Phosphorylation and Dephosphorylation Salinity stress induces protein phosphorylation

and dephosphorylation (Xiong et al., 2002). A GSK1/shaggy-like protein kinase, when overexpressed in Arabidopsis, caused increased anthocyanin synthesis and the transcription of NaCl stress-responsive genes as if there were a Na+ stress present (Piao et al., 2001). These plants also had increased salinity tolerance. The plants accumulated Na+ in the shoot in the same fashion as those overexpressing NHX1, which may indicate a common pathway is involved. Many other protein kinases and phosphatases have been implicated in the signaling pathway induced by salt stress (Trewavas and Malho, 1997; Knight and Knight, 2001), including recent reports showing the involvement of the mitogen-activated protein kinase (MAPK) signaling pathway (Hua et al., 2006; Alzwiy and Morris, 2007). Also, mutants of a kinase-associated protein phosphatase appeared to be involved in a salinity-signaling pathway separate from that of the well-characterized sos pathway (Manabe et al., 2008). The mutants were hypersensitive to salt stress, and known downstream receptor-like kinase family members were constitutively downregulated in the mutants. sos Mutant Pathway The sos (salt overly sensitive) mutants are recessive Arabidopsis mutants that

display sensitivity to Na+. The effect of Na+ on the mutants seems to be ionic rather than osmotic because the mutants are sensitive (unable to maintain root growth) to NaCl and LiCl and not to mannitol. The sos mutants 1, 2, and 3 are involved in a signaling pathway that is induced by salt stress, and several members of the signaling pathway have been identified (Qiu et al., 2004). The SOS1 protein has a long C-terminal tail, which extends into the cytoplasm of the cell and interacts with RCD1, a regulator of oxidative stress responses in Arabidopsis. This shows the sos pathway functions to relieve oxidative stress and indicates there is some level of cross talk between salinity stress and oxidative stress tolerance pathways (Katiyar-Agarwal et al., 2006). SOS3 is a myristoylated calcium-binding protein that responds to cytosolic Ca2+ increases (Liu and Zhu, 1997; Ishitani et al., 2000; Gong et al., 2004) and interacts with SOS2, a serine/threonine protein kinase (Halfter et al., 2000; Gong et al., 2004). Recently, it was shown that the SOS3-SOS2 interaction occurs in the root, while SOS2 interacts with the SOS3 homolog SOS3-LIKE CALCIUM BINDING PROTEIN8 (SCABP8)/CALCINUERIN B-LIKE10 (CBL10) in the shoot (Quan et al., 2007). SOS2 then interacts with SOS1, a Na+/H+ antiporter located in the plasma membrane of epidermal and stelar cells in the roots (Qiu et al., 2002; Shi et al., 2002), increasing SOS1 transcription and thence SOS1 activity (Shi et al., 2000; Qiu et al., 2002; Quintero et al., 2002). SOS1 may also be a Na+ sensor and provide feedback to the SOS pathway (Zhu, 2002). Sos2 and sos3 mutants accumulate more Na+ than wild-type plants and sos1 mutants accumulate less Na+ than wild-type plants indicating that SOS2 and SOS3 are likely to control other proteins besides SOS1 (Zhu et al., 1998). Sos1, sos2, and sos3 mutations also all affect K+ nutrition (Zhu et al., 1998). Sos4 encodes a pyridoxal kinase and may also be involved in the control of SOS1 (Zhu, 2002). The root tips of the sos5 mutant swell, and root growth is arrested under salt stress. SOS5 has been reported to encode a cell surface adhesion protein and is required for normal cell expansion (Shi et al., 2003). The sos pathway appears to be conserved in rice as homologs of SOS1, SOS2, and SOS3 have been identified (Martínez-Atienza et al., 2006). Response to salinity is a highly coordinated and complicated process involving the induction of transcription of many genes. It is important to identify the transcription factors, which coordinate the expression of the target genes (Chen et al., 2002; Xiong et al., 2002). The promoter regions, which are targets of these transcription factors, include the

Transcription Factors and Small RNAs

100

GENES FOR CROP ADAPTATION TO POOR SOIL

dehydration-responsive elements (DRE) (Dubouzet et al., 2003; Novillo et al., 2004) and ABAresponsive elements (ABRE). However, these elements are more likely to respond transcriptionally to osmotic changes than to Na+-related stresses. The role of zinc-finger proteins (Mukhopadhyay et al., 2004; Xu et al., 2008), H-protein promoter binding factors (Nagaoka and Takano, 2003), NAC-type transcription factors (Nakashima et al., 2007), calcium-binding transcription factors (Kim and Kim, 2006), basic leucine zipper (bZIP) (Xiang et al., 2008), and translation initiation factors (Rausellz et al., 2003) in regulation of salt-tolerance genes and downstream responses have also been examined. While constitutive overexpression of these genes often results in increased tolerance to abiotic stresses, it also frequently comes with growth inhibition under unstressed conditions (Liu et al., 1998). However, using a stress-inducible promoter to drive these genes may reduce the growth inhibition under unstressed conditions (Kasuga et al., 1999). The importance of small RNAs in the regulation of gene expression is only starting to be realized, and the impact this relatively newly discovered class of RNA is having on understanding plant transport regulation is already extremely important (Phillips et al., 2007; Sunkar et al., 2007). A pair of endogenous natural cis-antisense transcripts produces small interfering RNAs when Arabidopsis is salt stressed, and these siRNAs are involved in regulating the plant’s response to salinity stress (Borsani et al., 2005). Cell-specific Signaling Despite the specific responses different cell types have to salt stress, very little has been done to study cell types individually. Individual cell type-specific responses are likely to be involved in damage limitation while constitutive responses are likely to be involved in damage repair. Damage limitation is obviously the more desirable response to salt stress. One study examined the changes in cytosolic Ca2+ in response to a sudden and large Na+ stress and found distinctive oscillations in cytosolic Ca2+ in endodermal and pericycle cells (Kiegle et al., 2000). This information suggests the need for careful interpretation of the phenotypes resulting from constitutive overexpression of transgenes. Conclusion

Although significant progress has been made in elucidating the genes involved in contributing to salinity tolerance in crop plants, there is much left to learn. There are many genes from the gene families mentioned in this review (e.g., CHX, NHX, GLR) that have not been functionally described to this point. For example, there are nine HKT genes in rice, of which only three have been characterized to any extent (OsHKT2;1, OsHKT1;4, and OsHKT1;5). Additionally, there are large numbers of candidate genes being published in genomic studies of salt-stressed plants, which appear to be significantly regulated, but either have no known function associated with them or have not been placed into previously described salinity tolerance pathways. It is also evident that there is much less known about the genes involved in the osmotic tolerance component of salinity-stress tolerance. Further genetic studies with well-characterized parental plant populations will be necessary to uncover candidate genes for these important salinitytolerance components. References Ábrahám, E., Rigó, G., Székely, G., Nagy, R., Koncz, C., and Szabados, L. (2003) Light-dependent induction of proline biosynthesis by abscisic acid and salt stress is inhibited by brassinosteroid in Arabidopsis. Plant Mol Biol 51:363–372.

GENETIC DETERMINANTS OF SALINITY TOLERANCE IN CROP PLANTS

101

Achard, P., Renou, J.-P., Berthomé, R., Harberd, N.P., and Genschik, P. (2008) Plant DELLAs Restrain Growth and Promote Survival of Adversity by Reducing the Levels of Reactive Oxygen Species. Curr Biol 18:656–660. Agarie, S., Shimoda, T., Shimizu, Y., Baumann, K., Sunagawa, H., Kondo, A., Ueno, O., Nakahara, T., Nose, A., and Cushman, J.C. (2007) Salt tolerance, salt accumulation, and ionic homeostasis in an epidermal bladder-cell-less mutant of the common ice plant Mesembryanthemum crystallinum. J Exp Bot 58:1957–1967. Aharon, G.S., Apse, M.P., Duan, S., Hua, X., and Blumwald, E. (2003) Characterization of a family of vacuolar Na+/H+ antiporters in Arabidopsis thaliana. Plant Soil 253:245–256. Allen, G. and Sanders, D. (1994) Osmotic stress enhances the competence of Beta vulgaris vacuoles to respond to inositol 1,4,5-trisphosphate. Plant J 6:687–695. Alzwiy, I.A. and Morris, P.C. (2007) A mutation in the Arabidopsis MAP kinase kinase 9 gene results in enhanced seedling stress tolerance. Plant Sci 173:302–308. Amarasinghe, V. and Watson, L. (1989) Variation in salt secretory activity of microhairs in grasses. Aust J Plant Physiol 16:219–229. Amtmann, A., Fischer, M., Marsh, E.L., Stefanovic, A., Sanders, D., and Schachtman, D.P. (2001) The Wheat cDNA LCT1 Generates Hypersensitivity to Sodium in a Salt-Sensitive Yeast Strain. Plant Physiol 126:1061–1071. Amtmann, A. and Sanders, D. (1999) Mechanisms of Na+ uptake by plant cells. Adv Botanic Res 29:75–112. An, R., Chen, Q.-J., Chai, M.-F., Lu, P.-L., Su, Z., Qin, Z.-X., Chen, J., and Wang, X.-C. (2007) AtNHX8, a member of the monovalent cation:proton antiporter-1 family in Arabidopsis thaliana, encodes a putative Li+/H+ antiporter. Plant J 49:718–728. Apse, M., Aharon, G., Sneddon, W., and Blumwald, E. (1999) Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis. Science 285:1256–1258. Apse, M.P., Sottosanto, J.B., and Blumwald, E. (2003) Vacuolar cation/H+ exchange, ion homeostasis, and leaf development are altered in a T-DNA insertional mutant of AtNHX1, the Arabidopsis vacuolar Na+/H+ antiporter. Plant J 36:229–239. Ball, M.C. (1988) Salinity tolerance in the mangroves, Aegiceras corniculatum and Avicennia marina. 1. Water use in relation to growth, carbon partitioning and salt balance. Aust J Plant Physiol 15:447–464. Ballesteros, E., Blumwald, E., Donaire, J.P., and Belver, A. (1997) Na+/H+ antiporter activity in tonoplast vesicles isolated from sunflower roots induced by NaCl stress. Physiol Plant 99:328–334. Banuelos, M.A., Haro, R., Fraile-Escanciano, A., and Rodriguez-Navarro, A. (2008) Effects of Polylinker uATGs on the Function of Grass HKT1 Transporters Expressed in Yeast Cells. Plant Cell Physiol 49:1128–1132. Benito, B. and Rodriguez-Navarro, A. (2003) Molecular cloning and characterization of a sodium-pump ATPase of the moss Physcomitrella patens. Plant J 36:382–389. Berthomieu, P., Conéjéro, G., Nublat, A., Brackenbury, W.J., Lambert, C., Savio, C., Uozumi, N., Oiki, S., Yamada, K., Cellier, F., Gosti, F., Simonneau, T., Essah, P.A., Tester, M., Véry, A.-A., Sentenac, H., and Casse, F. (2003) Functional analysis of AtHKT1 in Arabidopsis shows that Na+ recirculation by the phloem is crucial for salt tolerance. EMBO J 22:2004–2014. Bhandal, I. and Malik, C. (1988) Potassium estimation, uptake, and its role in the physiology and metabolism of flowering plants. Int Rev Cytol 110:205–254. Blom-Zandstra, M., Vogelzang, S., and Veen, B. (1998) Sodium fluxes in sweet pepper exposed to varying sodium concentrations. J Exp Bot 49:1863–1868. Blumwald, E., Aharon, G.S., and Apse, M.P. (2000) Sodium transport in plant cells. Biochim Biophys Acta 1465:140–151. Bohnert, H.J., Ayoubi, P., Borchert, C., Bressan, R.A., Burnap, R.L., Cushman, J.C., Cushman, M.A., Deyholos, M., Fischer, R., Galbraith, D.W., Hasegawa, P.M., Jenks, M., Kawasaki, S., Koiwa, H., Kore-eda, S., Lee, B.-H., Michalowski, C.B., Misawa, E., Nomura, M., Ozturk, N., Postier, B., Prade, R., Song, C.-P., Tanaka, Y., Wang, H., and Zhu, J.-K. (2001) A genomics approach towards salt stress tolerance. Plant Physiol Biochem 39:295–311. Borsani, O., Zhu, J., Verslues, P.E., Sunkar, R., and Zhu, J.-K. (2005) Endogenous siRNAs Derived from a Pair of Natural cisAntisense Transcripts Regulate Salt Tolerance in Arabidopsis. Cell 123:1279–1291. Brini, F.C., Hanin, M., Mezghani, I., Berkowitz, G.A., and Masmoudi, K. (2007) Overexpression of wheat Na+/H+ antiporter TNHX1 and H+-pyrophosphatase TVP1 improve salt- and drought-stress tolerance in Arabidopsis thaliana plants. J Exp Bot 58:301–308. Briskin, D., Basu, S., and Assmann, S. (1995) Characterization of the red beet plasma membrane H+-ATPase reconstituted in a planar bilayer system. Plant Physiol 108:393–398. Briskin, D. and Reynolds-Niesman, I. (1991) Determination of H+/ATP stoichiometry for the plasma membrane H+-ATPase from red beet (Beta vulgaris L.) storage tissue. Plant Physiol 95:242–250. Byrt, C.S., Platten, J.D., Spielmeyer, W., James, R.A., Lagudah, E.S., Dennis, E.S., Tester, M., and Munns, R. (2007) HKT1;5-Like Cation Transporters Linked to Na+ Exclusion Loci in Wheat, Nax2 and Kna1. Plant Physiol 143:1918–1928.

102

GENES FOR CROP ADAPTATION TO POOR SOIL

Campbell, S.A. and Close, T.J. (1997) Dehydrins: genes, proteins, and association with phenotypic traits. New Phytol 137:61–74. Capell, T., Bassie, L., and Christou, P. (2004) Modulation of the polyamine biosynthetic pathway in transgenic rice confers tolerance to drought stress. PNAS 101:9909–9914. Carden, D.E. (1999) The cell physiology of barley salt tolerance. PhD thesis. University of Sussex, UK. Cheeseman, J.M. (1982) Pump-leak sodium fluxes in low salt corn roots. J Membr Biol 70:157–164. Cheeseman, J.M. (1988) Mechanisms of salinity tolerance in plants. Plant Physiol 87:547–550. Cheeseman, J.M., Bloebaum, P.D., and Wickens, L.K. (1985) Short term 22Na+ and 42K+ uptake in intact, mid-vegetative Spergularia marina plants. Physiol Plant 65:460–466. Cheffings, C.M. (2001) Calcium channel activity of a plant glutamate receptor homologue. In 12th International Workshop on Plant Membrane Biology, Madison, WI. Chen, T.H. and Murata, N. (2002) Enhancement of tolerance of abiotic stress by metabolic engineering of betaines and other compatible solutes. Curr Opin Plant Biol 5:250–257. Chen, W., Provart, N.J., Glazebrook, J., Katagiri, F., Chang, H.-S., Eulgem, T., Mauch, F., Luan, S., Zou, G., Whitham, S.A., Budworth, P.R., Tao, Y., Xie, Z., Chen, X., Lam, S., Kreps, J.A., Harper, J.F., Si-Ammour, A., Mauch-Mani, B., Heinlein, M., Kobayashi, K., Hohn, T., Dangl, J.L., Wang, X., and Zhu, T. (2002) Expression Profile Matrix of Arabidopsis Transcription Factor Genes Suggests Their Putative Functions in Response to Environmental Stresses. Plant Cell 14:559–574. Chen, Z., Newman, I., Zhou, M., Mendham, N., Zhang, G., and Shabala, S. (2005) Screening plants for salt tolerance by measuring K+ flux: a case study for barley. Plant Cell Environ 28:1230–1246. Chen, Z., Pottosin, I.I., Cuin, T.A., Fuglsang, A.T., Tester, M., Jha, D., Zepeda-Jazo, I., Zhou, M., Palmgren, M.G., Newman, I.A., and Shabala, S. (2007) Root Plasma Membrane Transporters Controlling K+/Na+ Homeostasis in Salt-Stressed Barley. Plant Physiol 145:1714–1725. Chung, J.-S., Zhu, J.-K., Bressan, R.A., Hasegawa, P.M., and Shi, H. (2008) Reactive oxygen species mediate Na+-induced SOS1 mRNA stability in Arabidopsis. Plant J 53:554–565. Clarkson, D.T. and Hanson, J.B. (1986) Proton fluxes and the activity of a stelar proton pump in onion roots. J Exp Bot 37:1136–1150. Clemens, S., Antonosiewicz, D.M., Ward, J.M., Schachtman, D.P., and Schroeder, J.I. (1998) The plant cDNA LCT1 mediates the uptake of calcium and cadmium in yeast. PNAS 95:12043–12048. Conti, L., Price, G., O’Donnell, E., Schwessinger, B., Dominy, P., and Sadanandom, A. (2008) Small Ubiquitin-Like Modifier Proteases OVERLY TOLERANT TO SALT1 and -2 Regulate Salt Stress Responses in Arabidopsis. Plant Cell 20:2894–2908. Cramer, G.R. (2002) Sodium-calcium interactions under salinity stress. In A. Läuchli, Lüttge, U., Ed., Salinity. Environmentplants-molecules. Kluwer, Dordrecht, pp 205–227. Cramer, G.R. (2003) Differential effects of salinity on leaf elongation kinetics of three grass species. Plant Soil 253:233–244. Cramer, G.R., Läuchli, A., and Polito, V. (1985) Displacement of Ca2+ by Na+ from the plasmalemma of root cells—a primary response to salt stress. Plant Physiol 79:207–211. Cuin, T.A., Betts, S.A., Chalmandrier, R., and Shabala, S. (2008) A root’s ability to retain K+ correlates with salt tolerance in wheat. J Exp Bot 59:2697–2706. Cuin, T.A., Miller, A., Laurie, S., and Leigh, R. (2003) Potassium activities in cell compartments of salt-grown barley leaves. J Exp Bot 54:657–661. Cuin, T.A. and Shabala, S. (2007) Compatible solutes reduce ROS-induced potassium efflux in Arabidopsis roots. Plant Cell Environ 30:875–885. Cuming, A.C., Cho, S.H., Kamisugi, Y., Graham, H., and Quatrano, R.S. (2007) Microarray analysis of transcriptional responses to abscisic acid and osmotic, salt, and drought stress in the moss, Physcomitrella patens. New Phytol 176:275–287. Davenport, R., James, R.A., Zakrisson-Plogander, A., Tester, M., and Munns, R. (2005) Control of Sodium Transport in Durum Wheat. Plant Physiol 137:807–818. Davenport, R.J. (1998) Mechanisms of toxic sodium influx in wheat. University of Cambridge. Davenport, R.J., Munoz-Mayor, A., Jha, D., Essah, P.A., Rus, A., and Tester, M. (2007) The Na+ transporter AtHKT1;1 controls retrieval of Na+ from the xylem in Arabidopsis. Plant Cell Environ 30:497–507. Davenport, R.J., Reid, R.J., and Smith, F.A. (1997) Sodium-calcium interactions in two wheat species differing in salinity tolerance. Physiol Plant 99:323–327. Davenport, R.J. and Tester, M. (2000) A Weakly Voltage-Dependent, Nonselective Cation Channel Mediates Toxic Sodium Influx in Wheat. Plant Physiol 122:823–834. DeBoer, A.H. (1999) Potassium translocation into the root xylem. Plant Biol 1:36–45.

GENETIC DETERMINANTS OF SALINITY TOLERANCE IN CROP PLANTS

103

Demidchik, V., Bowen, H.C., Maathuis, F.J.M., Shabala, S.N., Tester, M.A., White, P.J., and Davies, J.M. (2002) Arabidopsis thaliana root non-selective cation channels mediate calcium uptake and are involved in growth. Plant J 32:799–808. Demidchik, V., Essah, P.A., and Tester, M. (2004) Glutamate activates cation currents in the plasma membrane of Arabidopsis root cells. Planta 219:167–175. Demidchik, V. and Maathuis, F.J.M. (2007) Physiological roles of nonselective cation channels in plants: from salt stress to signalling and development. New Phytol 175:387–404. Demidchik, V. and Tester, M. (2002) Sodium Fluxes through Nonselective Cation Channels in the Plasma Membrane of Protoplasts from Arabidopsis Roots. Plant Physiol 128:379–387. Drew, M.C. and Dikumwin, E. (1985) Sodium exclusion from the shoots by roots of Zea mays (cv. LG11) and its breakdown with oxygen deficiency. J Exp Bot 36:55–62. Drew, M.C. and Läuchli, A. (1987) The role of the mesocotyl in sodium exclusion from the shoot of Zea mays L. (cv. Pioneer 3906). J Exp Bot 38:409–418. Du, J., Huang, Y.-P., Xi, J., Cao, M.-J., Ni, W.-S., Chen, X., Zhu, J.-K., Oliver, D.J., and Xiang, C.-B. (2008) Functional genemining for salt-tolerance genes with the power of Arabidopsis. Plant J 56:653–664. Dubcovsky, J., Santa Maria, G., Epstein, E., Luo, M.-C., and Dvorˇák J. (1996) Mapping of the K+/Na+ discrimination locus Kna1 in wheat. Theor Appl Genet 92:448–454. Dubouzet, J.G., Sakuma, Y., Ito, Y., Kasuga, M., Dubouzet, E.G., Miura, S., Seki, M., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2003) OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought-, high-salt- and cold-responsive gene expression. Plant J 33:751–763. Dym, O., Mevarech, M., and Sussman, J.L. (1995) Structural features that stabilize halophilic malate dehydrogenase from an Archaebacterium. Science 267:1344–1346. El-Hamdaoui, A., Redondo-Nieto, M., Torralba, B., Rivilla, R., Bonilla, I., and Bolaños, L. (2003) Influence of boron and calcium on the tolerance to salinity of nitrogen-fixing pea plants. Plant Soil 251:93–103. Elphick, C.H., Sanders, D., and Maathuis, F.J.M. (2001) Critical role of divalent cations and Na+ efflux in Arabidopsis thaliana salt tolerance. Plant Cell Environ 24:733–740. Essah, P.A., Davenport, R., and Tester, M. (2003) Sodium Influx and Accumulation in Arabidopsis. Plant Physiol 133: 307–318. Fernandez-Garcia, N., Lopez-Perez, L., Hernandez, M., and Olmos, E. (2009) Role of phi cells and the endodermis under salt stress in Brassica oleracea. New Phytol 181:347–360. Flowers, T., Flowers, S., Hajibagheri, M., and Yeo, A. (1990) Salt tolerance in the halophytic wild rice, Porteresia coarctata Tateoka. New Phytol 114:675–684. Flowers, T., Hajibagheri, M., and Yeo, A. (1991) Ion accumulation in the cell walls of rice plants growing under saline conditions—evidence for the Öertli hypothesis. Plant Cell Environ 14:319–325. Flowers, T.J. (2004) Improving crop salt tolerance. J Exp Bot 55:307–319. Flowers, T.J. and Dalmond, D. (1992) Protein synthesis in halophytes: the influence of potassium, sodium and magnesium in vitro. Plant Soil 146:153–161. Frank, W., Ratnadewi, D., and Reski, R. (2005) Physcomitrella patens is highly tolerant against drought, salt and osmotic stress. Planta 220:384–394. Fukuda, A., Chiba, K., Maeda, M., Nakamura, A., Maeshima, M., and Tanaka, Y. (2004a) Effect of salt and osmotic stresses on the expression of genes for the vacuolar H+-pyrophosphatase, H+-ATPase subunit A, and Na+/H+ antiporter from barley. J Exp Bot 55:585–594. Fukuda, A., Nakamura, A., Tagiri, A., Tanaka, H., Miyao, A., Hirochika, H., and Tanaka, Y. (2004b) Function, Intracellular Localization and the Importance in Salt Tolerance of a Vacuolar Na+/H+ Antiporter from Rice. Plant Cell Physiol 45:146–159. Fukuda, A. and Tanaka, Y. (2006) Effects of ABA, auxin, and gibberellin on the expression of genes for vacuolar H+-inorganic pyrophosphatase, H+-ATPase subunit A, and Na+/H+ antiporter in barley. Plant Physiol Biochem 44:351–358. Fukuda, A., Yazaki, Y., Ishikawa, T., Koike, S., and Tanaka, Y. (1998) Na+/H+ antiporter in tonoplast vesicles from rice roots. Plant Cell Physiol 39:196–201. Fukada-Tanaka, S., Inagaki, Y., Yamaguchi, T., Saito, N., and Iida, S. (2000) Colour-enhancing protein in blue petals. Nature 407:581. Galston, A.W. and Sawhnet, R.K. (1990) Polyamines in plant physiology. Plant Physiol 94:406–410. Garbarino, J. and DuPont, F.M. (1989) Rapid Induction of Na+/H+ Exchange Activity in Barley Root Tonoplast. Plant Physiol 89:1–4. Garcia, A., Rizzo, C.A., Ud-din, J., Bartos, S.L., Sendadhira, D., Flowers, T.J., and Yeo, A.R. (1997) Sodium and potassium transport to the xylem are inherited independently in rice, and the mechansim of sodium:potassium selectivity differs between rice and wheat. Plant Cell Environ 20:1167–1174.

104

GENES FOR CROP ADAPTATION TO POOR SOIL

Garg, A.K., Kim, J.-K., Owens, T.G., Ranwala, A.P., Choi, Y.D., Kochian, L.V., and Wu, R.J. (2002) Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses. PNAS 99:15898–15903. Gaxiola, R.A., Li, J., Undurraga, S., Dang, L.M., Allen, G.J., Alper, S.L., and Fink, G.R. (2001) Drought- and salt-tolerant plants result from overexpression of the AVP1 H1-pump. PNAS 98:11444–11449. Genc, Y., McDonald, G.K., and Tester, M. (2007) Reassessment of tissue Na+ concentration as a criterion for salinity tolerance in bread wheat. Plant Cell Environ 30:1486–1498. Gilliham, M. (2002) Regulation of ion loading to maize root xylem. PhD thesis. University of Cambridge. Gobert, A., Park, G., Amtmann, A., Sanders, D., and Maathuis, F.J.M. (2006) Arabidopsis thaliana Cyclic Nucleotide Gated Channel 3 forms a non-selective ion transporter involved in germination and cation transport. J Exp Bot 57:791–800. Gong, D., Guo, Y., Schumaker, K.S., and Zhu, J.-K. (2004) The SOS3 Family of Calcium Sensors and SOS2 Family of Protein Kinases in Arabidopsis. Plant Physiol 134:919–926. Gong, H.J., Randall, D.P., and Flowers, T.J. (2006) Silicon deposition in the root reduces sodium uptake in rice (Oryza sativa L.) seedlings by reducing bypass flow. Plant Cell Environ 29:1970–1979. Gong, Q., Li, P., Ma, S., Rupassara, S.I., and Bohnert, H.J. (2005) Salinity stress adaptation competence in the extremophile Thellungiella halophila in comparison with its relative Arabidopsis thaliana. Plant J 44:826–839. Gorham, J., Jones, R.G.W., and Bristol, A. (1990) Partial characterization of the trait for enhanced K+-Na+ discrimination in the D genome of wheat. Planta 180:590–597. Grabov, A. (2007) Plant KT/KUP/HAK Potassium Transporters: Single Family—Multiple Functions. Ann Botany 99:1035–1041. Halfter, U., Ishitani, M., and Zhu, J.-K. (2000) The Arabidopsis SOS2 protein kinase physically interacts with and is activated by the calcium-binding protein SOS3. PNAS 97:3735–3740. Haro, R., Bañuelos, M.A., Senn, M.E., Barrero-Gil, J., and Rodríguez-Navarro, A. (2005) HKT1 Mediates Sodium Uniport in Roots. Pitfalls in the Expression of HKT1 in Yeast. Plant Physiol 139:1495–1506. Harvey, D.M.R. (1985) The effect of salinity on ion concentrations within the root cell of Zea mays L. Planta 165:242–248. Hasegawa, P.M., Bressan, R.A., Zhu, J.-K., and Bohnert, H.J. (2000) Plant cellular and molecular responses to high salinity. Annu Rev Plant Physiol Plant Mol Biol 51:463–499. He, C., Yan, J., Shen, G., Fu, L., Holaday, A.S., Auld, D., Blumwald, E., and Zhang, H. (2005) Expression of an Arabidopsis Vacuolar Sodium/Proton Antiporter Gene in Cotton Improves Photosynthetic Performance Under Salt Conditions and Increases Fiber Yield in the Field. Plant Cell Physiol 46:1848–1854. Hien, D.T., Jacobs, M., Angenon, G., Hermans, C., Thu, T.T., Son, L.V., and Roosens, N. (2003) Proline accumulation and D1pyrroline-5-carboxylate synthetase gene properties in three rice cultivars differing in salinity and drought tolerance. Plant Sci 165:1059–1068. Hirayama, T., Ohto, C., Mizoguchi, T., and Shinozaki, K. (1995) A gene encoding a phosphatidylinositol-specific phospholipase C is induced by dehydration and salt stress in Arabidopsis thaliana. PNAS 92:3903–3907. Horie, T., Costa, A., Kim, T.H., Han, M.J., Horie, R., Leung, H.-Y., Miyao, A., Hirochika, H., An, G., and Schroeder, J.I. (2007) Rice OsHKT2;1 transporter mediates large Na+ influx component into K+-starved roots for growth. EMBO J 26:3003–3014. Horie, T., Horie, R., Chan, W.-Y., Leung, H.-Y., and Schroeder, J.I. (2006) Calcium Regulation of Sodium Hypersensitivities of sos3 and athkt1 Mutants. Plant Cell Physiol 47:622–633. Hu, Y. and Schmidhalter, U. (2005) Drought and salinity: A comparison of their effects on mineral nutrition of plants. J Plant Nutr Soil Sci 168:541–549. Hu, Y.C., Schnyder, H., and Schmidhalter, U. (2000) Carbohydrate deposition and partitioning in elongating leaves of wheat under saline soil conditions. Aust J Plant Physiol 27:363–370. Hua, Z.-M., Yang, X., and Fromm, M.E. (2006) Activation of the NaCl- and drought-induced RD29A and RD29B promoters by constitutively active Arabidopsis MAPKK or MAPK proteins. Plant Cell Environ 29:1761–1770. Huang, S., Spielmeyer, W., Lagudah, E.S., James, R.A., Platten, J.D., Dennis, E.S., and Munns, R. (2006) A Sodium Transporter (HKT7) Is a Candidate for Nax1, a Gene for Salt Tolerance in Durum Wheat. Plant Physiol 142:1718–1727. Huang, S., Spielmeyer, W., Lagudah, E.S., and Munns, R. (2008) Comparative mapping of HKT genes in wheat, barley, and rice, key determinants of Na+ transport, and salt tolerance. J Exp Bot 59:927–937. Ingram, J. and Bartels, D. (1996) The molecular basis of dehydration tolerance in plants. Annu Rev Plant Physiol Plant Mol Biol 47:377–403. Ishitani, M., Liu, J., Halfter, U., Kim, C.-S., Shi, W., and Zhu, J.-K. (2000) SOS3 Function in Plant Salt Tolerance Requires N-Myristoylation and Calcium Binding. Plant Cell 12:1667–1677. Jacoby, B. and Hanson, J.B. (1985) Controls on Na+-22 influx in corn roots. Plant Physiol 77:930–934.

GENETIC DETERMINANTS OF SALINITY TOLERANCE IN CROP PLANTS

105

James, R.A., Davenport, R.J., and Munns, R. (2006) Physiological Characterization of Two Genes for Na+ Exclusion in Durum Wheat, Nax1 and Nax2. Plant Physiol 142:1537–1547. James, R.A., Rivelli, A.R., Munns, R., and von Caemmerer, S. (2002) Factors affecting CO2 assimilation, leaf injury and growth in salt-stressed durum wheat. Funct Plant Biol 29:1393–1403. James, R.A., von Caemmerer, S., Condon, A.G., Zwart, A.B., and Munns, R. (2008) Genetic variation in tolerance to the osmotic stress component of salinity stress in durum wheat. Funct Plant Biol 35:111–123. Jang, I.-C., Oh, S.-J., Seo, J.-S., Choi, W.-B., Song, S.I., Kim, C.H., Kim, Y.S., Seo, H.-S., Choi, Y.D., Nahm, B.H., and Kim, J.-K. (2003) Expression of a Bifunctional Fusion of the Escherichia coli Genes for Trehalose-6-Phosphate Synthase and Trehalose-6-Phosphate Phosphatase in Transgenic Rice Plants Increases Trehalose Accumulation and Abiotic Stress Tolerance without Stunting Growth. Plant Physiol 131:516–524. Jefferies, R.L. (1973) The ionic relations of seedlings of the halophyte Triglochin maritima L. In A. WP, Ed., Ion transport in Plants. Academic, London, pp 297–321. Jeschke, W.D. (1984) K+/Na+ exchange at cellular membranes, intracellular compartmentation of cations, and salt tolerance. In T.R. Staples RC, Ed., Salinity tolerance in plants. Wiley, New York, pp 37–66. Kader, M.A., Lindberg, S., Seidel, T., Golldack, D., and Yemelyanov, V. (2007) Sodium sensing induces different changes in free cytosolic calcium concentration and pH in salt-tolerant and -sensitive rice (Oryza sativa) cultivars. Physiol Plant 130:99–111. Kang, J.S., Frank, J., Kang, C.H., Kajiura, H., Vikram, M., Ueda, A., Kim, S., Bahk, J.D., Triplett, B., Fujiyama, K., Lee, S.Y., von Schaewen, A., and Koiwa, H. (2008) Salt tolerance of Arabidopsis thaliana requires maturation of N-glycosylated proteins in the Golgi apparatus. PNAS 105:5933–5938. Kant, S., Kant, P., Raveh, E., and Barak, S. (2006) Evidence that differential gene expression between the halophyte, Thellungiella halophila, and Arabidopsis thaliana is responsible for higher levels of the compatible osmolyte proline and tight control of Na+ uptake in T. halophila. Plant Cell Environ 29:1220–1234. Karley, A.J., Leigh, R.A., and Sanders, D. (2000) Differential ion accumulation and ion fluxes in the mesophyll and epidermis of barley. Plant Physiol 122:835–844. Kasuga, M., Liu, Q., Miura, S., Yamaguchi-Shinozaki, K., and Shinozaki, K. (1999) Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nature Biotechnol 17:287–291. Katerji, N., Floorn, J.W., Hamdy, A., and Mastrorilli, M. (2003) Salinity effect on crop development and yield, analysis of salt tolerance according to several classification methods. Agricultural Water Management 62:37–66. Katiyar-Agarwal, S., Zhu, J., Kim, K., Agarwal, M., Fu, X., Huang, A., and Zhu, J.-K. (2006) The plasma membrane Na+/H+ antiporter SOS1 interacts with RCD1 and functions in oxidative stress tolerance in Arabidopsis. PNAS 103:18816– 18821. Kawasaki, S., Borchert, C., Deyholos, M., Wang, H., Brazille, S., Kawai, K., Galbraith, D., and Bohnert, H.J. (2001) Gene Expression Profiles during the Initial Phase of Salt Stress in Rice. Plant Cell 13:889–905. Kiegle, E., Gilliham, M., Haseloff, J., and Tester, M. (2000) Hyperpolarisation-activated calcium currents found only in cells from the elongation zone of Arabidopsis thaliana roots. Plant J 21:225–229. Kilian, J., Whitehead, D., Horak, J., Wanke, D., Weinl, S., Batistic, O., D’Angelo, C., Bornberg-Bauer, E., Kudla, J., and Harter, K. (2007) The AtGenExpress global stress expression data set: protocols, evaluation and model data analysis of UV-B light, drought and cold stress responses. Plant J 50:347–363. Kim, J. and Kim, H.-Y. (2006) Functional analysis of a calcium-binding transcription factor involved in plant salt stress signaling. FEBS Lett 580:5251–5256. Knight, H. and Knight, M.R. (2001) Abiotic stress signalling pathways: specificity and cross-talk. Trends Plant Sci 6:262–267. Knight, H., Trewavas, A.J., and Knight, M.R. (1997) Calcium signalling in Arabidopsis thaliana responding to drought and salinity. Plant J 12:1067–1078. Kolukisaoglu, Ü., Weinl, S., Blazevic, D., Batistic, O., and Kudla, J. (2004) Calcium Sensors and Their Interacting Protein Kinases: Genomics of the Arabidopsis and Rice CBL-CIPK Signaling Networks. Plant Physiol 134:43–58. Koyro, H.-W. and Stelzer, R. (1988) Ion concentrations in the cytoplasm and vacuoles of rhizodermis cells from NaCl treated Sorghum, Spartina and Puccinellia plants. J Plant Physiol 133:441–446. Kramer, D. (1983) The possible role of transfer cells in the adaptation of plants to salinity. Physiol Plant 58:549–555. Kramer, D., Läuchli, A., Yeo, A.R., and Gullasch, J. (1977) Transfer cells in roots of Phaseolus coccineus: ultrastructure and possible function in exclusion of sodium from the shoot. Ann Botany 41:1031–1040. Kronzucker, H.J., Szczerba, M.W., Moazami-Goudarzi, M., and Britto, D.T. (2006) The cytosolic Na+:K+ ratio does not explain salinity-induced growth impairment in barley: a dual-tracer study using 42K+ and 24Na+. Plant Cell Environ 29:2228–2237. Lacan, D. and Durand, M. (1996) Na+-K+ exchange at the xylem/symplast boundary. Its significance in the salt sensitivity of soybean. Plant Physiol 110:705–711.

106

GENES FOR CROP ADAPTATION TO POOR SOIL

Lacombe, B., Meyerhoff, O., Steinmeyer, R., Becker, D., and Hedrich, R. (2001) Role of Arabidopsis ionotropic glutamate receptors. In 12th Association de Canaux Ioniques, La Londe les Maures, France. Läuchli, A., James, R.A., Huang, C.X., McCully, M., and Munns, R. (2008) Cell-specific localization of Na+ in roots of durum wheat and possible control points for salt exclusion. Plant Cell Environ 31:1565–1574. Lefevre, I., Gratia, E., and Lutts, S. (2001) Discrimination between the ionic and osmotic components of salt stress in relation to free polyamine level in rice (Coryza sativa). Plant Science 161:943–952. Leng, Q., Mercier, R.W., Hua, B.-G., Fromm, H., and Berkowitz, G.A. (2002) Electrophysiological Analysis of Cloned Cyclic Nucleotide-Gated Ion Channels. Plant Physiol 128:400–410. Leung, J. and Giraudat, J. (1998) Abscisic acid signal transduction. Annu Rev Plant Physiol Plant Mol Biol 49:199–222. Li, J., Yang, H., Peer, W.A., Richter, G., Blakeslee, J., Bandyopadhyay, A., Titapiwantakun, B., Undurraga, S., Khodakovskaya, M., Richards, E.L., Krizek, B., Murphy, A.S., Gilroy, S., and Gaxiola, R. (2005) Arabidopsis H+-PPase AVP1 Regulates Auxin-Mediated Organ Development. Science 310:121–125. Lin, H.X., Zhu, M.Z., Yano, M., Gao, J.P., Liang, Z.W., Su, W.A., Hu, X.H., Ren, Z.H., and Chao, D.Y. (2004) QTLs for Na+ and K+ uptake of the shoots and roots controlling rice salt tolerance. Theor Appl Genet 108:253–260. Liu, J. and Zhu, J.-K. (1997) An Arabidopsis mutant that requires increased calcium for potassium nutrition and salt tolerance. PNAS 94:14960–14964. Liu, J. and Zhu, J.-K. (1998) A Calcium Sensor Homolog Required for Plant Salt Tolerance. Science 280:1943–1945. Liu, Q., Kasuga, M., Sakuma, Y., Abe, H., Miura, S., Yamaguchi-Shinozaki, K., and Shinozaki, K. (1998) Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA-binding domain, separate two cellular signal transduction pathways in drought- and low temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell 10:1391–1406. Lohaus, G., Hussmann, M., Pennewiss, K., Schneider, H., Zhu, J.-J., and Sattelmacher, B. (2000) Solute balance of a maize (Zea mays L.) source leaf as affected by salt treatment with special emphasis on phloem retranslocation and ion leaching. J Exp Bot 51:1721–1732. Lunde, C., Drew, D.P., Jacobs, A.K., and Tester, M. (2007) Exclusion of Na+ via Sodium ATPase (PpENA1) Ensures Normal Growth of Physcomitrella patens under Moderate Salt Stress. Plant Physiol 144:1786–1796. Maathuis, F.J.M. and Amtmann, A. (1999) K+ nutrition and Na+ toxicity: the basis of cellular K+/Na+ ratios. Ann Botany 84:123–133. Maathuis, F.J.M., Filatov, V., Herzyk, P., Krijger, G.C., Axelsen, K.B., Chen, S., Green, B.J., Li, Y., Madagan, K.L., SanchezFernandez, R., Forde, B.G., Palmgren, M.G., Rea, P.A., Williams, L.E., Sanders, D., and Amtmann, A. (2003) Transcriptome analysis of root transporters reveals participation of multiple gene families in the response to cation stress. Plant J 35:675–692. Maathuis, F.J.M. and Prins, H.B.A. (1990) Patch clamp studies on root cell vacuoles of a salt-tolerant and a salt-sensitive Plantago species. Plant Physiol 92:23–28. Maathuis, F.J.M. and Sanders, D. (2001) Sodium uptake in Arabidopsis thaliana is regulated by cyclic nucleotides. Plant Physiol 127:1617–1625. Magome, H., Yamaguchi, S., Hanada, A., Kamiya, Y., and Oda, K. (2008) The DDF1 transcriptional activator upregulates expression of a gibberellin-deactivating gene, GA2ox7, under high-salinity stress in Arabidopsis. Plant J 56:613–626. Malagoli, P., Britto, D.T., Schulze, L.M., and Kronzucker, H.J. (2008) Futile Na+ cycling at the root plasma membrane in rice (Oryza sativa L.): kinetics, energetics, and relationship to salinity tolerance. J Exp Bot 59:4109–4117. Malcolm, C.V., Lindley, V.A., O’Leary, J.W., Runciman, H.V., and Barrett-Lennard, E.G. (2003) Halophyte and glycophyte salt tolerance at germination and the establishment of halophyte shrubs in saline environments. Plant Soil 253:171–185. Manabe, Y., Bressan, R.A., Wang, T., Li, F., Koiwa, H., Sokolchik, I., Li, X., and Maggio, A. (2008) The Arabidopsis KinaseAssociated Protein Phosphatase Regulates Adaptation to Na+ Stress. Plant Physiol 146:612–622. Mansour, M.M.F. (2000) Nitrogen containing compounds and adaptation of plants to salinity stress. Biolog Plant 43:491–500. Marschner, H. (1995) Mineral nutrition of higher plants, 2nd edition. Academic Press, London. Martínez-Atienza, J., Jiang, X., Garciadeblas, B., Mendoza, I., Zhu, J.-K., Pardo, J.M., and Quintero, F.J. (2006) Conservation of the Salt Overly Sensitive Pathway in Rice. Plant Physiol 143:1001–1012. Matsushita, N. and Matoh, T. (1991) Characterization of Na+ exclusion mechanisms of salt-tolerant reed plants in comparison with salt-sensitive rice plants. Physiol Plant 83:170–176. Matsushita, N. and Matoh, T. (1992) Function of the shoot base of salt-tolerant reed (Phragmites communis Trinius) plants for Na+ exclusion from the shoots. Soil Sci Plant Nutr 38:565–571. McWhorter, C.G., Paul, R.N., and Ouzts, J.C. (1995) Bicellular trichomes of johnsongrass (Sorghum halepense) leaves— morphology, histochemistry, and function. Weed Sci 43:201–208. Mennen, H., Jacoby, B., and Marschner, H. (1990) Is sodium/proton antiport ubiquitous in plant cells? J Plant Physiol 137:180–183. Møller, I.S. and Tester, M. (2007) Salinity tolerance of Arabidopsis: a good model for cereals? Trends Plant Sci 12:534–540.

GENETIC DETERMINANTS OF SALINITY TOLERANCE IN CROP PLANTS

107

Moschou, P.N., Sanmartin, M., Andriopoulou, A.H., Rojo, E., Sanchez-Serrano, J.J., and Roubelakis-Angelakis, K.A. (2008) Bridging the Gap between Plant and Mammalian Polyamine Catabolism: A Novel Peroxisomal Polyamine Oxidase Responsible for a Full Back-Conversion Pathway in Arabidopsis. Plant Physiol 147:1845–1857. Mukhopadhyay, A., Vij, S., and Tyagi, A.K. (2004) Overexpression of a zinc-finger protein gene from rice confers tolerance to cold, dehydration, and salt stress in transgenic tobacco. PNAS 101:6309–6314. Munns, R. (1985) Na+, K+ and Cl− in xylem sap flowing to shoots of NaCl-treated barley. J Exp Bot 36:1032–1042. Munns, R. (1993) Physiological processes limiting plant growth in saline soils: some dogmas and hypotheses. Plant Cell Environ 16:15–24. Munns, R. (2002) Comparative physiology of salt and water stress. Plant Cell Environ 25:239–250. Munns, R., Guo, J., Passioura, J.B., and Cramer, G.R. (2000) Leaf water status controls day-time but not daily rates of leaf expansion in salt-treated barley. Aust J Plant Physiol 27:949–957. Munns, R. and Tester, M. (2008) Mechanisms of Salinity Tolerance. Annual Review of Plant Biology 59:651–681. Munns, R., Tonnet, L., Shennan, C., and Gardner, P.A. (1988) Effect of high external NaCl concentration on ion transport within the shoot of Lupinus albus. II. Ions in phloem sap. Plant Cell Environ 11:291–300. Murthy, M. and Tester, M. (1996) Compatible solutes and salt tolerance: misuse of transgenic tobacco. Trends Plant Sci 1: 294–295. Nagaoka, S. and Takano, T. (2003) Salt tolerance-related protein STO binds to a Myb transcription factor homologue and confers salt tolerance in Arabidopsis. J Exp Bot 54:2231–2237. Nakashima, K., Tran, L.-S.P., Nguyen, D.V., Fujita, M., Maruyama, K., Todaka, D., Ito, Y., Hayashi, N., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2007) Functional analysis of a NAC-type transcription factor OsNAC6 involved in abiotic and biotic stress-responsive gene expression in rice. Plant J 51:617–630. Nelson, D.E., Koukoumanos, M., and Bohnert, H.J. (1999) Myo-inositol-dependent sodium uptake in ice plant. Plant Physiol 119:165–172. Nishiyama, T., Fujita, T., Shin-I, T., Seki, M., Nishide, H., Uchiyama, I., Kamiya, A., Carninci, P., Hayashizaki, Y., Shinozaki, K., Kohara, Y., and Hasebe, M. (2003) Comparative genomics of Physcomitrella patens gametophytic transcriptome and Arabidopsis thaliana: Implication for land plant evolution. PNAS 100:8007–8012. Niu, X., Damsz, B., Kononowicz, A.K., Bressan, R.A., and Hasegawa, P.M. (1996) NaCl-induced alterations in both cell structure and tissue-specific plasma membrane H+-ATPase gene expression. Plant Physiol 111:679–686. Novillo, F., Alonso, J.M., Ecker, J.R., and Salinas, J. (2004) CBF2/DREB1C is a negative regulator of CBF1/DREB1B and CBF3/ DREB1A expression and plays a central role in stress tolerance in Arabidopsis. PNAS 101:3985–3990. Nublat, A., Desplans, J., Casse, F., and Berthomieu, P. (2001) sas1, an Arabidopsis Mutant Overaccumulating Sodium in the Shoot, Shows Deficiency in the Control of the Root Radial Transport of Sodium. Plant Cell 13:125–137. Ozturk, Z.N., Talamé, V., Deyholos, M., Michalowski, C.B., Galbraith, D.W., Gozukirmizi, N., Tuberosa, R., and Bohnert, H.J. (2002) Monitoring large-scale changes in transcript abundance in drought- and salt-stressed barley. Plant Mol Biol 48:551–573. Pate, J.S., Layzell, D.B., and McNeil, D.L. (1979) Modelling the transport and utilization of carbon and nitrogen in a nodulated legume. Plant Physiol 63:730–737. Peng, Y.-H., Zhu, Y.-F., Mao, Y.-Q., Wang, S.-M., Su, W.-A., and Tang, Z.-C. (2004) Alkali grass resists salt stress through high [K+] and an endodermis barrier to Na+. J Exp Bot 55:939–949. Perez-Alfocea, F., Balibrea, M.E., Alarcon, J.J., and Bolarin, M.C. (2000) Composition of xylem and phloem exudates in relation to the salt-tolerance of domestic and wild tomato species. J Plant Physiol 156:367–374. Phillips, J.R., Dalmay, T., and Bartels, D. (2007) The role of small RNAs in abiotic stress. FEBS Lett 581:3592–3597. Piao, H.L., Lim, J.H., Kim, S.J., Cheong, G.-W., and Hwang, I. (2001) Constitutive over-expression of AtGSK1 induces NaCl stress responses in the absence of NaCl stress and results in enhanced NaCl tolerance in Arabidopsis. Plant J 27:305– 314. Platten, J.D., Cotsaftis, O., Berthomieu, P., Bohnert, H., Davenport, R.J., Fairbairn, D.J., Horie, T., Leigh, R.A., Lin, H,-X., Luan, S., Mäser, P., Pantoja, O., Rodríguez-Navarro, A., Schachtman, D.P., Schroeder, J.I., Sentenac, H., Uozumi, N., Véry, A.-A., Zhu, J.-K., Dennis, E.S., and Tester, M. (2006) Nomenclature for HKT transporters, key determinants of plant salinity tolerance. Trends Plant Sci 11:372–374. Plett, D. (2008) Spatial and temporal alterations of gene expression in rice. PhD thesis. University of Adelaide, Adelaide. Poljakoff-Mayber, A. (1975) Morphological and anatomical changes in plants as a response to salinity. In GJ Poljakoff-Mayber, A., Ed., Plants in saline environments. Springer-Verlag, Berlin, pp 97–117. Qadir, M. and Oster, J.D. (2004) Crop and irrigation management strategies for saline-sodic soils and waters aimed at environmentally sustainable agriculture. Sci Total Environ 323:1–19. Qi, Z., Stephens, N.R., and Spalding, E.P. (2006) Calcium Entry Mediated by GLR3.3, an Arabidopsis Glutamate Receptor with a Broad Agonist Profile. Plant Physiol 142:963–971.

108

GENES FOR CROP ADAPTATION TO POOR SOIL

Qiu, Q.-S., Guo, Y., Dietrich, M.A., Schumaker, K.S., and Zhu, J.-K. (2002) Regulation of SOS1, a plasma membrane Na+/H+ exchanger in Arabidopsis thaliana, by SOS2 and SOS3. PNAS 99:8436–8441. Qiu, Q.-S., Guo, Y., Quintero, F.J., Pardo, J.M., Schumaker, K.S., and Zhu, J.-K. (2004) Regulation of Vacuolar Na+/H+ Exchange in Arabidopsis thaliana by the Salt-Overly-Sensitive (SOS) Pathway. J Biol Chem 279:207–215. Quan, R., Lin, H., Mendoza, I., Zhang, Y., Cao, W., Yang, Y., Shang, M., Chen, S., Pardo, J.M., and Guoa, Y. (2007) SCABP8/ CBL10, a Putative Calcium Sensor, Interacts with the Protein Kinase SOS2 to Protect Arabidopsis Shoots from Salt Stress. Plant Cell 19:1415–1431. Quarrie, S.A. and Mahmood, A. (1993) Improving salt tolerance in hexaploid wheat. In IPSR and JIC Annual Report 1992, p 4. Quintero, F.J., Ohta, M., Shi, H., Zhu, J.-K., and Pardo, J.M. (2002) Reconstitution in yeast of the Arabidopsis SOS signaling pathway for Na+ homeostasis. PNAS 99:9061–9066. Rabbani, M.A., Maruyama, K., Abe, H., Khan, M.A., Katsura, K., Ito, Y., Yoshiwara, K., Seki, M., Shinozaki, K., and YamaguchiShinozaki, K. (2003) Monitoring Expression Profiles of Rice Genes under Cold, Drought, and High-Salinity Stresses and Abscisic Acid Application Using cDNA Microarray and RNA Gel-Blot Analyzes. Plant Physiol 133:1755–1767. Rajendran, K., Tester, M., and Roy, S.J. (2009) Quantifying the three main components of salinity tolerance in cereals. Plant Cell Environ 32:237–249. Rausellz, A., Kanhonouz, R., Yenush, L., Serrano, R., and Rosy, R. (2003) The translation initiation factor eIF1A is an important determinant in the tolerance to NaCl stress in yeast and plants. Plant J 34:257–267. Reid, R.J. and Smith, F.A. (2000) The limits of sodium/calcium interactions in plant growth. Aust J Plant Physiol 27:709–715. Reinhardt, D.H. and Rost, T.L. (1995) Salinity accelerates endodermal development and induces an exodermis in cotton seedling roots. Environ Exper Bot 35:563–574. Ren, Z.-H., Gao, J.-P., Li, L.-G., Cai, X.-L., Huang, W., Chao, D.-Y., Zhu, M.-Z., Wang, Z.-Y., Luan, S., and Line, H.-X (2005) A rice quantitative trait locus for salt tolerance encodes a sodium transporter. Nature Biotechnol 37:1141–1146. Rengasamy, P. (2002) Transient salinity and subsoil constraints to dryland farming in Australian sodic soils: an overview. Aust J Exp Agric 42:351–361. Rengasamy, P. (2006) World salinization with emphasis on Australia. J Exp Bot 57:1017–1023. Roberts, S.K. (1998) Regulation of K+ channels in maize roots by water stress and ABA. Plant Physiol 116:145–153. Robinson, M., Véry, A.-A., Sanders, D., and Mansfield, T. (1997) How can stomata contribute to salt tolerance? Ann Botany 80:387–393. Roy, S.J., Gilliham, M., Berger, B., Essah, P.A., Cheffings, C., Miller, A.J., Davenport, R.J., Liu, L.H., Skynner, M.J., Davies, J.M., Richardson, P., Leigh, R.A., and Tester, M. (2008) Investigating glutamate receptor-like gene co-expression in Arabidopsis thaliana. Plant Cell Environ 31:861–871. Rubio, F., Gassmann, W., and Schroeder, J.I. (1995) Sodium-driven potassium uptake by the plant potassium transporter HKT1 and mutations conferring salt tolerance. Science 270:1660–1663. Rus, A., Baxter, I., Muthukumar, B., Gustin, J., Lahner, B., Yakubova, E., and Salt, D.E. (2006) Natural Variants of AtHKT1 Enhance Na+ Accumulation in Two Wild Populations of Arabidopsis. PLoS Genet 2:1964–1973. Rus, A., Lee, B.-h., Muñoz-Mayor, A., Sharkhuu, A., Miura, K., Zhu, J.-K., Bressan, R.A., and Hasegawa, P.M. (2004) AtHKT1 Facilitates Na+ Homeostasis and K+ Nutrition in Planta. Plant Physiol 136:2500–2511. Rus, A., Yokoi, S., Sharkhuu, A., Reddy, M., Lee, B.-h., Matsumoto, T.K., Koiwa, H., Zhu, J.-K., Bressan, R.A., and Hasegawa, P.M. (2001) AtHKT1 is a salt tolerance determinant that controls Na+ entry into plant roots. PNAS 98:14150–14155. Saavedra, L., Svensson, J., Carballo, V., Izmendi, D., Welin, B., and Vidal, S. (2006) A dehydrin gene in Physcomitrella patens is required for salt and osmotic stress tolerance. Plant J 45:237–249. Saneoka, H., Nagasaka, C., Hahn, D.T., Yang, W.-J., Premachandra, G.S., Joly, R.J., and Rhodes, D. (1995) Salt tolerance of glycinebetaine-deficient and -containing maize lines. Plant Physiol 107:631–638. Santa-María, G.E. and Epstein, E. (2001) Potassium/sodium selectivity in wheat and the amphiploid cross wheat X Lophopyrum elongatum. Plant Sci 160:523–534. Schachtman, D.P., Kumar, R., Schroeder, J.I., and Marsh, E.L. (1997) Molecular and functional characterization of a novel lowaffinity cation transporter (LCT1) in higher plants. PNAS 94:11079–11084. Schachtman, D.P. and Schroeder, J.I. (1994) Structure and transport mechanism of a high-affinity potassium uptake transporter from higher plants. Nature 370:655–658. Schroeder, J.I., Allen, G.J., Hugouvieux, V., Kwak, J.M., and Waner, D. (2001) Guard cell signal transduction. Annu Rev Plant Physiol Plant Mol Biol 52:627–658. Schubert, S. and Läuchli, A. (1990) Sodium exclusion mechanisms at the root surface of two maize cultivars. Plant Soil 123:205–209. Seki, M., Narusaka, M., Abe, H., Kasuga, M., Yamaguchi-Shinozaki, K., Carninci, P., Hayashizaki, Y., and Shinozaki, K. (2001) Monitoring the expression pattern of 1300 Arabidopsis genes under drought and cold stresses by using a full-length cDNA microarray. Plant Cell 13:61–72.

GENETIC DETERMINANTS OF SALINITY TOLERANCE IN CROP PLANTS

109

Shabala, S., Cuin, T.A., and Pottosin, I. (2007) Polyamines prevent NaCl-induced K+ efflux from pea mesophyll by blocking non-selective cation channels. FEBS Lett 581:1993–1999. Shabala, S., Demidchik, V., Shabala, L., Cuin, T.A., Smith, S.J., Miller, A.J., Davies, J.M., and Newman, I.A. (2006) Extracellular Ca2+ Ameliorates NaCl-Induced K+ Loss from Arabidopsis Root and Leaf Cells by Controlling Plasma Membrane K+Permeable Channels. Plant Physiol 141:1653–1665. Shepherd, U.H. and Bowling, D.J.F. (1979) Sodium fluxes in roots of Eleocharis uniglumis, a brackish water species. Plant Cell Environ 2:123–130. Shi, H., Ishitani, M., Kim, C., and Zhu, J.-K. (2000) The Arabidopsis thaliana salt tolerance gene SOS1 encodes a putative Na+/ H+ antiporter. PNAS 97:6896–6901. Shi, H., Kim, Y., Guo, Y., Stevenson, B., and Zhu, J.-K. (2003) The Arabidopsis SOS5 Locus Encodes a Putative Cell Surface Adhesion Protein and Is Required for Normal Cell Expansion. Plant Cell 15:19–32. Shi, H., Quintero, F.J., Pardo, J.M., and Zhu, J.-K. (2002) The Putative Plasma Membrane Na+/H+ Antiporter SOS1 Controls Long-Distance Na+ Transport in Plants. Plant Cell 14:465–477. Shomer-Ilan, A., Jones, G.P., and Paleg, L.G. (1991) In vitro thermal and salt stability of pyruvate kinase are increased by proline analogues and trigonelline. Aust J Plant Physiol 18:279–286. Sibole, J.V., Cabot, C., Poschenrieder, C., and Barcelo, J. (2003) Efficient leaf ion partitioning, an overriding condition for abscisic acid-controlled stomatal and leaf growth responses to NaCl salinization in two legumes. J Exp Bot 54:2111–2119. Silberbush, M. and Ben-Asher, J. (2001) Simulation study of nutrient uptake by plants from soilless cultures as affected by salinity buildup and transpiration. Plant Soil 233:59–69. Skopelitis, D.S., Paranychianakis, N.V., Paschalidis, K.A., Pliakonis, E.D., Delis, I.D., Yakoumakis, D.I., Kouvarakis, A., Papadakis, A.K., Stephanou, E.G., and Roubelakis-Angelakis, K.A. (2006) Abiotic Stress Generates ROS That Signal Expression of Anionic Glutamate Dehydrogenases to Form Glutamate for Proline Synthesis in Tobacco and Grapevine. Plant Cell 18:2767–2781. Sottosanto, J.B., Gelli, A., and Blumwald, E. (2004) DNA array analyzes of Arabidopsis thaliana lacking a vacuolar Na+/H+ antiporter: impact of AtNHX1 on gene expression. Plant J 40:752–771. Sottosanto, J.B., Saranga, Y., and Blumwald, E. (2007) Impact of AtNHX1, a vacuolar Na+/H+ antiporter, upon gene expression during short- and long-term salt stress in Arabidopsis thaliana. BMC Plant Biol 7:18. Stelzer, R. (1981) Ion localization in the leaves of Puccinellia peisonis. Zeitschrift für Pflanzenphysiologie Bodenkonse 103:27–36. Stephens, N.R., Qi, Z., and Spalding, E.P. (2008) Glutamate Receptor Subtypes Evidenced by Differences in Desensitization and Dependence on the GLR3.3 and GLR3.4 Genes. Plant Physiol 146:529–538. Storey, R., Schachtman, D.P., and Thomas, M.R. (2003) Root structure and cellular chloride, sodium and potassium distribution in salinized grapevines. Plant Cell Environ 26:789–800. Su, J. and Wu, R. (2004) Stress-inducible synthesis of proline in transgenic rice confers faster growth under stress conditions than that with constitutive synthesis. Plant Sci 166:941–948. Sugino, M., Hibino, T., Tanaka, Y., Nii, N., Takabe, T., and Takabe, T. (1999) Overexpression of DnaK from a halotolerant cyanobacterium Aphanothece halophytica acquires resistance to salt stress in transgenic tobacco plants. Plant Sci 146:81–88. Sulpice, R., Tsukaya, H., Nonaka, H., Mustardy, L., Chen, T.H.H., and Murata, N. (2003) Enhanced formation of flowers in salt-stressed Arabidopsis after genetic engineering of the synthesis of glycine betaine. Plant J 36:165–176. Sunarpi, Horie, T., Motoda, J., Kubo, M., Yang, H., Yoda, K., Horie, R., Chan, W.-Y., Leung, H.-Y., Hattori, K., Konomi, M., Osumi, M., Yamagami, M., Schroeder, J.I., and Uozumi, N. (2005) Enhanced salt tolerance mediated by AtHKT1 transporter induced Na+ unloading from xylem vessels to xylem parenchyma cells. Plant J 44:928–938. Sunkar, R., Chinnusamy, V., Zhu, J., and Zhu, J.-K. (2007) Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. Trends Plant Sci 12:301–309. Taji, T., Seki, M., Satou, M., Sakurai, T., Kobayashi, M., Ishiyama, K., Narusaka, Y., Narusaka, M., Zhu, J.-K., and Shinozaki, K. (2003) Comparative Genomics in Salt Tolerance between Arabidopsis and Arabidopsis-Related Halophyte Salt Cress Using Arabidopsis Microarray. Plant Physiol 135:1697–1709. Tester, M. and Davenport, R. (2003) Na+ tolerance and Na+ transport in higher plants. Ann Botany 91:503–527. Tester, M. and Leigh, R.A. (2001) Partitioning of nutrient transport processes in roots. J Exp Bot 52:445–457. Tracy, F.E., Gilliham, M., Dodd, A.N., Webb, A.A.R., and Tester, M. (2008) NaCl-induced changes in cytosolic free Ca2+ in Arabidopsis thaliana are heterogeneous and modified by external ionic composition. Plant Cell Environ 31:1063–1073. Trewavas, A.J. and Malho, R. (1997) Signal perception and transduction: the origin of the phenotype. Plant Cell 9:1181– 1195. Tyerman, S.D. and Skerrett, I.M. (1999) Root ion channels and salinity. Sci Hort 78:175–235.

110

GENES FOR CROP ADAPTATION TO POOR SOIL

Urano, K., Yoshiba, Y., Nanjo, T., Ito, T., Yamaguchi-Shinozaki, K., and Shinozaki, K. (2004) Arabidopsis stress-inducible gene for arginine decarboxylase AtADC2 is required for accumulation of putrescine in salt tolerance. Biochem Biophys Res Commun 313:369–375. Viehveger, K., Dordschbal, B., and Roos, W. (2002) Elicitor-activated phospholipase A2 generates lysophosphatidylcholines that mobilize the vacuolar H+ pool for pH signaling via the activation of Na+-dependent proton fluxes. Plant Cell 14:1509–1525. Vitart, V., Baxter, I., Doerner, P., and Harper, J.F. (2001) Evidence for a role in growth and salt resistance of a plasma membrane H+-ATPase in the root endodermis. Plant J 27:191–201. Volkov, V. and Amtmann, A. (2006) Thellungiella halophila, a salt-tolerant relative of Arabidopsis thaliana, has specific root ion-channel features supporting K+/Na+ homeostasis under salinity stress. Plant J 48:342–353. Volkov, V., Wang, B., Dominy, P.J., Fricke, W., and Amtmann, A. (2003) Thellungiella halophila, a salt-tolerant relative of Arabidopsis thaliana, possesses effective mechanisms to discriminate between potassium and sodium. Plant Cell Environ 27:1–14. Walia, H., Wilson, C., Condamine, P., Liu, X., Ismail, A.M., Zeng, L., Wanamaker, S.I., Mandal, J., Xu, J., Cui, X., and Close, T.J. (2005) Comparative Transcriptional Profiling of Two Contrasting Rice Genotypes under Salinity Stress during the Vegetative Growth Stage. Plant Physiol 139:822–835. Walia, H., Wilson, C., Wahid, A., Condamine, P., Cui, X., and Close, T.J. (2006) Expression analysis of barley (Hordeum vulgare L.) during salinity stress. Funct Integ Genomics 6:143–156. Wang, B., Davenport, R.J., Volkov, V., and Amtmann, A. (2006) Low unidirectional sodium influx into root cells restricts net sodium accumulation in Thellungiella halophila, a salt-tolerant relative of Arabidopsis thaliana. J Exp Bot 57:1161–1170. Wang, H., Miyazaki, S., Kawai, K., Deyholos, M., Galbraith, D.W., and Bohnert, H.J. (2003) Temporal progression of gene expression responses to salt shock in maize roots. Plant Mol Biol 52:873–891. Wang, Z.-l., Li, P.-h., Fredricksen, M., Gong, Z.-z., Kim, C.S., Zhang, C., Bohnert, H.J., Zhu, J.-K., Bressan, R.A., Hasegawa, P.M., Zhaoa, Y.-x., and Zhang, H. (2004) Expressed sequence tags from Thellungiella halophila, a new model to study plant salt-tolerance. Plant Sci 166:609–616. Watson, R., Pritchard, J., and Malone, M. (2001) Direct measurement of sodium and potassium in the transpiration stream of salt-excluding and non-excluding varieties of wheat. J Exp Bot 52:1873–1881. Wegner, L.H. and Raschke, K. (1994) Ion channels in the xylem parenchyma of barley roots. Plant Physiol 105:799–813. Wegner, L.H., Sattelmacher, B., Läuchli, A., and Zimmermann, U. (1999) Trans-root potential, xylem pressure, and cortical membrane potential of “low-salt” maize as influenced by nitrate and ammonium. Plant Cell Environ 22:1549–1558. Wei, J.-Z., Tirajoh, A., Effendy, J., and Plant, A.J. (2000) Characterization of salt-induced changes in gene expression in tomato (Lycopersicon esculentum) roots and the role played by abscisic acid. Plant Sci 159:135–148. Wei, W., Bilsborrow, P.E., Hooley, P., Fincham, D.A., Lombi, E., and Forster, B.P. (2003) Salinity induced differences in growth, ion distribution and partitioning in barley between the cultivar Maythorpe and its derived mutant Golden Promise. Plant Soil 250:183–191. White, P.J. (1996) The permeation of NH +4 through a voltage-independent K+ channel in the plasma membrane of rye roots. J Membr Biol 152:89–99. White, P.J. (1999) The molecular mechanism of sodium influx to root cells. Trends Plant Sci 4:245–246. White, P.J. and Broadley, M.R. (2001) Chloride in soils and its uptake and movement within the plant: a review. Ann Botany 88:967–988. White, P.J. and Davenport, R.J. (2002) The Voltage-Independent Cation Channel in the Plasma Membrane of Wheat Roots Is Permeable to Divalent Cations and May Be Involved in Cytosolic Ca2+ Homeostasis. Plant Physiol 130:1386–1395. Wilson, C. and Shannon, M.C. (1995) Salt-induced Na+/H+ antiport in root plasma membrane of a glycophytic and halophytic species of tomato. Plant Sci 107:147–157. Wimmers, L.E., Ewing, N.N., and Bennett, A.B. (1992) Higher plant Ca2+-ATPase: primary structure and regulation of mRNA abundance by salt. PNAS 89:9205–9209. Winter, E. (1982) Salt tolerance of Trifolium alexandrinum L. III. Effects of salt on ultrastructure of phloem and xylem transfer cells in petioles and leaves. Aust J Plant Physiol 9:227–237. Wolf, O., Munns, R., Tonnet, M.L., and Jeschke, W.D. (1991) The role of the stem in the partitioning of Na+ and K+ in salt-treated barley. J Exp Bot 42:697–704. Wu, C.-A., Yang, G.-D., Meng, Q.-W., and Zheng, C.-C. (2004) The Cotton GhNHX1 Gene Encoding a Novel Putative Tonoplast Na+/H+ Antiporter Plays an Important Role in Salt Stress. Plant Cell Physiol 45:600–607. Xiang, Y., Tang, N., Du, H., Ye, H., and Xiong, L. (2008) Characterization of OsbZIP23 as a Key Player of the Basic Leucine Zipper Transcription Factor Family for Conferring Abscisic Acid Sensitivity and Salinity and Drought Tolerance in Rice. Plant Physiol 148:1938–1952.

GENETIC DETERMINANTS OF SALINITY TOLERANCE IN CROP PLANTS

111

Xiong, L., Schumaker, K.S., and Zhu, J.-K. (2002) Cell signaling during cold, drought, and salt stress. Plant Cell Supplement 2002:S165–S183. Xu, D.-Q., Huang, J., Guo, S.-Q., Yang, X., Bao, Y.-M., Tang, H.-J., and Zhang, H.-S. (2008) Overexpression of a TFIIIA-type zinc finger protein gene ZFP252 enhances drought and salt tolerance in rice (Oryza sativa L.). FEBS Lett 582:1037–1043. Xu, D., Duan, X., Wang, B., Hong, B., Ho, T.-H.D., and Wu, R. (1996) Expression of a Late Embryogenesis Abundant Protein Gene, HVA1, from Barley Confers Tolerance to Water Deficit and Salt Stress in Transgenic Rice. Plant Physiol 110:249–257. Xu, J., Li, H.-D., Chen, L.-Q., Wang, Y., Liu, L.-L., He, L., and Wu, W.-H. (2006) A Protein Kinase, Interacting with Two Calcineurin B-like Proteins, Regulates K+ Transporter AKT1 in Arabidopsis. Cell 125:1347–1360. Xue, Z.-Y., Zhi, D.-Y., Xue, G.-P., Zhang, H., Zhao, Y.-X., and Xia, G.-M. (2004) Enhanced salt tolerance of transgenic wheat (Tritivum aestivum L.) expressing a vacuolar Na+/H+ antiporter gene with improved grain yields in saline soils in the field and a reduced level of leaf Na+. Plant Sci 167:849–859. Yadav, R., Flowers, T.J., and Yeo, A.R. (1996) The involvement of the transpirational bypass flow in sodium uptake by high- and low-sodium-transporting lines of rice developed through intravarietal selection. Plant Cell Environ 19:329–336. Yamaguchi, T., Apse, M.P., Shi, H., and Blumwald, E. (2003) Topological analysis of a plant vacuolar Na+/H+ antiporter reveals a luminal C terminus that regulates antiporter cation selectivity. PNAS 100:12510–12515. Yang, H., Knapp, J., Koirala, P., Rajagopal, D., Peer, W.A., Silbart, L.K., Murphy, A., and Gaxiola, R.A. (2007) Enhanced phosphorus nutrition in monocots and dicots over-expressing a phosphorus-responsive type I H+-pyrophosphatase. Plant Biotech J 5:735–745. Yeo, A. (1999) Predicting the interaction between the effects of salinity and climate change on crop plants. Sci Hort 78:159–174. Yeo, A.R. and Flowers, T.J. (1982) Accumulation and localisation of sodium ions within the shoots of rice (Oryza sativa) varieties differing in salinity resistance. Physiol Plant 56:343–348. Yeo, A.R. and Flowers, T.J. (1985) The absence of an effect of the Na/Ca ratio on sodium chloride uptake by rice. New Phytol 99:81–90. Yeo, A.R., Kramer, D., and Lauchli, A. (1977) Ion Distribution in Salt-stressed Mature Zea mays Roots in Relation to Ultrastructure and Retention of Sodium. J Exp Bot 28:17–29. Yeo, A.R., Yeo, M.E., and Flowers, T.J. (1987) The Contribution of an Apoplastic Pathway to Sodium Uptake by Rice Roots in Saline Conditions. J Exp Bot 38:1141–1153. Yokoi, S., Quintero, F.J., Cubero, B., Ruiz, M.T., Bressan, R.A., Hasegawa, P.M., and Pardo, J.M. (2002) Differential expression and function of Arabidopsis thaliana NHX Na+/H+ antiporters in the salt stress response. Plant J 30:529–539. Zahran, H.H., Marín-Manzano, M.C., Sánchez-Raya, A.J., Bedmar, E.J., Venema, K., and Rodríguez-Rosales, M.P. (2007) Effect of salt stress on the expression of NHX-type ion transporters in Medicago intertexta and Melilotus indicus plants. Physiol Plant 131:122–130. Zhang, H.-X. and Blumwald, E. (2001) Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit. Nature Biotechnol 19:765–768. Zhang, H.-X., Hodson, J.N., Williams, J.P., and Blumwald, E. (2001) Engineering salt-tolerant Brassica plants: Characterization of yield and seed oil quality in transgenic plants with increased vacuolar sodium accumulation. PNAS 98:12832–12836. Zhang, J.-S., Xie, C., Li, Z.-Y., and Chen, S.-Y. (1999) Expression of the plasma membrane H+-ATPase gene in response to salt stress in a rice salt-tolerant mutant and its original variety. Theor Appl Genet 99:1006–1011. Zhao, F., Wang, Z., Zhang, Q., Zhao, Y., and Zhang, H. (2006b) Analysis of the physiological mechanism of salt-tolerant transgenic rice carrying a vacuolar Na+/H+ antiporter gene from Suaeda salsa. J Plant Res 119:95–104. Zhao, F.-Y., Zhang, X.-J., Li, P.-H., Zhao, Y.-X., and Zhang, H. (2006a) Co-expression of the Suaeda salsa SsNHX1 and Arabidopsis AVP1 confer greater salt tolerance to transgenic rice than the single SsNHX1. Mol Breed 17:341–353. Zhu, J.-K. (2001) Plant salt tolerance. Trends Plant Sci 6:66 –71. Zhu, J.-K. (2002) Salt and drought stress signal transduction in plants. Annu Rev Plant Biol 53:247–273. Zhu, J.-K., Liu, J., and Xiong, L. (1998) Genetic Analysis of Salt Tolerance in Arabidopsis: Evidence for a Critical Role of Potassium Nutrition. Plant Cell 10:1181–1191. Zidan, I., Jacoby, B., Ravina, I., and Neumann, P.M. (1991) Sodium Does Not Compete with Calcium in Saturating Plasma Membrane Sites Regulating 22Na Influx in Salinized Maize Roots. Plant Physiol 96:331–334. Zörb, C., Noll, A., Karl, S., Leib, K., Yan, F., and Schubert, S. (2005) Molecular characterization of Na+/H+ antiporters (ZmNHX) of maize (Zea mays L.) and their expression under salt stress. J Plant Physiol 162:55–66.

5

Unraveling the Mechanisms Underlying Aluminum-dependent Root Growth Inhibition Paul B. Larsen

Introduction

While not necessarily recognized for its importance as a limiting factor for global agriculture, aluminum (Al) toxicity has profound consequences on crop growth and development throughout the world (von Uexkull and Mutert, 1995). Al is the third most abundant element in the earth’s crust and the most common metal. For the most part, Al exists in insoluble forms that have little to no impact on plant growth. This includes Al in the form of bauxite ore, which is the source of metallic aluminum that has become so critical to the industrialized world. In addition, Al can be found either as oxides or silicates, with Al being common in clay-based soils. Al is not considered to be toxic unless it is found in an acidic soil environment that is less than pH 5.0. In this type of environment, Al is solubilized to the Al3+ form, which has severe consequences for root growth and development. Acid soil is a pervasive global problem, with upward of 50% of the world’s agriculturally available land considered to be acidic. These areas include large swaths of land in South America, Africa, Europe, North America, Southeast Asia, and Australia, thus limiting agricultural productivity in these regions (von Uexkull and Mutert, 1995). It should be noted that Al is not considered to be a pollutant that is comparable to heavy metals that arise from human activity. Because of the profound abundance of Al, phytoremediation approaches that have been heavily promoted for correcting heavy metal contamination by using plants to remove the contaminant from soils will not work for addressing Al toxicity. Instead, modern agricultural practices have adopted the use of lime to raise the pH of affected soils. This approach is used in developed countries, yet this approach is not common in the Third World. Consequently, Al toxicity coupled with soil acidity should be considered to be a major detriment for agricultural productivity, which is of critical importance to a world that has a burgeoning population that desperately needs increased crop productivity to sustain it. Interestingly, the regions mentioned above in actuality represent some of the most biologically diverse regions, especially with regard to plant life. For example, the Amazonian rain forest is considered to be a highly Al toxic environment, yet clearly plant life thrives in this region. Consequently, it is apparent that many plants have adopted strategies for being able to survive and even thrive in such a harsh environment. It is not clear what mechanisms give such native plants the capability to grow in these regions, yet it points to the fact that Al toxicity from an agricultural standpoint is ultimately an issue that relates to increasing either resistance or tolerance in crop plants, which for the most part have not developed mechanisms that allow for growth in Al toxic environments. The clear challenge to scientists is to understand the molecular basis for Genes for Plant Abiotic Stress Editors Matthew A. Jenks and Andrew J. Wood © 2010 Blackwell Publishing ISBN: 978-0-813-81502-2

113

114

GENES FOR CROP ADAPTATION TO POOR SOIL

Al toxicity and subsequently identify and unravel the mechanisms that plants use to either exclude or tolerate Al. In the end, this work has and will continue to give new strategies for engineering crop plants that can grow and even thrive in soils that would currently be considered to be unproductive.

Mechanisms of Aluminum Toxicity

Regardless of the mechanisms of toxicity, it is clear that the root tip is the most sensitive tissue to Al (Ryan et al., 1993; Sivaguru and Horst, 1998). Studies have shown that application of Al to mature regions of the root have little to no effect on root growth, whereas addition of Al to the root tip only results in the normally observed root growth inhibition. It is not clear as to whether this root growth inhibition is dependent on blockage of cell division, cell elongation, or both although recent evidence argues that a main consequence of Al toxicity is loss of the root quiescent center (Rounds and Larsen, 2008), which will be discussed later. It should be noted that roots have two effective strategies by which to survive in an Al toxic environment. While these terms are often interchanged inappropriately, these strategies represent two distinct approaches, including Al resistance and Al tolerance. The former represents a strategy by which Al is prevented from entering the root either by exclusion associated with Al chelation or by alkalinization of the rhizosphere to reduce the amount of Al that exists in the toxic Al3+ form. In contrast, Al tolerance represents a strategy by which the root is capable of withstanding the effects of high levels of internalized Al. As might be expected, significant efforts and progress have been made with regard to Al resistance since exclusion of Al from root tissues is a far less complicated mechanism than what is predicted for Al tolerance. Since the phytotoxic form of Al is considered to be the trivalent cation species, it is predicted that Al toxicity arises from nonspecific interactions of Al3+ with a wide range of targets, many of which are negatively charged. These sites of toxicity are both apoplastic and symplastic, with these including representatives of all cellular biomolecules including polysaccharides, proteins, nucleic acids, and lipids. Much work has focused on attempting to identify which biochemical target is paramount with regard to promotion of Al-dependent root growth inhibition; yet until recently, there has been no convincing argument that one particular site of toxicity trumps the remaining sites in terms of importance. Clearly, based on the predicted complexity of Al toxicity, single gene mutations that significantly increase Al tolerance are not expected since, in essence, such changes should give only incremental changes in a plant’s capability to cope with internalized Al. Initially upon entering the root, Al binds noncovalently to negatively charged sites within the root cell wall (Ostatek-Boczynski et al., 1995). These sites include polygalacturonic acid, which is an important component of the cell wall, with Al3+ competing with cations such as Ca2+ for binding to these components. One visible consequence of treatment with Al is severe damage to the integrity of the root, due to actual physical tearing and sloughing off of the epidermal root tissue due to the rigidification of the cell wall. It is not obvious whether this damage is directly related to stoppage of root growth or simply represents a symptom of Al toxicity. Al is also believed to directly target the plasma membrane (Wagatsuma et al., 1995; Yermiyahu et al., 1997), both in terms of interacting with membrane lipids and binding to membrane-localized proteins. Lipid targets include phosphatidylinositol-4,5-bisphosphate, which serves as a precursor for generation of the IP3 second messenger (Jones and Kochian, 1995; Jones and Kochian, 1997). Interactions with lipids are of particular importance since this association is expected to result in lipid peroxidation, thus resulting in buildup of cellular free radicals and promotion of oxidative

UNRAVELING THE MECHANISMS UNDERLYING ALUMINUM-DEPENDENT ROOT GROWTH INHIBITION

115

stress. Binding of Al only to the root tips results in several effects including inhibition of transport activity, as evidenced by blockage of movement of solutes such as potassium and calcium (Huang et al., 1992; Huang et al., 1995; Liu and Luan, 2001). Additionally, Al toxicity results in disruption of H+ homeostasis, neutralization of the zeta (ζ) potential at the membrane surface (Olivetti et al., 1995; Papernik and Kochian, 1997), and inhibition of H+ flux mediated by the H+-ATPase (Ahn et al., 2001; Ahn et al., 2002). Other membrane protein targets include enzymes such as phospholipase A2 and phospholipase C (Ramos-Diaz et al., 2007), the latter of which is critical for production of the IP3 second messenger for mediation of specific signal transduction events, with in vitro studies showing that Al specifically inhibits phospholipase C (PLC) at levels of Al that are physiologically relevant (Haug et al., 1994; Jones and Kochian, 1995). Ultimately, Al is expected to negatively affect IP3-mediated signaling coupled with regulation of cytoplasmic Ca2+ (Jones et al., 1998a; Jones et al., 1998b, Shafer et al., 1994; Shi et al., 1993). In addition to this, Al inhibition of PLC activity results in a rapid decline in the capability of cells to produce phosphatidic acid (RamosDiaz et al., 2007), which is emerging as a critical signaling component in eukaryotes. It is not clear what effect inhibiting the generation of second messengers has on signaling pathways or cellular response, although it is predicted to be severe. It is not known how Al3+ crosses the plasma membrane, whether via ion transporters or adsorption endocytosis. However, Al accumulation in root tissue is rapid upon exposure (Lazof et al., 1994a). In soybean roots treated with AlCl3, Al accumulates in the symplasm of the three outer cortical cell layers after only 30 minutes of treatment (Lazof et al., 1994b). Rapid uptake is also observed in Arabidopsis roots, where significant levels of Al accumulate within 1 hour of exposure to moderate levels of Al (Larsen et al., 1996; Larsen et al., 1998). Finally, in single cells of Chara corallina, Al accumulation in the cytoplasm occurs within 30 minutes of exposure and is found in the tonoplast soon thereafter (Taylor et al., 2000). Although Al uptake is demonstrably rapid, it is not clear how Al speciates once inside the cell. As noted previously, the level of Al toxicity is directly related to the amount of Al that speciates to Al3+, with this speciation occurring only in an acidic environment. In contrast, intracellular pH is not considered to be acidic, thus leaving it unclear as to whether the Al3+ species occurs within the symplasm or whether some other form of Al is the culprit in toxicity within the cell. An alternative model to this is that Al localizes to the apoplasm, which is an environment that would likely be similar in acidity to the rhizosphere and promote Al3+ formation, may trigger production of some secondary compound that subsequently enters the cell, and causes damage that is inhibitory to root growth. Although it is not necessarily clear how Al specifically inhibits particular enzymes, such as PLC, the protein targets of Al3+ are predicted to be numerous. Interactions likely are through binding of Al3+ with acidic or polar amino acids, with these electrostatic interactions predicted to mask charges required for normal protein conformations. Additionally, Al3+ is likely to compete effectively with cofactors such as Mg2+ for binding to enzymes and other proteins. Displacement of such cofactors is predicted to have severe effects on protein function and enzymatic activity, since often times these cofactors are mechanistic requirements of the affected proteins. One well-studied system has been K+ transport in root hair and guard cells, where it has been shown that Al blocks K+ transport through a deleterious association with the cytoplasmic side of inward rectifying K+ channels (Liu and Luan, 2001). Other internal targets that have been intensively examined include components of the cytoskeleton (Blancaflor et al., 1998; Grabski and Schindler, 1995; Schwarzerova et al., 2002; Sivaguru et al., 1999). Al is thought to directly target specific constituents of the cytoskeletal network, including actin, through the displacement of other cations such as Mg2+. It is argued that this displacement

116

GENES FOR CROP ADAPTATION TO POOR SOIL

results in a severe change in the dynamics of the cytoskeleton, effectively reducing the capability of this microtubule network to depolymerize, which ultimately leads to its rigidification. Consequently, disorganization of the cytoskeletal network leads to gross alterations in cell shape, which often occurs when roots are exposed to extremely toxic levels of Al. Consistent with this, Al interactions cause a dramatic increase in rigor of the actin network in Al-treated soybean suspension culture (Grabski and Schindler, 1995). This has also been reported for Zea mays roots, where Al treatment causes an alteration in the organization of the cytoskeleton, being particularly profound in the region most sensitive to Al toxicity (Blancaflor et al., 1998; Sivaguru et al., 1999), and in tobacco cell lines, where perturbations in the cytoskeleton precede Al-induced reductions in cell viability (Schwarzerova et al., 2002). Promotion of oxidative stress by Al has received considerable attention in recent years since it has been shown that Al treatment leads to increased levels of reactive oxygen species (ROS) in roots (Achary et al., 2008; Boscolo et al., 2003). This is consistent with previous work focusing on either Al-inducible genes or Al-dependent increases in protein activity, both of which suggest that Al treatment leads to increased levels of proteins that function to control ROS levels (Ezaki et al., 1999; Ezaki et al., 2000, Ezaki et al., 2001; Richards and Gardner, 1994; Richards et al., 1998; Snowden and Gardner, 1993; Snowden et al., 1995). These Al-responsive factors include ascorbate peroxidase, glutathione-S-transferases, superoxide dismutase, and oxido-reductases that are known to be required for prevention of free radical buildup in cells following Al treatment. Interestingly, Al treatment has been shown in several systems to stimulate ROS production in the cell wall matrix. This ROS production, which is dependent on NADPH oxidase activity, results in a rapid accumulation of O2.−, H2O2 and .OH. Preliminary evidence argues that limitation of lipid peroxidation and buildup of free radicals during Al-induced oxidative stress can confer increased Al tolerance in roots and cell culture (Devi et al., 2003; Ezaki et al., 2000; Ezaki et al., 2001) with overexpression of Al-induced oxidative stress genes actually conferring a degree of Al resistance (Ezaki et al., 2001). While having a range of consequences, it is important to point out that Alinduced ROS has been linked to severe effects on DNA integrity, with ROS production resulting in DNA fragmentation (Achary et al., 2008). In fact, based on the conundrum of the speciation of Al away from toxic species once in the cytoplasm, it is attractive to hypothesize that Al-dependent apoplastic ROS may in fact be the mediators of Al responsive root growth inhibition. Although it is not clear as to its significance, Al-dependent DNA damage is emerging as a likely candidate for being a primary cause of Al responsive root growth inhibition (Achary et al., 2008; Banasik et al., 2005; Lankoff et al., 2006; Lima et al., 2007; Rounds and Larsen 2008). Al has been long speculated to interact directly with DNA, likely due to the predominance of negative charges found in the DNA double helix that are part of its phosphate backbone. In fact, Al has been found to interact with nuclei in the soybean root meristem within 30 minutes of Al exposure (Silva et al., 2000). In animals, Al has been linked to promotion of Alzheimer ’s disease, with this at least in part dependent on the condensation of chromatin structures through specific interactions with H1 zero histone linker proteins (Banasik et al., 2005; Lukiw et al., 1992). Interestingly, Al-dependent ROS buildup has been linked directly to genomic DNA fragmentation, suggesting that Al-dependent inhibition may be tied to this ROS-dependent DNA damage. Based on the numerous deleterious effects of Al, Al toxicity is a complex phenomenon resulting from interaction of Al with many biochemical targets, both symplastic and apoplastic. For us to understand Al tolerance, it will be necessary to dissect genetically the mechanisms underlying Al tolerance so that it will be possible to rank the relevance of each to the manifestation of Aldependent root growth inhibition. Based on such an analysis, it should allow for the design of strategies to engineer plants that will be capable of tolerating high levels of internalized Al. Such

UNRAVELING THE MECHANISMS UNDERLYING ALUMINUM-DEPENDENT ROOT GROWTH INHIBITION

117

an analysis has been undertaken with several plant species, most notably Arabidopsis and rice, with some progress in understanding the genetic basis for Al toxicity and tolerance having been made. For example, mutagenesis of Arabidopsis reveals at least eight distinct loci that are required for growth in an Al toxic environment (Larsen et al., 1996), suggesting that the mechanisms underlying Al-dependent root growth inhibition are highly complex.

Aluminum Resistance Mechanisms

Of course, the most effective means by which a plant can thrive in an Al-toxic environment is to prevent uptake of Al into its root tissue, which is also known as Al resistance. By not allowing Al to enter its tissue, Al-dependent damage should be minimal at best. This argument largely arises from the model in which Al toxicity is a highly complex phenomenon, suggesting that single gene changes would not result in substantial increases in Al tolerance. In contrast, prevention of Al uptake overrides the complexity of Al toxicity and provides a simple strategy by which to promote root growth in Al toxic environments. Al exclusion, which falls into the category of Al resistance, was first documented as an effective mechanism for preventing Al toxicity based on a study of snap beans (Miyasaka et al., 1991). In this study, two cultivars of snap beans that differed in growth in an Al-toxic environment were compared to establish the physiological basis for this change. Effectively, it was determined that the Al-resistant cultivar of snap beans had an enhanced capability to release citrate from the root into the rhizosphere, or root soil interface. Citrate is a major component of the citric acid cycle in eukaryotic catabolism, with this molecule representing a tri-carboxylic acid that is largely deprotonated and negatively charged at moderately low pHs. From this work, it was suggested that exudation of citrate leads to binding of it to Al3+ in the soil environment, with this likely making the chelated Al unavailable for entry into the root. In support of this finding, addition of citrate to the growth environment of the Al-sensitive snap bean cultivar resulted in an increase in the capability of this cultivar to grow in the presence of Al. From this work, further analysis was performed using near-isogenic lines of wheat that differed phenotypically only with regard to their growth in the presence of toxic levels of Al in an acidic environment (Delhaize et al., 1993a; Delhaize et al., 1993b; Ryan et al., 1995a; Ryan et al., 1995b). In support of previous work on snap beans, it was found that the near isogenic line of wheat that maintained root growth in the presence of Al had a significantly greater release of the di-carboxylic acid malate from its roots compared with the Al-sensitive line. From these results, it was concluded that Al resistance/exclusion was mediated by a single gene in wheat, which was subsequently labeled as Alt1. As with citrate, malate is a key component of the citric acid cycle that at acidic pH has an overall negative charge since its carboxylic acid groups are likely deprotonated. Interestingly, this increase in malate release was strictly in response to treatment with Al at low pH, with the two near-isogenic lines showing similar levels of malate release in the absence of Al. From these results, it was argued that the Alt1 locus likely encoded an Al-gated channel that was responsible for movement of cellular malate into the rhizosphere for chelation-based Al resistance (Ryan et al., 1997). At the time of the initial work with the near-isogenic lines of wheat, it was not clear whether the Alt1 locus represented a change in the kinetics of the release of malate or whether the increased Al resistance resulted from increased expression or reduced turnover of the malate transporter. The increase in malate release in the Al resistant line was not correlated with increased malate production, suggesting that the Alt1 locus did not impact the citric acid cycle nor did it alter intracellular compartmentalization of malate in the Al resistant line.

118

GENES FOR CROP ADAPTATION TO POOR SOIL

Consistent with these findings, work in maize, barley, and buckwheat showed that increased organic acid release by these species could also increase Al resistance for roots of each (Li et al., 2000; Ma et al., 1998; Ma, 2000; Ma et al., 2001; Pellet et al., 1995; Zheng et al., 1998). In each case, Al treatment resulted in release of Al-chelating organic acids, although the specific organic acid for each differed. As with snap beans, Al-resistant maize has been found to release high levels of citrate along with inorganic phosphate, the latter of which has been argued also to be involved in Al chelation or pH modification. It is still unclear as to whether phosphate release is important to Al resistance, although it could be argued that pH modification (i.e., alkalinization) could result in shifting the speciation of Al away from Al3+. As with wheat, Al resistance in barley arises from increased malate release, whereas increased resistance in buckwheat is associated with oxalic acid. In all of these cases, enhanced Al exclusion is directly dependent on release of Al-chelating organic acids, which are predicted to coordinate the Al3+ ion with their carboxylic acid groups that are likely unprotonated in an acidic environment. Following chelation, the fate of the bound Al is less clear. Although work with the near-isogenic line of wheat argues that the Al-resistant line accumulates far less Al than the Al-sensitive line (Delhaize et al., 1993a), it is unknown how binding of Al to an organic acid works to exclude Al. In contrast to this, other researchers have argued that chelation does not necessarily result in Al exclusion, but rather detoxifies Al prior to internalization, thus alleviating toxicity (Ma and Hiradate, 2000). As will be discussed for mechanisms of Al tolerance, evidence exists that internalized Al is in fact detoxified by chelation to organic acids such as citrate. Based on this, it is currently unclear as to whether Al-dependent organic acid release results only in exclusion of Al or if it is the initial step in detoxifying Al prior to internalization. Although none of the above scenarios appear to be dependent on increased organic acid biosynthesis, work in tobacco and papaya has argued that increased biosynthesis of citrate will in fact increase Al resistance (De la Fuente et al., 1997). In this study, bacterial citrate synthase, which is the enzyme responsible for catalyzing the conversion of oxaloacetate and malate to citrate in the citric acid cycle, was overexpressed resulting in increased citrate exudation into the rhizosphere coupled with increased Al resistance. Subsequent work has refuted these findings, with no discernible differences found in terms of internal citrate levels even following increasing levels of citrate synthase up to 100-fold in transgenic lines (Delhaize et al., 2001). Currently, it is unclear as to whether actual modification of rates of biosynthesis can have a substantial impact on organic exudation or Al exclusion. In recent years, intensive efforts have been made to identify the gene encoded by Alt1 in wheat since this was expected to make an exceptional molecular tool for genetic engineering of plants that have increased Al resistance. Through the use of a subtractive hybridization approach in which cDNAs from both the sensitive and resistant near isogenic lines of wheat were used, a gene that was more abundant in the tolerant line was identified (Sasaki et al., 2004). This gene, labeled as ALMT1, was found to encode a putative transmembrane domain protein with homologs in rice and Arabidopsis but not in animals. Although the gene sequences from the sensitive and tolerant lines were nearly identical, with only two amino acid substitutions, it was found that expression levels of ALMT1 were far greater in the tolerant line compared to the sensitive line. Further analysis revealed that ALMT1 expression was constitutive in both lines, with the expression being localized to the root tip. From these results, it is likely that the Alt1 dependent increase in Al resistance represents an increase in ALMT1 expression rather than a change in protein activity. Consistent with the role of ALMT1 in mediating Al resistance, it was found that the overexpression of ALMT1 represents a protein with transport activity. Electrophysiological analysis reveals that ALMT1 functions as a malate-specific transporter that is regulated by the presence of Al. Expression of ALMT1 in Xenopus oocytes revealed that ALMT1 only conducts malate upon

UNRAVELING THE MECHANISMS UNDERLYING ALUMINUM-DEPENDENT ROOT GROWTH INHIBITION

119

addition of AlCl3 but not LaCl3, the latter of which is another trivalent cation often used to determine whether phenomena are Al specific (Larsen et al., 1996; Yermiyahu et al., 1997). Additionally, it was found that ALMT1 is incapable of transporting citrate. From these analyzes, it was highly likely that ALMT1 overexpression represented the biochemical basis for increased malate exudation and the concomitant increase in Al resistance. In a subsequent analysis, it was found that overexpression of ALMT1 in barley resulted in a large increase in the capability of the transgenic barley to grow in the presence of normally inhibitory levels of Al, indicating that this approach represents an effective means to increase the capability of roots of crop plants to grow in an Al-toxic environment (Delhaize et al., 2004). Significant work has been performed since those findings with regard to the role of ALMT1 in Al resistance. For example, a confirmatory study was performed in Arabidopsis thaliana, in which a knockout mutant of AtALMT1 was identified and analyzed (Hoekenga et al., 2006). From this, it was found that AtALMT1 is required for normal Arabidopsis root growth in the presence of Al, further demonstrating the role of this factor in mediating Al exclusion. Unlike wheat ALMT1, AtALMT1 is Al inducible (Gabrielson et al., 2006) although it has been found that increased expression of this factor is not necessarily sufficient to prevent symptoms of Al toxicity especially in Arabidopsis mutants that have increased Al sensitivity (Gabrielson et al., 2006; Larsen et al., 2007). A similar mechanism of Al tolerance that is dependent on ALMT1 has also been described in rye (Collins et al., 2008). Current research has focused on the basis for increased ALMT1 expression in the Al-resistant line of wheat, with significant attention being directed toward comparison of the promoter regions of ALMT1 from both the sensitive and resistant lines (Sasaki et al., 2006), although it is still unclear as to what mediates the increased expression in the tolerant line. As presented earlier, other crop species have been found to rely on increased citrate export for mediating Al resistance, with this citrate expected to bind tightly to Al3+ in the rhizosphere. Consistent with this model, it has been found that Al resistance in sorghum is dependent on increased expression of a MATE (multidrug and toxic compound extrusion) transporter responsible for release of citrate from the root (Magalhaes et al., 2007). For this work, positional cloning was used to determine the nature of the Alt(SB) locus, which revealed that it encoded a citrate transporter that was highly abundant in the resistant line of sorghum. Analysis of SbMATE revealed that it is expressed to extremely high levels in the root tip of the Al resistant line of sorghum, with this protein being localized to the plasma membrane. Consistent with this localization pattern, increased SbMATE expression was found to be correlated with significant increases in citrate release by resistant roots compared to sensitive roots. Overexpression of SbMATE in Arabidopsis, both wild type and an AtALMT1 knockout, resulted in a modest increase in capability to grow in an Al-toxic environment in relative comparison to the parental controls, suggesting that SbMATE-dependent increases in citrate release may also serve as an effective strategy for increasing Al resistance in crop plants. In support of this work, an Al-resistant cultivar of barley was found to have increased expression of a root localized Al-activated MATE transporter that is responsible for citrate release (Furukawa et al., 2007). As with SbMATE, HvAACT1 is localized to the plasma membrane and is responsible for transport of citrate, but not malate, into the rhizosphere. Overexpression of HvAACT1 in tobacco results in an increased capability for root growth in the presence of Al. Additionally, similar findings have been recently reported for wheat, where it has been shown that, as seen for malate, increased citrate efflux can result in gains in Al resistance (Ryan et al., 2008). As with sorghum and barley, the increase in citrate efflux is correlated with increased expression of a MATE transporter that is expressed at greater levels in the resistant line compared to the sensitive line.

120

GENES FOR CROP ADAPTATION TO POOR SOIL

Interestingly, none of the described mechanisms of Al resistance that are dependent on enhanced release of organic acids seems to be dependent on changes in the kinetics of organic acid release, but rather results from increased expression of the genes of interest. It remains to be seen if any of the organic acid-dependent mechanisms of Al resistance indeed result from actual changes in transport activity, with this especially of interest since much of the current work on Al resistance for the most part is confirmatory with regard to the biochemical basis for increases in the effectiveness of these Al exclusion mechanisms. At one point, Arabidopsis mutants that had roots with increased resistance to Al were identified (Degenhardt et al., 1998; Larsen et al., 1998). While the work was largely abandoned due to the difficulty with regard to following the Al resistance phenotype during map-based cloning efforts, an alternative mechanism of Al resistance was described from this analysis. As discussed earlier, Al toxicity is dependent on the pH of the environment, with the toxic Al3+ species being favored in acidic environments. Although the majority of the alr (Al resistant) mutants had altered patterns of organic acid release, it was found from this work that one mutant had an increase in Al resistance that was actually dependent on enhanced proton uptake by the root, indicating a greater capability for modification of the rhizosphere pH resulting in its alkalinization (Degenhardt et al., 1998). Based on these findings, it was predicted that the increase in rhizosphere pH would result in reduced levels of Al3+ and consequently lead to reduced Al toxicity. Although this phenomenon has not been documented in studies of natural variation relating to Al resistance, it is clear that an enhanced capability to alkalinize the rhizosphere represents a viable strategy for modifying plants with an increased capability to grow in Al toxic soils.

Aluminum Tolerance Mechanisms

Although Al resistance appears to be mechanistically simple, it is much more difficult to predict how simple biochemical changes can result in anything more than an incremental change in Al tolerance, which relates to a plant’s capability to cope with internalized Al. Clearly, plants have developed strategies that are used to allow them to tolerate Al upon accumulation in the root. This is especially true of several plant species that are considered to be Al hyperaccumulators, including tea, buckwheat, and hydrangea. These species have been found to accumulate Al to extremely high levels, yet it is for the most part unclear as to how these and other plant species can cope with what is considered to be a highly promiscuous toxin. It is especially interesting to note that the color of the flowers of the hydrangea, which is a popular ornamental, is directly dependent on the level of internalized Al in the flowers (Ma et al., 1997). For example, if a hydrangea plant is grown in nonacidic soils, Al content in the plant tissue will be minimized, resulting in cream to pink colored flowers. In contrast, if a hydrangea plant is grown in an acidic Al-containing environment, the flowers will end up internalizing high levels of Al (upward of 3,000 mg/Kg of dry weight), thus resulting in a blue color. In this phenomenon, Al is believed to serve as a bridge molecule between delphinidin 3-glucoside and 3-caffeoylquinic acid, thus stabilizing this organic complex and promoting blue color formation. It is highly likely that many of the plant species that have become adapted through evolution to grow in an Al-toxic environment, such as found in South America and Africa, have biochemical alterations that allow them to cope with internalized Al. As with Al resistance, much of our understanding regarding Al tolerance focuses on the role of Al chelating organic acids. Although hydrangea leaves accumulate extremely high concentrations of Al, it has been found that this Al is actually in the form of an Al-citrate complex at a ratio of 1 : 1 that is believed to reduce the free levels of Al3+ and prevent it from binding to sites of toxicity

UNRAVELING THE MECHANISMS UNDERLYING ALUMINUM-DEPENDENT ROOT GROWTH INHIBITION

121

within the cell (Ma et al., 1997). Currently, it is not clear as to where in the cell this chelated Al is stored although it is predicted that it is localized to the vacuole. A similar mechanism is also found in buckwheat, although Al chelation and storage in this system seems to be more complex (Ma et al., 1998; Ma and Hiradate, 2000; Ma, 2000; Zheng et al., 1998). Initially, Al in the rhizosphere is predicted to enter the root by an as yet undefined path, after which it is chelated by three molecules of the dicarboxylic acid oxalate for every Al3+ ion. Following uptake and chelation, the Al : oxalic acid complex is transferred to the xylem where the three oxalic acids in the complex are replaced with one citric acid. Upon entering the xylem, the Al-citrate complex is subsequently transferred to leaf tissues, after which it is converted back to an Al : oxalic acid complex for storage in the vacuole. It is likely that this represents a common theme of Al tolerance in plants, with Al being removed from sensitive regions such as the root tip for storage elsewhere as an organic acid complex. Even so, this work likely oversimplifies the mechanisms of true Al tolerance without giving many clues as to the overriding cause of Al toxicity and Aldependent root growth inhibition.

Arabidopsis as a Model System for Aluminum Resistance, Tolerance, and Toxicity

Arabidopsis thaliana has served as a useful tool for examining the complexity of both Al resistance and tolerance. Unlike the aforementioned model systems, which have focused on the analysis of natural mechanisms of Al resistance, Arabidopsis gives the advantage of being able to screen for randomly generated mutants with changes in their capability to grow in an Al-toxic environment. This approach offers the opportunity to identify novel mechanisms that may not be revealed using traditional breeding approaches, since for example, some of the mechanisms identified may have deleterious pleiotropic effects or may affect the overall vitality of the plant. Additionally, the use of Arabidopsis allows for screening of thousands of plants in a small space and time frame, thus increasing the likelihood that mutations affecting the process of interest will be found. Coupled with the advances in our knowledge of Arabidopsis, including a fully sequenced genome, it becomes clear as to why use of Arabidopsis provides an opportunity to develop knowledge that may not necessarily arise from the study of natural variation. Although it has been argued that Arabidopsis is more sensitive to Al than agriculturally important plants, one cannot discount the potential for making discoveries in this model system that may be applicable to all plants. For example, it is clear from the work on ALMT1 that this factor is critical for malate release in both wheat and Arabidopsis (Sasaki et al., 2004; Hoekenga et al., 2006), indicating that each species likely relies on similar mechanisms for coping with Al in the environment. Interestingly, based on these similarities, it was just as likely that ALMT1 could have been discovered and studied first in Arabidopsis through the identification of a loss-of-function mutant that had increased Al sensitivity. Consequently, a mutagenesis approach in order to identify Arabidopsis mutants with altered growth in the presence of Al has utility to increasing our understanding of the mechanisms underlying Al resistance, tolerance, and toxicity.

Aluminum-sensitive Arabidopsis Mutants

Several years ago, a mutant screen was performed in order to identify Arabidopsis mutants that were hypersensitive to levels of Al that caused little to no root growth inhibition in wild type (Larsen et al., 1996; Larsen et al., 1997). It was predicted that these mutants would represent defects

122

GENES FOR CROP ADAPTATION TO POOR SOIL

in either Al-resistance or Al-tolerance mechanisms, indicating that the identified factors are required for mediation of these phenomena. This screen was designed in such a way as to enhance the likelihood that Al-specific mutants could be isolated. In this scheme, mutant roots were grown in a sterile Magenta box environment that had two nutrient gel layers, with the bottom layer having low to moderate levels of AlCl3 (pH 4.2) and the upper layer having no AlCl3. Mutant Arabidopsis roots that were capable of growth through the upper non–Al-containing layer but could not penetrate the lower Al-containing layer were identified and rescued for further analysis. From this screen, nine als (Al sensitive) mutants representing eight independent loci were isolated, suggesting that Al tolerance and resistance are complex traits in Arabidopsis. Most of these mutants represent changes in Al tolerance since indicators of Al response do not suggest that Al exclusion is altered. For example, both morin staining, which is a qualitative measure of internalized Al, and analine blue staining, which stains for levels of Al-dependent callose production (Horst et al., 1997; Llugany et al., 1994; Wissemeier et al., 1987), indicate that the Al hypersensitivity seen for almost all of the als mutants does not correlate with increased Al uptake or increased Al-dependent damage (Larsen et al., 1996).

The Role of ALS3 in Al Tolerance

Although many of these mutants represented difficult challenges with regard to isolation of the affected genes since their growth phenotypes were at best moderately severe, at least two have resulted in the identification and characterization of factors that are critical for allowing root growth in the presence of toxic levels of Al. Both of these, als1 and als3, do not appear to arise from alterations in Al exclusion as determined by the previously described analyzes with morin and analine blue (Larsen et al., 1996). Each is indistinguishable from wild type in the absence of Al, yet in the presence of subthreshold levels of Al in an acidic environment; both show moderate to severe root growth inhibition compared to wild type, which remains largely unaffected at this level of Al. This is especially severe for als3 loss-of-function mutants, which after three or four days of root growth in the presence of Al become completely inhibited, with their primary roots never being capable of resuming growth following rescue to a non-Al environment. This Al-dependent growth inhibition results in severe swelling of the root tip along with differentiation of the root tissue almost to the tip itself, as evidenced by production of root hairs at a point just behind the root tip. In conjunction with the severe effects on root growth, als3 loss-of-function mutants also have profound Al-dependent effects in the shoot, with leaf and rosette development being dramatically altered following Al exposure (Larsen et al., 1996; Larsen et al., 1997). These effects include severe inhibition of leaf expansion along with bifurcation of the shoot apical meristem, resulting in rosettes that appear to be two individual rosettes superimposed onto each other. In conjunction with the Al-dependent blockage of leaf development, inappropriate callose production occurs in the leaf promordia, which is a phenomenon that is not observed in the wild type. These results are specific to Al since treatment with other metals including a wide range of heavy metals and other trivalent cations fails to result in a growth phenotype for als3. Clearly, based on this analysis, als3 represents a lesion in an Al-tolerance factor that is required by Arabidopsis for both root and shoot growth in the presence of toxic levels of Al. Map-based cloning was subsequently employed to identify the nature of the mutation and the gene that was impacted. This exercise resulted in the determination that the als3-1 mutation was localized to the bottom half of Arabidopsis chromosome 2, with it ultimately being found to represent substitution of a T for a C in the coding sequence of gene At2g37330 (Larsen et al., 2005). Analysis of the protein product of this gene using predictive programs revealed that ALS3 encodes

UNRAVELING THE MECHANISMS UNDERLYING ALUMINUM-DEPENDENT ROOT GROWTH INHIBITION

123

a protein with seven putative membrane-spanning regions, suggesting that ALS3 is a membranelocalized protein involved with solute transport. Based on this protein sequence, the als3-1 mutation represents the substitution of a leucine for a serine in transmembrane domain 4 of the protein, suggesting that the als3-1 mutation disrupts protein conformation or protein activity. The phenotype of the als3-1 mutant is identical to T-DNA knockout mutants that represent T-DNA insertions in the ALS3 gene, indicating that this mutant likely represents a complete loss-of-function mutant. Interestingly, closely related homologs of ALS3 are found in both monocots and dicots, suggesting that an ALS3 mediated mechanism of Al tolerance may be universally found in plants. It remains to be determined whether mutations affecting ALS3 homologs in these other species will result in Al hypersensitivity. Although it is not currently clear as to how ALS3 functions in Al tolerance, several clues give an indication of its role in this process. Sequence analysis reveals that it is similar to a bacterial protein, ybbM, which is required for metal resistance in bacteria (Larsen et al., 2005). Interestingly, ybbM represents a partial ABC transporter that encodes just the transmembrane region of the protein. In general, ABC (ATP binding cassette) transporters each must have both a transmembrane region and an ATPase domain in order to function since ABC transporters actively move specific substrates across membranes (Davidson and Chen, 2004). ABC transporter proteins can either represent a full ABC transporter, which would have two membrane localized domains and two ATPase domains, or they can be half-type ABC transporters, which represent proteins with only single domains of each, with the half-type ABC transporters needing to dimerize to form a functional protein. In the case of ybbM, this encodes only one set of transmembrane domains although analysis of the bacterial genome reveals a predicted ATPase domain-containing protein as part of its operon. Essentially, this comparison suggests that ALS3 is an atypical ABC transporter that likely functions as part of a complex that would include an as yet unidentified ATPase domain that would be expected to provide the necessary energy for solute transport. Based on the similarities, it can be predicted that ALS3 is required for movement of either Al, possibly as an Al-organic acid complex, or some as yet unidentified chemical required to ameliorate the toxic effects of internalized Al. Further insight into its function can be gleaned from the biochemical, molecular, and cellular analysis of ALS3 (Larsen et al., 2005). Tissue localization indicates that ALS3 is primarily distributed throughout the plant in the vasculature, with ALS3 found exclusively in the phloem of this tissue (Figure 5.1). The phloem-based localization pattern is found throughout the plant from the region of differentiation just behind the root tip, throughout the root and leaf vasculature, and in several tissues of the flower. Interestingly, the leaf expression pattern shows that not only is ALS3 highly expressed in the phloem, but it is also abundant in the leaf hydathodes, which are responsible for the process of guttation that results in release of water from the hydathodes due to the water being forced out by root pressure. It is intriguing to consider that, since ALS3 expression in the vasculature represents an unbroken path from the root to the hydathodes, this expression pattern may indicate that a component of Al tolerance is represented by excretion of internalized Al via water of guttation. Consistent with this argument, localization of ALS3 is altered greatly following Al treatment. Although the vascular localization pattern remains the same with and without Al, there is a profound increase in ALS3 expression in the actual root tip, with ALS3 expressed throughout this region in the presence of Al. This further supports the argument that ALS3 is a component of some unidentified transport mechanism to remove Al away from the sensitive root tip and subsequently shuttle it to less sensitive tissues or to export the Al via the water of guttation. Other areas of ALS3 localization include the root epidermis, along with high levels of expression in several of the tissues of the flower including the stigma of the ovary, the complete anther, and the vasculature of the sepals and petals. Interestingly, the als3-1 mutation has been correlated with

124

Figure 5.1 Expression analysis of ALS3 in Arabidopsis. For this analysis, transgenic Arabidopsis T2 plants expressing an ALS3:GUS fusion under the control of the ALS3 promoter were analyzed for patterns of ALS3 expression, as evidenced by manifestation of GUS-dependent blue color. (A) 5d old transgenic seedlings grown in the absence of Al were examined for accumulation of ALS3:GUS in their roots. GUS activity was found primarily in the phloem and epidermis beginning at the region of root elongation. (B–E) Close-up of ALS3:GUS accumulation in the vasculature and epidermis of transgenic Arabidopsis roots, including a cross section of the mature region of a root (D). (F–G) Examination of ALS3:GUS activity in transgenic Arabidopsis roots exposed to either no (F) or 100 mM (G) AlCl3 (pH 4.2) for 24 hours in nutrient solution. (H) Patterns of ALS3:GUS activity in leaves of transgenic Arabidopsis show ALS3 expression in the phloem and hydathodes of the leaf. (I) Close-up view showing ALS3:GUS activity in the epithem of the hydathode, but not the water pore. (J) ALS3:GUS activity was also found in flowers including the floral vasculature, the junction between the filament and anther, and the stylar transmitting tract. For color detail, please see color plate section.

UNRAVELING THE MECHANISMS UNDERLYING ALUMINUM-DEPENDENT ROOT GROWTH INHIBITION

125

increased apoplastic accumulation of Al in mutant roots compared to the wild type, suggesting that loss of ALS3 function results in inappropriate buildup of Al due to failure to remove this away from the root tip. This buildup, which is evidenced by an increase in Al-sensitive hematoxylin staining, would likely then lead to the extreme response seen for als3-1 roots, including the terminal differentiation of the primary root due to the severe damage that would occur with Al toxicity. ALS3 is predicted to be a transmembrane protein suggesting that, as part of its function as an ABC transporter, it is responsible for movement of a specific substrate across a membrane that is related to Al tolerance. Subcellular localization analysis revealed that ALS3 is found at the plasma membrane, suggesting that ALS3 is responsible for movement of a substrate into or out of a cell. This further supports the argument that ALS3 represents a step in a likely complex pathway that is required for movement of Al away from the sensitive root tip to less sensitive regions such as leaves and flowers. As discussed previously, several lines of evidence support this role, including the inappropriate buildup of Al in roots of als3-1 compared to wild type. It can be argued that ALS3 is required for movement of Al into root cells for subsequent transfer to the phloem, with the Al subsequently moving to the aerial portions of the plant via the vasculature. Loss of ALS3 function would subsequently lead to inappropriate buildup of Al in the root tip and other sensitive tissues including the leaf primordia, leading to manifestation of the Al hypersensitivity phenotype seen for als3 loss-of-function mutants. Alternatively, it could be argued that ALS3 functions to transport some other substrate, possibly a growth regulating substance that is required to sustain growth in the presence of Al, or a chemical that is required for Al chelation. The former possibility is highly unlikely since als3 loss-of-function mutants do not demonstrate any obvious growth defects in the absence of Al, nor do they have increased sensitivity to a wide range of heavy metals or other trivalent cations (Larsen et al., 1997; Rounds and Larsen, 2008). This suggests that if ALS3 is required for transport of a growth regulator, this as yet unidentified substance is not required for normal growth and development nor is it required for tolerance to other stresses, with neither scenario being likely. The possibility that ALS3 is responsible for transport of a substance that chelates Al is intriguing, since as previously demonstrated, Al tolerance is strongly correlated with binding of internalized Al to citric and/or oxalic acid. This scenario also seems unlikely, though, since based on our knowledge of Al tolerance/ resistance, if ALS3 was responsible for movement of an Al-chelating substance, it would likely be facilitating movement of this substance out of the cell in order to promote Al chelation. There is very little evidence based on analysis of the als3 loss-of-function mutants that suggest that loss of ALS3 results in altered Al exclusion since there is no obvious change in overall Al levels compared to the wild type. Because of this, it seems that loss of ALS3 function leads to changes in Al redistribution rather than Al accumulation, with als3 loss-of-function mutants failing to properly move Al away from the Al-sensitive root tip. Much work remains to be done to understand the role of ALS3 in Al tolerance. For example, it is clear that ALS3 is only one component of a likely complex mechanism since it at best represents only one piece of the actual transporter, with a cognate ATPase domain still remaining to be discovered. Several candidate ATPase encoding genes have been studied in Arabidopsis to determine if loss-of-function mutants of these would result in increased Al sensitivity although to date none analyzed have been found to be required for growth in an Al toxic environment. The inability to identify an ATPase that functions with ALS3 could arise either from redundancy, with several of the ATPases having overlapping functions in Arabidopsis, or from the ATPase that is predicted to be involved not looking like a typical ATPase protein. Additionally, there is likely a protein complex that is required to reverse the effects of ALS3. Since ALS3 is predicted to either load or unload an Al-complex at the point of the phloem, it is

126

GENES FOR CROP ADAPTATION TO POOR SOIL

expected that a similar mechanism must exist to reverse this in order to deliver the Al-complex to its final destination, whether it be storage in leaves and flowers or exudation through water of guttation. Clearly, with the predicted complexity of this mechanism, identification of additional Arabidopsis mutants with increased Al sensitivity should reveal new components of this pathway.

ALS1 Encodes a Half-type ABC Transporter Required for Aluminum Tolerance

The initial screen for Arabidopsis mutants with increased Al sensitivity also resulted in the isolation of als1, which, although not as severe as als3, has roots that are capable of only limited growth in the presence of Al (Larsen et al., 1996). As with als3, als1 mutants are indistinguishable from wild type in the absence of Al, indicating that the ALS1 gene is not required for general root growth. Physiological analysis of als1 suggests that it represents a lesion affecting Al tolerance, since there is no indication that Al exclusion is negatively affected in the mutant. This was demonstrated both through quantification of Al uptake and production of Al induced callose, which is an effective indicator of Al stress. In each case, als1 mutants were identical to wild type, which is consistent with a change in Al tolerance rather than resistance. In support of ALS1 being responsible for mediation of Al tolerance, als1-1 mutant roots showed greater than wild-type expression of Alinducible genes following treatment with low levels of Al, including the genes AtALMT1, AtprxCb, and AtBCB (Larsen et al., 2007). Map-based cloning of the als1-1 mutation resulted in the determination that it represented a change in a previously uncharacterized half-type ABC transporter also known as AtTAP2 (At5g39040), with the ALS1 protein consisting of four predicted membrane spanning regions and one ATPase domain (Larsen et al., 2007). As previously stated, functional ABC transporters are comprised of two membrane-localized domains, which make up the pore through which the substrate is moved, and two ATPase domains, which function to hydrolyze ATP to provide the energy for active transport. Unlike ALS3, ALS1 is predicted to have all of the necessary motifs for ATPase function including the requisite Q, Walker A and B sequences, a Signature motif, and H motif. It is not clear whether ALS1 functions as a homodimer or a heterodimer with some as yet unidentified ABC transporter. Expression in yeast of ALS1 with or without ALS3 did not have an effect on the growth of yeast in the presence of Al, which may indicate that either required components have yet to be identified to re-create this Al tolerance mechanism in yeast or that ALS1 does not function or localize properly in yeast. The als1-1 mutation was found to result in the change of a glutamic acid to a lysine in a region between transmembrane loops (Larsen et al., 2007). Although it is not clear how this amino acid substitution affects protein function, it can be speculated that this change, which results in the swapping of a negatively charged side chain for a positively charged group, may lead to an alteration in protein folding or in capability for substrate transport. T-DNA insertion alleles of als1 give similar growth phenotypes in the presence of Al, indicating that the als1-1 mutation is severe in its effect on ALS1 function. As with ALS3, overexpression of ALS1 in Arabidopsis did not result in any discernible increase in Al tolerance, which could suggest that ALS1 is part of a complex pathway dependent on several factors for mediation of Al transport with overexpression of any one component not being sufficient to impact Al tolerance. Analysis of ALS1 in terms of localization gives some insight into its predicted function. Unlike ALS3, ALS1 is not Al inducible although it is expressed at moderate levels in all organs tested (Larsen et al., 2007). Histochemical analysis using an ALS1-GUS fusion revealed that ALS1 is localized mainly to the vasculature throughout the plant including the root and leaves (Figure 5.2).

Figure 5.2 Localization analysis for ALS1 in various organs of Arabidopsis. Transgenic Arabidopsis T2 plants expressing an ALS1:GUS fusion under the control of the ALS1 promoter were analyzed for patterns of ALS1 expression. (A) GUS activity found to occur primarily in the root tip and vasculature of five-day old transgenic seedlings. (B) Cross section of the mature region of an ALS1:GUS root shows that GUS activity is found in the ground tissue of the stele. (C) Analysis of a mature ALS1:GUS leaf shows that GUS activity is strictly localized to the vasculature. (D) Close-up of an ALS1:GUS leaf, which shows GUS activity in both the vasculature and a hydathode. (E) Pattern of GFP fluorescence in transgenic roots expressing ProACT2:GFP or ProACT2:ALS1:GFP. Arrows indicate areas where the vacuolar membrane wraps around the cell’s nucleus, with this analysis indicating that ALS1 is localized to the plasma membrane of the root. For color detail, please see color plate section.

127

128

GENES FOR CROP ADAPTATION TO POOR SOIL

Additionally, ALS1 is prominently expressed throughout the root tip, which is consistent with a role in detoxification of Al in this region. As with ALS3, the ALS1 localization pattern is continuous from the root tip to the leaf hydathodes suggesting that ALS1 may form a link in the movement of Al away from the sensitive root tip to less sensitive aerial tissues. Unlike ALS3, ALS1 is not found in either phloem or xylem elements but is actually localized to the ground tissue of the vasculature. It is not clear how this localization pattern relates to Al detoxification, although the subcellular localization pattern for ALS1 gives a clue as to its role in Al tolerance. Analysis of an ALS1-GFP fusion showed that ALS1 is specifically located at the vacuolar membrane of root cells, suggesting that ALS1 is required for movement of Al into the vacuole for Al detoxification. This is in contrast to ALS3, which is localized to the plasma membrane and is likely involved in movement of Al into or out of the cell. Based on these analyzes, it is possible that ALS1 and ALS3 function synergistically to localize internalized Al to less sensitive regions either intracellularly or through distribution to other tissues. ALS1 is likely part of a mechanism related to intracellular redistribution since it is found at the vacuolar membrane, suggesting that ALS1 is involved in movement of Al into the vacuole from the cytoplasm. Loss of ALS1 function would likely result in inappropriate buildup of Al in the cytoplasm and lead to increased toxicity. It is also possible yet unlikely that ALS1 is responsible for movement of some unknown compound out of the vacuole into the cytoplasm that would chelate internalized Al. ALS3 on the other hand is not predicted to be responsible for intracellular redistribution of Al but rather is likely to be necessary for long distance movement away from the root tip for storage in aerial tissues or exudation. This is predicted based on both the subcellular localization pattern of ALS3, which indicates that it is involved with movement of some substrate across the plasma membrane, and the determination that ALS3 is primarily found in the phloem sieve tube elements, suggesting that ALS3 functions to load some substrate into the vasculature. It is currently not clear what the role of ALS3 is in the root tip, although ALS3 could be part of a mechanism responsible for Al uptake into the symplasm for eventual symplastic export to other regions of the plant. On the flip side, ALS3 may function to transport Al out of the cell, both at the root tip and in the vasculature, with this serving two distinct purposes. At the root tip, ALS3 may pump Al out of the cytoplasm to reduce toxicity in this region, whereas in the vasculature, release of Al from the phloem may be carried out by ALS3 in order to facilitate movement of Al out of the vasculature and into ground tissue in aerial organs for storage. Hematoxylin staining of als3-1 mutant roots seems to suggest that ALS3 is required for Al internalization rather than Al export since loss of ALS3 function leads to a significant buildup of apoplastic Al in the zone of elongation in mutant roots. It should be noted that as with ALS1, ALS3 may be responsible for export of some unknown substrate out of the cell into the apoplast for amelioration of Al toxicity, although this seems less likely than a proposed direct involvement with Al movement.

Other Arabidopsis Factors Required for Aluminum Resistance/Tolerance

While Arabidopsis is a step removed from actual agriculturally relevant crops, it has served as an important model system for dissecting the mechanisms of Al tolerance and resistance. Clearly, mechanisms that allow roots to grow in an Al-toxic environment are complex. This is evidenced by the number of Arabidopsis mutants that have been identified as having increased Al sensitivity,

UNRAVELING THE MECHANISMS UNDERLYING ALUMINUM-DEPENDENT ROOT GROWTH INHIBITION

129

with at least eight distinct loci required for Al tolerance or resistance in Arabidopsis (Larsen et al., 1996). Quantitative Trait Locus (QTL) mapping has also been performed for Arabidopsis with regard to Al resistance/tolerance using recombinant inbred lines that were sorted based on their capability to grow in the presence of Al. Studies have revealed at least two distinct loci that condition the capability of growth in the presence of Al (Hoekenga et al., 2003; Kobayashi and Koyama, 2002), with at least five other regions that were found to be important for regulating Alresponsive root growth. Interestingly, one of these loci was located on the top of chromosome 1 in an area that is close to the location of AtALMT1, further suggesting the critical role that malate release plays in Al resistance in Arabidopsis. The other single factor QTL mapped very close to the location of ALS3 on chromosome 2, also indirectly supporting the importance of ALS3 to mediating root growth in the presence of Al. Nearly 45% of the variance in growth in the presence of Al for the recombinant inbred population can be accounted for by these two major QTLs, with the remainder being predicted to be related to malate release. It should be noted that this analysis is likely to represent an underestimation of the complexity of Al resistance and tolerance, since QTL analysis is dependent on the existence of discernible natural variation within a recombinant inbred population, with factors such as ALS1 possibly not having distinct enough changes between the two parent lines to result in a measurable difference. Another factor that has been tangentially related to Al resistance is the zinc finger protein STOP1, which is important for regulation of gene expression related to low pH tolerance and Al resistance (Iuchi et al., 2007). STOP1 was identified based on analysis of a mutant that was incapable of wild-type root growth in a low pH environment. Analysis of this factor revealed that it is a member of a two-gene family of poorly understood zinger finger proteins that likely serve as transcription factors, with it currently unclear as to how this factor is responsible for mediation of proton tolerance. Of interest in relation to Al resistance, loss of STOP1 function results in almost complete loss of expression of AtALMT1, which consequently leads to a severe reduction in malate release and ultimately lowers Al resistance. It is not clear as to the role of STOP1 in AtALMT1 expression since work has not been done to explore if there is a direct interaction between STOP1 and the promoter of AtALMT1.

Identification of Aluminum-tolerant Mutants in Arabidopsis

It has been proposed that based on the sheer volume of work pointing to the number of sites of toxicity that Al tolerance is a highly complex trait. Because of this complexity, it has been argued that analysis of Al tolerance, which is a phenomenon that is dependent on increased capability to cope with internalized Al, would be at best exceptionally difficult with the expectation being that genetic changes would only give incremental increases in Al tolerance. Recent work in Arabidopsis has shown that contrary to existing models, Al toxicity is far more simplistic than what was previously thought, and single gene changes can indeed produce profound increases in Al tolerance with regard to root growth (Gabrielson et al., 2006). This is not to say that the myriad number of demonstrated sites of Al binding do not have relevance to Al toxicity but rather that this recent work argues that certain aspects of Al toxicity are paramount for Al-dependent stoppage of root growth. Until recently, it has been extremely difficult to isolate mutations in Arabidopsis that give increases in Al tolerance. This is largely due to the high variability that one encounters in terms of

130

GENES FOR CROP ADAPTATION TO POOR SOIL

root growth, making it hard to distinguish clearly between wild-type roots and Al-tolerant roots in a mapping population. Although one can generally follow increased Al tolerance in a mapping population, this lack of precision based on root growth variability makes it nearly impossible to define a narrow enough mapping window for actual isolation of the mutation. This ultimately led to the abandonment of the map-based cloning exercises relating to alr104 and alr128 since precise mapping populations could not be generated for either, thus resulting in determination of only a rough genomic location for each (Degenhardt et al., 1998; Larsen et al., 1998). With the isolation of the als3-1 mutant and its subsequent analysis, a new approach for isolation of Al-tolerant mutants emerged. As stated earlier, the effect of Al on root growth of als3 loss-of-function mutants is severe, with root growth effectively stopping in the presence of levels of Al that have only mild to moderate effects on wild-type roots. This severe effect of Al on als3 mutant root growth has given an opportunity to dissect Al tolerance in Arabidopsis through the identification of suppressor mutations that block the Al hypersensitivity seen for als3-1 (Gabrielson et al., 2006). Most importantly, the clear differences seen in the presence of Al for als3-1 roots compared to roots of als3-1 suppressor mutants gives the opportunity to perform successfully a map-based cloning approach to isolate the mutations of interest and develop new knowledge of Al tolerance. For this approach, als3-1 seeds were mutagenized with EMS, and M2 seedlings were screened for mutants that had roots that were capable of strong to moderate root growth in the presence of levels of Al that were severely inhibitory to als3-1 root growth. From this screen, more than 40 mutants that were capable of reversing the severe Al-dependent root growth phenotype of als3-1 were recovered, indicating that each of these mutants had a single genetic change that resulted in either an increase in Al resistance or tolerance (Gabrielson et al., 2006). It is predicted that mutations that cause enhanced Al exclusion, increased capability to alkalinize the rhizosphere, or a greater capability to tolerate internalized Al would result in suppression of the Al hypersensitivity found in als3-1 roots. Of the 40 recovered suppressor mutants, 11 of these showed profound increases in root growth even in comparison to wild type, suggesting that such mutations may represent strategies for increasing Al tolerance in crop plants. During the initial period of this work, three of the most promising als3-1 suppressor mutants were chosen for further characterization with the goal of isolating the respective mutations. Physiological characterization indicated that these particular suppressor mutants, labeled as alt (Al tolerant) mutants, had increased root growth due to changes that positively impacted Al tolerance rather than Al exclusion or rhizosphere alkalinization (Gabrielson et al., 2006). Characterization included quantification of Al uptake compared to wild type and als3-1, with this analysis showing comparable levels of Al in the alt mutants, wild type, and als3-1 following Al treatment. Additionally, callose deposition, which is an indicator of Al stress, occurred similarly in the roots of the alt mutants compared to wild type and als3-1, indicating that the alt mutants were experiencing similar levels of Al toxicity as seen for the parent lines. Finally, Al responsive gene expression occurred normally in the alt mutants compared to wild type and als3-1 following Al treatment, lending further support to the likelihood that the increased root growth seen for the alt mutants results from an alteration in a mechanism that allows for the mutant roots to cope better with internalized Al rather than a mechanism of Al resistance being enhanced. Genetic analysis of one of the three alt mutants initially studied revealed that when isolated from the als3-1 mutation, this mutation results in root growth that is far more vigorous than wild-type roots, indicating that this mutation has utility for increasing Al tolerance in other plant species (Gabrielson et al., 2006) (Figure 5.3). This is especially true since many of these alt mutations are dominant, meaning that the mutant versions of these genes can be introduced directly into other plants to confer increased Al tolerance. At the time of the initial study, it was argued that the

UNRAVELING THE MECHANISMS UNDERLYING ALUMINUM-DEPENDENT ROOT GROWTH INHIBITION

131

AlCl3

alt1-1

als3-1

Col-0

1.5 mM alt1-1

als3-1

Col-0

0.75 mM alt1-1

als3-1

Col-0

0 mM

Figure 5.3 Growth of alt1-1;als3-1 in an Al toxic environment. Col-0 wt, als3-1, and alt1-1;als3-1 seedlings were grown for 7 days on solid nutrient media supplemented with 0, 0.75 mM, or 1.50 mM AlCl3 (pH 4.2). Whereas the als3-1 mutant is highly sensitive to levels of Al that have little effect on wild-type root growth, introduction of the alt1-1 mutation into the als3-1 background results in complete suppression of the als3-1 Al hypersensitivity phenotype. Interestingly, even at very high levels of Al, the alt1-1 mutation results in root growth that is significantly greater than what is seen for wt, suggesting that the alt1-1 mutation may be useful for engineering Al tolerance in agriculturally relevant plants.

increase in Al tolerance seen for one of these mutants was likely due to an increased capability to raise the pH of the growth environment (Gabrielson et al., 2006). This study was done with an alt1;als3-1 double mutant, with the growth environment supplemented with a buffer that works effectively at pH 4.2 (Degenhardt et al., 1998). It was found at the time of the study that addition of Homo-PIPES buffer to the growth medium effectively eliminated the suppression of the als3-1 phenotype by the alt1 mutation, suggesting that alt1 functions to increase rhizosphere pH. As will be discussed, this result ended up being complicated by the inclusion of the als3-1 mutation in the alt1 mutant background. An alternative and more likely explanation is that in the absence of a buffered growth medium, over time the healthy growing roots of wild-type and alt1;als3-1 seedlings are capable of growth in the presence of low levels of Al not only because of a latent tolerance to Al but also because of a gradual root-mediated increase in the pH of their growth environment due to phenomena such as nutrient uptake. In the presence of Homo-PIPES, the capacity of roots to modify the pH of the growth environment would be abolished, thus increasing the long-term

132

GENES FOR CROP ADAPTATION TO POOR SOIL

toxicity of Al since it would be forced to stay in the Al3+ form for the course of the experiment. It is likely that during the study in question, the presence of the als3-1 mutation in the alt1 background coupled with the strict maintenance of the pH of the growth medium at 4.2 resulted in a severe reduction in the previously observed increase in Al tolerance seen for alt1 mutant roots. As will be discussed, based on the identified nature of the alt1 mutation, it is likely that this latter scenario explains the negative effects that Homo-PIPES buffer has on the Al tolerance phenotype of alt1.

The Nature of the alt1 Mutations

As stated, the identification of Al-tolerant mutations in an als3-1 background facilitated the actual isolation of the mutations since bona fide populations for map-based cloning could be generated. By having such populations, it has been possible to refine the genetic windows in which these mutations lie to narrow regions, which subsequently has allowed for the identification of candidate genes. Of the three suppressor mutants originally described (Gabrielson et al., 2006), each was found to be located at a similar location on chromosome 5 of the Arabidopsis genome, suggesting that each is allelic. Currently, it is not clear how many of the remaining als3-1 suppressor mutations represent alleles of alt1, although at least one of these has been found to represent a mutation at a locus different than alt1 (Nezames and Larsen, unpublished results). To isolate the dominant alt1 mutation, a mapping population was generated by crossing alt1;als3-1 (Col-0 background) to als3-1 (La-0 background), with the latter line generated through several generations of backcrossing. It was necessary to take this approach since it was only possible to follow the alt1 phenotype consistently if it was analyzed in an als3-1 background. If the als3-1 mutation had not been included in this mapping approach, it would have been difficult to accurately discriminate between wild-type and alt1 roots since, as previously described, variation due to unrelated factors such as timing of germination also impacts root length. Although somewhat counter-intuitive, it was necessary to isolate F2 individuals from this population that resembled als3-1 roots rather than those that had the suppressor phenotype, with this resulting from the dominant nature of the alt1 mutations. From this mapping exercise, it was found that the alt1-1 and alt1-2 mutations represented nucleotide substitutions and consequent amino acid changes in a previously characterized gene, At5g40820, which encodes a factor responsible for detection of DNA damage and control of the cell cycle in response to this damage (Culligan et al., 2004; Culligan et al., 2006; McSherry et al., 2007; Vespa et al., 2005). Homologs of this factor, which is known as ATR (ATAXIA TELANGIECTASIAMUTATED AND RAD3-RELATED), are found in all eukaryotes including yeast. It is related to ATM (ATAXIA TELANGIECTASIA-MUTATED), which is named for the human disorder (Ataxiatelangiectasia, also known as Boder-Sedgwick syndrome or Louis-Bar syndrome). When Ataxiatelangiectasia Mutated (ATM) function is compromised in humans, it leads to neural degeneration and accumulation of chromosomal damage that causes poor coordination (ataxia), capillary dilation (telangiectasia), weakening of the immune system, and a significant increase in the likelihood of developing cancer. Little is known about ATR (encoding Ataxia-telangiectasia Mutated and Rad3-related Kinase) in humans due to loss-of-function mutations in this gene resulting in lethality. Because of this, ATM has been more intensively studied and has been found to be a nuclear localized protein required for monitoring and responding to DNA damage (Lavin, 2008). ATM and ATR both have kinase activities and are expected to phosphorylate a target protein in response to DNA damage in order to halt progression of the cell cycle to allow for repair of this damage. Loss of ATM function leads to accumulation of DNA damage coupled with normal cell cycle progression

UNRAVELING THE MECHANISMS UNDERLYING ALUMINUM-DEPENDENT ROOT GROWTH INHIBITION

133

in spite of this damage, thus resulting in proliferation of compromised cells. Consequently, those individuals with Ataxia telangiectasia have a higher likelihood of cancer due to a reduced capability to correct DNA damage. Additionally, both ATR and ATM have been implicated in telomere homeostasis (Vespa et al., 2005), with loss of these factors leading to rapid telomere shortening. Both ATM and ATR from Arabidopsis represent highly complex proteins, with little currently known about the biochemical function of their various domains. AtATR encodes a protein that is 2,702 amino acids in length with a predicted molecular weight of 302 kiloDalton (kD). AtATM is similar in size and structure, which is consistent with overlapping functions of each in monitoring DNA integrity. AtATR can be dissected into several domains, many of which have not had a function attributed to them. Within the amino-terminus is what is described as an UME domain, which is named after three protein groups in which it is found including UVSB PI-3 kinase, MEI-41, and ESR1. This domain is predicted to be required for protein-protein interactions, which is consistent with the expectation that AtATR and AtATM serve as scaffolding proteins for large complexes required for monitoring DNA integrity. The alt1-1 mutation was found to represent an amino acid substitution in the UME domain (G1098E), suggesting that this mutation may disrupt association of AtATR with a partner protein (Rounds and Larsen, 2008). Additionally, a significant portion of the amino-terminus of AtATR is comprised of HEAT motifs that make an armadillo-like fold (ARM), which is comprised of two curving alpha helices that make up a superhelical structure predicted to scaffold other large biomolecules including proteins. Finally, the carboxy-terminus is made up of several domains that have predicted serine-threonine kinase activity with several of these domains being similar to those found in Phosphatidylinositol kinase (PIK)-related kinases and Phosphatidylinositol 3- and 4-kinases. The alt1-2 mutation is found in a domain related to phosphatidylinositol 3- and 4-kinases, resulting in an amino acid substitution (L2553F) that likely leads to steric hindrance due to introduction of a phenylalanine next to another bulky phenylalanine (Rounds and Larsen, 2008). Currently, it is not clear how either amino acid substitution affects AtATR function since an AtATR substrate has not been identified. Although both mutations are dominant, it is likely that each represents a negative effect on AtATR function with the dominant nature of these resulting from the mutant forms competing with the wild-type form of AtATR for participation in a protein complex, thus disrupting the overall activity of the DNA damage assessment machinery. Further clues to the nature of the alt1 mutations have come from comparison to a complete lossof-function atr mutant (Culligan et al., 2004; Culligan et al., 2006; Rounds and Larsen, 2008). It has been previously demonstrated that loss of AtATR function leads to Arabidopsis roots that are hypersensitive to both ionizing radiation and Hydroxyurea, both of which are agents of DNA damage. Exposure to either of these stresses leads to increased rates of DNA fragmentation in the case of ionizing radiation or poisoning of the replication fork by Hydroxyurea. Presumably, loss of AtATR function leads to a failure to recognize and respond to the damaging effects of these agents and consequently results in compromised cells that are no longer capable of division. Interestingly, this response is opposite to what is seen for the alt1 mutants in the presence of Al, which results in increased Al tolerance (Gabrielson et al., 2006). In order to determine if the alt1 mutants were similar to an atr complete loss-of-function mutant, a double mutant consisting of a T-DNA insertion mutant for atr (atr-2) and als3-1 (atr-2;als3-1) was generated and it was determined that loss of AtATR function could restore the growth of als3-1 roots in the presence of Al. Loss of AtATR function suppressed the als3-1 Al hypersensitivity phenotype, suggesting that the two alt1 mutations compromise AtATR function (Rounds and Larsen, 2008). In support of this argument, both alt1-1 and alt1-2 were tested for their capability to grow in the presence of Hydroxyurea, which is a replication fork poison that severely inhibits root growth for an atr

134

GENES FOR CROP ADAPTATION TO POOR SOIL

loss-of-function mutant. Consistent with the analysis of an atr-2;als3-1 double mutant, both alt1-1 and alt1-2 were severely inhibited following treatment with Hydroxyurea indicating that both alleles, although dominant, represent deleterious mutations that reduce AtATR function (Rounds and Larsen, 2008). As stated, the dominant nature of each of these mutations likely arises from the mutant versions of AtATR still being produced, with these defective versions poisoning the protein complex that AtATR participates in with regard to monitoring DNA integrity. As with Al toxicity, loss of AtATR function also positively impacts the capability of roots to grow in the presence of both nickel (Ni) and cadmium (Cd), each of which are predicted to have negative consequences on DNA integrity. This increase in tolerance is most notable with regard to growth in the presence of Ni, which results in a significant increase in tolerance for both alt1-1 and alt1-2 mutant roots compared to wild type. In contrast, the level of Cd tolerance varies between alt1-1 and alt1-2, with alt1-1 mutant roots being far more Cd resistant than either alt1-2 or atr-2. This is consistent with the predicted severity of each allele since alt1-2 is likely more deleterious to AtATR function since its growth pattern in the presence of Hydroxyurea is more comparable to atr-2 than alt1-1 (Rounds and Larsen, 2008). Interestingly, whereas reduced AtATR function leads to increased tolerance for Al, Ni, and Cd, the opposite is found for response to ionizing radiation and Hydroxyurea, both of which lead to increased root growth inhibition in atr-2. This strongly argues that Al, Ni, and Cd are causing cellular damage that is distinct from that caused by ionizing radiation and Hydroxyurea, with AtATR mediating different response mechanisms for each stress. It should be noted that the alt1 mutations may not only be useful for generating transgenic crops that can grow in Al toxic environments, but these also may be useful for increasing tolerance to Ni and Cd in polluted environments. Although this may not necessarily be beneficial to agriculture since crop plants are not normally grown in this type of environment, such an approach may be useful for engineering plants for phytoremediation, since clearly plants that are to be used for this approach must not only be capable of hyperaccumulating heavy metals but also must be capable of coping with these metals once internalized. It is also possible that introduction of the alt1 mutations into plants such as switchgrass may increase the capability of these plants to grow in suboptimal environments, which may increase biomass for production of biofuel. Although improvement of phytoremediation strategies may be one outcome of this work, Al cannot be removed from growth environments since it is so abundant in soils. Consequently, for plants to be engineered to grow in Al-toxic environments, they must be modified in a way that allows them either to exclude environmental Al or to tolerate Al once it is internalized. AtATR serves as a checkpoint for cells that have accumulated DNA damage, with this damage, if great enough, leading to cell cycle arrest (Culligan et al., 2004; Culligan et al., 2006). Previous work with AtATR has used a GUS-based marker for cell cycle progression in which the mitotic destruction box for CyclinB1;1 was fused to the GUS reporter. During normal cell cycle progression, CyclinB1;1 is rapidly degraded as a cell transitions from the G2 phase. It has been found that following stress that leads to DNA damage, progression out of the G2 phase is blocked by AtATR leading to a buildup of cells trapped in this phase and subsequent loss of cell cycle progression. This CyclinB1;1 reporter system was introgressed into both als3-1 and alt1-1 to determine how Al toxicity affects cell cycle progression (Figure 5.4). Interestingly, it was found that treatment with low levels of Al greatly increased the amount of GUS staining in both wild type and als3-1, indicating that one consequence of Al treatment is an increase in time required to transit out of the G2 phase of the cell cycle, which likely leads to reduced root growth rates. In contrast, treatment with similar levels of Al did not result in a concomitant increase in GUS staining of roots of alt1-1, suggesting that vigorous root growth in this mutant in the presence of Al occurs because the cell cycle is not affected by Al toxicity due to the loss of AtATR function (Rounds and Larsen, 2008).

135

Figure 5.4 Al-dependent root growth inhibition results from inhibition of cell cycle progression and loss of the quiescent center. (A) Treatment with inhibitory levels of AlCl3 causes a disproportionate increase in the number of cells trapped in the G2 phase of the cell cycle. A cell cycle progression marker representing a truncated version of the CDS of CyclinB1;1, including a predicted mitotic destruction box, fused to the GUS reporter gene was introgressed into both als3-1 and alt1-1. Col-0 wt, als3-1 and alt1-1 lines carrying this CyclinB1;1::GUS reporter were grown in the absence or presence of increasing concentrations of AlCl3 in a soaked gel environment (pH 4.2), following which seedlings were stained for GUS activity. Growth in the absence of AlCl3 resulted in minimal levels of GUS staining, indicating that few cells were trapped in the G2 phase under these conditions. Addition of AlCl3 resulted in a profound increase in GUS staining in roots of Col-0 wt and als3-1, showing that a consequence of Al treatment is an increase in the number of cells trapped in the G2 phase of the cell cycle. Addition of highly inhibitory levels of AlCl3 resulted in complete loss of GUS activity in als3-1 roots, suggesting that cells in the root tip of als3-1 were completely differentiated and not undergoing cell division. This is supported by development of lateral roots immediately behind the primary root tip of Al-treated als3-1. In contrast to Col-0 wt and als3-1, alt1-1 roots had only basal levels of GUS activity even with high AlCl3, indicating that alt1-1 mutant roots do not arrest the cell cycle in response to Al, which likely results from reduced AtATR activity in the alt1-1 mutant. (B) Evans’ blue staining indicates that even though root growth is severely arrested, als3-1 roots are viable following Al treatment. Col-0 wt, als3-1, and alt1-1 seedlings grown in the absence or presence of AlCl3 were stained with the vital stain Evans blue. Since Evans blue staining was minimal in all samples tested, as evidenced by lack of blue color in treated roots, it is likely that Al-dependent root growth inhibition does not result from tissue death but rather arises from terminal differentiation of the root tip. (C) Al treatment results in loss of the quiescent center (QC) in Arabidopsis roots. A GUSbased marker for the root quiescent center, QC46, was introgressed into als3-1 and alt1-1. Col-0 wt, als3-1, and alt1-1 carrying the QC46 GUS promoter trap were grown either in the absence or presence of 1.50 mM AlCl3 in a soaked gel environment (pH 4.2), after which seedlings were stained for GUS activity to assess the status of the QC. In the absence of Al, all samples had normal staining at the position of the QC in the root tip, suggesting that all samples maintained a viable QC. In contrast, treatment with levels of Al that cause severe root growth inhibition resulted in the loss of GUS activity in both Col-0 wt and als3-1 roots. This suggests that Al forces the QC in both of these lines to fully differentiate, with terminal differentiation due to the loss of stem cells in the QC likely resulting in root growth inhibition following Al treatment. Alt1-1 roots had normal GUS activity at the position of the QC even after treatment with Al, which indicates that AtATR activity is required to trigger the differentiation of the QC in the presence of Al. For color detail, please see color plate section.

136

GENES FOR CROP ADAPTATION TO POOR SOIL

Following treatment with levels of Al that are highly inhibitory to wild-type roots, it was found that there was a profound increase in GUS staining for wild-type roots in conjunction with severe reorganization of the root tip, in which the zone of differentiation was now found almost immediately behind the root tip. Analysis of als3-1 roots at this Al concentration surprisingly revealed no GUS staining, which likely arises from the complete arrest of cell division in als3-1 following treatment with high levels of Al. Consistent with this, Evan’s blue staining, which is used to detect whether cells remain viable, showed that als3-1 roots were indeed still alive but growth was completely arrested, with als3-1 roots being terminally differentiated as evidenced by development of root hairs at the root tip (Figure 5.4). This correlates well with the previous observation that Al treatment leads to irreversible root growth inhibition for the primary root of als3-1, with this likely resulting from an Al-dependent permanent loss of cell division in these roots. As found following treatment with low levels of Al, alt1-1 roots treated with levels of Al that are highly inhibitory to wild type showed little to no GUS staining, indicating that even in the presence of highly toxic concentrations of Al, alt1-1 roots are incapable of halting cell division due to the loss of AtATR function (Rounds and Larsen, 2008). Consequently, alt1-1 roots maintain vigorous root growth in the presence of Al since these mutant roots can no longer block cell division in response to Al toxicity. Further insights into the role of AtATR in mediating Al-dependent root growth inhibition have come from analysis of the effect of Al toxicity on the quiescent center. Plant growth is dependent on cell division at a meristem that represents a pool of stem cells that is known as the quiescent center (QC) in roots. Failure to maintain the QC leads to loss of progenitor cells and consequently loss of root growth. GUS-based markers for the QC are available, and one of these was introgressed into either an als3-1 or alt1-1 background, after which it was determined how Al toxicity affects maintenance of the QC (Figure 5.4). Treatment with levels of Al that are highly inhibitory to wildtype roots lead to loss of GUS staining for both wild type and als3-1, indicating that one severe consequence of Al toxicity is loss of the QC. In contrast, treatment of alt1-1 mutant roots with the same level of Al had no effect on the QC, with these roots staining normally for the QC. Based on these results, AtATR functions to force the stem cells of the QC to differentiate due to the toxic effects of Al, with loss of these progenitor cells leading to irreversible stoppage of root growth. Loss of AtATR function is predicted to result in loss of the mechanism required for detecting the toxic effects of Al, thus preventing the forced differentiation of the stem cells and consequently resulting in maintenance of the QC and sustaining root growth even in the presence of toxic levels of Al. Surprisingly, even though roots of alt1-1 are experiencing the toxic effects of Al, they are incapable of responding to this damage, which argues that Al-dependent root growth inhibition does not necessarily result from actual Al toxicity but rather from the response of the plant to this damage. It is currently unclear to which aspect of Al toxicity AtATR responds, since as previously described, Al toxicity is predicted to be extremely complex. AtATR is known to be responsible for detection of DNA damage and required for telomere homeostasis. Although there is no evidence that Al treatment leads to enhanced telomere shortening (Rounds and Larsen, 2008), several studies have indicated that one consequence of Al treatment is an increase in DNA fragmentation (Achary et al., 2008, Lankoff et al., 2006; Rounds and Larsen, 2008). It is not clear if this increase in fragmentation is due to a direct interaction between Al and DNA, which has been shown to occur due to the highly negative nature of DNA, or from damage that results from Al-dependent increases in ROS. It is likely based on the predicted function of AtATR that Al-responsive DNA damage is paramount with regard to the manifestation of Al-

UNRAVELING THE MECHANISMS UNDERLYING ALUMINUM-DEPENDENT ROOT GROWTH INHIBITION

137

dependent root growth inhibition, with mutations that block detection of this preventing this inhibition. Based on this work, a model for the cause of Al-dependent root growth inhibition can be proposed. Whereas it was previously believed that this was a highly complicated phenomenon based on the predicted number of sites of Al toxicity, it appears that in fact stoppage of root growth following Al treatment is inherently simple and is actually a function of the response of the plant to toxicity rather than the toxicity itself. It is likely that following Al treatment, internalized Al leads to accumulation of DNA damage. In a wild-type root, AtATR is responsible for detecting and/or responding to this damage with this response leading to slowing or stoppage of cell division, as evidenced by accumulation of CyclinB1;1 in Al-treated roots. Ultimately, Al-dependent damage becomes so great that the cells of the QC become compromised and are deemed no longer appropriate for serving as progenitor cells. This subsequently leads to AtATR forced differentiation of these stem cells and loss of the necessary pool of cells for continuation of cell division and root growth. Consistent with this, Al treatment leads to terminal differentiation of the root since cell maturation continues even though cell division is eliminated. Loss of AtATR function leads to failure to detect the Al-dependent damage and loss of capability to trigger the cells of the QC to differentiate, which subsequently results in maintenance of the QC and sustains cell division and root growth even though the QC is compromised. Based on this model, it can be argued that the toxic effects of Al are not necessarily deleterious to short-term survival of plants in the presence of Al, which raises the ultimate question of why plants have this AtATR-dependent mechanism. It is possible that while Al toxicity may not have an immediate consequence on plant fitness, long-term growth of plants in an Al-containing environment may lead to accumulation of genetic defects that if passed on to subsequent generations, will lead to reductions in the vigor and survivability of the population. Because of this, it is possible that AtATR-dependent root growth inhibition following Al treatment is responsible not for protecting the individual, which ultimately is sacrificed due to blockage of root growth, but in actuality it is in place to safeguard progeny from the cumulative negative effects of growing in an environment that promotes genetic lesions. Clearly work remains to be done to determine the relationship of AtATR to Al toxicity, including determining exactly what type of damage AtATR is detecting following Al treatment. As stated earlier, one possibility is that Al treatment leads to an increase in DNA fragmentation, although this is unlikely the root cause of Al-dependent growth inhibition since the response of AtATR loss-of-function mutants to Al stress and ionizing radiation, which is known to cause DNA breakage, is opposite. Additionally, the response of these mutants is also opposite when comparing Al stress and Hydroxyurea-mediated root growth inhibition, which is a replication fork poison to which AtATR loss-of-function mutants are hypersensitive. Al toxicity is known to result in promotion of reactive oxygen species (ROS) formation, particularly in the apoplast, with this ROS subsequently entering the cell and affecting several targets (Achary et al., 2008). It has been proposed that one severe consequence of Al-dependent ROS is DNA damage, including oxidation of specific bases resulting in the production of aberrant bases such as 8-hydroxy-2′-deoxyguanosine. Such an increase in ROS is consistent with studies showing that Al treatment leads to increased expression of several ROS-related genes including peroxidases, suggesting that roots actively attempt to ameliorate the stress related to increases in Al-dependent ROS. It remains to be determined what the link is between Al-dependent DNA damage and AtATR, although it is expected that further analysis of the remaining als3-1 suppressor mutants should provide new insights into what is clearly a complex mechanism that promotes root growth inhibition following Al treatment.

138

GENES FOR CROP ADAPTATION TO POOR SOIL

Conclusions

Although the mechanisms of Al toxicity, resistance, and tolerance remain unclear, significant progress regarding understanding how plants cope with environmental Al and how Al promotes root growth inhibition has been made in recent years. Originally considered to be highly complex to the point that it was thought to be almost impossible to attribute Al-dependent root growth inhibition to one phenomenon or a limited number of phenomena, it is becoming clear that this in actuality represents a relative simplistic mechanism that can ultimately be overcome by defects in the pathway necessary for responding to this stress. Ultimately, such advances in our knowledge not only shed new light onto the mechanisms underlying Al toxicity, but also give new strategies for modifying agriculturally important plants to allow them to grow in the presence of Al. It is expected that by continuing to analyze the existing Arabidopsis als3-1 suppressor mutants, greater knowledge of the mechanisms underlying Al toxicity and tolerance will be gained, thus giving new insights into how to engineer Al-tolerant plants. If this approach for increasing Al tolerance is combined with our knowledge related to enhancing Al resistance through increasing malate or citrate exudation, it is expected that plants that not only are more capable of excluding Al but also of tolerating whatever Al may enter the root will be generated, thus resulting in plants that would be expected to thrive even in severely Al-toxic environments.

References Achary, V.M.M., Jena, S., Panda, K.K., and Panda, B.B. (2008) Aluminium-induced oxidative stress and DNA damage in root cells of Allium cepa L. Ecotoxicology and Environmental Safety 70:300–310. Ahn, S.J., Sivaguru, M., Osawa, H., Chung, G.C., and Matsumoto, H. (2001) Aluminum inhibits the H+-ATPase activity by permanently altering the plasma membrane surface potentials in squash roots. Plant Physiology 126:1381–1390. Ahn, S.J., Sivaguru, M., Chung, G.C., Rengel, Z., and Matsumoto, H. (2002) Aluminum-induced growth inhibition is associated with impaired efflux and influx of H+ across the plasma membrane in root apices of squash (Cucurbita pepo). J Exp Bot 53:1959–1966. Banasik, A., Lankoff, A., Piskulak, A., Adamowska, K., Lisowska, H., and Wojcik, A. (2005) Aluminum-induced micronuclei and apoptosis in human peripheral-blood lymphocytes treated during different phases of the cell cycle. Environ Toxicol 20:402–406. Blancaflor, E.B., Jones, D.L., and Gilroy, S. (1998) Alterations in the cytoskeleton accompany aluminum-induced growth inhibition and morphological changes in primary roots of maize. Plant Physiology 118:159–172. Boscolo, P.R., Menossi, M., and Jorge, R.A. (2003) Aluminum-induced oxidative stress in maize. Phytochemistry 62:181–89. Collins, N.C., Shirley, N.J., Saeed, M., Pallotta, M., and Gustafson J.P. (2008) An ALMT1 gene cluster controlling aluminum tolerance at the Alt4 locus of rye (Secale cereale L.). Genetics 179:669–682. Culligan, K., Tissier, A., and Britt, A. (2004). ATR regulates a G2-phase cell-cycle checkpoint in Arabidopsis thaliana. Plant Cell 16:1091–1104. Culligan, K.M., Robertson, C.E., Foreman, J., Doerner, P., and Britt, A.B. (2006) ATR and ATM play both distinct and additive roles in response to ionizing radiation. Plant J 48:947–961. Davidson, A.L. and Chen, J. (2004) ATP-binding cassette transporters in bacteria. Annual Review of Biochemistry 73: 241–68. De la Fuente, J.M., Ramirez-Rodriguez, V., Cabrera-Ponce, J.L., and Herrera-Estrella, L.M. (1997) Aluminum tolerance in transgenic plants by alteration of citrate synthesis. Science 276:1566–1568. Degenhardt, J., Larsen, P.B., Howell, S.H., and Kochian, L.V. (1998). Aluminum resistance in the Arabidopsis mutant alr-104 is caused by an aluminum-induced increase in rhizosphere pH. Plant Physiology 117:19–27. Delhaize, E., Craig, S., Beaton, C.D., Bennet, R.J., Jagadish, V.C., and Randall, P.J. (1993a) Aluminum tolerance in wheat (Triticum aestivum L.) I. Uptake and distribution of aluminum in root apices. Plant Physiology 103:685–693. Delhaize, E., Ryan, P.R., and Randall, P.J. (1993b) Aluminum tolerance in wheat (Triticum aestivum L.) II. Aluminum-stimulated excretion of malic acid from root apices. Plant Physiology 103:695–702.

UNRAVELING THE MECHANISMS UNDERLYING ALUMINUM-DEPENDENT ROOT GROWTH INHIBITION

139

Delhaize, E., Hebb, D.M., and Ryan, P.R. (2001) Expression of a Pseudomonas aeruginosa citrate synthase gene in tobacco is not associated with either enhanced citrate accumulation or efflux. Plant Physiology 125:2059–67. Delhaize, E., Ryan, P.R., Hebb, D.M., Yamamoto, Y., Sasaki, T., and Matsumoto, H. (2004) Engineering high-level aluminum tolerance in barley with the ALMT1 gene. PNAS USA 101:15249–54. Devi, S.R., Yamamoto, Y., and Matsumoto, H. (2003) An intracellular mechanism of aluminum tolerance associated with high antioxidant status in cultured tobacco cells. J Inorg Biochem 97:59–68. Ezaki, B., Sivaguru, M., Ezaki, Y., Matsumoto, H., Gardner, R.C. (1999). Acquisition of aluminum tolerance in Saccharomyces cerevisiae by expression of the BCB or NtGDI1 gene derived from plants. FEMS Microbiol Lett 171:81–87. Ezaki, B., Gardner, R.C., Ezaki, Y., and Matsumoto, H. (2000) Expression of aluminum-induced genes in transgenic Arabidopsis plants can ameliorate aluminum stress and/or oxidative stress. Plant Physiology 122:657–666. Ezaki, B., Katsuhara, M., Kawamura, M., and Matsumoto, H. (2001) Different mechanisms of four aluminum (Al)-resistant transgenes for Al toxicity in Arabidopsis. Plant Physiology 127(3):918–927. Furukawa, J., Yamaji, N., Wang, H., Mitani, N., Murata, Y., Sato, K., Katsuhara, M., Takeda, K., and Ma, J.F. (2007) An aluminum-activated citrate transporter in barley. Plant Cell Physiol 48:1081–1091. Gabrielson, K.M., Cancel, J.D., Morua, L.F., and Larsen, P.B. (2006) Identification of dominant mutations that confer increased aluminum tolerance through mutagenesis of the Al-sensitive Arabidopsis mutant, als3-1. J Exp Bot 57:943–951. Grabski, S. and Schindler, M. (1995) Aluminum induces rigor within the actin network of soybean cells. Plant Physiology 108:897–901. Haug, A., Shi, B., and Vitorello, V. (1994) Aluminum interaction with phosphoinositide-associated signal transduction. Archives of Toxicology 68:1–7. Hoekenga, O.A., Vision, T.J., Shaff, J.E., Monforte, A.J., Lee, G.P., Howell, S.H., and Kochian, L.V. (2003) Identification and characterization of aluminum tolerance loci in Arabidopsis (Landsberg erecta x Columbia) by quantitative trait locus mapping. A physiologically simple but genetically complex trait. Plant Physiology 132:936–948. Hoekenga, O.A., Maron, L.G., Pineros, M.A., Cancado, G.M., Shaff, J., Kobayashi, Y., Ryan, P.R., Dong, B., Delhaize, E., Sasaki, T., Matsumoto, H., Yamamoto, Y., Koyama, H., and Kochian, L.V. (2006) AtALMT1, which encodes a malate transporter, is identified as one of several genes critical for aluminum tolerance in Arabidopsis. PNAS USA 103:9738–9743. Horst W.J., Puschel, A.-K., and Schmohl, N. (1997) Induction of callose formation is a sensitive marker for genotypic aluminum sensitivity in maize. Plant Soil 192:23–30. Huang, J.W., Grunes, D.L., and Kochian, L.V. (1992) Aluminum effects on the kinetics of calcium uptake into cells of the wheat root apex. Quantification of calcium fluxes using a calcium-selective vibrating microelectrode. Planta 188:414–421. Huang, J.W., Pellet, D.M., and Kochian, L.V. (1995) Aluminum interactions with voltage-dependent calcium transport in plasma membrane vesicles isolated from roots of aluminum sensitive and resistant wheat cultivars. Plant Physiology 110:561–569. Iuchi, S., Koyama, H., Iuchi, A., Kobayashi, Y., Kitabayashi, S., Kobayashi, Y., Ikka, T., Hirayama, T., Shinozaki, K., and Kobayashi, M. (2007) Zinc finger protein STOP1 is critical for proton tolerance in Arabidopsis and coregulates a key gene in aluminum tolerance. PNAS USA 104:9900–9905. Jones, D.L. and Kochian, L.V. (1995) Aluminum inhibition of the inositol 1,4,5-triphosphate signal transduction pathway in wheat roots: A role in aluminum toxicity? Plant Cell 7:1913–1922. Jones, D.L. and Kochian, L.V. (1997) Aluminum interaction with plasma membrane lipids and enzyme metal binding sites and its potential role in Al cytotoxicity. FEBS Letters 400:51–57. Jones, D.L., Kochian, L.V., and Gilroy, S. (1998a) Aluminum induces a decrease in cytosolic calcium concentration in BY-2 tobacco cell cultures. Plant Physiology 116:81–89. Jones, D.L., Gilroy, S., Larsen, P.B., Howell, S.H., and Kochian, L.V. (1998b) Effect of aluminum on cytoplasmic Ca2+ homeostasis in root hairs of Arabidopsis thaliana (L.). Planta 206:378–387. Kobayashi, Y. and Koyama, H. (2002) QTL analysis of Al tolerance in recombinant inbred lines of Arabidopsis thaliana. Plant Cell Physiol 43:1526–1533. Lankoff, A., Banasik, A., Duma, A., Ochniak, E., Lisowska, H., Kuszewski, T., Gäz´ dz´ , S., and Wojcik, A. (2006) A comet assay reveals that aluminum induces DNA damage and inhibits the repair of radiation-induced lesions in human peripheral blood lymphocytes. Toxicol Lett 161:27–36. Larsen, P.B., Tai C.-Y., Kochian, L.V., and Howell, S.H. (1996) Arabidopsis mutants with increased sensitivity to aluminum. Plant Physiology 110:743–751. Larsen, P.B., Kochian, L.V., and Howell, S.H. (1997) Al inhibits both shoot development and root growth in als3, an Al-sensitive Arabidopsis mutant. Plant Physiology 114:1207–1214. Larsen, P.B., Degenhardt, J., Tai, C.-Y., Stenzler, L.M., Howell, S.H., and Kochian, L.V. (1998) Aluminum-resistant Arabidopsis mutants that exhibit altered patterns of aluminum accumulation and organic acid release from roots. Plant Physiology 117:9–18.

140

GENES FOR CROP ADAPTATION TO POOR SOIL

Larsen, P.B., Geisler, M.J., Jones, C.A., Williams, K.M., and Cancel, J.D. (2005). ALS3 encodes a phloem-localized ABC transporter-like protein that is required for aluminum tolerance in Arabidopsis. Plant Journal 41:353–363. Larsen, P.B., Cancel, J., Rounds, M., and Ochoa, V. (2007) Arabidopsis ALS1 encodes a root tip and stele localized half type ABC transporter required for root growth in an aluminum toxic environment. Planta 225:1447–1458. Lavin, M.F. (2008) Ataxia-telangiectasia: from a rare disorder to a paradigm for cell signalling and cancer. Nat Rev Cell Mol Biol 9:759–769. Lazof, D.B., Goldsmith, J.G., Rufty, T.W., and Linton, R.W. (1994a) Rapid uptake of aluminum into cells of intact soybean root tips. Plant Physiology 106:1107–1114. Lazof, D.B., Rincon, M., Rufty, T.W., MacKown, C.T., and Carter, T.E. (1994b) Aluminum accumulation and associated effects on 15NO3− influx in roots of two soybean genotypes differing in Al tolerance. Plant Soil 164:291–297. Li, X.F., Ma, J.F., and Matsumoto, H. (2000) Pattern of aluminum-induced secretion of organic acids differs between rye and wheat. Plant Physiology 123:1537–1544. Liu, K. and Luan, S. (2001) Internal aluminum block of plant inward K+ channels. Plant Cell 13:453–1466. Lima, P.D.L., Leite, D.S., Vasconcellos, M.C., Cavalcanti, B.C., Santos, R.A., Costa-Lotufo, L.V., Pessoa, C., Moraes, M.O., and Burbano, R.R. (2007) Genotoxic effects of aluminum chloride in cultured human lymphocytes treated in different phases of cell cycle. Food and Chemical Toxicology 45:1154–1159. Llugany, M., Massot, N., Wissemeier, A.H., Poschenrieder, C., Horst, W.J., and Barcelo, J. (1994) Aluminum tolerance of maize cultivars as assessed by callose production and root elongation. Z. Pflanzenernaehr Bodenkd 157:447–451. Lukiw, W.J., Krishnan, B., Wong, L., Kruck, T.P., Bergeron, C., and Crapper McLachlan, D.R. (1992) Nuclear compartmentalization of aluminum in Alzheimer ’s disease (AD). Neuobiol Aging 13:115–121. Ma, J.F., Hiradate, S., Nomoto, K., Iwashita, T., and Matsumoto, H. (1997) Internal detoxification mechanism of Al in hydrangea. Identification of Al form in the leaves. Plant Physiology 113:1033–1039. Ma, J.F., Hiradate, S., and Matsumoto, H. (1998) High aluminum resistance in buckwheat. Plant Physiology 117:753–759. Ma, J.F. and Hiradate, S. (2000). Form of aluminum for uptake and translocation in buckwheat (Fagopyrum esculentum Moench). Planta 211:355–60. Ma, J.F. (2000) Role of organic acids in detoxification of aluminum in higher plants. Plant Cell Physiology 4:383–390. Ma, J.F., Ryan, P.R., and Delhaize, E. (2001) Aluminium tolerance in plants and the complexing role of organic acids. Trends Plant Sci 6:273–278. Magalhaes, J.V., Liu, J., Guimarares, C.T., Lana1, U.G.P., Alves, V.M.C., Wang, Y.H., Schaffert, R.E., Hoekenga, O.A., Pinieros, M.A., Shaff, J.E., Klein, P.E., Carneiro, N.P., Coelho, C.M., Trick, H.N., and Kochian, L.V. (2007) A gene in the multidrug and toxic compound extrusion (MATE) family confers aluminum tolerance in sorghum. Nature Genetics 39:1156–1161. McSherry, T.D., Kitazono, A.A., Javaheri, A., Kron, S.J., and Mueller, P.R. (2007) Non-catalytic function for ATR in the checkpoint response. Cell Cycle 6:2019–2030. Miyasaka, S.C., Buta, J.G., Howell, R.K., and Foy, C.D. (1991) Mechanism of aluminum tolerance in snapbeans. Root exudation of citric acid. Plant Physiology 96:737–743. Olivetti, G.P., Cumming, J.R., and Etherton, B. (1995) Membrane potential depolarization of root cap cells precedes aluminum tolerance in snapbean. Plant Physiology 96:737–743. Ostatek-Boczynski, Z., Kerven, G.L., and Blamey, F.P.C. (1995) Aluminium reactions with polygalacturonate and related organic ligands. Plant and Soil 171:41–45. Papernik, L.A. and Kochian, L.V. (1997) Possible involvement of Al-induced electrical signals in Al tolerance in wheat. Plant Physiology 115:657–667. Pellet, D.M., Grunes, D.L., and Kochian, L.V. (1995) Organic acid exudation as an aluminum tolerance mechanism in maize (Zea mays L.). Planta 196:788–795. Ramos-Diaz, A., Brito-Argaez, L., Munnik, T., and Hernanez-Sotomayor, S.M.T. (2007) Aluminum inhibits phosphatidic acid formation by blocking the phospholipase C pathway. Planta 225:393–401. Richards, K.D. and Gardner, R.C. (1994) The effect of aluminium treatment on wheat roots: expression of heat shock, histone and SHH genes. Plant Science 98:37–45. Richards, K.D., Schott, E.J., Sharma, Y.K., Davis, K.R., and Gardner, R.C. (1998) Aluminum induces oxidative stress genes in Arabidopsis thaliana. Plant Physiology 116:409–418. Rounds, M.A. and Larsen, P.B. (2008) Aluminum-dependent root-growth inhibition in Arabidopsis results from AtATR-regulated cell-cycle arrest. Current Biology 18:1495–1500. Ryan, P.R., diTomaso, J.M., and Kochian, L.V. (1993) Aluminum toxicity in roots: an investigation of spatial sensitivity and the role of the root cap. J Exp Botany 44:437–446. Ryan, P.R., Delhaize, E., and Randall, P.J. (1995a) Characterisation of Al-stimulated efflux of malate from the apices of Al-tolerant wheat roots. Planta 196:103–110.

UNRAVELING THE MECHANISMS UNDERLYING ALUMINUM-DEPENDENT ROOT GROWTH INHIBITION

141

Ryan, P.R., Delhaize, E., and Randall, P.J. (1995b) Malate efflux from root apices and tolerance to aluminum are highly correlated in wheat. Australian Journal of Plant Physiology 22:531–536. Ryan, P.R., Skerret, M., Findlay, G.P., Delhaize, E., and Tyerman, S.D. (1997) Aluminum activates an anion channel in the apical cells of wheat roots. PNAS USA 94:6547–6552. Ryan, P.R., Raman, H., Gupta, S., Horst, W.J., and Delhaize, E. (2008) A second mechanism for aluminum resistance in wheat relies on the constitutive efflux of citrate from roots. Plant Physiology (in press). Sasaki, T., Yamamoto, Y., Ezaki, B., Katsuhara, M., Ahn, S.J., Ryan, P.R., Delhaize, E., and Matsumoto, H. (2004) A wheat gene encoding an aluminum-activated malate transporter. Plant J 37:645–53. Sasaki, T., Ryan, P.R., Delhaize, E., Hebb, D.M., Ogihara, Y., Kawaura, K., Noda, K., Kojima, T., Toyoda, A., Matsumoto, H., and Yamamoto, Y. (2006) Sequence upstream of the wheat (Triticum aestivum L.) ALMT1 gene and its relationship to aluminum resistance. Plant Cell Physiol 47:1343–1354. Schwarzerova, K., Zelenkova, S., Nick, P., and Opatrny, Z. (2002) Aluminum-induced rapid changes in the microtubular cytoskeleton of tobacco cell lines. Plant Cell Physiol 43:207–216. Shafer, T.J., Nostrandt, A.C., Tilson, H.A., and Mundy, W.R. (1994) Mechanisms underlying AlCl3 inhibition of agoniststimulated inositol phosphate accumulation. Biochemical Pharmacology 47:1417–1425. Shi, R., Chou, K., and Haug, A. (1993) Aluminum impacts elements of the phosphoinositide signalling pathway in neuroblastoma cells. Molecular and Cellular Biochemistry 121:109–118. Silva, I.R., Smyth, T.J., Moxley, D.F., Carter, T.E., Allen, N.S., and Rufty, T.W. (2000) Aluminum accumulation at nuclei of cells in the root tip. Fluorescence detection using lumogallion and confocal laser scanning microscopy. Plant Physiology 123:543–552. Sivaguru, M. and Horst, W.J. (1998) The distal part of the transition zone is the most aluminum-sensitive apical root zone of Zea mays L. Plant Physiology 116:155–163. Sivaguru, M., Baluska, F., Volkmann, D., Felle, H.H., and Horst, W.J. (1999) Impacts of aluminum on the maize cytoskeleton: short term effects on the distal part of the transition zone. Plant Physiology 119:1–10. Snowden, K.C. and Gardner, R.C. (1993) Five genes induced by aluminum in wheat (Triticum aestivum L.) roots. Plant Physiology 103:855–861. Snowden, K.C., Richards, K.D., and Gardner, R.C. (1995) Aluminum-induced genes: induction by toxic metals, low calcium, and wounding and pattern of expression in root tips. Plant Physiology 107:341–348. Taylor, G.J., McDonald-Stephens, J.L., Hunter, D.B., Bertsch, P.M., Elmore, D., Rengel, Z., and Reid, R.J. (2000) Direct measurement of aluminum uptake and distribution in single cells of Chara corallina. Plant Physiology 123:987–996. Vespa, L., Couvillion, M., Spangler, E., and Shippen, D.E. (2005) ATM and ATR make distinct contributions to chromosome end protection and the maintenance of telomeric DNA in Arabidopsis. Genes Dev 19:2111–2115. von Uexkull, H.R. and Mutert, E. (1995) Global extent, development and economic impact of acid soils. Plant and Soil 171:1–15. Wagatsuma, T., Ishikawa, S., Obata, H., Tawaraya, K., and Katohda, S. (1995) Plasma membrane of younger and outer cells is the primary specific site for aluminum toxicity in roots. Plant and Soil 171:105–112. Wissemeir, A.H., Klotz, F., and Horst, W.J. (1987) Aluminum induced callose synthesis in roots of soybean (Glycine max L.). Journal of Plant Physiology 129:487–492. Yermiyahu, U., Rytwo, G., Brauer, D.K., and Kinraide, T.B. (1997) Binding and electrostatic attraction of lanthanum (La3+) and aluminum (Al3+) to wheat root plasma membranes. Journal of Membrane Biology 159:239–252. Zheng, S.J., Ma, J.F., and Matsumoto, H. (1998) High aluminum resistance in buckwheat. I. Al-induced specific secretion of oxalic acid from root tips. Plant Physiology 117:745–751.

6

Genetic Determinants of Phosphate Use Efficiency in Crops Fulgencio Alatorre-Cobos, Damar López-Arredondo, and Luis Herrera-Estrella

Introduction

Low phosphate availability is one of the major constraints for plant productivity worldwide. Therefore, the use of phosphate fertilizer is a necessary practice to increase crop yield. Phosphate ore, named also phosphate rock, is the basic source of all phosphate materials. Currently, total reserves of this nonrenewable natural resource are estimated at 45.78 billion tons; however, with the current and increasing fertilizer use, phosphate rock deposits are depleting rapidly (Cisse and Mrabet, 2004). This situation has contributed to discussions about a future “phosphate crisis” (Abelson, 1999). The negative consequences of this crisis (smaller phosphate deposits occurring in more challenging environments and mined with higher costs) have raised the urgent need to obtain crop varieties with reduced fertilizer requirements to produce high yields. In this chapter, we first analyze the importance of improving crop nutrition to increase food production and the role of phosphate in plant metabolism. Then we present current knowledge about phosphate sensing and signaling pathways during phosphate starvation in plants, describe some molecular determinants that control the phosphate acquisition and discuss the use of this knowledge to design new strategies to improve phosphate use efficiency.

Why Improve Crop Nutrition and the Relationship with World Food Security?

According to previous projections, world population is growing by up to 160 people per minute; and consequently, by 2050 the world’s population likely will be 10 billion (Hoisington et al., 1999; Cakmak, 2002). This increase is anticipated to occur mainly in developing countries located in Africa, Latin America, and Asia; regions with serious problems regarding food production and availability, water scarcity, and nutritional disorders/malnutrition (Cakmak, 2002). Malnutrition has long been recognized as a major public health problem. Currently, inadequate food availability is the main cause for undernourished people, suffering from micronutrient deficiencies such as iron (Fe), zinc (Zn), iodine (I), and vitamin A deficiencies, which result in serious impairments of human health and development (Pinstrup-Andersen et al., 1999; Underwood, 2000; Welch and Graham, 2000). Genes for Plant Abiotic Stress Editors Matthew A. Jenks and Andrew J. Wood © 2010 Blackwell Publishing ISBN: 978-0-813-81502-2

143

144

GENES FOR CROP ADAPTATION TO POOR SOIL

The problems described above strongly suggest that to feed the growing world population, a massive increase in food production will be necessary. This challenge will have to be met, mainly, by developing countries, and a pivotal role will be played by diverse crops, especially cereal grains such as rice, maize, and, wheat. In this context, The International Model for Policy Analysis of Agricultural Commodities and Trade (IMPACT) has estimated that global cereal production should increase by 56% between 1997 and 2050. By the year 2020, almost 96% of the world’s rice consumption, about 70% of the world’s wheat consumption, and almost 60% of the world’s maize consumption will be in developing countries (Hoisington et al., 1999; Rosegrant et al., 2002; Rosegrant and Cline, 2003). The availability of additional arable and fertile land to achieve the required increased food production will be a major constraint; it is estimated that the area used for crop production will expand by only around 10% (Rosegrant et al., 2001; Cakmak, 2002) with some areas lost to agriculture mainly due to urbanization and water scarcity. In addition, soil degradation in the form of erosion, nutrient depletion, acidity, salinization, depletion of organic matter, and poor drainage will further compromise soil productivity causing a decline in global crop production (Scherr, 1999). According to Scherr (1999), nearly 40% of the agricultural land has been affected by soil degradation, particularly in sub-Saharan Africa and Central America. Therefore, to meet the increasing demand for food, the main goals of agricultural plant sciences are to increase productivity per unit of land area, to improve crop quality in order to alleviate malnutrition and, not less important, to develop a more environmentally friendly agriculture. Currently, large amounts of mineral fertilizers are applied to improve crop nutrition and increase crop yield, however, this has adverse effects on the environment. Some strategies such as applying manure, using rotational cropping systems based in legumes, and recycling crop residues are recommended to improve soil fertility, enhance nutrient use efficiency, and decrease fertilizer usage (FAO, 2000; Yadav et al., 2000; Timsina and Connor, 2001). However, these strategies will not be sufficient to sustain agricultural production at the level required to meet future food production needs. Current advances in plant genome sequencing and understanding of molecular signaling pathway mediating abiotic stress responses, as well as genetic engineering, will provide new and useful tools to meet crop improvement and world food security.

Phosphorus and Crops: Phosphorus as an Essential Nutrient and Its Supply as a Key Component to Crop Yield

Plants are the primary producers in the ecosystems, and as such, they need adequate levels of mineral nutrients to achieve optimal growth and productivity. There are 17 essential elements required for plant growth (Asher, 1991). Carbon (C), hydrogen (H), and oxygen (O) are considered nonmineral nutrients and represent about 95% of plant biomass; nitrogen (N), calcium (Ca), potassium (K), magnesium (Mg), phosphorus (P), and sulfur (S) are considered to be macronutrients since each represents 0.1% or more of the total dry weight of plants (Kochian, 2000); boron (B), chlorine (Cl), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo), nickel (Ni), and zinc (Zn) are considered to be micronutrients since plants require them in less than 0.01% dry weight. Among the nutrients required for plant growth, N, P, and K are the main components of fertilizers and represent a significant percentage of the agricultural production costs. In this chapter, we will focus on discussing current knowledge about the mechanisms that regulate P uptake and use efficiency. P is an essential element for plant growth and reproduction (Raghothama, 1999; White and Hammond, 2008). Its concentration in plants ranges from 0.1 to 0.5% dry weight (Vance et al.,

GENETIC DETERMINANTS OF PHOSPHATE USE EFFICIENCY IN CROPS

145

2003). Plants acquire P from the soil solution by the root system as orthophosphates (Pi), mainly in the form of H 2 PO−4 and to a lesser extent as HPO−42 . The concentration of Pi in the soil solution is generally between 0.1 to 10 micrometer (μM), a very low level compared with that required in the plant that ranges between 5 to 10 milliMole (mM) (Schachtman et al., 1998; Hinsinger, 2001; Vance et al., 2003; Kirkby and Johnston, 2008). Despite the fact that P is quite abundant in many soils, Pi is one of the most unavailable and inaccessible macronutrients. Pi availability is one of the most limiting factors for plant growth and productivity in both natural and agricultural ecosystems. Pi availability is affected by several factors, mainly by its low diffusion rate (10−12 to 10−15 m2 s−1), its interaction with inorganic components in the soil, its rapid conversion by microbial activity into organic forms not readily available for plant uptake and temperature and moisture into the soil (Kirkby and Johnston, 2008). Aluminum (Al) and iron (Fe) ions, which predominate in acid soils of the world, interact strongly with Pi to form insoluble compounds not easily assimilated by plants. Pi also interacts with calcium (Ca) and magnesium (Mg) ions occurring in alkaline soils, causing Pi precipitation. These interactions are influenced by soil solution pH, because Pi availability is high only at pHs between 6.5 and 7.5 (Marschner, 1995; Schachtman et al., 1998; Raghothama, 1999; Hinsinger, 2001; Vance et al., 2003). Some estimates indicate that 5.7 billion hectares throughout the world contain low levels of available Pi that compromise optimal development of plants, and consequently, crop production (Hinsinger, 2001; Cakmak, 2002). P plays a key role in many essential cellular processes, including nucleic acid and phospholipids synthesis, membrane structure and stability, energy generation, glycolysis, respiration, enzyme activation/inactivation, and signaling cascades. Over their growth period, plants have a variable demand for nutrients. In the early stages of growth, the rate of nutrient uptake per unit length of root is much higher than at later stages of development, as demonstrated by Mengel and Barber (1974) in the maize plant, for which P uptake rate per unit length of root is 10 times higher in 20day-old plants than in 30-day-old plants. It is known that during the reproductive phase, plants allocate between 15 and 60% of their P to reproductive tissues, an indispensable process for plant persistence, especially in low Pi environments (Güsewell, 2004). In crop plants for which total biomass or grain yield is a key component of productivity, the Pi supply must be maintained at adequate levels. It is estimated that a suboptimal P nutrition can lead to yield losses in the range of 10 to 15% of the maximal yield (Raghothama, 1999; Shenoy and Kalagudi, 2005). To overcome this situation, the application of Pi-containing fertilizers is a common practice in intensive agriculture. Some projections indicate that to increase yield capacity of crop plants, and in turn to ensure global food demand in 2020, fertilizer use should increase from 144 million tons in 1990 to 208 million tons in 2020 (Bumb and Baanante, 1996; FAO, 2000). However, Byrnes and Bumb (1998) consider that the increase in crop production will also cause an increment in nutrient depletion from soils, increasing fertilizer consumption from 208 million to 300 million tons (Byrnes and Bumb, 1998; Cakmak, 2002). Despite excessive amounts of Pi fertilizers currently used, crop yield is not as high as could be expected. This has been associated with a high Pi fixation in soil and a low recovery (10–15%) by crops of the total Pi applied. This recovery rate could reach 50% if periodical and timely applications are carried out (Syers et al., 2007).

Phosphorus and Plant Metabolism: Regulatory and Structural Functions

Phosphorus (P) is an essential macronutrient for all living organisms as has been explained above (Poirier and Bucher, 2002). P plays several cellular functions that are essential for cellular opera-

146

GENES FOR CROP ADAPTATION TO POOR SOIL

tion. One key function of P is as a structural component of nucleic acids (DNA and RNA) and phospholipids. It has been observed that in Pi-starved plants, DNA and RNA concentrations decrease, which has a consequence on plant growth rate, because more DNA and RNA are required in rapidly growing tissue (Niklas, 2008; Raven, 2008). Additionally, when plants are starved of Pi, phospholipids are hydrolyzed and replaced by nonphosphorous lipids such as galactolipids (monogalactosyl diacylglycerol, digalactosyl diacylglycerol) and sulfolipids to maintain membrane structure and functionality (Sulphoquinovosyl diacylglycerol); this adaptive response releases Pi for other cellular functions and contributes to tissue Pi economy (Ohta et al., 2000; Somerville et al., 2000; Staehelin and Newcomb, 2000; Cruz-Ramírez et al., 2006; Kobayashi et al., 2006). Pi is also a component of many phosphorylated compounds that participate as intermediates of metabolic pathways, such as the Calvin cycle, glycolysis, amino acid and nucleotide metabolism, osmoprotectants synthesis (glycine-betaine, mannitol, pinitol), and secondary metabolism (Bray et al., 2000; Coruzzi and Last, 2000; Croteau et al., 2000; Dennis and Blakeley, 2000). Moreover, there are many compounds that must be phosphorylated/dephosphorylated to be compartmentalized in cellular organelles to perform their physiological roles. Pi also participates in the regulation of a diverse array of proteins by phosphorylation/dephosphorylation reactions (Raven, 2008). Aside from its global metabolic and structural functions, Pi plays a critical role in energy transfer as part of adenosine triphosphate (ATP). ATP and its derivatives, ADP and AMP, are involved in all aspects of energy transfer in every aspect of plant growth and development, for example, in photosynthesis and respiration, nucleic acid biosynthesis, cytoskeletal rearrangements, and membrane transport. Pi is also required as NADPH in biosynthetic reactions and signaling cascades, as well as a substrate and regulatory factor in oxidative metabolism. Additionally, phospholipids also act as substrates for the production of biochemical signals, such as inositol triphosphate (IP3) and jasmonate among others, involved in the plant responses to pathogen infection (Hammond-Kosack and Jones, 2000). As expected, Pi homeostasis is critical to maintain normal plant metabolism (Lauer et al., 1989). When plants are in Pi-optimal conditions, exceeding Pi is stored in the vacuole, which in response to Pi deprivation, is transported across the tonoplast membrane back to the cytoplasm (Marschner, 1995; Raghothama, 1999). In addition, in Pi-deprived plants alternative metabolic pathways requiring lower phosphorylated intermediates are activated (Plaxton and Carswell, 1999; Vance et al., 2003).

Phosphate Starvation: Adaptations to Phosphate Starvation and Current Knowledge about Phosphate Sensing and Signaling Networks during Phosphate Stress

Plants have evolved a series of highly specialized mechanisms that are triggered by Pi deficiency. These mechanisms are based on metabolic and developmental processes required to mobilize Pi from the soil and to increase P uptake, assimilation, and use efficiency. In general, plants grown in Pi-deficient soils present changes in the development of the root system architecture that enhances lateral root proliferation and higher root hair density and elongation, leading to an increased capacity of soil exploration (Bates and Lynch, 1996; Raghothama, 1999; Ma et al., 2001; López-Bucio et al., 2002). Some species, such as white lupine (Lupinus albus) respond to Pi scarcity by developing specialized root structures known as proteoid roots (Dinkelaker et al., 1995; Keerthisinghe et al., 1998; Vance et al., 2003; Lambers et al., 2006), which

GENETIC DETERMINANTS OF PHOSPHATE USE EFFICIENCY IN CROPS

147

are cluster roots consisting of massive numbers of secondary/tertiary rootlets covered by root hairs (Kirkby and Johnston, 2008). In addition to developmental adaptations, several biochemical processes are activated in plants to increase Pi availability. Many plant species, particularly legumes, excrete low-molecular weight organic acids and release protons into the rhizosphere, contributing to Pi solubilization from the soil (Dinkelaker et al., 1989; Dinkelaker et al., 1995; Zhang et al., 1997; Raghothama, 1999; Gerke et al., 2000; Lambers et al., 2006). Enhanced production of phosphate scavenging and recycling enzymes, such as phosphatases and nucleases also occurs. Extra-cellular phosphatases are involved in releasing Pi from organic compounds from the soil and intracellular phosphatases in remobilizing Pi from senescent plant tissues (Plaxton and Carswell, 1999). In Arabidopsis, an acid phosphatase, AtACP5, and a vacuolar purple acid phosphatase, AtPAP26, are induced under Pi-deficient conditions (del Pozo et al., 1999; Veljanovski et al., 2006). Ribonucleases and nucleotide-phosphodiesterase are also induced in Pi-deprived plants. These enzymes allow the complete utilization of Pi from nucleic acids present in organic matter (Bariola et al., 1994; Bariola et al., 1999; Abel et al., 2000). Phospholipases also play an important role in remobilizing P into the cells. Phospholipases PLC5, PLDZ1, and PLDZ2 participate in the hydrolysis of membrane phospholipids, which are replaced by galactolipids and sulfolipids (Li et al., 2005; Nakamura et al., 2005; Cruz-Ramírez et al., 2006; Kobayashi et al., 2006). In response to Pi starvation, plants show a remarkable flexibility in adjusting metabolic rates and using alternative metabolic pathways (Vance et al., 2003). It has been reported that the levels of ATP and ADP decrease during Pi deficiency, whereas pyrophosphate (PPi) levels increase, which under low Pi conditions may function as an energy donor (Ashihara et al., 1988). In fact, enzymes that do not require Pi or ATP as substrate are activated, and are involved in “bypass reactions” in glycolytic pathways, thus permitting carbon metabolism to proceed (Poirier and Bucher, 2002; Plaxton, 2004). A decrease in the rate of photosynthesis and activation of an alternative respiration pathway also occurs (Poirier and Bucher, 2002). High-throughput techniques for gene expression analysis used in Arabidopsis thaliana, Lupinus albus, Oryza sativa, and Zea mays produced a comprehensive survey of global gene expression in response to Pi deprivation, showing that multiple genes are upregulated and downregulated. Through microarray analysis, Wu and others (2003) reveal that of 6,172 analyzed genes, 1,800 genes show changes of two magnitude orders under stress conditions. The affymetrix gene chip (ATH1) applied by Misson and others (2005) represents a more complete analysis because they evaluate the spacio-temporal expression of 22,810 genes, almost all of the Arabidopsis genome, of which 612 appear induced and 254 repressed under Pi deficiency. A functional classification of analyzed genes shows they are involved in some metabolic routes, ion transport, signal transduction, and growth and developmental processes. Morcuende and others (2007) used affymetrix ATH1 arrays and large-scale real-time reverse transcription PCR of ∼2,200 transcription factor (TF) genes and other gene families. Of the TFs analyzed, 31 of them appear to be candidates to orchestrate the transcriptional changes observed in response to Pi starvation, including members of the WRKY, basic leucine zipper (bZIP), bHLH, MYB, and other transcription factor families. PHR1 (phosphate starvation response) is a MYB-like TF, first discovered in Arabidopsis, which regulates the expression of numerous Pi starvation responsive genes in different plant species. The expression of AtPHR1 itself is independent of plant Pi status (Rubio et al., 2001). Pi-responsive genes located downstream of AtPHR1, including genes encoding Pi transporters, RNases, phosphatases, protein kinases, transcription factors, and an array of metabolic enzymes, contain the

148

GENES FOR CROP ADAPTATION TO POOR SOIL

GNATATNC motif in their promoters (Rubio et al., 2001; Franco-Zorrilla et al., 2004; Hammond et al., 2004; Misson et al., 2004). In addition to PHR1, other TFs involved in Pi signal transduction have been identified and characterized. OsPTF1, a bHLH TF identified in rice which expression is Pi starvation induced in roots, has been associated with tolerance to Pi starvation (Yi et al., 2005). Arabidopsis WRKY75 is Pi deprivation induced TF that has been proposed to act as a positive regulator of some Pi-starved induced genes and as a negative regulator of root development (Devaiah et al., 2007a). The Arabidopsis ZAT6 (Zinc finger 6) is induced during Pi starvation and regulates the expression of Pi-starved induced genes through the modulation of internal P content by regulating the root system architecture (Devaiah et al., 2007b). Reverse and forward genetic approaches have allowed the identification of other components of the Pi signal transduction network (Figure 6.1). A small ubiquitin-like modifier (SUMO) E3 ligase (SIZ1), which has expression that is increased by Pi deficiency, was proposed to act as a positive regulator of PHR1, through sumoylation (Miura et al., 2005). The microRNA miR399 gene family, whose expression is upregulated by P starvation is also regulated via the PHR1-dependent pathway and is involved in modulating the expression of PHO2, a ubiquitin E2 conjugating enzyme that plays an important role in Pi homeostasis (Franco-Zorrilla et al., 2004; Aung et al., 2006; Bari et al., 2006; Chiou et al., 2006; Shin et al., 2006; Franco-Zorrilla et al., 2007). PHO2 acts as a negative regulator of the expression of genes involved in Pi uptake (Aung et al., 2006; Bari et al., 2006; Chiou et al., 2006). The TPSI1/Mt4/At4 family of noncoding transcripts has been proposed to attenuate the responses mediated by miR399, acting as mimicry targets that sequester miR399 (Franco-Zorrilla et al., 2007). All of the elements noted earlier describe a signaling pathway occurring in Pi-starved plants. However, because the expression of some genes involved is not affected in phr1 mutant plants, it is hypothesized that their regulation is mediated via a PHR1-independent pathway. Recently, it has become apparent that the magnitude of Pi starvation responses arises from the interplay of systemic signals derived from total Pi into the plant and, of local signals dependent of external Pi concentrations (Figure 6.1) (Franco-Zorrilla et al., 2004; Ticconi and Abel, 2004). Both signal pathways appear to regulate morphological, physiological, and biochemical plant adaptations to Pi deficiency (Bates and Lynch, 1996; Burleigh and Harrison, 1999; Martín et al., 2000). This notion is supported by a wide range of studies, which include divided roots experiments and others using Arabidopsis mutants such as pdr2 (Pi deficiency response) and lpi (low phosphorus insensitive), which clearly suggest that lateral root and root hair elongation and proliferation are under local signal control, and that gene expression is controlled by systemic signals (Bates and Lynch, 1996; Burleigh and Harrison, 1999; Martín et al., 2000; Ticconi et al., 2004; SánchezCalderón et al., 2006). In fact, it has been proposed that decreased primary root growth in Pi starvation is involved in the localized sensing of soil Pi by root meristem and cap cells, resulting in cessation of cell division (Sánchez-Calderón et al., 2005; Svistoonoff et al., 2007). A series of elegant experiments have also demonstrated that the balance of phytohormones plays an important role in the adaptive responses to low Pi availability. Particularly, the concentration, transport, and/ or sensitivity to auxin, ethylene and cytokinin have been implicated in the remodeling of root morphology (López-Bucio et al., 2002; Ticconi et al., 2004; Franco-Zorrilla et al., 2005; LópezBucio et al., 2005). In Arabidopsis, an increase in auxin sensitivity mediated by the TRANSPORT INHIBITOR RESPONSE1 (TIR1) auxin receptor was proposed to regulate the increase in lateral root formation observed in Pi-deprived seedlings (López-Bucio et al., 2002; Pérez-Torres et al., 2008). Cytokinins are thought to be negative regulators of root growth (Werner et al., 2003). Similar roles of plant hormones have been postulated in cluster root formation in white lupine (Neumann et al., 2000).

149

Figure 6.1 Pi signaling pathways in Arabidopsis thaliana. In plants, molecular, metabolic, and morphological changes observed in response to external Pi availability seem to be determinate by local and systemic Pi signaling pathways. Under a high Pi regimen, Pi uptake occurs through low-affinity Pi transporters; under this condition, PHO2, a Pi homeostasis modulator, is induced and could negatively regulate the expression of high-affinity Pi transporters. A similar role has also been suggested for SPX proteins. In contrast, when plants grow under Pi starvation, several signaling pathways are triggered, all of them focused to enhance Pi uptake and optimize internal Pi use. To date, it is unknown how low external Pi availability is perceived and how this signal is transmitted. The PHR1 signaling pathway is the route best described for the activation of Pi-responsive genes. PHR1, a transcriptional factor sumoylated by SIZ1, positively regulates a large sub-set to Pi responsive genes, including the Mir399 gene family, which in turn can systemically modulate PHO2 expression. Other transcriptional factors (bHLH32, WRKY75, ZAT6) recently reported modulated subsets of Pi-responsive genes or control, together with hormones (auxins, cytokinins, and ethylene) and sugar root growth (ZAT6). The expression of the auxin receptor TIR1 is induced by Pi deprivation and plays a role in the increased lateral root density observed under this condition. For color detail, please see color plate section.

150

GENES FOR CROP ADAPTATION TO POOR SOIL

Nutrient Use Efficiency

Selection of Pi use efficient crops, capable of growing on low fertility soils and with reduced fertilizer, is being carried out through several approaches. Traditional selection based on phenotypic information has produced promising results and its recent combination with molecular markerassisted breeding will likely accelerate the production of Pi-use efficient genotypes, especially for cereals. Moreover, progress in the deciphering of molecular networks that regulate Pi homeostasis has allowed the production of transgenic plants with improved Pi uptake and use efficiencies. Strategies to improve Pi use and acquisition efficiency can be different; however, all consider nutrient use efficiency as a central concept.

Defining Phosphorus Use Efficiency (PUE)

Nutrient use efficiency (NUE) is a term with several definitions, but it is generally described as the measurement of the capacity of a plant to acquire and use nutrients for production of timber, crops, or forage (Gourley et al., 1993). Specifically, phosphorus-use efficiency (PUE) is defined as the ability of crop genotypes to function well under Pi deficiency (Shenoy and Kalagudi, 2005). Such ability can be expressed as a ratio of output (total plant P, grain P, biomass yield, grain yield) and input (total P, soil P or Pi fertilizer applied) (Shenoy and Kalagudi, 2005; Pathak et al., 2008). Depending on the parameters used to quantify PUE, the ratio obtained can be an integrative index (economic yield related to the total available soil P), which can reveal the Pi uptake efficiency or the efficiency to produce grain or total plant dry matter (Pathak et al., 2008). On the other hand, when the dynamics between Pi acquisition and Pi utilization needs to be established, then PUE is considered to be the equivalent to the inverse of P concentration (Gourley et al., 1993; Begum and Islam, 2005). These different indexes show the broad interpretation given to the PUE concept, but each of them, finally, refers to any of its two components: Pi acquisition and Pi internal use efficiency (Gourley et al., 1993; Good et al., 2004; Shenoy and Kalagudi, 2005). Some physiological and biochemical mechanisms and plant morphological traits controlling both components have been analyzed; however, the genetic determinants that modulate PUE are now being dissected through studies of Pi starvation responses. In the following sections, we will review Pi acquisition and use, describing some molecular determinants for each.

Genetic Determinants for the Phosphate Acquisition

Pi acquisition is an indispensable process, which must begin as soon as seed germination occurs and the radical system is established, or when nutrient storage contained in cotyledon leaves is depleted. Mechanisms involved in Pi uptake have been recently grouped in two pathways: (1) the direct uptake pathway, where the plants access Pi through direct contact between the root and the soil solution, and (2) the mycorrhizal uptake pathway, where phosphorus is taken and released into plant cells via fungal hyphae (Smith et al., 2003; Karandashov and Bucher, 2005). Pi uptake from the soil through the mycorrhizal pathway is realized against negative gradient of concentration and active symporters are needed. Once inside the fungal cytoplasm, Pi is translocated to intraradical mycelium and transferred to the plant cell. An interchange of Pi and carbohydrates occurs in

GENETIC DETERMINANTS OF PHOSPHATE USE EFFICIENCY IN CROPS

151

the interface formed by fungal arbuscular membrane and plant periarbuscular membrane. Pi supply obtained in this way can be sufficient or only a small contribution to Pi requirements of colonized plants (Karandashov and Bucher, 2005; Javot et al., 2007). In intensified agriculture, where the Pi fertilizer application is a common and necessary practice, the participation of the mycorrhizal pathway in Pi uptake has been significantly diminished; the excessive Pi applied in the soil drastically reduces the colonization of roots by AM fungi (Wissuwa et al., 2008). Constant selection of crop cultivars under these conditions has caused a progressive loss of alleles that contribute to the plant-fungi symbiosis among almost all modern varieties (Javot et al., 2007; Wissuwa et al., 2008), leading in a way that Pi uptake in these materials is mainly carried out through a direct phosphate uptake pathway. Although the study of the mechanisms that regulate the interaction between plant roots and mycorrhizal fungi is an important topic of research that could provide important insights for crop improvement, in this chapter we will concentrate only on the plant responses to Pi deprivation.

Direct Phosphate Uptake Pathway

In this pathway, an electrochemical gradient must be generated across the plasma membrane for Pi uptake, which is coupled to cotransport cations, often H+, and it is mediated by Pi transporters localized at the plasma membrane (Schachtman et al., 1998; Daram et al., 1999; Bucher, 2007). Pioneering work carried out by Emmanuel Epstein and later contributions demonstrated that Pi uptake is a dual-phasic process that follows Michaelis-Menten kinetics (Epstein, 1976; Aung et al., 2006). In this way, depending on external Pi concentration, low or high-affinity transport mechanisms operate. Plant Pi transporters assisting in one or the other system have been identified experimentally or predicted with the recent massive sequencing of plant genomes, and grouped into three families: Pht1, Pht2, and Pht3 (Muchhal et al., 1996; Smith et al., 1997; Okumura et al., 1998; Daram et al., 1999; Rausch and Bucher, 2002). In Arabidopsis, the Pht1 family comprises eight high-affinity transporters encoded by genes mainly expressed in roots and highly responsive to Pi starvation (Mudge et al., 2002; Misson et al., 2004). When Pi is limited, the uptake of this nutrient is a concerted action of ARAth;Pht1;2 and ARAth;Pht1;3 (Shin et al., 2004), whereas ARAth;Pht1;1 and ARAth;Ph1;4 control the majority of Pi influx to the cell in high-Pi regimens (Shin et al., 2004). ARAth;Pht1;1 appears to have a major involvement in Pi uptake taking into account that this transporter can compensate for the function of ARAth;Ph1;4 (Misson et al., 2004; Shin et al., 2004). Orthologous genes to ARAth;Pht1;1 have been identified in other species such as Hordeum vulgare, Solanum tuberosum, Medicago truncatula, Oriza sativa, and Zea mays (Daram et al., 1998; Harrison et al., 2002; Paszkowski et al., 2002; Rae et al., 2003; Glassop et al., 2005; Nagy et al., 2005; Nagy et al., 2006; Liu et al., 2008), sharing the same expression pattern and its basic role in Pi uptake. In maize, no Pi transporter with exclusive expression in roots has been reported; however, ZEAma;Pht1;1, ZEAma;Pht1;2, and ZEAma;Pht1;3 have been suggested as key components in the direct phosphate uptake pathway (Nagy et al., 2006). How the expression and activity of these Pi transporters, especially under Pi stress, are regulated is unknown. Information about this is scarce, and the molecular signaling pathways that mediate such actions are not completely elucidated. However, their regulation is presumed complex because positive and negative transcriptional programs seem to act in concert to modulate the Pi homeostasis in plants. Heterologous expression of some Pi high-affinity transporters of dicots in monocots,

152

GENES FOR CROP ADAPTATION TO POOR SOIL

and vice versa (Koyama et al., 2005; Park et al., 2007; Tittarelli et al., 2007), indicate that cis-acting elements and trans-acting factors are conserved in both types of angiosperms; and therefore, the transcriptional control of these genes must be conserved (Tittarelli et al., 2007). The PHR1-binding site, also named P1BS, and sequence related to it, have been found in promoters of Pi transporters from different species, suggesting that Pi acquisition is modulated by PHR1; for instance, in Hordeum vulgare, six Pht1 genes responsive to low Pi, harbor P1BS-like elements (Schümann et al., 2004; Schünmann et al., 2004). Although, PHR1 seems to play a major role in the regulation of Pht1 genes, the participation of other TF and its cognate binding sites is quite possible. In 2001, Mukatira and others identified new elements (TATCA(/T)A(/T)q1 on regulatory regions of ARAthPht1;1 and ARAthPht1;4. Recently, a comparison of Arabidopsis, rice, barley, and wheat revealed new conserved promoter motifs in Pi responsive genes (SGCCGGCS, CTATNTATA, ATAAGTC), although transcriptional pathways potentially involved remain uncovered (Tittarelli et al., 2007). Negative transcriptional regulation on Pi transporter expression has also been proposed. Specific interactions between the ARAthPht1;4 promoter and nuclear protein factors obtained from Pisufficient plants suggest that Pi-responsive genes are under a negative regulation (Mukatira et al., 2001). SPX domain (SYG1/Pho81/XPHR1) has been strongly related with signaling transduction pathway in Saccharomyces cerevisiae, Neurospora crassa, mouse, and human (Spain et al., 1995; Lenburg and O’Shea, 1996; Peleg et al., 1996; Battini et al., 1999). In Arabidopsis and rice, 20 and 6 proteins, respectively, harboring SPX domains have been identified. Some genes encoding for this protein class are induced by Pi starvation (in Arabidopsis, AtSPX1, AtSPX2, and AtSPX3, and in rice, OrSPX1). Functional analysis of them resulted in assigning SPX genes (AtSPX3 and OrSPX1) a role in a negative feedback, which seems to modulate Pi homeostasis and the expression of Pth genes (Duan et al., 2008; Wang et al., 2009). It was proposed that AtSPX3 represses ARA;Pht1;4 and modulates Pi allocation, since spx3 plants show an enhanced expression of this transporter and higher Pi shoot concentrations (Duan et al., 2008). In rice, suppression of OsSPX1 resulted in the upregulation of OsPT2 and OsPT8 transporter genes under Pi-sufficient conditions, leading to alter Pi uptake (Wang et al., 2009; Mukatira et al., 2001; Miura et al., 2005; Chen et al., 2008; Duan et al., 2008; Wang et al., 2009). The existence of negative mechanisms of regulation, however, does not preclude the action of a positive transcriptional control, but suggests a mechanism finely coordinated to maintain adequate cellular Pi levels, particularly when this nutrient is limited. Knowledge about the function of the Pht2 and Pht3 families is more limited. Pht2 transporters belong to a Pi low-affinity transport system according functional analysis of ARAth;Pht2;1 (Daram et al., 1999). This gene is not responsive to Pi starvation, and is orthologous to the potato, SOLtu;Pht2;1, which encodes a Pi transporter found in the inner envelope of plastids (Daram et al., 1999; Rausch et al., 2004). In both species, this gene is regulated by light and developmental signals as well as by Pi sink strength in plant organs (Rausch et al., 2004). According to these data, the Pht2 family could be involved in the uptake and intercellular movement of Pi but not in Pi remobilization. In plastids, these transporters participate in photosynthesis by importing Pi and counterbalancing an ADP/ATP exchange via ADP/ATP translocator to maintain Pi homeostasis (Rausch et al., 2004). In Arabidopsis, three genes have been identified as members of the Pht3 family, which have been described as mitochondrial phosphate transporters (Takabatake et al., 1999; Rausch and Bucher, 2002). The detail functions of Pht3 transporters in response to environmental stress and their molecular regulation are still poorly understood (Takabatake et al., 1999). ARAth;Pht3;2 was reported as an induced gene in shoots and roots by Pi starvation (Misson et al., 2005; Morcuende et al., 2007; Müller et al., 2007).

GENETIC DETERMINANTS OF PHOSPHATE USE EFFICIENCY IN CROPS

153

PHR1 and Its Central Role in Pi Uptake

Genetic determinants for Pi uptake have been discovered in studies of plant Pi starvation responses. Several transcriptome analyzes carried out in Arabidopsis, maize, rice, common bean, and soybean have revealed a great variety of Pi-responsive genes with potential regulatory roles related to Pi homeostasis (Misson et al., 2005; Wasaki et al., 2006; Hernández et al., 2007; Morcuende et al., 2007; Calderon-Vazquez et al., 2008; Guo et al., 2008). From several studies, it has become clear that PHR1 is a key regulator that modulates Pi acquisition in Arabidopsis, common bean, and rice (Rubio et al., 2001; Nilsson et al., 2007; Valdés-López et al., 2008; Zhou et al., 2008). The participation of AtPHR1 in Pi uptake was initially suggested by the reduced Pi leaf content in phr1 Arabidopsis plants (Rubio et al., 2001). Recently, AtPHR1 was overexpressed in Arabidopsis and an enhanced Pi acquisition was reported (Nilsson et al., 2007). Under Pi starvation, total free shoot Pi content was 4-fold in the PHR1 overexpressing lines as compared to that accumulated in control plants, whereas in Pi-optimal conditions, a 1.5-fold increase in Pi content was observed. This increased Pi uptake in PHR1 overexpressing plants can be explained at least in part by the finding that the ARAth;Pht1;4, ARAth;Pht1;7, ARAth;Pht1;8, and ARAth;Pht1;9 Pi transporter genes are overexpressed in these transgenic lines (Nilsson et al., 2007). PHR1 is conserved in vascular plants, and its regulatory function is being explored in cereals and legumes. OsPHR1 and OsPHR2, two AtPHR1-like genes, were identified in rice (Zhou et al., 2008). Expression pattern analyzes, overexpression, and silencing showed that both transcriptional activators are involved in Pi-signaling. Excessive Pi accumulation in shoots in OsPHR2-overexpressing plants grown under Pi-sufficient conditions confirmed that OsPHR2 is a key factor mediating the Pi uptake and that it is functionally homologous to AtPHR1. Enhanced Pi acquisition in this case was also associated with an upregulation of Pi transporter genes, specifically OsPT9, whose expression was 40-fold higher in roots of OsPHR2-overexpressing plants than in wild-type plants (Zhou et al., 2008). In the common bean, orthologous to AtPHR1, PvPHR1, has been reported also, (Valdés-López et al., 2008), where it plays an essential role in regulating Pi transport. The relationship between Pi homeostasis and transcriptional control by PHR1 on Pi transporters is being clearly delimitated in monocot and dicot plants. However, it is important to note that PHR1 plays an important role in Pi acquisition by modulating the expression of genes encoding secreted phosphohydrolases, which release Pi from organic molecules present in the soil (Bariola et al., 1994; del Pozo et al., 1999; Köck et al., 2006; Nilsson et al., 2007). Finally, PHR1 also appears to participate in Pi homeostasis by controlling Pi translocation, specifically mediating the mir399/ PHO2 pathway (Aung et al., 2006; Bari et al., 2006; Lin et al., 2008).

Genetic Determinants for Pi Acquisition by Modulating Root System Architecture

Pi is highly reactive and thus has low mobility in the soil, and is usually found in Pi-rich patches in the upper soil strata. Under Pi stress conditions, the plant needs to reach these Pi-rich patches to secure an adequate Pi supply, which could be facilitated by altering its root developmental program. Alterations in cell length, root meristem activity, root hair density and elongation, and lateral root formation and elongation are changes in root architecture that are frequently observed in Pi-deprived plants (Bates and Lynch, 1996; Ma et al., 2001; Williamson et al., 2001; LópezBucio et al., 2002; Sánchez-Calderón et al., 2005; Lai et al., 2007). The capacity to respond to Pi stress through changes in RSA is directly associated with Pi acquisition and is highly variable among plants, even within genotypes of the same species, as it is has been reported for maize,

154

GENES FOR CROP ADAPTATION TO POOR SOIL

wheat, rice, barley, sorghum, common bean, and soybean (Gahoonia and Nielsen, 1996; Bhadoria et al., 2001; Wissuwa and Ae, 2001; Singh et al., 2003; Shimizu et al., 2004; Beebe et al., 2006; Zhang et al., 2009). In maize, for example, Pi-efficient genotypes exhibit enhanced crown, brace, and lateral growth (Alves et al., 2001; Gaume et al., 2001; Zhu and Lynch, 2004), whereas in common bean, higher Pi-efficient materials have shallower basal roots (Beebe et al., 2006). In rice, a larger root system is also associated with tolerance to Pi deficiency (Wissuwa and Ae, 2001; Shimizu et al., 2004) because a higher root elongation is positively correlated with an enhanced tillering ability and shoot P content (Shimizu et al., 2008). On the other hand, variation among wheat and barley genotypes is explained by differences in hair root length and formation (Gahoonia and Nielsen, 1996; Gahoonia et al., 1999). Some genetic determinants that control these changes of RSA are known to mediate the relationship between hormone-related genes and root development under Pi stress and analysis of quantitative trait loci (QTL) associated with root traits. QTL and Root System Architecture

In Arabidopsis, three QTL controlling root growth, specifically primary root length (PRL), in response to Pi starvation, were recently mapped: LPR1, LPR2, and LPR3 (Low Phosphate Response). LPR1 and LPR2 are specifically involved with the control of PRL under Pi starvation, whereas LPR3 has a role in PRL under high Pi conditions (Reymond et al., 2006). LPR1 is expressed in the root tip, and like its paralog LPR2, encodes a multicopper oxidase (MCO), whose role in Pi signaling pathway during Pi deprivation has not yet been clearly defined (Svistoonoff et al., 2007). It has been suggested that LPR proteins could determinate PRL by modifying the activity and/or distribution pattern of hormone-like compounds in the root, and this in turn could trigger the root morphological changes usually observed under Pi starvation (arrested growth of primary root, proliferation of lateral roots and root hairs) (Svistoonoff et al., 2007). For maize, Zhu and others (2005) reported the generation of 169 recombinant inbred lines (RIL) using B73 (nonefficient) and Mo17 (efficient) hybrids; they identified five QTLs controlling lateral root length, one linked with lateral root number, one associated with root hair length, and six with Pi seed content (Zhu et al., 2005). In the common bean, a similar approach was also used, and 26 individual QTLs were found for Pi accumulation, which were also associated with root traits (Beebe et al., 2006). In rice, QTLs affecting Pi absorption mechanisms have been extensively studied (Wissuwa and Ae, 2001; Shimizu et al., 2004; Shimizu et al., 2008). The qREP-6 QTL for root elongation and the qTNP-6 QTL for tiller number have been fine-mapped recently; and when identified, they could be useful in the rice genetic improvement for adaptability to soils with low Pi availability (Shimizu et al., 2008). The large number of QTL associated with root traits involved with Pi efficiency in different plant species shows the complexity of this important agronomic trait. Hormone-related Genes and Root Development

Modulation of changes observed in RSA in response to Pi starvation by hormone-related genes has been suggested (López-Bucio et al., 2002; Franco-Zorrilla et al., 2004). Recently, identification and molecular description of several transcriptional modulators involved with Pi homeostasis through control of RSA or hormone sensing have been reported (Devaiah et al., 2007a, 2007b; Pérez-Torres et al., 2008). ZAT6 is a TF induced in Pi-deprived Arabidopsis plants that negatively controls root development. In ZAT6 overexpressing transgenic plants, a significant decrease in PRL, an enhanced lateral

GENETIC DETERMINANTS OF PHOSPHATE USE EFFICIENCY IN CROPS

155

root length, and higher Pi content in shoots and roots is observed, independent of Pi regimen (Devaiah et al., 2007b). However, a reduced expression of ZAT6 in the auxin-resistant mutants aux1-7, axr1-3, axr2-1, and ax4-1, under Pi starvation suggests that this TF could be an intermediary, Pi-responsive regulator, of the changes in RSA caused by low Pi availability through an auxindependent pathway (Devaiah et al., 2007b). In root branching, the establishment of lateral root primordium and its emergence is determined by auxin action; however, the mechanism (auxin synthesis, distribution, or sensitivity) responsible for the increase of root lateral density under Pi starvation has remained uncovered (López-Bucio et al., 2002). Recently, Pérez-Torres and others (2008) showed that Pi starvation induces the expression of TIR1 auxin receptor (Dharmasiri et al., 2005; Kepinski and Leyser, 2005), and that this in turn increases auxin sensitivity in perycicle cells, which finally leads to higher root lateral formation in Pi-deprived plants. This study also showed that constitutive expression of TIR1 in Arabidopsis increases lateral root formation 75% compared to wild-type plants. Although Pi content was not quantified, these data are very interesting taking into account the close relationship between root branching and Pi acquisition efficiency. Changes of RSA under Pi deprivation by altered gibberellic acid (GA) biosynthesis have also been recently reported in Arabidopsis. MYB62 is TF induced by Pi starvation that has been proposed to be a negative regulator of expression of GA biosynthesis genes. MYB62-overexpressed plants have a reduced level of GA synthesis with a higher Pi content that was accounted for by an altered RSA (reduced PRL in high Pi and decreased lateral root length in high and low Pi) (Devaiah et al., 2009).

Genetic Determinants Involved with Phosphorus Utilization Efficiency

Pi internal utilization efficiency is calculated using as parameters crop yield and plant Pi concentration. Both parameters are closely linked to Pi loading to the vascular system and its translocation within the plant. Deficient Pi loading seriously compromises plant growth and seed production (Poirier et al., 1991), whereas Pi translocation, especially from leaves, is essential during grain filling in cereals (Manske et al., 2000). Known genes involved in these processes, Pi loading and translocation, are described below.

PHO1: The Pi Loading Control

Unlike the low diffusion of Pi in the soil, inside the plants, this nutrient is highly mobile. Once Pi is absorbed by root epidermal cells, it moves toward the root stele either radially through the apoplastic pathway or cell to cell via plasmodesmata to the cells surrounding the xylem. These latter cells release Pi into the xylem, which is then translocated to the Pi sink aboveground tissues (Clarkson, 1993; Rausch and Bucher, 2002). Xylem loading of Pi had been studied in barley and maize, however, the isolation of Arabidopsis mutants affected in this process allowed dissecting it from Pi uptake (Poirier et al., 1991). The pho1 (phosphate 1) Arabidopsis mutant is characterized by a dramatic reduction in shoot Pi content (95%) compared to the wild type, in spite of the fact that its Pi uptake is not affected (Poirier et al., 1991). PHO1 (AtPHO1) encodes a protein with two domains, named SPX and EXS, potentially involved in phosphate transporter or sensing (Hamburger et al., 2002). PHO1 shows no homology to any previously described solute transporter, including Pi transporters, suggesting that Pi is not charged to the xylem by uniporters or ion selective channels (Hamburger et al., 2002). In Arabidopsis, AtPHO1 belongs to a gene family with 11 members and forms a clade together

156

GENES FOR CROP ADAPTATION TO POOR SOIL

AtPHO1;1, the only homologous gene able to complement to pho1 mutant (Wang et al., 2004; Stefanovic et al., 2007). AtPHO1 seems to function exclusively in cells of the root vascular system and the lower part of hypocotyls (Hamburger et al., 2002; Wang et al., 2004), whereas AtPHO1;1 is active in the stele of roots and shoots (Wang et al., 2004; Stefanovic et al., 2007). AtPHO1 and AtPHO2 are induced by Pi starvation, which is in line with their roles in Pi homeostasis. However, they seem to be regulated by distinct signaling pathways, since AtPHO1;1 induction requires AtPHR1 but the transcriptional control of AtPHO1 remains unknown and no P1BS or P1BS-like elements are present in its promoter region (Stefanovic et al., 2007).

Mir399/PHO2 Pathway Role in Whole-plant Pi Homeostasis

Pi distribution between shoots and roots, as well as Pi uptake, are involved in the maintenance of Pi homeostasis. When synchrony between both processes fails, an increased Pi accumulation with toxic effects can be observed in roots or shoots. In contrast to the pho1 phenotype, the Arabidopsis pho2 mutant shows an excessive Pi accumulation in leaves with unaltered Pi levels in roots (Delhaize and Randall, 1995). Positional mapping of this mutant revealed that PHO2 (AtPHO2) is a ubiquitin-conjugating E2 enzyme predominantly expressed in the vascular tissue of Pi-sufficient plants (Aung et al., 2006; Bari et al., 2006). Under this condition, AtPHO2 negatively controls the expression of some genes related with Pi uptake (ARAth;Pht1;1 and ARAth;Pht;4) and Pi internal redistribution (ARAth;Pht1;8 and ARAth;Pht;9; ARAthPht3;2), possibly through ubiquitination of as yet unknown TFs (Aung et al., 2006; Bari et al., 2006). When Pi starvation occurs, AtPHO2 expression decreases and as a consequence Pi uptake and translocation is favored. This suppression is specifically carried out by mir399s-mediated cleavage of AtPHO2 transcripts (Aung et al., 2006; Bari et al., 2006). To maintain whole-plant homeostasis, mir399s are transported through the phloem conduit and exert their biological role on AtPHO2 in a systemic manner (Lin et al., 2008; Pant et al., 2008). In Arabidopsis and rice, mir399s upregulation by Pi deprivation is controlled by AtPHR1 and OsPHR2, respectively, and suggests that this regulatory network is common to monocot and dicot plants (Zhou et al., 2008). However, mir399s targets or action mechanisms seem to be different in both species. Osmir399 does not mediate the degradation of OsPHO2 transcripts (Zhou et al., 2008). This observation suggests the existence of a translational repression mediated by this family of noncoding microRNAs, as previously proposed by Bari and others (2006).

Genetic Engineering to Improve the Phosphate Use Efficiency

The potential phosphate crisis and the need for more sustainable agriculture have created the necessity to develop novel plant varieties with increased Pi use and acquisition efficiency. Among the options to achieve this goal, plant genetic engineering provides important alternatives. Pi use efficiency improvement through this strategy has taken advantage of progress in the gene cloning and transfer systems, however, their achievements depend mainly on the research focused in the identification of genes and signaling cascades involved in plant response to Pi starvation. To date, transgenic approaches based on the overexpression of genes directly associated with Pi transport, Pi release from external sources, and Pi homeostasis have produced promising results that suggest that Pi uptake and biomass production can be increased under Pi limiting conditions. Some examples of these approaches with successful results are shown in Table 6.1.

Table 6.1 Enhanced phosphate use efficiency via manipulation of genes involved with Pi use, acquisition and homeostasis Gen*

Gene product

Gene source

Target plant

Phenotype observed in transgenic plants

Refs**

NtPT1

High affinity phosphate transporter

Tobacco

Rice

•

Pi uptake increased by 24% and 37% at low Pi (32 μ) and high Pi (320 μ), respectively. Pi shoot content increased by 41% and 55% under low and high Pi, respectively.

1

Higher PEPC activity in roots and shoots Enhanced oxalate exudation No increase in plant Pi concentration Significant increase in the dry matter yield

2

High phosphatase activity

3

•

PEPC

Phosphoenolpyruvate

Maize

Rice

Carboxylase

• • • •

OsACP1

Acid phosphatase

Rice

Arabidopsis

•

CS

Citrate synthase

Pseudomonas aeruginosa

Tobacco

•

MtPAP1

Purple acid phosphatas

Medicado truncatula

Arabidopsis

Increased levels of synthesis and excretion of citrate • Enhanced shoot biomass (15%) and fruit dry weight (35%) • Higher Pi content in transgenic plants than wild-type plants • •

ex : phyA

Phytase containing a signal peptide sequence

Aspergillus niger Daucus carota

Arabidopsis

• • •

4 5

High acid phosphatase activity (4.6–9.9-fold) Increased biomass production (2-fold) and total P content in transgenic plants grown with phytate

6

High total root phytase activity (20-fold) Higher total shoot P content (∼15-fold) Higher plant growth

7

DcCS

Mitocondrial citrate synthase

Daucus carota

Arabidopsis

•

Increased citrate synthase activity (3-fold) • Higher citrate excreted by roots (2.5-fold) • Increase in shoot P content (20–30%)

8

AtPHR1

MYB-related transcription factor

Arabidopsis

Arabidopsis

•

Increased Pi shoot accumulation (4-fold)

9

OsPHR2

AtPHR1-like gene

Rice

Rice

•

Increased Pi shoot accumulation (2–2.5-fold)

10

OsPTF1

Transcription factor

Rice

Rice

•

Higher tillering ability, root and shoot biomass and P content (20–30%)

11

Mir399

Non-encoding microRNA

Arabidopsis

Arabidopsis

•

Impaired Pi translocation between shoots and roots Excessive Pi accumulation in shoots

12

•

* Overexpressed genes using the CaMV 35S promoter, with the exception of NtPT1 and PEPC, which used the maize Ubi and PEPC promoter, respectively. ** Refs: 1) Park et al., 2007; 2) Begum et al., 2005; 3) Hur et al., 2007; 4) de la Fuente et al., 1997; 5) López-Bucio et al., 2000; 6) Xiao et al., 2006; 7) Richardson et al., 2001; 8) Koyama et al., 2000; 9) Nilsson et al., 2007; 10) Zhou et al., 2008; 11) Yi et al., 2005; 12) Chiou et al., 2006

157

158

GENES FOR CROP ADAPTATION TO POOR SOIL

Excessive of Pi accumulation, observed in shoots when molecular determinants of Pi homeostasis have been overexpressed, could be overcome using root specific and low Pi-inducible promoters, especially those expressed in root epidermis and root hairs (for example, ARAth;Pht1;1 and ARAth;Pht;4). Genetic manipulation of root traits involved directly with Pi uptake is another viable option, although poorly explored, to enhance the Pi use efficiency. Several genes involved in regulating root hair formation and elongation are already known in Arabidopsis (for review, see Ishida et al., 2008; Schiefelbein et al., 2009), and mapping of QTLs controlling root hair length have been already carried out in maize (Zhu et al., 2005). Future cloning of genes responsible for these maize QTL will provide new targets to improve Pi uptake by increasing the absorptive surface area and increasing soil exploration. In addition, recent identification of transcriptional factors (ZAT6, OsPHR2) that control root system architecture under Pi starvation and the specific role of TIR1 in lateral root formation offer other possibilities to change the RSA; for example, overexpression of TIR1 o ZAT6 into a low phosphorus insensitive (lpi) or a low phosphate root1 (lpr1) background, where growth of the primary root is not arrested under Pi starvation (SánchezCalderón et al., 2005; Devaiah et al., 2007b; Svistoonoff et al., 2007; Pérez-Torres et al., 2008) could have a dramatic effect on RSA, potentially, since a higher lateral root production could be obtained on larger primary roots in Pi-deprived plants. Crops with these morphological changes could possess improved adaptation to Pi-limiting conditions, with a lower Pi fertilizer demand and larger yields.

Conclusions

Higher crop yields with lower production costs and minimum negative impacts on the environment are currently in demand. The desire is to increase food production to alleviate hunger in developing countries under a sustainability framework. In this way, nutrient use improvement has a central role because nutrient soil availability and excessive fertilizer application are two principal problems for crop productivity. High-throughput techniques for gene expression analysis together with large-scale forward genetic screenings and substantial progress in the identification of genetic determinants controlling Pi use and acquisition efficiency have yielded valuable tools for the future production of novel plant genotypes with an enhanced Pi uptake and utilization capacity. However, there are still many unknown aspects regarding the mechanisms by which plants respond to Pi deprivation and sense Pi availability. The mechanisms that regulate Pi homeostasis are complex, and this complexity must be taken into account in the design of Pi-efficient plant genotypes.

References Abel, S., Nürnberger, T., Ahnert, V., Krauss, G.J., and Glund, K. (2000) Induction of an extracellular cyclic nucleotide phosphodiesterase as an accessory ribonucleolytic activity during phosphate starvation of cultured tomato cells, Plant Physiology 122, 543–552. Abelson, P.H. (1999) A potential phosphate crisis, Science 283, 2015. Alves, V.M.C., Parentoni, S.N., Vasconcellos, C.A., Bahia-Filho, A.F.C., Pitta, G.V.E., and Schaffert, R.E. (2001) Mechanisms of phosphorus efficiency in maize, in Plant Nutrition-Food security and sustainability of agro-ecosystems through basic and applied research (Eds. W.J. Horst, M.K. Schenk, A. Bürkert, N. Claassen, H. Flessa, W.B. Frommer, H. Goldbach, H.-W. Olfs, V. Römheld, B. Sattelmacher, U. Schmidhalter, S. Schubert, N. v. Wirén, and L. Wittenmayer), Kluwer Academic Publishers, The Netherlands, pp. 566–567.

GENETIC DETERMINANTS OF PHOSPHATE USE EFFICIENCY IN CROPS

159

Asher, C.J. (1991) Beneficial elements, functional nutrients and possible new essential elements, in Micronutrients in agriculture (Eds. J.J. Mortwedt, F.R. Coz, L.M. Shuman, and P.P. Welch), Soil Science Society of America, Madison, Wisconsin, pp. 703–723. Ashihara, H., Xiao-Ni, L., and Toshiko, U. (1988) Effect of inorganic phosphate on the biosynthesis of purine and pyrimidine nucleotides in suspension-cultured cells of Catharanthus roseus, Annals of Botany 61, 225–232. Aung, K., Lin, S.-I., Wu, C.-C., Huang, Y.-T., Su, C.-L., and Chiou, T.-J. (2006) Pho2, a phosphate overaccumulator, is caused by a nonsense mutation in a microRNA399 target gene, Plant Physiology 41, 1000–1011. Bari, R., Pant, B.D., Stitt, M., and Scheible, W.-R. (2006) PHO2, microRNA399, and PHR1 define a phosphate-signaling pathway in plants, Plant Physiology 141, 988–999. Bariola, P.A., Howard, C.J., Taylor, C.B., Verburg, M.T., Jaglan, V.D., and Green, P.J. (1994) The Arabidopsis ribonuclease gene RSN1 is tightly controlled in response to phosphate limitation, The Plant Journal 6, 673–685. Bariola, P.A., MacIntosh, G.C., and Green, P.J. (1999) Regulation of S-like ribonuclease levels in Arabidopsis: antisense inhibition of RSN1 or RSN2 elevates anthocyanin accumulation, Plant Physiology 119, 331–342. Bates, T.R. and Lynch, J.P. (1996) Stimulation of root hair elongation in Arabidopsis thaliana by low phosphorus availability, Plant, Cell and Environment 19, 529–538. Battini, J.L., Rasko, J.E.J., and Miller, A.D. (1999) A human cell-surface receptor for xenotropic and polytropic murine leukemia viruses: possible role in G protein-coupled signal transduction, Proceeding of the National Academy of Sciences USA 96, 1385–1390. Beebe, S.E., Rojas-Pierce, M., Yan, X., Blair, M.W., Pedraza, F., Muñoz, F., Tohme, J., and Lynch, J.P. (2006) Quantitative trait loci for root architecture traits correlated with phosphorus acquisition in common bean, Crop Science 46, 413–423. Begum, H.H. and Islam, M.T. (2005) Role of synthesis and exudation of organic acids in phosphorus nutrition in plants in tropical soils, Biotechnology 4, 333–340. Bhadoria, P.B.S., Singh, S., and Claassen, N. (2001) Phosphorus efficiency of heat, maize and groundnut grown in low phosphorussupplying soil, in Plant Nutrition—Food security and sustainability of agro-ecosystems through basic and applied research (Eds. W.J. Horst, M.K. Schenk, A. Bürkert, N. Claassen, H. Flessa, W.B. Frommer, H. Goldbach, H.-W. Olfs, V. Römheld, B. Sattelmacher, U. Schmidhalter, S. Schubert, N. v. Wirén, and L. Wittenmayer), Kluwer Academic Publisher, The Netherlands, pp. 530–531. Bray, E.A., Bailey-Serres, J., and Weretilnyk, E. (2000) Responses to Abiotic stresses, In Biochemistry and Molecular Biology of Plants (Eds. B.B. Buchanan, W. Gruissem, and R.L. Jones), ASPP, Rockville, MD, pp. 1158–1203. Bucher, M. (2007) Functional biology of plant phosphate uptake at root and mycorrhiza interfaces, New Phytologist 173, 11–26. Bumb, B.L. and Baanante, C.A. (1996) The role of fertilizer in sustaining food security and protecting the environment to 2020, 2020 Vision Discussion Papers 17. (Ed. International Food Policy Research Institute, IFPRI), Washington, DC. Burleigh, S.H. and Harrison, M.J. (1999) The down-regulation of Mt4-like genes by phosphate fertilization occurs systematically and involves phosphate translocation to the shoots, Plant Physiology 119, 241–248. Byrnes, B.H. and Bumb, B.L. (1998) Population growth, food production and nutrient requirements, in Nutrient use in Crop production (Ed. Z. Rengel), The Haworth Press, New York, pp. 1–27. Cakmak, I. (2002) Plant nutrition research: Priorities to meet human needs for food in sustainable ways, Plant and Soil 247, 3–24. Calderon-Vazquez, C., Ibarra-Laclette, E., Caballero-Perez, J., and Herrera-Estrella, L. (2008) Transcript profiling of Zea mays roots reveals gene responses to phosphate deficiency at the plant- and species-specific levels, Journal of Experimental Botany 59, 2479–2497. Chen, Z.-H., Jenkins, G.I., and Nimmo, H.G. (2008) Identification of an F-Box protein that negatively regulates Pi starvation responses, Plant and Cell Physiology 49, 1902–1906. Chiou, T.-J., Aung, K., Lin, S.-I., Wu, C.-C., Chiang, S.-F., and Su, C.-l. (2006) Regulation of phosphate homeostasis by microRNA in Arabidopsis, The Plant Cell 18, 412–421. Cisse, L. and Mrabet, T. (2004) World phosphate production: Overview and prospects, Phosphorus Research Bulletin 15, 21–25. Clarkson, D.T. (1993) Roots and delivery of solutes to the xylem, Philosophical transactions of the Royal Society of London 341, 5–17. Coruzzi, G. and Last, R. (2000) Amino acids, in Biochemistry and Molecular Biology of Plants (Eds. B.B. Buchanan, W. Gruissem, and R.L. Jones), ASPP, Rockville, MD, pp. 358–419. Croteau, R., Kutchan, T.M., and Lewis, N.G. (2000) Natural Products (Secondary metabolites), in Biochemistry and Molecular Biology of Plants (Eds. B.B. Buchanan, W. Gruissem, and R.L. Jones), ASPP, Rockville, MD, pp. 250–1318. Cruz-Ramírez, A., Oropeza-Aburto, A., Razo-Hernández, F., Ramírez-Chávez, E., and Herrera-Estrella, L. (2006) Phospholipase DZ2 plays an important role in extraplastidic galactolipid biosynthesis and phosphate recycling in Arabidopsis roots, Proceedings of the National Academy of Sciences USA 103, 6765–6770.

160

GENES FOR CROP ADAPTATION TO POOR SOIL

Daram, P., Brunner, S., Rausch, C., Steiner, C., Amrhein, N., and Bucher, M. (1999) Pht2;1 encodes a low-affinity phosphate transporter from Arabidopsis, The Plant Cell 11, 2153–2166. Daram, P., Brunner, S., Persson, B.L., Amrhein, N., and Bucher, M. (1998) Functional analysis and cell-specific expression of a phosphate transporter from tomato, Planta 206, 225–233. de la Fuente, J.M., Ramírez-Rodriguez, V., and Cabrera-Ponce, J.L. (1997) Aluminum tolerance in transgenic plants by alteration of citrate synthesis, Science 276, 1566–1568. del Pozo, J.C., Allona, I., Rubio, V., Leyva, A., de la Peña, A., Aragoncillo, C., and Paz-Ares, J. (1999) A type 5 acid phosphatase gene from Arabidopsis thaliana is induced by phosphate starvation and by some other types of phosphate mobilising/oxidative stress conditions, The Plant Journal 19, 579–589. Delhaize, E. and Randall, P. (1995) Characterization of a phosphate-accumulator mutant of Arabidopsis thaliana, Plant Physiology 107, 207–213. Dennis, D.T. and Blakeley, S.D. (2000) Carbohydrate metabolism, in Biochemistry and Molecular Biology of Plants (Eds. B.B. Buchanan, W. Gruissem, and R.L. Jones), ASPP, Rockville, MD, pp. 630–675. Devaiah, B.N., Madhuvanthi, R., Karthikeyan, A.S., and Raghothama, K.G. (2009) Phosphate starvation responses and gibberellic acid biosynthesis are regulated by the MYB62 transcription factor in Arabidopsis, Molecular Plant 2, 43–58. Devaiah, B.N., Karthikeyan, A.S., and Raghothama, K.G. (2007a) WRKY75 transcription factor is a modulator of phosphate acquisition and root development in Arabidopsis, Plant Physiology 143, 1789–1801. Devaiah, B.N., Nagarajan, V.K., and Raghothama, K.G. (2007b) Phosphate homeostasis and root development in Arabidopsis are synchronized by the zinc finger transcription factor ZAT6, Plant Physiology 145, 147–159. Dharmasiri, N., Dharmasiri, S., and Estelle, M. (2005) The F-box protein TIR1 is an auxin receptor, Nature 435, 441–445. Dinkelaker, B., Hengeleer, C., and Marshchner, H. (1995) Distribution and function of proteoid roots and other root clusters, Botanica Acta 108, 183–200. Dinkelaker, B., Römheld, V., and Marschner, H. (1989) Citric acid excretion and precipitation of calcium citrate in the rhizosphere of white lupin (Lupinus albus L), Plant, Cell and Environment 12, 285–292. Duan, K., Yi, K., Dang, L., Huang, H., Wu, W., and Wu, P. (2008) Characterization of a sub-family of Arabidopsis genes with the SPX domain reveals their diverse functions in plant tolerance to phosphorus starvation, The Plant Journal 54, 965–975. Epstein, E. (1976) Kinetics of ion transport and the carrier concept, in Encyclopedia of plant physiology (Eds U. Lüttge and M.G. Pitman), Springer-Verlag, Berlin, pp.70–94. FAO. (2000) Fertilizer requirements in 2015 and 2030. FAO, Rome. Franco-Zorrilla, J.M., Valli, A., Todesco, M., Mateos, I., Puga, M.I., Rubio-Somoza, I., Leyva, A., Weigel, D., García, J.A., and Paz-Ares, J. (2007) Target mimicry provides a new mechanism for regulation of microRNA activity, Nature Genetics 39, 1033–1037. Franco-Zorrilla, J.M., Martín, A.C., Leyva, A., and Paz-Ares, J. (2005) Interaction between phosphate-starvation, sugar, and cytokinin signaling in Arabidopsis and the roles of cytokinin receptors CRE1/AHK4 and AHK3, Plant Physiology 138, 847–857. Franco-Zorrilla, J.M., González, E., Bustos, R., Linhares, F., Leyva, A., and Paz-Ares, J. (2004) The transcriptional control of plant responses to phosphate limitation, Journal of Experimental Botany 55, 285–293. Gahoonia, T.S., Nielsen, N.E., and Lyshede, O.B. (1999) Phosphorus (P) acquisition of cereal cultivars in the field at three levels of P fertilization, Plant and Soil 211, 269–281. Gahoonia, T.S. and Nielsen, N.E. (1996) Variation in acquisition of soil phosphorus among wheat and barley genotypes, Plant and Soil 178, 223–230. Gaume, A., Mächler, F., De Leon, C., Narro, L., and Frossard, E. (2001) Low-P tolerance by maize (Zea mays L.) genotypes: Significance of root growth, and organic acids and acid phosphatase root exudation, Plant and Soil 228, 253–264. Gerke, J., Biessner, L., and Römer, W. (2000) The quantitative effect of chemical phosphate mobilization by carboxylate anions on P uptake by a single root. 1. The basic concept and determination of soil parameters, Journal of Plant Nutrition and Soil Science 163, 207–212. Glassop, D., Smith, S., and Smith, F. (2005) Cereal phosphate transporters associated with the mycorrhizal pathway of phosphate uptake into roots, Planta 222, 688–698. Good, A.G., Shrawat, A.K., and Muench, D.G. (2004) Can less yield more? Is reducing nutrient input into the environment compatible with maintaining crop production? Trends in Plant Science 9, 597–605. Gourley, C.J.P., Allan, D.L., and Ruselle, M.P. (1993) Defining phosphorus efficiency in plants, Plant and Soil 155, 289–192. Güsewell, S. (2004) N : P ratios in terrestrial plants: Variation and functional significance, New Phytologist 164, 243–266. Guo, W., Zhang, L., Zhao, J., Liao, H., Zhuang, C., and Yan, X. (2008) Identification of temporally and spatially phosphatestarvation responsive genes in Glycine max, Plant Science 175, 574–584.

GENETIC DETERMINANTS OF PHOSPHATE USE EFFICIENCY IN CROPS

161

Hamburger, D., Rezzonico, E., Petétot, J.M.-C., Somerville, C., Poirier, Y., and Adam, L. (2002) Identification and characterization of the Arabidopsis PHO1 gene involved in phosphate loading to the xylem, Plant Physiology 14, 889–902. Hammond, J.P., Broadley, M.R., and White, P.J. (2004) Genetic responses to phosphorus deficiency, Annals of Botany 94, 323–332. Hammond-Kosack, K. and Jones, J.D.G. (2000) Response to plant pathogens. In Biochemistry and Molecular Biology of Plants (Eds. B.B. Buchanan, W. Gruissem, and R.L. Jones), ASPP, Rockville, MD, pp. 1102–1156. Harrison, M.J., Dewbre, G.R., and Liu, J. (2002) A phosphate transporter from Medicago truncatula involved in the acquisition of phosphate released by arbuscular mycorrhizal fungi, The Plant Cell 14, 2413–2429. Hernández, G., Ramírez, M., Valdés-López, O., Tesfaye, M., Graham, M.A., Czechowski, T., Schlereth, A., Wandrey, M., Erban, A., Cheung, F., Wu, H.C., Lara, M., Town, C.D., Kopka, J., Udvardi, M.K., and Vance, C.P. (2007) Phosphorus stress in common bean: Root transcript and metabolic responses, Plant Physiology 144, 752–767. Hinsinger, P. (2001) Bioavailability of soil inorganic P in the rhizosphere as affected by root-induced chemical changes: A review, Plant and Soil 237, 173–195. Hoisington, D., Khairallah, M., Timothy, R., Ribaut, J.-M., Skovmand, B., Taba, S., and Warburton, M. (1999) Plant genetic resources: What can they contribute toward increased crop productivity? Proceedings of the National Academy of Sciences USA 96, 5937–5943. Hur, Y.J., Lee, H.G., Jeon, E.J., Lee, Y.Y., Nam, M.H., Yi, G., Eun, M.Y., Nam, J., Lee, J.H., and Kim, D.H. (2007) A phosphate starvation-induced acid phosphate from Oryza sativa: Phosphate regulation and transgenic expression, Biotechnology Letters 29, 829–835. Ishida, T., Kurata, T., Okada, K., and Wada, T. (2008) A genetic regulatory network in the development of trichomes and root hairs, Annual Review of Plant Biology 59, 365–386. Javot, H., Pumplin, N., and Harrison, M.J. (2007) Phosphate in the arbuscular mycorrhizal simbiosis: Transport properties and regulatory roles, Plant, Cell and Environment 30, 310–322. Karandashov, V. and Bucher, M. (2005) Symbiotic phosphate transport in arbuscular mycorrhizas, Trends in Plant Science 10, 22–29. Keerthisinghe, G., Hocking, P.J., Ryan, P.R., and Delhaize, E. (1998) Effects of phosphorus supply on the formation and function of proteoid roots of white lupin (Lupinus albus L.), Plant, Cell and Environment 21, 467–478. Kepinski, S. and Leyser, O. (2005) The Arabidopsis F-box protein TIR1 is an auxin receptor, Nature 435, 446–451. Kirkby, E.A. and Johnston, A.E.J. (2008) Soil and fertilizer phosphorus in relation to crop nutrition, in The Ecophysiology of plant-phosphorus interactions (Eds. P. J. White and J. P. Hammond), Springer, Dordrecht, The Netherlands, pp. 177–223. Kobayashi, K., Masuda, T., Takamiara, K.-i., and Ohta, H. (2006) Membrane lipid alteration during phosphate starvation is regulated by phosphate signaling and auxin/cytokinin cross-talk, The Plant Journal 47, 238–248. Kochian, L.V. (2000) Molecular physiology of mineral nutrient acquisition, transport and utilization. In Biochemistry and Molecular Biology of Plants, B. Buchanan, W. Gruisem, and R. Jones, Eds. ASPP, Rockville, MD, pp. 1204–1249. Köck, M., Stenzel, I., and Zimmer, A. (2006) Tissue-specific expression of tomato ribonuclease LX during phosphate starvationinduced root growth, Journal of Experimental Botany 57, 3717–3726. Koyama, T., Ono, T., Shimizu, M., Jinbo, T., Mizuno, R., Tomita, K., Mitsukawa, N., Kawazu, T., Kimura, T., Ohmiya, K., and Sakka, K. (2005) Promoter of Arabidopsis thaliana phosphate transporter gene drives root-specific expression of transgene in rice, Journal of Bioscience and Bioengineering 99, 38–42. Koyama, H., Kawamura, A., Kihara, T., Hara, T., Takita, E., and Shibata, D. (2000) Overexpression of mitochondrial citrate synthase in Arabidopsis thaliana improved growth on a phosphorus-limited soil, Plant and Cell Physiology 41, 1030–1037. Lai, F., Thacker, J., Li, Y., and Doerner, P. (2007) Cell division activity determines the magnitude of phosphate starvation responses in Arabidopsis, The Plant Journal 50, 545–556. Lambers, H., Shane, M.W., Cramer, M.D., Pearse, S.J., and Veneklaas, E.J. (2006) Root structure and functioning for efficient acquisition of phosphorus: matching morphological and physiological traits, Annals of Botany 98, 603–713. Lauer, M., Blevins, D., and Gracz, S. (1989) 31P-nuclear magnetic resonance determination of phosphate compartmentation in leaves of reproductive soybeans (Glycine max L.) as affected by phosphate, Plant Physiology 89, 1331–1336. Lenburg, M.E. and O’Shea, E.K. (1996) Signaling phosphate starvation, Trends in Biochemical Sciences 21, 383–387. Li, W., Li, M., Zhang, W., Welti, R., and Wang, X. (2005) The plasma membrane-bound phospholipase Dδ enhances freezing tolerance in Arabidopsis thaliana, Nature Biotechnology 22, 427–433. Lin, S.-I., Chiang, S.-F., Lin, W.-Y., Chen, J.-W., Tseng, C.-Y., Wu, P.-C., and Chiou, T.-J. (2008) Regulatory network of microRNA399 and PHO2 by systemic signaling, Plant Physiology 147, 732–746. Liu, J., Versaw, W.K., Pumplin, N., Gomez, S.K., Blaylock, L.A., and Harrison, M.J. (2008) Closely related members of the Medicago truncatula PHT1 phosphate transporter gene family encode phosphate transporters with distinct biochemical activities, The Journal of the Biological Chemistry 283, 24673–24681.

162

GENES FOR CROP ADAPTATION TO POOR SOIL

López-Bucio, J., Hernández-Abreu, E., Sánchez-Calderón, L., Pérez-Torres, A., Rampey, R.A., Bartel, B., and Herrera-Estrella, L. (2005) An auxin transport independent pathway is involved in phosphate stress-induced root architectural alterations in Arabidopsis. Identification of BIG as a mediator of auxin pericycle cell activation, Plant Physiology 137, 681–691. López-Bucio, J., Sánchez-Calderón, L., Nieto-Jacobo, M.F., Simpson, J., and Herrera-Estrella, L. (2002) Phosphate availability alters architecture and causes changes in hormone sensitivity in the Arabidopsis root system, Plant Physiology 129, 244–256. López-Bucio, J., Martínez de la Vega, O., Guevara-García, A., and Herrera-Estrella, L. (2000) Enhanced phosphorus uptake in transgenic tobacco plants that overproduce citrate, Nature Biotechnology 18, 450–453. Ma, Z., Bielenberg, D.G., Brown, K.M., and Lynch, J.P. (2001) Regulation of root hair density by phosphorus availability in Arabidopsis thaliana, Plant, Cell and Environment 24, 459–467. Manske, G.G.B., Ortiz-Monasterio, J.I., Van Ginkel, M., González, R.M., Rajaram, S., Molina, E., and Vlek, P.L.G. (2000) Traits associated with improved P-uptake efficiency in CIMMYT’s semidwarf spring bread wheat grown on an acid Andisol in Mexico, Plant and Soil 221, 189–204. Marschner H. (1995) Mineral nutrition of higher plants, 2nd Edition, Academic Press, London. Martín, A.C., del Pozo, J.C., Iglesias, J., Rubio, V., Solano, R., de la Peña, A., Leyva, A., and Paz-Ares, J. (2000) Influence of cytokinins on the expression of phosphate starvation responsive genes in Arabidopsis, The Plant Journal 24, 559–567. Mengel, D.B. and Barber, S.A. (1974) Rate of nutrient uptake per unit of corn root under field conditions, Agronomy Journal 66, 399–402. Misson, J., Raghothama, K.G., Jain, A., Jouhet, J., Block, M.A., Bligny, R., Ortet, P., Creff, A., Somerville, S., Rolland, N., Doumas, P., Nacry, P., Herrera-Estrella, L., Nussaume, L., and Thibaud, M.-C. (2005) A genome-wide transcriptional analysis using Arabidopsis thaliana Affymetrix gene chips determined plant responses to phosphate deprivation, Proceedings of the National Academy of Sciences USA 102, 11934–11939. Misson, J., Thibaud, M.-C., Bechtold, N., Raghothama, K., and Nussaume, L. (2004) Transcriptional regulation and functional properties of Arabidopsis Pht1;4, a high affinity transporter contributing greatly to phosphate uptake in phosphate deprived plants, Plant Molecular Biology 55, 727–741. Miura, K., Rus, A., Sharkhuu, A., Yokoi, S., Karthikeyan, A.S., Raghothama, K.G., Baek, D., Koo, Y.D., Jin, J.B., Bressan, R.A., Yun, D.-J., and Hasegawa, P.M. (2005) The Arabidopsis SUMO E3 ligase SIZ1 controls phosphate deficiency responses, Proceedings of the National Academy of Sciences USA 102, 7760–7765. Morcuende, R., Bari, R., Gibon, Y., Zheng, W., Pant, B.D., Bläsing, O., Usadel, B., Czechowski, T., Udvardi, M.K., Stitt, M. and Scheible, W.-R. (2007) Genome-wide reprogramming of metabolism and regulatory networks of Arabidopsis in response to phosphorus, Plant, Cell and Environment 30, 85–112. Muchhal, U.S., Pardo, J.M., and Raghothama, K.G. (1996) Phosphate transporters from the higher plant Arabidopsis thaliana, Proceedings of the National Academy of Sciences USA 93, 10519–10523. Mudge, S.R., Rae, A.L., Diatloff, E., and Smith, F.W. (2002) Expression analysis suggests novel roles for members of the Pht1 family of phosphate transporters in Arabidopsis, The Plant Journal 31, 341–353. Müller, R., Morant, M., Jarmer, H., Nilsson, L., and Nielsen, T.H. (2007) Genome-wide analysis of the Arabidopsis leaf transcriptome reveals interaction of phosphate and sugar metabolism, Plant Physiology 143, 156–171. Mukatira, U.T., Liu, C., Varadarajan, D.K., and Raghothama, K.G. (2001) Negative regulation of phosphate starvation-induced genes, Plant Physiology 127, 1854–1862. Nagy, R., Vasconcelos, M.J.V., Zhao, S., McElver, J., Bruce, W., Amrhein, N., Raghothama, K.G., and Bucher, M. (2006) Differential regulation of five Pht1 phosphate transporters from maize (Zea mays L.), Plant Biology 8, 186–197. Nagy, R., Karandashov, V., Chague, V., Kalinkevich, K., Tamasloukht, M.B., Xu, G., Jakobsen, I., Levy, A.A., Amrhein, N., and Bucher, M. (2005) The characterization of novel mycorrhiza-specific phosphate transporters from Lycopersicon esculentum and Solanum tuberosum uncovers functional redundancy in symbiotic phosphate transport in solanaceous species, The Plant Journal 42, 236–250. Nakamura, Y., Awai, K., Masuda, T., Yoshioka, Y., Takamiya, K.-i., and Ohta, H. (2005) Novel phosphatidylcholine-hydrolyzing phospholipase C induced by phosphate starvation in Arabidopsis, The Journal of Biological Chemistry 280, 7469–7476. Neumann, G., Massonneau, A., Langlade, N., Dinkelaker, B., Hengeler, C., Römheld, V., and Martinoia, E. (2000) Physiological aspects of cluster root function and development in phosphorus-deficient white lupin (Lupinus albus L.), Annals of Botany 85, 909–919. Niklas, K.J. (2008) Carbon/nitrogen/phosphorus allometric relations across species, in The Ecophysiology of Plant-Phosphorus Interactions (Eds. White, P.J. and Hammond, J.P., Springer, Dordrecht, The Netherlands, pp. 9–30. Nilsson, L., Müller, R., and Nielsen, T.H. (2007) Increased expression of the MYB-related transcription factor, PHR1, leads to enhanced phosphate uptake in Arabidopsis thaliana, Plant, Cell and Environment 30, 1499–1512. Ohta, H., Awai, K., and Takamiya, K. (2000) Glyceroglycolipids of photosynthetic organisms. Their biosynthesis and evolutionary origin, Trends Glycoscience Glycotechnology 12, 241–253.

GENETIC DETERMINANTS OF PHOSPHATE USE EFFICIENCY IN CROPS

163

Okumura, S., Mitsukawa, N., Shirano, Y., and Shibata, D. (1998) Phosphate transporter gene family of Arabidopsis thaliana, DNA Research 5, 261–269. Pant, B.D., Buhtz, A., Kehr, J., and Sheible, W.-R. (2008) MicroRNA399 is a long-distance signal for the regulation of plant phosphate homeostasis, The Plant Journal 53, 731–738. Park, M.R., Baek, S.-H., De los Reyes, B.G., and Yun, S.J. (2007) Overexpression of a high-affinity phosphate transporter gene from tobacco (NtPT1) enhances phosphate uptake and accumulation in transgenic rice plants, Plant and Soil 292, 259–269. Paszkowski, U., Kroken, S., Roux, C., and Briggs, S.P. (2002) Rice phosphate transporters include an evolutionarily divergent gene specifically activated in arbuscular mycorrhizal symbiosis, Proceedings of the National Academy of Sciences USA 99, 13324–13329. Pathak, R.R., Ahmad, A., Lochab, S., and Raghuram, N. (2008) Molecular physiology of plant nitrogen use efficiency and biotechnological options for its enhancement, Current Science 94, 1394–1403. Peleg, Y., Aramayo, R., Kang, S., Hall, J.G., and Metzenberg, R.L. (1996) NUC-2, a component of the phosphate-regulated signal transduction pathway in Neurospora crassa, is an ankyrin repeat protein, Molecular Genetics and Genomics 252, 709–716. Pérez-Torres, C.-A., López-Bucio, J., Cruz-Ramírez, A., Ibarra-Laclette, E., Dharmasiri, S., Estelle, M., and Herrera-Estrella, L. (2008) Phosphate availability alters lateral root development in Arabidopsis by modulating auxin sensitivity via a mechanism involving the TIR1 auxin receptor, The Plant Cell 20, 3258–3272. Pinstrup-Andersen, P., Pandya-Lorch, R., and Rosegrant, M.W. (1999) World food prospects: Critical issues for the early twentyfirst century. 2020 Vision Food Policy Report (Ed International Food Policy Research Institute, IFPRI), pp. 5–32. Washington, DC. Plaxton, W.C. (2004) Plant response to stress: Biochemical adaptations to phosphate deficiency. Encyclopedia of Plant and Crop Science, 976–980. Plaxton, W.C. and Carswell, M.C. (1999) Metabolic aspects of the phosphate starvation response in plants, in Plant responses to environmental Stress: From Phytohormones to genome reorganization. (Ed. H.R. Lerner), New York, pp. 349–372. Poirier, Y. and Bucher, M. (2002) Phosphate transport and homeostasis in Arabidopsis, in The Arabidopsis book (Eds. C.R. Somerville and E.M. Meyerowitz), American Society of Plant Biologists, Rockville, MD, USA, doi/10.1199/tab.0072, http:// www.aspb.org/publications/Arabidopsis/. Poirier, Y., Thoma, S., Somerville, C., and Schiefelbein, J. (1991) A mutant of Arabidopsis deficient in xylem loading of phosphate, Plant Physiology 97, 1087–1093. Rae, A.L., Cybinski, D.H., Jarmey, J.M., and Smith, F.W. (2003) Characterization of two phosphate transporters from barley; evidence for diverse function and kinetic properties among of the Pht1 family, Plant Molecular Biology 53, 27–36. Raghothama, K.G. (1999) Phosphate acquisition, Annual Review of Plant Physiology and Plant Molecular Biology 50, 665–93. Rausch, C., Zimmermann, P., Amrhein, N., and Bucher, M. (2004) Expression analysis suggests novel roles for the plastidic phosphate transporter Pht2;1 in auto- and heterotrophic tissues in potato and Arabidopsis, The Plant Journal 39, 13–28. Rausch, C. and Bucher, M. (2002) Molecular mechanisms of phosphate transport in plants, Planta 216, 23–27. Raven, J.A. (2008) PHOSPHORUS AND THE FUTURE, in The Ecophysiology of plant-phosphorus interactions (Eds. P.J. White and J.P. Hammond), Springer, Dordrecht, The Netherlands, pp. 271–283. Reymond, M., Svistoonoff, S., Loudet, L., Nussaume, L., and Desnos, T. (2006) Identification of QTL controlling root growth response to phosphate starvation in Arabidopsis thaliana, Plant, Cell and Environment 29, 115–125. Richardson, A.E., Hadobas, P.A., and Hayes, J.E. (2001) Extracellular secretion of Aspergillus phytase from Arabidopsis roots enables plants to obtain phosphorus from phytate, The Plant Journal 25, 641–649. Rosegrant, M.W. and Cline, S. (2003) Global food security: Challenge and policies, Science 302, 1917–1918. Rosegrant, M.W., Meijer, S., and Cline, S.A. (2002) International Model for Policy Analysis of Agricultural Commodities and Trade (IMPACT): Model description, (Ed. International Food Policy Research Institute, IFPRI), Washington, DC. Rosegrant, M.W., Paisner, M.S., Meijer, S., and Witcover, J. (2001) 2020 Global Food Outlook; Trends, Alternatives, and Choices. A 2020 Vision for Food, Agriculture, and the Environment Initiative. (Ed. International Food Policy Research Institute), Washington, DC. Rubio, V., Linhares, F., Solano, R., Martín, A.C., Iglesias, J., Leyva, A., and Paz-Ares, J. (2001) A conserved MYB transcription factor involved in phosphate starvation signaling both in vascular plants and in unicellular algae, Genes and Development 15, 2122–2133. Sánchez-Calderón, L., López-Bucio, J., Chacón-López, A., Gutiérrez-Ortega, A., Hernández-Abreu, E., and Herrera-Estrella, L. (2006) Characterization of low phosphorus insensitive mutants reveals a crosstalk between low phosphorus-induced determinate root development and the activation of genes involved in the adaptation of Arabidopsis to phosphorus deficiency, Plant Physiology 140, 879–889.

164

GENES FOR CROP ADAPTATION TO POOR SOIL

Sánchez-Calderón, L., López-Bucio, J., Chacón-López, A., Cruz-Ramírez, A., Nieto-Jacobo, F., Dubrovsky, J.G., and HerreraEstrella, L. (2005) Phosphate starvation induces a determinate developmental program in the roots of Arabidopsis thaliana, Plant, Cell and Environment 46, 174–184. Schachtman, D.P., Reid, R.J., and Ayling, S.M. (1998) Phosphorus uptake by plants: From soil to cell, Plant Physiology 116, 447–453. Scherr, S.J. (1999) Soil degradation. A threat to developing-country food security by 2020? Food, Agriculture, and the Environmental Discussion Paper 27. (Ed International Food Policy Research Institute), Washington, DC. Schiefelbein, J., Kwak, S-H., Wieckowski, Y., Barron, C., and Bruex, A. (2009) The gene regulatory network for root epidermal cell-type pattern formation in Arabidopsis, Journal of Experimental Botany doi:10.1093/jxb/ern339. Schümann, P.H.D., Richardson, A.E., Vickers, C.E., and Delhaize, E. (2004) Promoter analysis of the barley Pht1;1 phosphate transporter gene identifies regions controlling root expression and responsiveness to phosphate deprivation, Plant Physiology 136, 4205–4214. Schünmann, P.H.D., Richardson, A.E., Smith, F.W., and Delhaize, E. (2004) Characterization of promoter expression patterns derived from the Pht1 phosphate transporter genes of barley (Hordeum vulgare L.), Journal of Experimental Botany 55, 855–865. Shenoy, V.V. and Kalagudi, G.M. (2005) Enhancing plant phosphorus use efficiency for sustainable, Biotechnology Advances 23, 501–513. Shimizu, A., Kato, K., Komatsu, A., Motomura, K., and Ikehashi, H. (2008) Genetic analysis of root elongation induced by phosphorus deficiency in rice (Oryza sativa L.): Fine QTL mapping and multivariate analysis of related traits, Theoretical and Applied Genetics 117, 987–996. Shimizu, A., Yanagihara, S., Kawasaki, S., and Ikehashi, H. (2004) Phosphorus deficiency-induced root elongation and its QTL in rice (Oryza sativa L.), Theoretical and Applied Genetics 109, 1361–1368. Shin, H., Shin, H.-S., Chen, R., and Harrison, M.J. (2006) Loss of At4 function impacts phosphate distribution between the roots and the shoots during phosphate starvation, The Plant Journal 45, 712–726. Shin, H., Shin, H.-S., Dewbre, G.R., and Harrison, M.J. (2004) Phosphate transport in Arabidopsis: Pht1;1 and Pht1;4 play a major role in phosphate acquisition from both low- and high-phosphate environments, The Plant Journal 39, 629–642. Singh, S.P., Terán, H., Muñoz, C.G., Osorno, J.M., Takegami, J.C., and Thung, M.D.T. (2003) Low soil fertility tolerance in landraces and improved common bean genotypes, Crop Science 43, 110–119. Smith, S.E., Smith, F.A., and Jakobsen, I. (2003) Mycorrhizal fungi can dominate phosphate supply to plants irrespective of growth responses, Plant Physiology 133, 16–20. Smith, F.W., Ealing, P.M., Dong, B., and Delhaize, E. (1997) The cloning of two Arabidopsis genes belonging to a phosphate transporter family, The Plant Journal 11, 83–92. Somerville, C., Browse, J., Jaworski, J.G., and Ohlrogge, J.B. (2000) Lipids. In Biochemistry and Molecular Biology of Plants (Eds. B.B. Buchanan, W. Gruissem, and R.L. Jones), Rockville, MD, pp. 456–527. Spain, B.H., Koo, D., Ramakrishnan, M., Dzudzor, B., and Colicelli, J. (1995) Truncated forms of a novel yeast protein suppress the lethality of a G protein a subunit deficiency by interacting with the b subunit, The American Society for Biochemistry and Molecular Biology 270, 25435–25444. Staehelin, L.A. and Newcomb, E.H. (2000) Membrane structure and membranous organelles. In Biochemistry and Molecular Biology of Plants (Eds. B.B. Buchanan, W. Gruissem, and R.L. Jones), ASPP, Rockville, MD, pp. 2–50. Stefanovic, A., Ribot, C., Rouached, H., Wang, Y., Chong, J., Belbahri, L., Delessert, S., and Poirier, Y. (2007) Members of the PHO1 gene family show limited functional redundancy in phosphate transfer to the shoot, and are regulated by phosphate deficiency via distinct pathways, The Plant Journal 50, 982–994. Svistoonoff, S., Creff, A., Reymond, M., Sigoillot-Claude, C., Ricaud, L., Blanchet, A., Nussaume, L., and Desnos, T. (2007) Root tip contact with low-phosphate media reprograms plant root architecture, Nature Genetics 39, 794–798. Syers, J.K., Johnston, A.E., and Curtis, D. (2007) Efficiency of soil and fertilizer phosphorus use: Reconciling changing concepts of soil phosphorus behaviour with agronomic information. (Ed. FAO). Vienna. Takabatake, R., Hata, S., Taniguchi, M., Kouchi, H., Sugiyama, T., and Izui, K. (1999) Isolation and characterization of cDNAs encoding mitochondrial phosphate transporters in soybean, maize, rice, and Arabidopsis, Plant Molecular Biology 40, 479–486. Ticconi, C.A. and Abel, S. (2004) Short on phosphate: Plant surveillance and countermeasures, Trends in Plant Science 9, 548–555. Ticconi, C.A., Delatorre, C.A., Lahner, B., Salt, D.E., and Abel, S. (2004) Arabidopsis pdr2 reveals a phosphate-sensitive checkpoint in root development, The Plant Journal 37, 801–814. Timsina, J. and Connor, D.J. (2001) Productivity and management of rice-wheat cropping systems: Issues and challenges, Field Crops Research 69, 93–132.

GENETIC DETERMINANTS OF PHOSPHATE USE EFFICIENCY IN CROPS

165

Tittarelli, A., Milla, L., Vargas, F., Morales, A., Neupert, C., Meisel, L.A., Salvo-G, H., Peñaloza, E., Muñoz, G., Corcuera, L.J., and Silva, H. (2007) Isolation and comparative analysis of the wheat TaPT2 promoter: Identification in silico of new putative regulatory motifs conserved between monocots and dicots, Journal of Experimental Botany 58, 2573–2582. Underwood, B.A. (2000) Overcoming micronutrient deficiencies in developing countries: Is there a role for agriculture, Food Nutrition Bulletin 21, 356–360. Valdés-López, O., Arenas-Huertero, C., Ramírez, M., Girard, L., Sánchez, F., Vance, C.P., Reyes, J.L., and Hernández, G. (2008) Essential role of MYB transcription factor: PvPHR1 and microRNA: PvmiR399 in phosphorus-deficiency signalling in common bean roots, Plant, Cell and Environment 31, 1834–1843. Vance, C.P., Uhde-Stone, C., and Allan, D.L. (2003) Phosphorus acquisition and use: Critical adaptations by plants for securing a nonrenewable resource, New Phytologist 157, 147–447. Veljanovski, V., Vanderbeld, B., Knowles, V.L., Snedden, W.A., and Plaxton, W.C. (2006) Biochemical and molecular characterization of AtPAP26, a vacuolar purple acid phosphatase up-regulated in phosphate-deprived Arabidopsis suspension cells and seedlings, Plant Physiology 142, 1282–1293. Wang, C., Ying, S., Huang, H., Li, K., Wu, P., and Shou, H. (2009) Involvement of OsSPX1 in phosphate homeostasis in rice, The Plant Journal 57, 895–904. Wang, Y., Ribot, C., Rezzonico, E., and Poirier, Y. (2004) Structure and expression profile of the Arabidopsis PHO1 gene family indicates a broad role in inorganic phosphate homeostasis, Plant Physiology 135, 400–411. Wasaki, J., Shinano, T., Onishi, K., Yonetani, R., Yazaki, J., Fujii, F., Shimbo, K., Ishikawa, M., Shimatani, Z., Nagata, Y., Hashimoto, A., Ohta, T., Sato, Y., Miyamoto, C., Honda, S., Kojima, K., Sasaki, T., Kishimoto, N., Kikuchi, S., and Osaki, M. (2006) Transcriptomic analysis indicates putative metabolic changes caused by manipulation of phosphorus availability in rice leaves, Journal of Experimental Botany 57, 2049–2059. Welch, R.M. and Graham, R.D. (2000) A new paradigm for world agriculture: productive, sustainable, nutritious, healthful food systems, Food Nutrition Bulletin 21, 361–366. Werner, T., Motyka, V., Laucou, V., Smets, R., Van Onckelen, H., and Schmülling, T. (2003) Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity, The Plant Cell 15, 2532–2550. White, P.J. and Hammond, J.P. (2008) Phosphorus nutrition of terrestrial plants, in The Ecophysiology of Plant-Phosphorus Interactions (Eds. P.J. White and J.P. Hammond), Springer, Dordrecht, The Netherlands, pp. 51–81. Williamson, L.C., Ribrioux, S.P.C.P., Fitter, A.H., and Leyser, H.M.O. (2001) Phosphate availability regulates root system architecture in Arabidopsis, Plant Physiology 126, 875–882. Wissuwa, M., Mazzola, M., and Picard, C. (2008) Novel approaches in plant breeding for rhizosphere-related traits, Plant and Soil, DOI 10.1007/s11104-008-9693-2. Wissuwa, M. and Ae, N. (2001) Genotypic variation for tolerance to phosphorus deficiency in rice and the potential for its exploitation in rice improvement, Plant Breeding 120, 43–48. Wu, P., Ma, L.G., Hou, X.L., Wang, M.Y., Wu, Y.R., Liu, F.Y., and Deng, X.W. (2003) Phosphate starvation triggers distinct alterations of genome expression in Arabidopsis roots and leaves, Plant Physiology 132, 1260–1271. Xiao, K., Katagi, H., Harrison, M., and Wang, Z.-Y. (2006) Improved phosphorus acquisition and biomass production in Arabidopsis by transgenic expression of a purple acid phosphatase gene from M. truncatula, Plant Science 170, 191–202. Yadav, R.L., Dwivedi, B.S., and Pandey, P.S. (2000) Rice-wheat cropping system: Assessment of sustainability under green manuring and chemical fertilizer inputs, Field Crops Research 65, 15–30. Yi, K., Wu, Z., Zhou, J., Du, L., Guo, L., Wu, Y., and Wu, P. (2005) OsPTF1, a novel transcription factor involved in tolerance to phosphate starvation in rice, Plant Physiology 138, 2087–2096. Zhang, D., Cheng, H., Geng, L., Kan, G., Cui, S., Meng, Q., Gai, J., and Yu, D. (2009) Detection of quantitative trait loci for phosphorus deficiency tolerance at soybean seedling stage, Euphytica DOI 10.1007/s10681-009-9880-0. Zhang, F.S., Ma, J., and Cao, Y.P. (1997) Phosphorus deficiency enhances root exudation of low-molecular weight organic acids and utilization of sparingly soluble inorganic phosphates by radish (Rhapanus sativus L.) and rape (Brassica napus L.), Plant and Soil 196, 261–264. Zhou, J., Jiao, F., Wu, Z., Li, Y., Wang, X., He, X., Zhong, W., and Wu, P. (2008) OsPHR2 is involved in phosphate-starvation signaling and excessive phosphate accumulation in shoots of plants, Plant Physiology 146, 1673–1686. Zhu, J., Kaeppler, S.M., and Lynch, J.P. (2005) Mapping of QTL controlling root hair length in maize (Zea mays L.) under phosphorus deficiency, Plant and Soil 270, 299–310. Zhu, J. and Lynch, J.P. (2004) The contribution of lateral rooting to phosphorus acquisition efficiency in maize (Zea mays) seedlings, Functional Plant Biology 31, 949–958.

7

Genes for Use in Improving Nitrate Use Efficiency in Crops David A. Lightfoot

Introduction

Nitrogen (N) is a major factor in plant growth and crop yield (Marschner, 1995). The growth and development of plants are often profoundly affected by the form and abundance of the nitrogen supply because the form of nitrogen significantly alters intracellular metabolisms. Restricted or inappropriate nitrogen supply or form alters development including shoot-to-root ratio, root development, seed development, and the rate of senescence. Activities of enzymes of primary metabolism respond to N supply, but so do the enzymes of photosynthesis, secondary metabolism, and metabolic control (Figure 7.1). Since N fertilizers are rapidly depleted from most soil types and symbiotic nitrogen fixation in many legumes ceases in mid-season, all field crops have some degree of dependence on organic or inorganic nitrogenous fertilizer (Marschner, 1995). Nitrogen must often be supplied to the soils surrounding growing crops in excess, before or during the growing season. Nitrogen that is not assimilated can contaminate the environment (Cherfas, 1990; Burkholder et al., 1992; David et al., 1997) and have negative effects on human health in food and water (Tannenbaum et al., 1978; Mirvish, 1985; Moller, 1990; Duncan et al., 1998). Therefore, nitrogen use by crops has to be optimized. Nitrogen-use efficiency (NUE) is the metric commonly targeted for improvement by breeders and biotechnologists in this field (Lightfoot et al., 2007, 2008). Enhanced NUE by plants should also enable crops to be cultivated under low nitrogen availability, slow release fertilizers, stress conditions, or poor soil quality (Hirel et al., 2007). NUE is defined as the percentage of fertilizer N recovered by a crop. NUE is the product of many components (Moll et al., 1982). On a coarse scale NUE is determined by nitrogen productivity (NP) and the mean residence time of nitrogen (MRT). In turn they are expressions of (1) N uptake efficiency; (2) the fraction of N translocated to the seed; (3) the translocation index; (4) soil supply efficiency; and (5) developmental efficiency. Consequently, on a fine scale, thousands of genes and hundreds of regulatory networks contribute to NUE from seed germination to final harvest in the plant and hundreds more to the microbial activity in the soil. Improvement of cereal NUE could enable practices directed toward reducing groundwater contamination by nitrates in Illinois and the USA (Lightfoot et al., 1999; Ameziane et al., 2000). The decreased undesirable environmental effects and reduced dietary nitrates could decrease several human and animal health problems. For brevity this chapter focuses on genes and regulatory networks with major effects on NUE with an emphasis on patented technologies near commercialization and pending patents. Genes for Plant Abiotic Stress Editors Matthew A. Jenks and Andrew J. Wood © 2010 Blackwell Publishing ISBN: 978-0-813-81502-2

167

168 Threonine

Lysine

Valine

Leucine

Serine

Fumarate

Malate

Tyrosine

Oxalosuccinate

Isocitrate

Microbial activity, NO3 NH4

Alpha-Ketoglutarate Succinate

Shikimate

Ornithine

Citrulline

Arginine

Anthranilate

Proline

Glutamate

Glutamine

Histidine

16 Fatty acids

Alanine

DAHP

9 Amines

Tryptophan

Chorismate

Phenylalanine

5 Amines

Prephenate Erythrose 4phosphate

Citrate

Pyruvate

Oxaloacetate

Acetyl-CoA

Pyruvate

Phosphoenolpyruvate

6-phosphoglycerate

Dihydroxyacetone-P

Glycealdehyde-3-P

Fructose 1,6-bisphosphate

Fructose phosphate

Glucose

Figure 7.1 Metabolite changes related to NUE in GDH transgenic roots. Metabolites in blue boxes were not detected. Metabolites in red boxes were used as internal standards and therefore detected. Metabolites in blue boxes were increased 2–3 fold. Metabolites in black boxes were detected and not changed. Metabolites in green boxes were decreased. For color detail, please see color plate section.

Methionine

Cystathionine

Homoserine

Isoleucine

Aspartate

Glycine

Cysteine

O-Acetylserine

Aspartate 4-semialdehyde

0

1–2

2–3

3–4

4–5

5–10

10–15

Asparagine

Low

High

Fold Increase

GENES FOR USE IN IMPROVING NITRATE USE EFFICIENCY IN CROPS

169

The Two Forms of NUE: Regulation of Nitrogen Partitioning and Yield in Crops

Ironically, the biochemical basis of the regulation of nitrogen uptake, partitioning, and the tolerance of high concentrations are not well understood (von Wiren et al., 2000; Coruzzi and Bush 2001). However, genetically and phenotypically carbon and nitrogen partitioning and yield are clearly interrelated (Limami et al., 1999; Specht et al., 1999). Nitrogen use efficiency (NUE) is one expression of this coordinated regulation. NUE under N limitation is the ability to yield well with the available resources. NUE under excess fertilizer N is the ability to assimilate all available N. The genes underlying both forms of NUE are very different; on the one hand assimilation will be cardinal and the other regulation will be key. The lack of satisfactory cell free assays and easily quantifiable substrate changes have hindered progress in understanding the molecular biology of nitrogen regulation, in contrast to nitrogen assimilation where these assays are available (Kaiser et al., 1998; Lightfoot et al., 1999). However, the whole plant responses to nitrogen limitation and excess are clear and easy to score (Moll et al., 1982; Doehlert et al., 1991; Marschner 1995). These phenotypic changes include several that are very clear, like plant size, leaf chlorosis, and early senescence. Such phenotypes can be used in a genetic method to isolate the important genes underlying NUE and derive an understanding of their function and biochemistry.

Mutants as Tools to Isolate Important Plant Genes

Gene function identification by mutagenesis of A. thaliana is an established protocol, and hundreds of mutants in a variety of plant processes have been defined, mapped, and genes isolated (Huala et al., 2001). Recently the development of TILLING programs for many crops (Cooper et al., 2008) promise an abundant supply of new mutants. Many mutants in mineral nutrition, root development, and disease resistance have been isolated and mapped in Arabidopsis. Gene isolation by positional cloning has been reported for many mutated genes. However, mutants of relevance to NUE are rare, only the assimilation enzymes, gluR and gsr1 lesions have been reported (Table 7.1). Reviewing these will be informative. Mutants in GS and GOGAT are lethal because of the fluxes through photorespiration (Coruzzi et al., 1999). Mutants in the mitochondria located NADH-dependent GDH enzymes (there are two) tend to be less resistant to abiotic stresses in both A. thaliana and Zea mays (Pryor, 1990; Melo-Oliveira et al., 1996; Miyashita and Good 2008). Mutants in the NADPH GDH enzymes found in plant plastids and cytosol do not have clear phenotypes (Hershey et al., 2009). Mutants in aspartate and asparagine synthesis and transport are deleterious, underlining the role in transport and storage (Brears et al., 1993; Coruzzi et al., 1999). Mutants in nitrite reductase are lethal because nitrite is very toxic. In contrast mutants in nitrate reductase are either neutral or can increase NUE (Oh et al., 1980). Barley with 10% of the usual NR activity was just as productive as the wild type. This phenomenon has been repeated in other plant species. Nitrate reductase, it seems, is produced in massive excess to a well-fertilized plant’s needs, probably a holdover from the predomesticated era. Reducing the metabolic load of unnecessary protein production can increase NUE. Two regulatory mutants were well characterized. Gsr1 was a lesion that caused increased susceptibility to methylamine (Meyer, 2007). Different nitrogen sources provided different degrees of protection from the toxicity of methylamine. Glutamine was more protective than

170

Fd-GOGAT – Ferredoxin dependent glutamate synthase

GS2 – Chloroplastic Glutamine synthetase

AMT – Ammonium transporters

NR – Nitrate reductase isoform 2 NiR – Nitrite reductase

NR – Nitrate reductase isoform 1

O. sativa N. tabacum N. tabacum N. tabacum

A. thaliana

CaMV 35S Rbc SSU1 CaMV 35S CaMV 35S

CaMV 35S

S. oleracea

O. sativa N. tabacum P. sativa N. tabacum

CaMV 35S

N. tabacum

Zea mays G. max N. tabacum

NMS mutation RAB17 CaMV 35S CaMV 35S

H. vulgaris Porphyra sp. N. tabacum N. tabacum

L. sativa N. plum … ifolia H. vulgaris Z. mays S. tuberosum N. plum … ifolia A. thaliana

RAB17 UBI CaMV 35S

CaMV 35S CaMV 35S

plumbaginifolia N. tabacum N. tabacum

A. thaliana Z. mays O. sativa

CaMV 35S

N.

N. tabacum

N. tabacum

CaMV 35S,rol D

N. plumbaginifolia

A. thaliana

CaMV 35S

A. thaliana

Target plant

Nrt1.1 – High affinity nitrate transporter 1 Nrt2.1 – High affinity nitrate transporter 2

Promoter(s)

Gene source

Gene and paralog/mutant allele number

No phenotype, with just 10% of NR activity Improved NUE, yield with N limitation Reduced nitrate levels Increased NiR activity, no phenotypic difference Increased NiR activity, no phenotypic difference Higher NiR activity, higher nitrite accumulation Increased NUE Increased NUE Improved photorespiration capacity, and increased resistance to photo-oxidation Enhanced photorespiration, salt tolerance Enhanced growth rate Co-suppression reduced growth rate Diurnal changes in NH3 assimilation

Reduced nitrate content, chlorate sensitivity Nitrite accumulation in high nitrate supply

Increase in constitutive nitrate uptake but not the induced form Increased nitrate influx under low N conditions 3–4 fold drop in NR protein and activity, no change in NR transcript Increased NR activity, biomass, drought stress

Phenotype observed

Hoshida et al. (2000) Chichkova et al. (2001) Migge et al. (2000) Ferrario-Mery et al. (2000)

Loussaert et al. (2008) Loussaert et al. (2008) Kozaki & Takeba (1996)

Takahashi et al. (2001)

Crete et al. (1997)

Oh et al. (1980) Loussaert et al. (2008) Djannane et al. (2002) Crete et al. (1997)

Curtis et al. (1999) Lea et al. (2004)

Ferrario-Mery et al. (1998)

Fraisier et al. (2000) Vincentz & Caboche (1991)

Liu et al. (1999)

Table 7.1 Selected transgenic and mutant lines with effects on N transport, primary N assimilating genes and secondary N metabolism (adapted from Pathak et al., 2008).

171

GDH2 AS1 – Glutamine dependent asparagine synthetase

GDH1

A. thaliana T. aestivum Z. mays L. esculentum A. thaliana L. esculentum

CaMV 35S CaMV 35S CaMV 35S CaMV 35S CaMV 35S CaMV 35S::MIT 2A11

E. coli C. sorokiana C. sorokiana A. nidulans A. thaliana L. esculentum lys/ala90 CaMV 35S CaMV 35S

Z. mays

OsUBI

E. coli

A. thaliana P. sativum

N. tabacum

CaMV 35S

E. coli

A. thaliana N. tabacum

S. tuberosum

O. sativa M. sativa

O. sativa CaMV 35S

P. sativum L. japonicas Populus sp.

CaMV 35S CaMV 35S CaMV 35S

G. max M. sativa P. sylvestris O. sativa M. sativa

N. tabacum L. corniculatus L. japonicus T. aestivum N. tabacum N. tabacum M. sativa N. tabacum

CAMV35S CaMV 35S rol D Rbc SSU1 CaMV 35S CaMV 35S CaMV 35S CaMV 35S

P. vulgaris G. max G. max P. vulgaris M. sativa M. sativa G. max P. sativa

NADH-GOGAT– NADH-dependent glutamate synthase GDH – Glutamate dehydrogenase gdhA

N. tabacum

CAMV35S::MITATPase

P. vulgaris

Target plant

GS1 – Cytosolic glutamine synthetase

Promoter(s)

Gene source

Gene and paralog/mutant allele number

Enhanced seed protein Slightly increased biomass and increased level of free asparagines, plants required N source

Growth inhibited, insoluble GS in mitochondria Inceased herbicide tolerance Accelerated senescence Decrease in biomass Enhanced capacity to accumulate nitrogen Enhanced growth under N starvation Herbicide tolerance No increase in GS activity Enhanced growth, leaf-soluble protein, ammonia levels No change in whole plant N Higher biomass and leaf proreins Enhanced growth rate, leaf chlorophyll, total soluble protein Enhanced grain filling, increased grain weight Higher total C and N content, increased dry weight Increased biomass, nutritional value N assim., NUE, WUE, herbicide tolerance, amino acid and sugar content Increased N assimilation, biomass and sugar content herbicide tolerance, grain biomass, amino acids Increased biomass and herbicide tolerance Increased biomass Increased biomass Higher aminacid concentrstions Increased abiotic stress tolerance Twice GDH activity, higher mRNA abundance and twice glutamate concentration

Phenotype observed

Continued

Lam et al. (2003) Harrison et al. (2003)

Nadzan et al. (2007)

Lightfoot (unpublished) Schmidt & Miller (2009) Schmidt & Miller (2009) Kisaka & Kida (2005) Coruzzi et al. (1999) Kisaka et al. (2007)

Ameziane et al. (2000) Mungur et al (2005, 2006) Lightfoot et al. (1999) Lightfoot et al. (2007) Lightfoot et al. (1999)

Fei et al. (2003) Suarez et al. (2003) Gallardo et al (1999) Manetal (2005) Yamaya et al. (2002) Shoenbeck (2000)

Hemon et al. (1990) Vincent et al. (1997) Limami et al. (1999) Habash et al. (2001) Fuentes et al. (2001) Donn et al. (1984) Ortega et al. (2001) Oliveira et al. (2002)

Hemon et al. (1990)

172 Zea mays

A. thaliana

Dof1 – Transcription factor

bZIP – Transcription factor MYB – Transcription factor glycosyl hydrolase family 9

HAP3 – Transcription factor and interacting 14 : 3 : 3 proteins

Zinc Finger C3HC4 NF-YB Plant nuclear factor Y A. thaliana Z. mays A. thaliana

A. thaliana

CaMV 35S OsRACT CaMV 35S

CaMV 35S

A. thaliana Z. mays Z. mays G. max G. hirsutum

A. thaliana

A. thaliana

A. thaliana

CaMV 35S

A. thaliana

ANR1 – MADS transcription factor GLB1 – PII regulatory protein 35S C4PDK

Z. mays G. max A. thaliana

OsACT1 AtACT7 CaMV 35S

E. coli

POT – Proton-dependent oligopeptide transport (PTR class) HMP – Flavohemoglobin

A. thaliana A. thaliana Zea mays A. thaliana

mutants RAB17 RAB17 CaMV 35S Null mutant

A. thaliana A. thaliana Z. mays A. thaliana

iGluR – Glutamate receptors

B. napus S. bicolor O. sativa

btg26

H. vulgaris OsANT1

N. tabacum

CaMV 35S

S. bicolor

AspAT – Mitochondrial aspartate aminotransferase AlaAT – Alanine aminotransferase

Target plant

Promoter(s)

Gene source

Continued

Gene and paralog/mutant allele number

Table 7.1

Improved Improved Improved Improved Improved

NUE and WUE NUE and WUE NUE and seed yield NUE and WUE NUE, WUE and yield

Improved NUE, plant size, vegetative growth, growth rate, light inducible seedling vigor and/or biomass

Growth rate, increased anthocyanin production in low N Enhanced growth rate under N limited conditions, increase in amino acid content

decreased NO, increased NUE, biomass, chlorophyll and yield in the field Lateral root induction and elongation

Yields constant to 50% less N fertilizer Increased NUE Excess N increased biomass, grain yield metabolites and total nitrogen content, indicating increased nitrogen uptake efficiency. Growth inhibited or enhanced, kainate resistant Increased NUE Increased NUE and yield Increased NUE, biomass and protein in seed, MSX resistance in mutants

Increased AspAT, PEPCase activity

Phenotype observed

Dotson et al. (2009)

Nelsen et al. (2007)

Nadzan et al. (2007)

Yanagisawa et al. (2004)

Zhang et al. (2003)

Zhang and Forde (1998)

Basra et al. (2007)

Dotson et al. (2009) Schneeberger et al. (2008)

Coruzzi et al. (1998)

Good et al. (2007)

Sentoku et al. (2000)

GENES FOR USE IN IMPROVING NITRATE USE EFFICIENCY IN CROPS

173

Methylamine GS

activity

Methylglutamine signal

perturbation

nitrogen starvation responses Figure 7.2 The model for metabolism perturbation inferred from responses to methylamine of wild-type and sensitive mutant gsr1 of Arabidopsis. Altered were chloroplast function, proton motive force (PMF), nitrogen metabolism gene transcript abundances, enzyme activities including glutamine synthetase (GS), and responses to exogenous nitrogen and carbon (Meyer et al., 2006).

glutamate, nitrate, and nitrate mixed with ammonium more than ammonium. The lesion in gsr1 interfered with photosynthesis emphasizing the control of C metabolism by the plant’s N status. The lesion appears to map to a region of chromosome 5 encoding an amine oxidase suggesting a mechanism for the lesion in detoxification. The gene underlying a methylamine-resistant mutant in tobacco has not yet been determined but appears to be related to nitrate assimilation or transport and shoot root partitioning (Godon et al., 1996). Lesions in putative glutamate receptors are known (Coruzzi et al., 1998; Hershey et al., 2009). These lesions provide alterations in growth and NUE as well as tolerance of second messaging inhibitors like Kadaic acid. See Figure 7.2. Several transporters provide for alterations in NUE because all forms of N are accumulated against a steep concentration gradient (Table 7.1). Ammonium in particular is hard to accumulate, and may pass in and out of cells several times before assimilation is successful. Consequently lesions in ammonium and nitrate transport are deleterious and do not improve NUE (Liu et al., 1999; Rawat et al., 1999; Gupta et al., 2008). The lesion in proton-dependent peptide transport is different (Schneeberger et al., 2008). Resistance to herbicides targeted to the enzyme GS-like MSX is provided by the lack of these transporters and NUE can be increased. These transporters will be particularly important to assimilating the fertilizer N from root-associated microbes (Garcia et al., 2009).

174

GENES FOR CROP ADAPTATION TO POOR SOIL

Transcript Analysis

It has been discovered that large numbers of genes are involved in processes like NUE by clustering during microarrays in crop plants. To obtain functional information on these genes, efficient expression monitoring methods have been developed (Wang et al., 2000; Meyer et al., 2006; Goldman et al., 2009). Rapid and simultaneous differential expression analysis of independent biological samples indicates activity. Using the expression profiles, gene regulation perturbations in transgenic and mutant plants can be monitored and function inferred providing a central platform for plant functional genomics. However, microarrays of relevance to NUE are rare; only gsr1 and a few enzyme lesions have been reported. Transcript abundance measurements have led to the patenting of sets of protein families (Pfams) implicated in NUE. For example, Goldman and others (2009) have applied for a patent for 157 protein families implicated in NUE by microarray then tested for activity in transgenics with one or more positive results from assays of NUE, WUE, yield, enhanced tolerance to salt, cold, and heat, and enhanced level of oil and/or protein in seed. Schnable and Dash (2008) found a smaller number from analyzes in maize. However, it can be concluded from the findings that genes in many pathways affect NUE.

Metanomic Tools for Extending Functional Genomics

In the postgenome sequence era, the determination of gene function(s) and relationships to pathways will be the focus. Multi-parallel analysis of mRNA abundance and their protein products will suggest functions but not direct information on biological function. The multiplicity of gene interactions and metabolic network changes engineered by mutation are not always predictable, and many changes are cryptic. Metabolic profiling can link phenotype to biochemistry (Fiehn et al., 2000). The methods are fast, reliable, sensitive, and automated due to improvements in mass spectrometry (MS). However, to date they are commonly used by chemists but rarely directly applied to biological phenomena in plants (Katona et al., 1999; Adams et al., 1999 Fiehn et al., 2000; Mungur et al. 2005). A range of analytical techniques that are available for MS including gas chromatography (GC) have become suitable for analysis of complex biological samples because of the development of ionization techniques, electrospray ionization, and matrix-assisted laser desorbtion ionization (Blackstock, 2000). Electron impact quadropole mass spectrometry has proven to be a robust technology for metabolite profiling (Katona et al., 1999, Adams et al., 1999 Fiehn et al., 2000), and libraries of compound identities have been developed for plants at mass accuracy of 0.01d, often by MS-MS fragmentation. However, the mass accuracy of ion cyclotron MS in a Fourier Transformed MS format provides for mass accuracy to 0.0001d. This accuracy allows for unequivocal identification of a larger number of compounds in fewer analyzes. Figure 7.1 shows the effects of glutamate dehydrogenase (GDH) on metabolic pathways related to NUE in tobacco roots. FT-ICR-MS detected 2,012 ions reproducible in two to four ionization protocols (Mungur et al., 2005). There were 283 ions in roots and 98 ions in leaves that appeared to significantly change abundance due to the measured GDH activity. About 58% of ions could not be used to infer a corresponding metabolite. From the 42% of ions that inferred known metabolites many amino acids, organic acids, amines, and sugars increased and many fatty acids and amines decreased (Figure 7.1). These changes were profound and underlay the ability of the GDH transgene to cause increased NUE ammonium assimilation and nutritional

GENES FOR USE IN IMPROVING NITRATE USE EFFICIENCY IN CROPS

175

Table 7.2 Pfam implicated in NUE by –omics approaches. (2-oxoacid_dh, ADH_N, ADH_zinc_N, AP2, AUX_IAA, Aa_trans, Abhydrolase.sub.–1, Acyl_transf.sub.–1, Aldedh, Aldo_ ket_red, Alpha-amylase, Aminotran.sub.–1.sub.–2, Aminotran.sub.–3, Ammonium_transp, Arm, Asn_synthase, BAG, BSD, Beta_elim_lyase, Biotin_lipoyl, Brix, Bromodomain, C1.sub.–4, CTP_transf.sub.–2, Catalase, CcmH, Chal_sti_synt_C, Cyclin_C, Cyclin_N, Cys_Met_Meta_PP, DAO, DIM1, DPBB.sub.–1, DRMBL, DUF167, DUF231, DUF250, DUF6, DUF783, DUF962, E2F_TDP, E3_binding, EBP, Enolase_C, Enolase_N, F420_oxidored, FAD_binding.sub.–2, FA_ desaturase, FKBP_C, FTCD_N, Fe_bilin_red, Fer4, GAF, GATase.sub.–2, GIDA, GSHPx, Gpi16, HGTP_anticodon, HI0933_like, HLH, HMG_CoA_synt, HWE_HK, Hamlp_like, HhH-GPD, Homeobox, Hpt, Iso_dh, K-box, LEA.sub.–4, LRRNT.sub.–2, LRR.sub.–1, Ldh.sub.–1_C, Ldh.sub.–1_N, Lectin_legA, Lectin_legB, Lipase_GDSL, MFS.sub.–1, MIP, MatE, Metalloenzyme, Methyltransf.sub.–1, Methyltransf.sub.–12, Molybdop_Fe4S4, Molybdopterin, Molydop_binding, Mov34, MtN3_slv, Myb_DNA-binding, NAD_Gly3P_dh_N, NAD_binding.sub.–2, NIR_SIR, NIR_SIR_ferr, NPH3, NTP_ transferase, Nuc_sug_transp, PA, PAR1, PFK, PGI, PGK, PGM_PMM_I, PGM_PMM_II, PGM_PMM_III, PGM_PMM_IV, PP2C, PTR2, Peptidase_C26, Phi.sub.–1, Phytochrome, Pkinase, Pkinase_Tyr, Pollen_allerg.sub.–1, Pribosyltran, Proteasome, Pyr_redox, Pyr_redox.sub.–2, Pyr_redox_dim, RNA_pol_L, RNA_pol_Rpb6, RRM.sub.–1, RRN3, Radical_SAM, Ras, Response_reg, Rhodanese, Ribosomal_S8e, Rieske, SAC3_GANP, SBDS, SET, SRF-TF, SURF5, Skp1, Skp1_POZ, Ssl1, Sterol_desat, Sugar_tr, TCP, ThiF, Transaldolase, UQ_con, Ubie_methyltran, WD40, WRKY, adh_short, bZIP.sub.–1, bZIP. sub.–2, cNMP_binding, iPGM_N, p450, tRNA-synt.sub.–2b, ubiquitin, zf-A20, zf-AN1, zf-B_box, zf-C.sub.2H.sub.2, zf-C3HC4, zf-CCCH)

value (Lightfoot et al., 1999). The changes in core metabolism looked very similar to the changes reported for N-sink altered opaque mutants of Z. mays (Mungur et al., 2005). The C skeleton map of Figure 7.1 may not be the best way to look at the interactome. Two other views, the nitrogen fate map for N assimilation and the protein-protein interaction are also useful. In all three views, it is clear that N-assimilation is at the nexus of many pathways. In fact pyruvate and glutamate are at the center of metabolism being related by 2.7 metabolisms on average to the other metabolites in the cell. Still it is surprising that modest changes in glutamate concentrations in the cytoplasm had so many effects. Equally surprising was the observation that the transcriptome was not altered (Mungur, 2004). It should be expected that analysis of the proteome and metabolome promise will identify new genes useful for altering NUE that are missed by mutation, over-expression, and TA screens. Metabolic analysis was also applied to the alanine amidotransferase (AAT) transgenic plants with improved NUE (Table 7.2; Goldman et al., 2009). Rice (Oryza sativa L.) was genetically engineered by introducing a barley AlaAT (alanine aminotransferase) cDNA driven by a rice tissuespecific promoter (OsAnt1; Shrawat et al., 2008). This modification increased the biomass and grain yield significantly in comparison with control plants when plants were well supplied with nitrogen. Compared with controls, transgenic rice plants also demonstrated significant changes in key metabolites and total nitrogen content, indicating increased nitrogen uptake efficiency. Metabolites altered included many of those reported for GDH.

Transgenics Lacking A Priori Evidence for NUE

Table 7.2 summarizes some recent examples of transgenic approaches lacking a priori evidence of involvement in NUE to improving NUE. These approaches also showed that NUE and WUE are closely interrelated. The connection between C metabolism and N metabolism underlie this association. These protein family and gene lists are very interesting, being mixtures of assimilation enzyme

176

GENES FOR CROP ADAPTATION TO POOR SOIL

transport factors and esoteric proteins. Proteins like gluconases, catalases, and lyases are hard to place in relation to known pathways underlying NUE. However, the preponderance of proteins involved in TA control and protein degradation provides clues to the effect. Regulatory pathways in plants are hierarchical with about three layers (Century et al., 2008). The enzymes on the basal layer are each controlled by several middle layer regulators. In turn these are regulated by a few proteins sensitive to key environmental cues. The relationships are reciprocal to a change in a lyase that might alter the activity of a protein in a well-characterized NUE pathway. Interactome analysis promised much in this area in the next few years.

Microbial Activity

It has been known for many years that each molecule of fertilizer N applied to a field will be metabolized by six to seven different microbes before it is assimilated by the target crop plant (Marschner, 1995). Consequently crop plants have been bred to optimize the NUE given current fertilization practices and soil microbial compositions. The area of nitrification and denitrification was recently revolutionized by the identification of slow-growing Archae as major factors (De La Torre et al., 2008). Many studies of plant NUE will have to be revised in view of these unrecognized variables in experiments. See Figure 7.3. New enhanced-release fertilizers, also known as slow-release nitrogen fertilizers, are starting to be used to increase nitrogen use efficiency (NUE) and extend N availability over a growing plant season (Garcia et al., 2009). They appear to change the microbial populations in soil. Their use will provide new opportunities for the genetic improvement of crops. Methylene urea (MU), one type of slow-release nitrogen, may play a major role as an environmentally safe source of N fertilizers because of its low leaching potential. MU is used widely in industrialized countries at a rate of 220,000 metric tons per year. However, the NUE of plants depends on the biological activity of microbes in the soil, specifically, their capacity to convert organics into plant available ammonium and nitrate. The process of decomposition of the MU is driven by both biological and abiotic factors in the soil environment (Koivunen and Horwarth, 2004). Only a few species of bacteria are able to degrade MU in soil including but not limited to Ochrobactrum anthropi, Ralstonia paucula, and Agrobacterium tumefaciens. The bacteria synthesize enzymes capable of hydrolyzing MU in a nitrogen form available for the plants. The enzymes are all classified as MDUase but are different for each bacterial species and catalyze a common set of chemical reactions. MU hydrolysis leads to three different products: formaldehyde, urea, and ammonium. Formaldehyde is toxic to plants. To improve the NUE by the plants and diminish the N leached by runoff, the processes responsible for N degradation are important (Koivunen and Horwarth, 2004; De La Torre et al., 2008). Soil quality before and after the use of N fertilizers should be compared by physical, chemical, and biological methods (Garcia et al., 2009). NUE is very sensitive to specific soil characteristics as well as possible interactions and thus can reflect changes in soil quality. Biomass, community structures, and specific functions of soil microorganisms appear to be of major importance for NUE and could serve as sensitive soil quality indicators (SQI). The microbiological characteristics of a soil reflect and integrate chemical, physical, and biological soil properties over time, since microbial soil communities strongly depend on the conditions of the habitat they colonize. Therefore, microbiological characteristics of a soil may provide indicators for NUE improvements.

3.5

(H) Shannon Index

3 2.5 control 2 urea 1.5 MU 1 0.5 0 15 days

42 days

90

cm

70

50

control MU

30

urea

10

7

14

21

28

35

42

days

Figure 7.3 Microbial populations are altered by N fertilization. Panel A. Shannon indexes (H) for characterizing soil bacterial diversity in soil treated with different N sources show differences that develop over time. Panel B. The alterations contribute to plant growth. For color detail, please see color plate section.

177

178

GENES FOR CROP ADAPTATION TO POOR SOIL

Nodule Effects and Mycorrhizal Effects

A not surprising observation has been that ineffective nodules reduce legume yield during drought (Sinclair et al., 2006). What is surprising is that agronomists often use N fertilizer to inhibit nodule formation, and nodules are heavily suppressed when nitrate is present. Breeders therefore have been selecting against the nodulation trait. This practice should be discouraged. Breakthroughs in understanding of the molecular control of nodule formation have also shown that these genes are derivatives of genes altering the microbial community around roots (Zhu et al., 2006). Future studies matching root genotypes to microbial populations should provide for improved crops having massive changes in NUE.

Water Effects

Drought tolerance genes provide for greater NUE because assimilation continues longer and biomass increases (Pennisi, 2008; Lightfoot, 2008). Both the NF-YB1 gene (Nelson et al., 2007) and the gdhA gene signal the cell and plant to maintain photosynthesis when water first becomes limiting and helps photosynthesis recover fast when water again appears (rainfall). Glutamate, an amino acid implicated in signaling and homeostasis (Forde and Lea, 2007), was modified to create drought-tolerant crops, first in tobacco (Ameziane et al., 2000; Mungur et al., 2006) then in maize (Lightfoot et al., 2007). What was most interesting about the plants was that although total free amino acid concentrations doubled, glutamate did not, leading to the hypothesis that the drought tolerance was caused by signaling of sufficiency that caused an increase in compatible solutes (water holding molecules). Mungur and others (2005) showed that many hundreds of metabolites changed in abundance in shoots and roots, but no transcript abundances were altered. Therefore, the NUE/WUE technologies should not be used in geological drought prone areas.

Conclusions

Understanding of the control of NUE is at a new beginning. Surprising effects of individual protein abound showing that understanding of the processes behind NUE is preliminary. The potential for gain remains massive. Most of the gradual increase in crop yield in major well-fertilized crops is likely an expression of NUE and WUE. However, to date the improvements of NUE provided by genes like GDH and AAT remain to be commercialized. The integration of techniques made by systems biology holds the promise of new breakthroughs. Can crop yields be moved on in quantum leaps by NUE manipulation? The very large yield gains from weed control provided by herbicide resistance technologies suggest that NUE and WUE were improved in those crops. Can NUE be directly manipulated? If the soil’s microbial community is considered and properly measured, that may finally be possible. A reduction in run-off N will be of benefit for environmental health and human health. At the same time as the human population is growing toward its climax and food becomes scarcer, crops that do not compete with crops for food are needed for biofuels. Growth on marginal lands requires the second form of NUE, that is, growth with low or no inputs. Here we must reverse centuries of crop breeding on NUE in a few years. The directed manipulation of NUE must be applied to both fields if this planet is to sustain a human population of 12 billion.

GENES FOR USE IN IMPROVING NITRATE USE EFFICIENCY IN CROPS

179

References Adams, M.A., Chen, Z.L., Landman, P., and Collmer, T.D. (1999). Simultaneous determination by capillary gas chromatography of organics acids, sugars, and sugar alcohols in plant tissue extracts as their trimethylsilyl derivatives. Anal. Biochem. 266, 77–84. Ameziane, R., Bernhard, K., and Lightfoot, D.A. (2000) Expression of the Escherichia coli glutamate dehydrogenase gene in Nicotiana tabacum causes resistance to the herbicide phosphinothricin and exogenous ammonium chloride. Plant and Soil 221, 45–57. Basra, A., Edgerton, M., Lee, G.J., Lu, M., Lutfiyya, L.L., Wu, W., and Wu, X. (2007) Plants containing a heterologous flavohemoglobin gene and methods of use thereof, United States Patent Application 20070074312. Blackstock, W. (2000) Trends in automation and mass spectrometry for proteomics. Proteomics a Trends in Guide, p 12–17. Brears, T., Liu, C., Knight, T.J., and Coruzzi, G.M. (1993) Ectopic overexpression of asparagine synthetase in transgenic tobacco. Plant Physiol. 103, 1285–1290. Burkholder, J.M., Noga, E.J., Hobbs, C.H., and Glasgow, Jr., H.B. (1992). New “phantom” dinoflagellate is the causative agent of major estuarine fish kills. Nature 358, 407–410. Century, K., Reuber, T.L., and Ratcliffe, O.J. (2008) Regulating the regulators: The future prospects for transcription-factor-based agricultural biotechnology products. Plant Physiol. 147, 20–9. Cherfas, J. (1990) The fringe of the ocean—under siege from land. Science 248, 163–165. Chichkova, S., Arellano, J., Vance, C.P., and Hernandej, G. (2001) Transgenic tobacco plants that overexpress alfalfa NADH glutamate synthase have higher carbon and nitrogen content. J. Exp. Bot. 52, 2079–2087. Cooper, J.L., Till, B.J., Laport, R.G., Darlow, M.C., Kleffner, J.M., Jamai, A., El-Mellouki, T., Liu, S., Ritchie, R., Nielsen, N., Bilyeu, K.D., Meksem, K., Comai, L., and Henikoff, S. (2008) TILLING to detect induced mutations in soybean. BMC Plant Biol. 8, 9–18. Coruzzi, G. and Brears, M. (1999) Transgenic plants that exhibit enhanced nitrogen assimilation. United States Patent 5,955,651. Coruzzi, G. and Bush, D.R. (2001) Nitrogen and carbon nutrient and metabolite signaling in plants. Plant Physiol. 125, 61–64. Coruzzi, G., Oliveira, I., Lam, H.M., and Hsieh, M.H. (1998) Plant glutamate receptors. United States Patent, 5,824–867. Crete, P., Caboche, M., and Meyer, C. (1997) Nitrite reductase expression is regulated at the post-transcriptional level by the nitrogen source in Nicotiana plumbaginifolia and Arabidopsis thaliana. Plant J. 11, 625–634. Curtis, I.S., Power, J.B., and de Llat, A.A.M. (1999) Expression of chimeric nitrate reductase gene in transgenic lettuce reduces nitrate in leaves. Plant Cell Rep. 18, 889–896. David, M.B., Gentry, L.E. Kovacic, D.A., and Smith K.M. (1997) Nitrogen balance in and export from an agricultural watershed. J. Env. Qual 26, 1038–1048. De la Torre, J.R., Walker, C.B., Ingalls, A.E., Könneke, M., and Stahl, D. (2008) Cultivation of a thermophilic ammonia oxidizing archaeon synthesizing crenarchaeol. Environ Microbiol 10, 810–818. Djannane, S., Chauvin, J.E., and Meyer, C. (2002) Glasshouse behavior of eight transgenic potato clones with a modified nitrate reductase expression under two fertilization regimes. J. Exp. Bot. 53, 1037–1045. Doehlert, D. and Lambert, R.J. (1991) Metabolic Characteristics associated with starch, protein and oil deposition in developing maize kernels. Crop Sci. 31, 151–157. Donn, G., Tischer, J., Smith, A., and Goodman, H.M. (1984) Herbicide-resistant alfalfa cells: An example of gene amplification in plants. J. Mol. Appl. Genet. 2, 621–635. Dotson, S.B., Edgerton, M.D., Wu, J., Xie, Z.,, Lee, G.J., Adams, T.R., and Nelson, D.E. (2009). Transgenic plants with enhanced agronomic traits, United States Patent Application 20090049573 February 19. Duncan, D., Dykhuisen, R., Frazer, A., MacKenzie, H., Golden, M., Benjamin N., and Leifert C. (1998) Human health effects of nitrate. Gut 42, 334–340. Fei, H., Chaillou, S., Mahon, J.D., and Vessey, J.K. (2003) Overexpression of a soyabean cytosolic glutamine synthetase gene linked to organ-specific promoters in pea plants grown in different concentrations of nitrate. Planta, 216, 467–474. Fiehn, O., Kopka, J., Dormann, P., Altmann, T., Trethewey, R., and Willmitzer, L. (2000) Metabolite profiling for plant functional genomics. Nature Biotechnology 18, 1157–1161. Ferrario-Méry, S., Valadier, M.H., and Foyer, C. (1998) Overexpression of nitrate reductase in tobacco delays drought-induced decreases in nitrate reductase activity and mRNA. Plant Physiol. 117, 293–302. Ferrario-Méry, S., Valadier, M.H., Godefroy, N., Miallier, D., Hirel, B., Foyer, C.H., and Suzuki, A. (2002) Diurnal changes in ammonia assimilation in transformed tobacco plants expressing ferredoxin-dependent glutamate synthase mRNA in the antisense orientation. Plant Sci. 163, 59–67. Forde, B.G. and Lea, P.J. (2007) Glutamate in plants: metabolism, regulation, and signalling. J. Exp. Bot. 58, 2339–2358.

180

GENES FOR CROP ADAPTATION TO POOR SOIL

Fraisier, V., Gojon, A., Tillard, P., and Daniel-Vedele, F. (2000). Constitutive expression of a putative high affinity nitrate transporter in Nicotiana plumbaginifolia: Evidence for post transcriptional regulation by a reduced nitrogen source. Plant J. 23, 489–496. Fuentes, S.I., Allen, D.J., Ortiz-Lopez, A., and Hernández, G. (2001) Over-expression of cytosolic glutamine synthetase increases photosynthesis and growth at low nitrogen concentrations. J. Exp. Bot. 52, 1071–1081. Gallardo, F., Fu, J., Canton, F.R., Garcia-Gutierrez, A., Canovas, F.M., and Kirby, E.G. (1999) Expression of a conifer glutamine synthetase in transgenic poplar. Planta 210, 19–26. Garcia-Teijeiro, R., Lightfoot, D.A., and Hernandez, J.D. (2009) Effect of a modified urea fertilizer on soil microbial populations around maize (Zea mays L.) roots. Comm Soil Sci Plant Anal 40, 2152–2168. Godon, C., Krapp, A., Leydecker, M.T., Daniel-Vedele, F., and Caboche, M. (1996) Methylammonium-resistant mutants of Nicotiana plumbaginifolia are affected in nitrate transport. Mol Gen Genet. 250, 357–366. Goldman, B., Darveaux, S., Cleveland, B., Abad, J., and Sayed, M.S. (2009) Genes and uses for plant improvement. United States Patent Application 20090070897 March 12. Good, A.G. and others. (2007) Engineering nitrogen use efficiency with alanine aminotransferase. Can. J. Bot. 85, 252–262. Gupta, R., Liu, J., Dhugga, K.S., and Simmons, C.R. (2008) Manipulation of Ammonium Transporters (AMTS) to Improve Nitrogen Use Efficiency in Higher Plants. United States Patent Application 20080222753. Habash, D.Z., Massiah, A.J., Rong, H.L., Wallsgrove, R.M. and Leigh, R.A. (2001) The role of cytosolic glutamine synthetase in wheat. Ann. Appl. Biol. 2001, 138, 83–89. Harrison, J., Crescenzo, M.P., and Hirel, B. (2003) Does lowering Glutamine synthetase activity in nodules modify nitrogen metabolism and growth of Lotus japonicus L. Plant Physiol 133, 253–262. Hemon, P, Robbins, M.P., and Cullimore, J.V. (1990) Targeting of glutamine synthetase to the mitochondria of transgenic tobacco. Plant Mol Biol. 15, 895–904. Hershey, H.P., Simmons, C.R., and Loussaert, D. (2007) Genes for Enhancing Nitrogen Utilization Efficiency in Crop Plants. United States Patent Application 20070300323 December 27. Hershey, H.P., Frank, M.J., Simmons, C.R., and Turano, F.J. (2009) Glutamate receptor associated genes and proteins for enhancing nitrogen utilization efficiency in crop plants. United States Patent Application 20090025102 January 22. Hirel, B., Le Gouis, J., Ney, B., and Gallais, A. (2007) The challenge of improving nitrogen use efficiency in crop plants: Towards a more central role for genetic variability and quantitative genetics within integrated approaches. J. Exp. Bot. 58, 2369–2387. Hoshida, H., Tanaka, Y., Hibino, T., Hayashi, Y. and Tanaka, A. (2000) Enhanced tolerance to salt stress in transgenic rice that overexpresses chloroplast glutamine synthetase. Plant Mol. Biol. 43, 103–111. Huala, E., Dickerman, A.W., Garcia-Hernandez, M., Weems, D., Reiser, L., LaFond, F., Hanley, D., Kiphart, D., Zhuang, M., Huang, W., Mueller, L.A., Bhattacharyya, D., Bhaya, D., Sobral, B.W., Beavis, W., Meinke, D.W., Town, C.D., Somerville, C., and Rhee, S.Y. (2001) The Arabidopsis information resource (TAIR): a comprehensive database and web-based information retrieval, analysis, and visualization system for a model plant. Nucleic Acids Res. 29, 102–105. Kaiser, B.N., Finnegan, P.M., Tyerman, S.D., Whitehead, L.F., Bergersen, F.J., Day, D.A., and Udvardi, M.K. (1998) Characterization of an ammonium transport protein from the peribacteroid membrane of soybean nodules. Science 281, 1202–1206. Katona, Z.F., Sass, P., and Molnar-Perl, I. (1999) Simultaneous determination of sugars, sugar alcohols in acids and amino acids in apricots by gas chromatography mass spectrometry. J. Chromatography A 847, 91–102. Kisaka, H. and Kida, T. (2005) Method of producing transgenic plants having improved amino acid composition. United States Patent, 6, 969, 782. Kisaka, H., Kida, T., and Miwa, T. (2007) Transgenic tomato plants that overexpress a gene for NADH-dependent glutamate dehydrogenase (legdh1). Breed. Sci. 57, 101–106. Koivunen, M. and Horwarth, W.R. (2004) Effect of management history and temperature on the mineralization of methylene urea in soil. Nutrient Cycling in Agro Ecosystems. 68 (1), 25–35. Kozaki, A. and Takeba, G. (1996). Photorespiration protects C3 plants from photooxidation. Nature 384, 557–560. Lam, H.M., Wong, P., Chan, H.K., Yam, K.M., Chow, C.M., and Corruzi, G.M. (2003) Overexpression of the ASN1 gene enhances nitrogen status in seeds of Arabidopsis. Plant Physiol. 132, 926–935. Lea, U.S., ten Hoopen, F., Provan, F., Kaiser, W.M., Meyer, C., and Lillo, C. (2004) Mutation of the regulatory phosphorylation site of tobacco nitrate reductase results in constitutive activation of the enzyme in vivo and nitrite accumulation. Plant J. 35, 566–573. Lightfoot, D.A. (2008) Blue Revolution Brings Risks and Rewards. Science 321, 771–772. Lightfoot, D.A. Long L.M., and Vidal, M.E. (1999) Plants containing the gdhA gene and methods of use thereof. US Patent # 5,998,700.

GENES FOR USE IN IMPROVING NITRATE USE EFFICIENCY IN CROPS

181

Lightfoot, D.A., Bernhardt, K., Mungur R., Nolte S., Ameziane, R., Colter, A., Jones, K., Iqbal, M.J., Varsa, E.C., and Young, B.G. (2007) Improved drought tolerance of transgenic Zea mays plants that express the glutamate dehydrogenase gene (gdhA) of E. coli. Euphytica 156, 106–115. Limami, A., Phillipson, B., Ameziane, R., Pernollet, N., Jiang, Q., Roy, R., Deleens, E., Chaumont-Bonnet, M., Gresshoff, P.M., and Hirel, B. (1999) Does root glutamine synthetase control plant biomass production in Lotus japonicus L.? Planta. 209, 495–502. Liu, K.H., Huang, C.Y., and Tsay, Y.F. (1999) CHL1 is a dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake. Plant Cell 11, 865–874. Loussaert, D.F., O’Neill, D., Simmons, C.R., and Wang, H. (2008) Nitrate reductases from red algae, compositions and methods of use thereof. United States Patent Application 20080313775, December 18. Man, H.M., Boriel, R., El-Khatib, R., and Kirby, E.G. (2005) Characterization of transgenic poplar with ectopic expression of pine cytosolic glutamine synthetase under conditions of varying nitrogen availability. New Phytol. 167, 31. Marschner, H. (1995) Mineral Nutrition of Higher Plants. Academic Press, London, UK. Melo-Oliveira, R., Oliveira, I.C., and Coruzzi, G.M. (1996) Arabidopsis mutant analysis and gene regulation define a nonredundant role for glutamate dehydrogenase in nitrogen assimilation. Pro Natl Acad Sci USA 43, 4718–4723. Meyer, R. (1997) A genetic analysis of response to ammonium and methylammonium in Arabidopsis thaliana. MS Thesis. SIUC, Carbondale, IL, p. 118. Meyer, R., Yuan, Z., Afzal, J., Iqbal, J., Zhu, M., Garvey, G., and Lightfoot, D.A. (2006) Identification of Gsr1: A locus inferred to regulate gene expression in response to exogenous glutamine. Euphytica 151, 291–302. Migge, A., Carrayol, E., Hirel, B., and Becker, T.W. (2000) Leaf specific overexpression of plastidic glutamine synthetase stimulates the growth of transgenic tobacco seedlings. Planta 2, 252–260. Mirvish S.S. (1985) Gastric cancer and salivary nitrate and nitrite. Nature 315, 461–462. Miyashita, Y. and Good, A.G. (2008) NAD(H)-dependent glutamate dehydrogenase is essential for the survival of Arabidopsis thaliana during dark-induced carbon starvation. J Exp Bot. 59, 667–680. Moll, R.H., Kamprath, E.J., and Jackson, W.A. (1982) Analysis and interpretation of factors which contribute to efficiency to nitrogen utilization. Agron. J. 74, 562–564. Moller, H., Landt, J., Pederson, E., Jensen, P., Autrup, H., and Jensen, O.M. (1990) Endogenous nitrosation in relation to nitrate exposure from drinking water and diet in a Danish rural population. Cancer Res. 49, 3117–3121. Mungur, R. (2004) Plants altered by the Escherichia coli glutamate dehydrogenase gene. MS Thesis, SIUC Carbondale. Mungur, R., Wood, A.J., and Lightfoot, D.A. (2006) Water potential during water deficit in transgenic plants: Nicotiana tabacum altered by the Escherichia coli glutamate dehydrogenase gene. Plant Growth Regulation 50, 231–238. Mungur, R., Glass, A.D.M., Goodenow, D.B., and Lightfoot, D.A. (2005) Metabolic fingerprinting in transgenic Nicotiana tabacum altered by the Escherichia coli glutamate dehydrogenase gene. J. Biomed. Biotech. 2, 198–214. Nadzan, G., Schneeberger, R., and Feldmann, K. (2007) Nucleotide sequences and corresponding polypeptides conferring improved nitrogen use efficiency characteristics in plants. United States Patent Application 20070169219. Nelson, D.E., Repetti, P.P., Adams, T.R., Creelman, R.A., Wu, J., Warner, D.C., Anstrom, D.C., Bensen, R.J., Castiglioni, P.P., Donnarummo, M.G., et al. (2007) Plant nuclear factor Y (NF-Y) B subunits confer drought tolerance and lead to improved corn yields on water-limited acres. Proc. Natl. Acad. Sci .USA 104, 16450 –16455. Oh, J.Y., Warner, R.L., and Kleinhofs, A. (1980) Effect of nitrate reductase-deficiency upon growth, yield and protein in barley. Crop Sci. 20, 487–490. Oliveira, I.C., Brears, T., Knight, T.J., Clark, A., and Coruzzi, G.M. (2002) Overexpression of cytosolic glutamate synthetase. Relation to nitrogen, light, and photorespiration. Plant Physiol. 129, 1170 –1180. Ortega, J.L., Temple, S.J., and Sengupta-Gopalan, C. (2001) Constitutive overexpression of cytosolic glutamine synthetase (GS1) gene in transgenic alfalfa demonstrates that GS1 be regulated at the level of RNA stability and protein turnover. Plant Physiol. 126, 109–121. Pathak, R.R., Ahmad, A., Lochab, S., and Raghura, N. (2008) Molecular physiology of plant nitrogen use efficiency and biotechnological options for its enhancement. Current Science 94, 1394–1399. Pennisi, E. (2008) The Blue Revolution, Drop by drop, gene by gene. Science 320, 171–173. Prins, W.H. (1983) Effect of a wide range of nitrogen applications on the herbage nitrate content in long term fertilizer trials in all-grass swards. Fert. Res. 4, 101–113. Pryor, A. (1990) A maize glutamate dehydrogenase null mutant is cold temperature sensitive. Maydica 35, 367–372. Quillere, I., Dufosse, C., Roux, Y., Foyer, C.H., Caboche, M., and Morot-Gaudry, J.F. (1994) The effects of deregulation of NR gene expression on growth and nitrogen metabolism of Nicotiana plumbaginifolia plants. J. Exp. Bot. 45, 1205–1211. Rawat, S.R., Silim, S.N., Kronzucker, H.J., Siddiqi, M.Y., and Glass, A.D. (1999). AtAMT1 gene expression and NH4+ uptake in roots of Arabidopsis thaliana: evidence for regulation by root glutamine levels. Plant J. 19, 143–152.

182

GENES FOR CROP ADAPTATION TO POOR SOIL

Schmidt, R.R. and Miller, P. (2009) Polypeptides and polynucleotides relating to the α- and β-subunits of glutamate dehydrogenases and methods of use. United States Patent 7,485,771. Schnable, P.S. and Dash, S. (2008) Plant genes involved in nitrate uptake and metabolism. United States Patent Application 20080141390. Schneeberger, R., Margolles-Clark, E., Park, J.-H., Jankowski, B., and Bobzin, S.C. (2008) Modulating plant nitrogen levels Patent: US 7335510-A 1 26-FEB-2008. Schoenbeck, M.A. et al. (2000) Decreased NADH-glutamate synthase activity in nodules and flowers of alfalfa (Medicago sativa L.) transformed with an antisense glutamate synthase transgene. J. Exp. Bot. 51, 29–39. Sentoku, N., Tanignchi, M., Sugiyama, T., Ishimaru, K., Ohsugi, R., Takaiwa, F., and Toki, S. (2000) Analysis of the transgenic tobacco plants expressing Panicum miliaceum aspartate aminotransferase genes. Plant Cell Rep. 19, 598–603. Shrawat, A.K., Carroll, R.T., DePauw, M., Taylor, G.J., and Good, A.G. (2008) Genetic engineering of improved nitrogen use efficiency in rice by the tissue-specific expression of alanine aminotransferase. Plant Biotechnology Journal 6, 722–732. Sinclair, T.R., Purcell, L.C., King, C.A., Sneller, C.H., Chen, P., and Vadez, V. (2006) Drought tolerance and yield increase of soybean resulting from improved symbiotic N2 fixation. Field Crops Research 101, 68–71. Specht, J.E., Hume, D.J., and Kumudini, S.V. (1999) Soybean Yield Potential—A Genetic and Physiological Perspective. Crop Science 39, 1560–1571. Suarez, R., Márquez, J., Shishkova, S., and Hernández, G. (2003) Overexpression of alfalfa cytosolic glutamine synthetase in nodules and flowers of transgenic Lotus japonicus plants. Physiol. Plant. 117, 326–336. Takahashi, M., Sasaki, Y., Ida, S., and Morikawa, H. (2001) Nitrite reductase gene enrichment improves assimilation of NO2 in Arabidopsis. Plant Physiol. 126, 731–741. Tannenbaum, S.R., Fett, D., Young, V.R., Land, P.D., and Bruce, W.R. (1978) Nitrite and nitrate are formed by endogenous synthesis in the human intestine. Science 200, 1487–1491. Vincent, R., et al. (1997) Over expression of a soybean gene encoding cytosolic glutamine synthetase in shoots of transgenic Lotus cornicultus L. plants triggers changes in ammonium and plant development. Planta 201, 424–433. Vincentz, M. and Caboche, M. (1991) Constitutive expression of nitrate reductase allows normal growth and development of Nicotiana plumbaginifolia plants. EMBO J. 10, 1027–1035. von Wiren, N., Gazzarrini, S., Gojon, A., and Frommer, W.B. (2000) The molecular physiology of ammonium uptake and retrieval. Curr Opin Plant Biol. 3, 254–261. Wang, R., Guegler, K., LaBrie, S.T., and Crawford, N.M. (2000) Genomic analysis of a nutrient response in Arabidopsis reveals diverse expression patterns and novel metabolic and potential regulatory genes induced by nitrate. Plant Cell. 12, 1491–1510. Yamaya, T., Obara, M., Nakajima, H., Sasaki, S., Hayakawa, T., and Sato, T. (2002) Genetic manipulation and quantitative-trait loci mapping for nitrogen recycling in rice. J. Exp. Bot. 53, 917–925. Yanagisawa, S., Akiyama, A., Kisaka, H., Uchimiya, H., and Miwa, T. (2004) Metabolic engineering with Dof1 transcription factor in plants: Improved nitrogen assimilation and growth under low-nitrogen conditions. Proc. Natl. Acad. Sci. USA 101, 7833–7838. Zhang, H. and Forde, B.G. (1998) An Arabidopsis MADS box gene that controls nutrient-induced changes in root architecture. Science 279, 407–409. Zhang, Y., Dickinson, J.R., Paul, M.J., and Halford, N.J. (2003) Molecular cloning of an Arabidopsis homologue of GCN2, a protein kinase involved in co-ordinated response to amino acid starvation. Planta 217, 668–675. Zhu, H., Riely, B.K., Burns, N.J., and Ané, J.M. (2006) Tracing non-legume orthologs of legume genes required for nodulation and arbuscular mycorrhizal symbioses. Genetics 172, 2491–2499.

Section 3 Genes for Plant Tolerance to Temperature Extremes

Genes for Plant Abiotic Stress Editors Matthew A. Jenks and Andrew J. Wood © 2010 Blackwell Publishing ISBN: 978-0-813-81502-2

8

Genes and Gene Regulation for Low-temperature Tolerance Mantas Survila, Pekka Heino, and E. Tapio Palva

Introduction

Plants as sessile and poikilothermic organisms are constantly exposed to and need to survive variable and often unfavorable environmental conditions. Consequently plant growth and productivity are greatly affected by various abiotic and biotic stress factors. Abiotic stresses, including extremes in temperature, drought, and high salinity, are in fact the principal causes of crop failure worldwide, and a threat to the agricultural industry. The still ongoing growth of the human population accentuates this problem as agricultural production needs to be expanded to more unsuitable marginal land. Low temperature (LT) presents a severe environmental challenge to plant growth and distribution in temperate and subtropical regions. Plants growing in these areas are regularly exposed to low temperature during their growth season. For annual herbaceous plants, unscheduled frosts early in spring or during summer can severely affect the growth and survival of plants. In addition to this, perennial herbaceous and woody plants also need to survive the often drastically lower temperatures during the winter months. Therefore, tolerance to LT and especially to freezing temperatures has a particular importance for both agriculture and forestry. All plants have optimal temperature ranges for their proper growth and development, as well as minimum and maximum temperatures for survival (Levitt, 1980). According to their responses to low temperature, plants can be classified into three broad categories: chilling sensitive, chilling tolerant but freezing sensitive, and freezing tolerant. Chilling-sensitive plants, such as rice (Oryza sativa) and maize (Zea mays), can be irreparably damaged when the temperature drops below 10 °C, mainly due to the loss in both membrane integrity and compartmentalization of the intracellular organelles. In chilling-tolerant-but-freezing-sensitive plants, like potato (Solanum tuberosum), no primary injury occurs at temperatures above 0 °C. However, when the temperature drops below the freezing point (generally somewhat below 0 °C), most of the liquid water in the plant freezes, leading to lethal injury. Due to the presence of more efficient ice nucleates and lower solute concentration in the apoplast as compared to the cytoplasm, this freezing invariably occurs extracellularly. As the temperature is lowered further, additional water is drawn from the cells to the growing intercellular ice, causing dehydration of the cells. Plants that survive the initial formation of ice are considered freezing tolerant and their ultimate level of tolerance is essentially determined by their ability to withstand such freeze-induced cellular dehydration. See Figure 8.1. The damage caused by freezing is mainly due to membrane perturbations. Freeze-induced cellular dehydration can cause multiple forms of damage, which can severely compromise the structure and function of the membranes (Steponkus et al., 1993; Uemura and Steponkus, 1997; Uemura et al., 2006). In addition to the cellular dehydration, additional factors contribute to the resulting damage during freezing. Growth of extracellular ice crystals exposes cells and tissues to mechanical stress, Genes for Plant Abiotic Stress Editors Matthew A. Jenks and Andrew J. Wood © 2010 Blackwell Publishing ISBN: 978-0-813-81502-2

185

Figure 8.1 An outline of processes leading to cold acclimation and development of freezing tolerance (see text for details). For color detail, please see color plate section.

186

GENES AND GENE REGULATION FOR LOW-TEMPERATURE TOLERANCE

187

and low temperatures per se can disrupt the function of macromolecular complexes and denature proteins. Furthermore, the reactive oxygen species (ROS) generated during LT exposure, especially in conditions of high light intensities, can severely damage the different macromolecular structures of the cell (McKersie and Bowley 1998). Most plants native to temperate regions have the ability to respond to low, non-freezing temperature (LT) by activating a cold-acclimation program leading to enhanced freezing tolerance (FT). In annual and many overwintering herbaceous plants, LT exposure is sufficient to trigger full acclimation, regardless of the photoperiod. In annual herbaceous plants, cold acclimation is a relatively fast process (for example, in the model plant Arabidopsis, a clear increase in FT can be achieved after only one day of LT exposure). In addition to LT, FT in Arabidopsis can be increased by application of the plant hormone abscisic acid (ABA) (Chen et al., 1983; Mäntylä et al., 1995). This, together with the facts that ABA levels increase during cold acclimation and ABA deficient mutants of Arabidopsis are impaired in cold acclimation suggest that this hormone plays a role in development of freezing tolerance (reviewed in Heino and Palva 2003). Additional environmental cues are required for winter survival, especially for woody perennials. (For reviews see Welling and Palva, 2008 and Heino et al., 2009.) Trees and other perennials undergo such adaptive changes in late stages of summer and early autumn. The most obvious changes are morphological and involve the partial or total loss of aerial organs, such as leaves and shoots, and the formation of specialized organs, such as buds and tubers. However, more subtle changes occur in the remaining, overwintering plant parts, allowing them to turn from a freezing-sensitive to a freezing-tolerant state. In overwintering woody plants, acclimation is normally a two-step process. Shortening of the photoperiod is the first cue of the coming winter, and this triggers growth cessation and development of dormancy and leads to moderate increase in freezing tolerance. Subsequent exposure to low temperature then induces the second stage of acclimation leading to tolerance against the winter temperatures (reviewed in Welling and Palva, 2006). The ability to cold acclimate is a quantitative trait involving a large number of genes. In fact, several studies have indicated that cold acclimation is associated with massive reprogramming of gene expression. Through transcriptome profiling by using microarrays or serial analysis of gene expression (SAGE) in Arabidopsis, it has been estimated that 5 to 25% of the genes show altered expression patterns during LT exposure (Kreps et al., 2002; Hannah et al., 2005; Robinson and Parkin, 2008). The induction of specific LT responsive genes results in a large amount of physiological, metabolic, and developmental alterations that determine the ultimate level of tolerance achieved during acclimation. These changes include alterations in the levels of phytohormones and antioxidants, production of compatible solutes and protective proteins, including chaperons as well as proteins with currently unknown functions (reviewed for example in Thomashow, 1999; Xin and Browse, 2000; Aalto et al., 2006). In the last decade significant progress has been made in studies of the signaling pathways that control the LT responsive transcriptome as well as in characterization of transcription factors (TF) that mediate the activation of LT responsive genes. The elucidation of the signal pathways and the detailed function of the TFs and small RNAs in configuration of the LT transcriptome have been described in detail in recent, excellent reviews, and we encourage the readers to familiarize themselves with these. This chapter is focusing on genes and proteins that are involved in FT development during cold acclimation, mainly in Arabidopsis. The regulatory networks that lead to expression of those genes are also described, and the implication of these findings to crop improvement is discussed.

188

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

Protective Mechanisms Induced during Cold Acclimation

The ability of plants to acquire FT involves extensive reprogramming of gene expression and consequently, metabolic adjustments and induction of protective mechanisms (Thomashow, 1999; Cook et al., 2004). The imposition of a temperature stress on a plant leads to the modification of metabolism in two ways that are important for survival: •

•

First, modifications that adjust the cellular metabolism to the consequences of fluctuations in temperature, such as changes in the structure and catalytic properties of enzymes and membrane metabolite transporters (Kubien et al. 2003). Second, modifications that are directly linked with enhanced tolerance mechanisms such as production of metabolites that can function as antioxidants, osmolytes (reduce cellular dehydration), chelating agents (neutralize potentially toxic levels of metals and inorganic ions), or as compatible solutes (stabilize enzymes, membranes and other cellular components) (Thomashow, 1999).

Plant survival also requires activation of additional protective systems, such as structural alterations in membranes and production of several classes of protective proteins (Fig. 8.1).

Alterations in Membrane Structure

Cellular membranes are known to be the primary sites of injury during a freeze-thaw cycle, and multiple forms of membrane damage such as expansion-induced-cell lysis, fracture lesions and lamellar-to-hexagonal-II phase transition have been shown to be caused by freeze-induced cellular dehydration (Steponkus et al., 1993; Uemura and Steponkus, 1997; Uemura et al., 2006). Thus, a key function of cold acclimation is to provide protection and stability to the cellular membranes (Cossins, 1994; Guy and Li, 1998). Indeed, cold acclimation has been shown to prevent expansioninduced lysis and the formation of hexagonal II phase lipids in rye and other plants (Steponkus et al., 1993; Uemura and Steponkus, 1997). Membrane fluidity is essential for its function and dependent on the composition and degree of fatty acid unsaturation. Therefore, the relative proportion of unsaturated fatty acids strongly influences the fluidity of the membrane in a temperature-dependent manner (Uemura and Steponkus, 1997); and consequently, proportion of unsaturated fatty acids has been reported to increase during cold acclimation in Arabidopsis (Uemura et al., 1995; Welti et al., 2002; reviewed in Wang et al., 2006). Additionally, phospholipid composition appears to affect FT and ensure functionality of the membrane at LT (Murata et al., 1992). Phospholipids are subject to hydrolysis by phospholipases during the normal course of plant growth and development as well as in response to plant stresses including freezing, drought, pathogen attack, and wounding (Yoshida, 1979; Young et al., 1996; Frank et al., 2000; Zien et al., 2001). Phospholipases D and C (PLD, PLC) are major phospholipidcatabolizing enzymes in plants and play an important role in membrane lipid hydrolysis and in mediation of plant responses to a wide range of stresses. PLD hydrolyzes phospholipids to phosphatidic acid (PA) and a head group, whereas PLC hydrolyzes phosphatidylinositol 4,5bisphosphate (PIP2) into diacyl glycerol (DAG) and inositol 1,4,5-triphosphate (IP3) (Alexandre et al., 1990). Both PLC and PLD have been shown to be activated in 15 seconds of cold treatment in Arabidopsis suspension cultures, although the exact mechanism of activation is not known (Ruelland et al., 2002).

GENES AND GENE REGULATION FOR LOW-TEMPERATURE TOLERANCE

189

Phospholipase D anchors the microtubules to plasma membrane, so its activation might lead to a conformational change in the cytoskeleton (Gardiner et al. 2001), which could lead to actin filament rearrangements and activation of mechanosensitive Ca2+ channels. The PA produced through PLD activity can directly affect the stability of bilayers as it has been shown to favor nonbilayer formation, leading to impaired membrane stability and integrity. However, transgenic Arabidopsis plants, in which the most common form of PLD, PLDα1, was silenced by antisense suppression exhibited decreased PA level during freezing stress, which was correlated to an increase in FT (Welti et al., 2002; Rajashekar et al., 2006). However, the suppression of PLDα1 in Arabidopsis also lead to an increased accumulation of osmolytes (Rajashekar et al., 2006), which can help to stabilize the structure of membranes and macromolecules during freeze-induced dehydration (Santarius, 1992). Thus, the high concentrations of osmolytes in PLDα1-deficient plants may, at least in part, explain their enhanced freezing tolerance.

Solute Biosynthesis

The accumulation of sucrose and other simple sugars that typically occur during cold acclimation was originally coupled with the stabilization of membranes as these molecules can protect membranes against freeze-induced damage in vitro (Anchordoguy et al., 1987; Strauss and Hauser, 1986). The role of sugars in abiotic stress tolerance and their direct interaction with lipid bilayers preventing membranes from detrimental changes have been substantiated (Hincha et al., 2006). Both monoand disaccharides interact with lipid membranes and are effective against abiotic stresses (Ohtake et al., 2006). According to the (most widely accepted) water replacement hypothesis, sugars can substitute water during drying, reduce Tm (melting temperature) and form an amorphous carbohydrate glass with high melting temperature (Tg—devitrification/glass transition temperature) (Oliver et al., 2002). Therefore, sugars not only inhibit the fusion of biological membranes by interacting directly with the polar headgroups of membrane phospholipids (through hydrogen bonds) essentially replacing the water molecules upon dehydration, but also inhibit the fluid-to-gel membrane phase transition at low hydration, thereby stabilizing the native structures of the lipid bilayers (Ohtake et al., 2006). The accumulation of soluble sugar is concomitant with an increase in several enzymatic activities involved in carbohydrate metabolism: amylases, sucrose synthase, sucrose phosphate synthase, fructose-1, 6-biphosphatase, sedoheptulose-1,7-biphosphotase, Rubisco, and accumulation of free sugars involve an enhancement of all photosynthetic pathways (Oquist, Hurry, and Huner, 1993, Hurry et al., 1994; Hurry et al., 1995). However, the presence of certain sugar components in specific regions of the plant may not be sufficient for whole plant survival under stress conditions. For example, glucose can interact directly with lipid headgroups, but has a low Tg value and devitrifies at relatively low temperatures, not preventing membrane fusion during dehydration (Oliver et al., 2002). This indicates that alternate mechanisms and physical parameters may also determine the ability of different sugars in membrane protection against fusion during drying (Hincha et al., 2007). Polysaccharides have paramount importance in sugar vitrification mechanisms and possess higher Tg values than simple sugars. Tg values increase with increasing molecular weight (Oliver et al., 2002) and degree of higher polymerization of sugars (DP) (Slade and Levine, 1991). Fructans are fructose-based polymers, synthesized from sucrose by fructosyltransferases, which can stabilize membranes by direct H-bonding to the phosphate and choline groups of membrane lipids, resulting in a reduced water outflow from the dry membranes (Valluru and Van den Ende, 2008). High DP fructans are more efficient in stabilizing biological membranes under stress (Vereyken et al., 2003), however, a mixture of both high DP and lower DP fructans and fructose (formed after

190

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

partial hydrolysis by cold stress-induced fructan exohydrolases) provides superior membrane protection. The low DP fructans could provide the necessary sugar–lipid interaction and decrease in Tm, while the high DP fructans could provide the high Tg (Crowe et al., 1997). Maltose accumulation during cold acclimation has also been shown to contribute to freezing tolerance. Kaplan and Guy (2004) demonstrated that an Arabidopsis gene, BMY8, encoding a βamylase localized in chloroplast stroma, is specifically induced in response to low temperature. By suppressing the expression of the BMY8 gene by RNA interference, they were able to demonstrate that the accumulation of maltose was significantly reduced with a concomitant increase in freezing sensitivity of the photosystem II in the transgenic plants (Kaplan et al., 2006). In addition to providing protection to the membranes, sugars have a role in protection against other consequences of freeze-induced dehydration, such as osmotic adjustment. Because the vapor pressure of ice is much lower than water at any given temperature, ice formation in the apoplast establishes a vapor-pressure gradient between the apoplast and surrounding cells. The unfrozen cytoplasmic water migrates down the gradient from the cell cytosol to the apoplast, which causes dehydration of the cells. The accumulation of various osmotically active, small hydrophilic compounds, such as sugars, in cold-acclimated plant cells limits dehydration by reducing the difference in vapor pressure between extracellular and intracellular spaces. In addition to the osmotic adjustment, accumulation of soluble sugars and other solutes decrease the freezing point of cytoplasmic water, thus preventing intracellular ice formation at freezing temperatures, and provide direct protection for different proteins and macromolecules of the cell (Mahajan and Tuteja, 2005). The osmotic adjustment of dehydrating cells is not completely explained by the accumulation of soluble sugars (Guinchard et al., 1997). Other osmolytes such as free amino acids, glycinebetaine (N-N-N trimethylglycine), and polyamines contribute to lowering water potential within the protoplast during freeze-induced dehydration. Proline and polyamines have cryoprotective effects on the plasma membrane in a manner similar to free sugars. An increase in proline content during cold acclimation has been found in a number of species (Koster and Lynch, 1992; Savoure et al., 1997). Although proline may simply be a form of nitrogen storage, its role in osmoregulation has long been suspected (Delauney and Verma, 1993). Increasing proline production by overexpression of δ1-pyrroline-5-carboxylate synthetase or suppressing catabolism significantly enhanced osmotolerance of transgenic tobacco (Kavi et al., 1995) or freezing tolerance in Arabidopsis (Nanjo et al., 1999), respectively. The biosynthesis of glycinebetaine and polyamines is stimulated by a number of environmental stresses, including cold (Koster and Lynch, 1992; Holmström et al., 2000; Cook et al., 2004; Kaplan et al., 2004). Two genes encoding S-adenosyl-L-methionine decarboxylase have been found to be upregulated by cold in a hardy Solanum species, S. sogarandinum (Rorat et al., 1997). This enzyme takes part in the biosynthesis of spermine and spermidine. In Arabidopsis, putrescine is produced through the arginine decarboxylate (ADC) pathway and one of the two ADC genes, ADC1 has been shown to be cold responsive (Hummel et al., 2004). Recently, Cuevas and others (2008) have demonstrated that Arabidopsis adc1 and adc2 mutants with impaired putrescine biosynthesis have reduced tolerance to freezing demonstrating that accumulation of this polyamine is required for cold acclimation.

Production of Protective Proteins

In addition to the physical and biochemical restructuring of cell membranes and induction solute biosynthesis, plants also produce proteins that are believed to protect the cellular structures and macromolecules during freezing. The ability of plants to control water content and phase in their tissues is probably one of the most important factors in freezing survival, especially for perennial

GENES AND GENE REGULATION FOR LOW-TEMPERATURE TOLERANCE

191

plants. Extracellular freezing is controlled by two types of proteins: ice nucleating proteins (INP) and antifreeze proteins (AFP). Ice nucleators initiate extracellular ice formation at temperatures below −1.5 °C, while antifreeze proteins modify the rate of growth and morphology of ice crystals (Goldstein and Nobel, 1994). AFPs have been identified in winter rye (Griffith et al., 1992; Marentes et al., 1993; Hon et al., 1994; Antikainen et al., 1996) and carrot (Smallwood et al., 1999). AFPs have the abilities to bind onto the surface of ice and inhibit its growth, lower the ice nucleation temperature, and inhibit the recrystallization of ice (Knight et al., 1995; Smallwood et al., 1999 reviewed in Griffith et al., 2005). During cold acclimation, AFPs are secreted into apoplast of the leaves and crown, and their accumulation has been shown to correlate with increased freezing tolerance in rye, wheat, and barley (Griffith et al., 1992; Marentes et al., 1993; Antikainen et al., 1996; Griffith and Yaish, 2004). There is emerging evidence that plants produce several different cryoprotective proteins during cold acclimation. These proteins are believed to protect the cellular membranes as well as the macromolecular structures in the cytoplasm during a freeze-thaw cycle. Alterations in the profile of plasma membrane proteins have been detected during cold acclimation (Kawamura and Uemura, 2003; Uemura et al., 2006); and LT responsive, plasma membrane localized, or associated, proteins have been shown to contribute to the increased freezing tolerance (Puhakainen et al., 2004; Uemura et al., 2006). In addition, COR15am in Arabidopsis, a soluble protein localized in the chloroplast, stabilizes the inner chloroplast envelope membrane against fusion with the plasma membrane during freeze-induced dehydration (Steponkus et al., 1998). Similarly, osmotin-like protein, lectins, and cryoprotectin are cold-regulated proteins that are able to stabilize membranes during a freezethaw cycle. Osmotin-like protein from Solanum dulcamara has been shown to stabilize isolated protoplast from Brassica oleracea during a freeze-thaw cycle (Newton and Duman, 2000). Lectins are sugar-binding proteins and because thylakoid membranes contain galactolipids, galacto-specific lectins were studied for their effect on membrane stability. Lectins were found to bind to thylakoid membranes, and in some cases exert a cryoprotective effect (Hincha et al., 1997a). Lectins bind to the galactolipid headgroups, which leads to reduced fluidity at the membrane surface and reduced solute permeability and membrane damage during freezing and thawing, respectively (Hincha et al., 1997a). Cryoprotectin is a plant lipid-transfer protein (LTP) homologue that stabilizes membranes during freezing, and consequently, cryoprotectin has been suggested to be a specialized LTP homologue that protects membranes during freezing, due to a binding mechanism precluding lipid transfer activity (Hincha et al., 2001). Recently, the SFR2 protein has also been shown to protect the chloroplast membranes during freezing in Arabidopsis (Fourrier et al., 2008). The SFR2 was first identified in a genetic screen for freezing sensitivity in Arabidopsis (Warren et al., 1996) and the cloning of the gene mutated in the sfr2-1 mutant revealed that SFR2 encodes a β-glucosidase (Thorlby et al., 2004). The sfr2-1 mutant is sensitive to freezing, indicating that the function of the SFR2 is essential for cold acclimation. The mechanism by which the β-glucosidase activity of SFR2 is mediating the protection of membranes is currently unknown. A large and ubiquitous group of stress target genes are genes encoding late embryogenesis abundant (LEA) proteins. LEA proteins were initially identified in cotton, where they accumulated during late embryogenesis (Dure et al., 1981), and they have since been found in developing seeds of many different plant species, but also in vegetative tissues, especially under stress conditions (Ingram and Bartels, 1996). LEA proteins are a diverse group of proteins, which has, according to specific amino acid sequence motifs been divided into different subgroups (Dure et al., 1989; Bray et al., 1993). In a recent analysis, the 51 LEA proteins identified in Arabidopsis were divided into nine groups, according to their Pfam nomenclature (Hundertmark and Hincha, 2008). Common features for all LEA proteins appear to be high hydrophilicity, lack of any defined structure, and

192

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

heat stability (Wise and Tunnacliffe, 2004). LEA proteins have been shown to stabilize proteins during freezing (Hara et al., 2001; Bravo et al., 2003) and suggested to act in processes like ion sequestration (Kruger et al., 2002; Alsheikh et al., 2005) and scavenging radicals (Hara et al., 2003). However, only in a few cases has the function of specific LEA proteins been characterized. For a comprehensive description of LEA proteins and their genes, we refer to Hundertmark and Hincha (2008). Dehydrins are probably the best-characterized group of LEA proteins, and they have been demonstrated to be involved in development of freezing tolerance (reviewed in Rorat, 2006; Aalto et al., 2006). Dehydrin encoding genes have been demonstrated to be responsive to low temperature in several plant species (Danyluk et al., 1998; Bravo et al., 1999; Nylander et al., 2001). Arabidopsis harbors 10 dehydrin genes in total, 5 of which have been demonstrated to be LT responsive (Hundertmark and Hincha, 2008). A dehydrin protein accumulates at the plasma membrane during cold acclimation (Puhakainen et al., 2004a) particularly in tissues more sensitive to freeze damage (Danyluk et al., 1998), and dehydrins can protect proteins, like lactate dehydrogenase against freeze damage (Kazuoka and Oeda, 1994; Goyal et al., 2005). Several studies with transgenic plants ectopically expressing dehydrins have also indicated their direct involvement in increased freezing tolerance. A citrus dehydrin, when expressed in tobacco, increased the cold tolerance (Hara et al., 2003), and expression of a wheat dehydrin increased the freezing tolerance of strawberry (Houde et al., 2004). In addition, simultaneous overexpression of two different Arabidopsis dehydrins led to improved freezing tolerance of transgenic Arabidopsis plants (Puhakainen et al., 2004). Recently, it was demonstrated that plants can actively and directly protect the plasma membrane during a freeze/thaw cycle (Yamazaki et al., 2008). It is well known that in animal cells, membrane damage can be repaired in a Ca2+-dependent manner by using a complex mechanism, where synaptotagamin is in a central role (McNeil and Kirchhausen, 2005). The amount of a synaptotagamin homolog, SYT1, in the Arabidosis plasma membrane has been shown to increase in response to low temperature (Kawamura and Uemura, 2003), and this increase is derived from LT responsive expression of the SYT1 gene (Yamazaki et al., 2008) Furthermore, by using SYT1 T-DNA insertion and RNA interference (RNAi)-lines, where the expression of the SYT1 gene was suppressed, Yamazaki and others (2008) were able to demonstrate that Ca2+-dependent synaptotagamin was essential for development of full freezing tolerance (Yamazaki et al., 2008) and suggested that it was involved in resealing of membranes after damage generated during freezing. One of the common themes in abiotic stresses in plants is the generation of reactive oxygen species (ROS). The stress-induced production of ROS, including superoxide radicals (O2−), hydrogen peroxide (H2O2) and hydroxyl radicals (OH·), is derived from altered chloroplast and mitochondria metabolism during stress. LT exposure, especially in connection with high light, is also leading to excessive production of ROS, which can greatly damage cellular structures. Plants use low molecular mass antioxidants and employ a diverse array of enzymes such as superoxide dismutases (SOD), catalases (CAT), ascorbate peroxidases (APX), glutathionine reductases and glutathionine peroxidases (GPX) to scavenge ROS (Mittler, 2002). In Arabidopsis, genes encoding all of these enzymes have been shown to be responsive to LT (Soitamo et al., 2008, reviewed in Mittler et al., 2004).

Regulation of Gene Expression Signal Transduction in Cold Acclimation

To initiate the cold acclimation process, plants need to perceive the LT signal and transduce it to nucleus, resulting in reprogramming of LT responsive transcriptome. Currently, no direct evidence

GENES AND GENE REGULATION FOR LOW-TEMPERATURE TOLERANCE

193

exists for a specific receptor for low temperature in plants, but several molecules have been suggested to carry this function (reviewed in Heino and Palva, 2003). Theoretically, low temperature can be sensed by any compartment of the cell. LT-induced alteration in conformation of a membrane localized or soluble protein can alter the function or localization of the protein thus leading to initial transmission of the cold signal. Membrane receptors and ion channels in the plasma membrane provide classes of putative temperature sensors in plants and both mechanosensitive Ca2+- channels, whose activity is modulated by low temperature (Ding and Pickard, 1993) and receptor kinases transcriptionally responsive to low temperature (Hong et al., 1997; Kreps et al., 2002) have been identified. In the prokaryote Synecocystis the cold receptor appears to be Hik33, a histidine kinase functioning as the sensor in a two-component regulatory system (Suzuki et al., 2000). Hik33 has two hydrophobic helices that span the plasma membrane and form a dimer, whose structure may be influenced by the physical characteristics of lipids in the plasma membrane. The physical characteristic of lipids is affected by temperature; and when the temperature is decreasing, the membranes become more rigid (Murata and Los, 1997). The increase in membrane rigidity activates Hik33, and the histidine residue in the kinase domain of Hik33 is phosphorylated. This phosphate group is then transferred to the histidine kinase Hik19, and finally to the response regulator Rer1. This phospho-relay from Hik33 to Rer1 regulates the expression of a desaturase gene DesB that, in turn, regulates the level of unsaturation of fatty acids in membrane lipids. Futhermore, Hik33 regulates the expression of a set of osmotic and cold-stress-inducible genes, which leads to alterations in photosynthesis, oxidative stress responses and membrane functions (Mikami et al., 2002). A histidine kinase gene (AHK1/AtHK1) responsive to changes in osmolarity due to cold, salinity, and dehydration stress has been isolated from Arabidopsis. However, the function of the AHK1 appears to be in sensing alterations in osmolarity rather than temperature (Urao et al., 1999; Tran et al., 2007). Overexpression of the AHK1 in yeast could complement the sln1 and sho1 mutations demonstrating that AHK1 can function as an osmosensor in yeast cells (Urao et al., 1999). Transgenic Arabidopsis plants overexpressing the AHK1 were more tolerant to drought, and conversely the ahk1 mutants exhibited increased sensitivity to drought (Tran et al., 2007). During drought stress the ahk1 mutant also showed reduced accumulation of mRNAs for stress and ABA responsive genes (Tran et al., 2007). Recently, another histidine kinase gene in Arabidosis, AHK3, was also shown to be cold responsive (Tran et al., 2007). The analysis of the ahk3 mutant demonstrated that it exhibited hypersensitivity to ABA, indicating that AHK3 acts as a negative regulator in ABA signaling in Arabidopsis (Tran et al., 2007). Analogously to Synecocystis, plasma membrane rigidification appears to be involved in cold signaling in plants. Treatment of alfalfa suspension cultures and Brassica seedlings by either the membrane rigidifier DMSO or the actin microfilament destabilizer cytochalasin D (CD) resulted in cold acclimation and expression of the LT responsive genes even at normal growth temperature. Conversely, treatment with the membrane fluidizer benzyl alcohol (BA) or the actin filament stabilizer jasplakinolide (JK) were shown to prevent cold acclimation and induction of the genes at 4 °C (Örvar et al., 2000; Sangwan et al., 2001). Furthermore, treatment of Brassica seedlings with gadolinium, an antagonist of mechanosensitive Ca2+-channels, inhibited gene activation after cold, DMSO, or CD treatment. Consequently, a model was proposed, where low temperature causes rigidification of the plasma membrane, which leads to reorganization of the cytoskeleton and activation of a mechanosensitive Ca2+-channel. The resulting Ca2+-influx to the cytoplasm would then act as a second messenger in cold signaling (Sangwan et al., 2001). Recently, studies with the Arabidopsis fad2 mutant, defective in oleate desaturase gene, are providing support for this model (Vaultier et al., 2006). By comparing the activation temperatures for diacylglycerol kinase, an

194

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

enzyme known to be activated within seconds from the perception of the LT signal, in the fad2 mutant and in wild-type Arabidopsis, Vaultier and others (2006) were able to show that the activation took place at significantly higher temperature in the fad2 mutant, indicating that membrane rigidification is indeed involved in the perception of the cold signal (Vaultier et al., 2006). Ca2+ has long been known to act as an important second messenger in plant responses to environmental cues (Trewavas and Malho, 1997). In response to low temperature, Ca2+-influxes have been observed within seconds (Knight et al., 1996), and inhibition of this influx is compromising cold acclimation (Monroy and Dhindsa, 1995; Tähtiharju et al., 1997; Sangwan et al., 2001). Therefore, the early events in cold acclimation include temperature-dependent calcium influx to the cytosol (Plieth et al., 1999). However, preventing the influx of apoplastic Ca2+ through the plasma membrane caused only a partial inhibition of LT responsive gene activation in Arabidopsis, indicating that Ca2+ release from intracellular compartments is also involved in the acclimation process (Knight, 1996; Tähtiharju, 1997). Inositol trisphosphate (IP3) and cyclic adenosine 5′-diphosphate ribose (cADPR) are signaling molecules that mediate Ca2+-release from endoplasmic reticulum in animal cells. In plants the main intracellular source appears to be the vacuole (Munnik et al., 1998) and both IP3 and cADPR have been shown to release calcium from plant vacuoles (Allen et al., 1995). Both IP3 and cADPR have also been implicated as regulators for Ca2+-influx in response to low temperature (Knight et al., 1996; Xiong et al., 2001). In tomato, cADPR can mediate activation of ABA and LT response genes, indicating that Ca2+-release from intracellular stores is also involved in cold acclimation. IP3 is produced by hydrolysis of PIP2, which is synthesized by phosphatidylinositol 4-phosphate 5-kinase. An osmotic stress and ABA responsive gene encoding this enzyme has been identified in Arabidopsis (Mikami et al., 1998). Hydrolysis of PIP2 is mediated by PLC and activation of this enzyme in early stages of LT treatment indicates that IP3 production is involved in cold signaling (Ruelland et al., 2002). A direct connection between phosphoinositide metabolism and stress signal transduction was demonstrated by Xiong and others (2001). They isolated an Arabidopsis mutant, FIERY1, defective in inositol polyphosphate 1-phosphatase. The mutant plants had significantly higher IP3 content and IP3 was also accumulating during stress and ABA treatment, leading to impairment of the stress responses (Xiong et al., 2001). Their results clearly indicate that IP3 is involved in mediating low temperature and ABA signaling and indicate that a critical issue of tolerance development would be the ability to attenuate the IP3 signal, which otherwise could lead to disturbances in Ca2+-homeostasis (Xiong et al., 2001). Analogously, the sac9 mutation in Arabidopsis, disrupting a gene for phosphoinositide phosphatase, contains higher levels of both PIP2 and IP3 and exhibits hypersensitivity to freezing stress (Williams et al., 2005). The signal initiated by Ca2+-influx is generally mediated through Ca2+-binding proteins acting as Ca2+-sensors. In plants the main Ca2+-sensors are calmodulin (CaM), Ca2+-dependent protein kinases (CDPK) and calcineurin B-like (CBL) proteins. CaM is a highly conserved protein that has been considered to be the primary sensor for changes in cytosolic Ca2+ levels in all eukaryotes (Rudd and Frankling-Tong, 2001; Ludvig et al., 2005). The involvement of CaM in LT responses was initially demonstrated in alfalfa and Arabidopsis, where CaM antagonist was shown to prevent cold acclimation and reduce expression of cold-regulated genes (Monroy et al., 1993; Tähtiharju et al., 1997). In alfalfa LT treatment also activated a CaM encoding gene MsCK1 (Monroy and Dhindsa, 1995). CaM has also been shown to directly regulate the activity of a family of transcription factors (calmodulin-binding transcription activators, CAMTAs) (Bouché et al., 2002) and, interestingly, CAMTAI and CAMTA3 have recently been shown to be involved in development of freezing tolerance during cold acclimation (Doherty et al., 2009). However, the role of CaM in mediating the cold signal appears to be more complex, as indicated by the negative effect of CaM overexpression to the expression of LT responsive genes (Townley and Knight, 2002). CaM also appears to be involved in LT responsive alterations in metabolism. The Ca2+/CaM dependent NAD kinase activity

GENES AND GENE REGULATION FOR LOW-TEMPERATURE TOLERANCE

195

increased tremendously after 60 and 120 minutes of cold treatment (Ruiz et al., 2002). NAD kinase catalyzes the phosphorylation of NAD+ to NADP+, thus regulating cellular metabolic pathways, and the increase in the kinase activity was directly proportional with the increase in foliar proline content in response to cold, indicating a role in regulation of proline accumulation (Ruiz et al., 2002). Plants also harbor Ca2+-sensors, distantly related to CaM and called calmodulin-like (CML) proteins (McCormack and Braam, 2003; McCormack et al., 2005). One of the CML proteins in Arabidopsis, AtCML9, was recently demonstrated to be transcriptionally responsive to ABA and various abiotic stresses, including cold, and the salt stress mediated induction of the CML9 gene was demonstrated to take place through an ABA-dependent pathway (Magnan et al., 2008). T-DNA insertion lines harboring insertions in the CML9 gene exhibited hypersensivity to ABA and enhanced tolerance to osmotic stress and drought indicating that CML9 might act as a negative regulator of ABAdependent processes during abiotic stresses (Magnan et al., 2008). CBL-proteins are plant specific Ca2+-sensors that are related to the regulatory subunit B of calcineurin, an important regulatory protein phosphatase in animal and yeast cells (Klee et al., 1998). In these cells calcineurin B is binding to and regulating the activity of the catalytic subunit calcineurin A. Interestingly, in plant cells CBLs are not regulating phosphatase activities. Instead they are interacting with a specific class of protein kinases, CBL-interacting protein kinases (CIPK), and regulating their activity (Luan et al., 2002). Arabidopsis genome harbors at least 10 genes encoding CBL-proteins and 25 genes encoding CIPKs (Luan et al., 2002). It appears that the interaction network between different CBLs and CIPKs is complex and provides the functional specificity as well as redundancy for the responses mediated by these sensors (Luan, 2008). The involvement of CBL proteins in stress responses was initially demonstrated by Liu and Zhu (1998), who showed that a CBL protein, SOS3/CBL4, is mediating salt stress signaling in Arabidopsis by interacting and activating SOS2/CIPK24, which then phosphorylates SOS1, a Na+/H+ antiporter (Shi et al., 2000). CBL1 gene has been shown to be responsive to several abiotic stresses, including low temperature (Kudla et al., 1999), and it has been shown to interact with CIPK1 to regulate osmotic stress responses (D’Angelo et al., 2006). A T-DNA insertion mutant of CBL1 exhibits altered expression of LT responsive genes, but no effect on freezing tolerance was detected (Albrecht et al., 2003), and in fact CBL1 has been suggested to act as a negative regulator of cold response in Arabidopsis (Cheong et al., 2003). CIPK1 has been suggested to form a convergence point for ABA-dependent and ABA-independent stress responses. CIPK1 can interact with both CBL1 and CBL9, and during osmotic stress CBL1/CIPK1 and CBL9/CIPK1 complexes are mediating ABAindependent and ABA-dependent responses, respectively (D’Angelo et al., 2006). Another CIPK, CIPK3, has been shown to form a cross-talk node between cold and ABA response pathways (Kim et al., 2003). The disruption of the CIPK3 gene affects both cold and ABA responsive gene expression but has no effect on drought responses (Kim et al., 2003). Mitogen-activated protein kinase (MAPK) cascades are also indicated in mediating of the LT signal. MAPK cascade is composed of three sequentially acting protein kinases: a MAP kinase kinase kinase (MAPKKK) that phosphorylates and activates a MAP kinase kinase (MAPKK), which then activates an MAPK. All of the components in the MAP kinase cascades are encoded by multigene families in Arabidopsis, and different members of the families have diverse roles in plant responses to the environment. A MAPKKK, MEKK1, is transcriptionally induced during LT treatment (Mizoguchi et al., 1996), and a cascade consisting of MEKK1, MKK2, and MPK4/MPK6 has been shown to form part of the LT signaling network (Teige et al., 2004). In alfalfa cells, a stress-activated MAP kinase (SAMK) is activated by cold. During LT treatment, the activation of the SAMK was detected at 10 minutes, reached maximum at 60 minutes, and returned to basal level at 6 hours. This activation of SAMK could be mimicked by membrane rigidification and microfilament destabilization, and was dependent on calcium influx. It was, however, prevented by

196

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

antagonists of CDPKs, suggesting that the cold signal in alfalfa is sensed by changes in membrane fluidity, causing rearrangement of cytoskeleton, followed by Ca2+ influx into cytoplasm, and activation of CDPKs followed by MAPKs (Sangwan et al., 2002). In addition to kinases, protein phosphatases (PP) have been implicated in LT signaling. Expression of cold-responsive genes is enhanced at normal growth temperatures by PP1 and PP2A inhibitors (Monroy et al., 1998). Also, the activity of PP2A was decreased by 85% within one hour of LT treatment, suggesting that PP2A might be an early target for cold inactivation. PP2A has been shown to regulate the activity of several kinases, including MAPKs and second messenger-dependent kinases in mammalian cells. Therefore, inactivation of PP2A by low temperature could increase the kinase activities in the cell, thus promoting signal transduction (Monroy et al., 1998). Two related protein phosphatases 2C, ABI1 and AtPP2CA, have been implicated as negative regulators of ABA signaling (Tähtiharju and Palva, 2001). Both protein phosphatases, ABI1 and AtPP2CA, are cold inducible, but only the AtPP2CA is strongly expressed during the whole acclimation process. AtPP2CA was shown to modulate the expression of several cold-inducible genes such as LTI78 and RAB18 in ABA-independent and ABA-dependent pathways, respectively (Tähtiharju and Palva, 2001). Production of excessive amounts of ROS is detrimental to the cells. However, subtle alterations in the level of ROS are also acting as signals in plants. The Arabidopsis fro1 (frostbite1) mutant, which has constitutively higher levels of ROS, compared to the wild-type, exhibits impaired expression of LT responsive genes and hypersensitivity to chilling and freezing (Lee et al. 2002). FRO1 encodes the Fe–S subunit of complex I (NADH dehydrogenase) of the respiratory electron transfer chain in mitochondria, and its disruption leads to high levels of ROS generation (Lee et al., 2002). The high level of ROS might then interfere with cold signaling.

Transcriptional Regulation of Gene Expression

Plant cold-acclimation responses are highly integrated into cellular functions at all levels. Despite the complexity of the pathways and networks involved in this process, recent studies of gene expression, RNA profiling, proteomics, and metabolomics, are starting to reveal some details of this complexity. Cold acclimation leads to massive physiological and molecular alterations in plants. The transcriptional reprogramming taking place during LT exposure is not only leading to production of protein that protects the cells from freezing damage but also to large alterations in the metabolite profile of the cells (Kaplan et al., 2004; Cook et al., 2004). Out of the 434 polar metabolites measured, close to 75% were shown at increased levels during cold acclimation (Kaplan et al., 2004; Cook et al., 2004). Cold acclimation is mediated by several parallel, but converging, signal transduction pathways, whose activation led to alterations in the gene expression profile, part of which are LT specific and part shared with other stresses (Chinnusamy et al., 2004; Shinozaki and Shinozaki, 2006; Nakashima et al., 2009). (It is noteworthy to mention that even if there are controversial theories regarding the direct involvement of ABA in the cold acclimation process, this chapter is including discussions about the ABA pathway as an essential component of the acclimation process.) In broad terms, the pathways leading to altered gene expression can be divided into two distinct groups, depending on the involvement of the phytohormone abscisic acid (ABA) on their activation. In recent years there has been a wealth of studies to dissect these pathways. These studies, which, until recently, have mainly been concentrating on the distal ends of the pathways, have led to the identification of specific cis elements in promoters of LT responsive genes as well as transcription factors that bind

GENES AND GENE REGULATION FOR LOW-TEMPERATURE TOLERANCE

197

to these sequences in an LT-dependent manner. Current models of transcriptional regulation of cold-acclimation-related genes include several transcription factors, each activating a partially overlapping set of target genes (Zhu et al., 2007; Nakashima et al., 2009). In general, low temperature is inducing massive alterations in gene expression. By using microarrays, the part of the total transcriptome that is affected by low temperature in Arabidopsis has been estimated to be between 4% (Lee et al., 2005) and 20% (Hannah et al., 2005). The differences among the estimates are most likely derived from experimental differences between the analyzes. Recently, Robinson and Parkin (2008) have used serial analysis of gene expression (SAGE) to analyze the changes in the Arabidopsis transcriptome during cold acclimation. Their results indicate that 6% of the genes are differentially expressed. An emerging view from this analysis is that only a few of the genes that appear to be downregulated in cold are encoding proteins that are involved in transcription, whereas this class is enriched among the upregulated genes (Lee et al., 2005; Robinson and Parking, 2008). Temporal analysis of transcriptome changes also indicate that the early stage of cold acclimation is including activation of several genes encoding different types of transcription factors (Lee et al., 2005; Robinson and Parking, 2008), indicating the involvement of different regulons in cold acclimation. Several of the LT responsive genes harbor in their promoter regions one or more copies of a cis element called C-repeat/dehydration-responsive element/low temperature response element (CRT/DRE/LTRE) (Baker et al., 1994; Yamaguchi-Shinozaki and Shinozaki, 1994; Nordin et al., 1993). This element, with a core sequence of CCGAC, is the binding site for a family of cold-specific transcriptional activators known as CBFs or DREB1s (Stockinger et al., 1997; Liu et al., 1998). CBFs/DREB1s belong to the large, plant-specific, APETALA2/ETHYLENE RESPONSE FACTOR family of transcription factors, and they control ABA-independent expression of genes in response to cold stress (Liu et al., 1998). CBFs/DREB1s were originally isolated as members of a small gene family consisting of three genes CBF1-3/DREB1A-C, CBF1 being identical to DREB1B and CBF2 and CBF3 with DREB1C and DREB1A, respectively (Liu et al., 1998; Gilmour et al., 1998). An additional CBF homolog, CBF4 has been isolated from Arabidopsis. However, CBF4 appears not to regulate cold responses, but instead to mediate drought responses in Arabidopsis (Haake et al., 2002), similarly to two other related transcription factors, DREB2A and DREB2B (Liu et al., 1998). Ectopic expression of CBFs/DREB1s (hereafter called CBFs) in transgenic plants has been shown to activate genes harboring the CRT-elements in their promoter regions even at warm temperatures and to confer improved freezing, drought, and salt tolerance (Jaglo-Ottosen et al., 1998; Liu et al., 1998; Kasuga et al., 1999; Hsieh et al., 2002). Recently, it has been demonstrated that the CBFs do not have an equal function during cold acclimation, but instead, they appear to determine different overlapping parts of the CBF regulon (Novillo et al., 2007). Transcript profiling of transgenic Arabidopsis plants overexpressing CBFs demonstrate that CBF targets include genes encoding transcription factors RAP2.1 and RAP2.7, which might further define CBF subregulons (Fowler and Thomashow, 2002; Vogel et al., 2005). The role of the CBF regulon in configuration of the LT transcriptome has been estimated by using transgenic plants that overproduce CBFs, and 12–28% of the cold-induced genes have been assigned to the CBF regulon (Fowler and Thomashow, 2002; Vogel et al., 2005). Interestingly, 84% of the genes that were highly induced (more than 15 fold) with LT were shown to be members of the CBF regulon (Vogel et al., 2005). The proteins that are encoded by the genes belonging to the CBF regulon include several transcription factors, signal transduction proteins, like protein kinases and phosphatases, putative cryoprotective/LEA proteins, enzymes involved in the sugar metabolism and ROS scavenging (Fowler and Thomashow, 2002). The important contribution of the CBF regulon to cold

The CBF Regulon

198

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

acclimation is also reflected in the contribution of the CBF regulon to the LT metabolome. Out of the 325 metabolites that were found to increase in Arabidopsis during LT exposure, 79% were also upregulated in normal temperature in transgenic plants overexpressing CBF3 (Cook et al., 2004). The CBF genes themselves are transiently responsive to low temperature and expressed only in early stages of cold acclimation (Gilmour et al., 1998). The level of cold induction of the CBF genes appears also to be related to temperature changes; the lower the temperature, the higher the level of CBF transcripts (Zarka et al., 2003). Interestingly, it has recently been demonstrated that the CBF genes have an additional expression window during a freeze/thaw cycle. When the plant tissues are frozen and thawed after cold acclimation, the amounts of CBF transcripts are increased during the thawing period (Welling and Palva 2008). This indicates that the CBF-controlled processes need to be reactivated during the period when plants are recovering from the stress (Welling and Palva, 2008). The CBF-family appears to be ubiquitous in the plant kingdom in regulation of abiotic stress responses, and CBF orthologs have been identified in several plant species, including rice, Brassica, wheat, rye, barley, populus, and birch (Jaglo et al., 2001; Choi et al., 2002; Benedict et al., 2006; Welling and Palva, 2008). In many cases, the ectopic expression of these genes in transgenic Arabidopsis has led to expression of CBF target genes and enhanced tolerance, indicating that the encoded proteins are functional transcription factors binding to CRT-elements and activating gene expression (Dubouzet et al., 2003; Qin et al., 2004; Zhang et al., 2004; Ito et al., 2006). Conversely, the LT responsive genes from other species can be activated by Arabidopsis CBFs, as indicated by the constitutive expression of the birch Bpdhn36 gene promoter in transgenic Arabidopsis overexpressing CBF1 (Puhakainen et al., 2004b) and activation of the cold acclimation program in transgenic populous ectopically expressing the CBF1 gene from Arabidopsis (Benedict et al., 2006). Since CBF transcripts start to accumulate within 15 minutes of plants’ exposure to cold, it was proposed (Gilmour et al., 1998) that a transcription factor, present in the cell already at normal growth temperature, recognize the CBF promoters and induce CBF expression upon activation by cold stress. Indeed, a transcription factor ICE1 (INDUCER OF CBF EXPRESSION1) has been isolated and characterized in Arabidopsis (Chinnusamy et al., 2003). ICE1 was identified as a constitutively expressed, nuclearly localized MYC-type basic helix–loop–helix transcription factor, which binds to MYC recognition elements in the CBF3 promoter and triggers the expression of CBF3 during cold acclimation. The ice1 mutant was found to be defective in induction of CBF3 and was more sensitive to chilling and incapable of cold acclimation (Chinnusamy et al., 2003). Constitutive overexpression of ICE1 was found to enhance the expression of CBF3, CBF2, and their target genes during LT exposure, and the transgenic plants exhibited increased freezing tolerance (Chinnusamy et al., 2003). Interestingly, cold exposure was required for activating the CBF gene expression, indicating that cold-stress-induced post-translational modification is necessary for ICE1 activation (Chinnusamy et al., 2003). Recently, it was reported that activation of ICE1 is mediated by LT-dependent phosphorylation of the protein (Chinnusamy et al., 2007). The effect of the ICE1 mutation on the CBF genes was mainly evident for CBF3, indicating that additional mechanisms might exist for the induction of the other CBFs (Chinnusamy et al., 2003). Recently, an ICE1 paralog, ICE2, was identified in Arabidopsis (Fursova et al., 2009). The predicted protein shows extensive similarity to ICE1, but the ICE2 gene (At1g12860) contains an additional predicted exon in the 5′-end of the gene. However, this exon is not detected in any available cDNA sequence derived from ICE2 (Fursova et al., 2008). Overexpression of the gene, without the first exon, resulted in enhanced tolerance to freezing and increased expression of the CBF1 gene (Fursova et al., 2008). Whether the ICE2 indeed encodes a functional ICE1 paralog remains to be seen. ICE1 is also predicted to mediate to be required for the expression of the constitutively expressed HOS9 gene (Zhu et al., 2004) in

GENES AND GENE REGULATION FOR LOW-TEMPERATURE TOLERANCE

199

Arabidopsis (Benedict et al., 2006). HOS9 is apparently active in normal growth temperature, indicating that ICE1 can also be active without low temperature, and temperature signal is only shifting the activity of ICE1 toward the CBF genes. In this respect, it is highly interesting that ICE1 has recently been demonstrated to play a key role in differentiation of guard cells (Kanaoka et al., 2008). The transient nature of CBF expression during LT exposure indicates an active repression mechanism for the genes. The expression of CBFs can be repressed either by their own gene products or the products of their downstream target genes. Novillo and others (2004) have shown that the expression of CBF1 and CBF3 is negatively regulated by CBF2. On the other hand, CBF2 appears to be repressed by CBF3, indicating that CBF expression involves self-regulated feedback loops (Chinnusamy et al., 2003). However, recent data from Novillo and others (2007) indicate that CBF2 expression is not affected by CBF1 or CBF3, indicating that the autoregulation of CBFs is only mediated by CBF2. Guo and others (2002) have shown that the expression of the CBF target genes in los1-1 mutant is highly reduced, but the transcription of CBF genes themselves are superinduced in response to low temperature, showing that LOS1 could participate in the feedback regulation of the CBF genes. LOS1 encodes a translation elongation factor, required for translation in low temperature indicating that either CBFs or products of their target genes are needed for repression of the CBF expression (Guo et al., 2002). Xiong and others (2002) identified an Arabidopsis mutant, fry2 (FIERY2) allelic to the clp1 mutant (Koiwa et al. 2002), showing enhanced levels of mRNAs corresponding to CBF2 and cold stress-responsive genes. The enhanced expression is restricted to genes that are regulated by CBFs indicating that FRY2 is involved in a specific repression mechanism for the CBFs (Xiong et al., 2002). FRY2 encodes a phosphatase, specific for the conserved heptapeptide in the C-terminal domain of RNA polymerase II (CTD). Dephosphorylation of CTD is essential for promoter clearance and proper processing of the mRNA transcript, indicating that this repression is taking place during transcription. Interestingly, even if the CBFs and their target genes were superinduced in the fry2 mutant, the plants were impaired in cold acclimation (Xiong et al., 2002). Analogously with the fry2 mutant, Arabidopsis fry1 (FIERY1) mutants exhibited enhanced expression of CBFs and their target genes but showed impaired cold tolerance (Xiong et al., 2001). CBF genes are also negatively regulated by a transcription factor MYB15 (an R2R3MYB family protein) in Arabidopsis. MYB15 has a low constitutive expression but is cold responsive, and the MYB15 protein can bind to the MYB recognition elements present in the promoters of the CBFs (Agarwal et al., 2006). MYB15 T-DNA insertion mutant lines show enhanced expression of CBFs during cold acclimation and enhanced freezing tolerance, whereas transgenic Arabidopsis overexpressing MYB15 show a decreased expression of CBFs and a reduction in freezing tolerance. Thus, MYB15 is an upstream transcription factor that negatively regulates the expression of CBFs (Agarwal et al., 2006) (Figure 8.2). Interestingly, ICE1 can negatively regulate MYB15 as indicated from the increased MYB15 transcript level in ice1 mutant compared with wildtype plants under cold stress (Agarwal et al., 2006). Yeast two-hybrid and in vitro pull-down assays showed that MYB15 can interact with ICE1, but the functional significance of ICE1–MYB15 interaction in cold acclimation remains to be determined (Agarwal et al., 2006). A cold-induced C2H2-type zinc finger transcription factor, ZAT12, also appears to function as a negative regulator of CBFs, due to the fact that ZAT12 overexpression leads to decreased expression of CBFs (Vogel et al., 2005). Another C2H2-type zinc-finger transcription factor, ZAT10, has been indicated as a negative regulator of CBF target genes (Lee et al., 2002). The gene encoding ZAT10 appears to be positively regulated by CBFs, as indicated by the enhanced and reduced expression of ZAT10 in CBF3 overexpressing and ice1 mutant plants, respectively (Maruyama et al., 2004; Chinnusamy et al., 2003). ZAT10 is negatively regulated by LOS2, a bifunctional enolase that apparently binds to the MYC recognition sequence in the ZAT10 promoter (Lee et al., 2002).

200

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

Figure 8.2 Control of the CBF regulon in Arabidopsis (see text for details). For color detail, please see color plate section.

The ICE1-CBF regulatory system is also controlled/modified by altering the stability of ICE1 by alternative ubiquitination/sumoylation (Lee et al., 2001; Miura et al., 2007). Ubiquitin E3 ligases are ubiquitinating proteins, thus providing substrate specificity for regulated proteolysis by the 26S proteasome pathway and small ubiquitin-like modifier (SUMO) proteins are a family of small proteins that are covalently attached to protein substrates by SUMO E3 ligases. Sumoylation is preventing ubiquitinylation, thus protecting proteins from degradation (Ulrich 2005). In Arabidopsis, the hos1 mutation showing enhanced expression of CBFs and their target genes during LT treatment, has been shown to be in a gene encoding a RING finger ubiquitin E3 ligase (Lee et al., 2001). Transgenic Arabidopsis plants overexpressing HOS1 show a substantial reduction in the level of the ICE1, and a reduction in levels of the CBFs and their downstream genes as well as hypersen-

GENES AND GENE REGULATION FOR LOW-TEMPERATURE TOLERANCE

201

sitivity to freezing stress (Dong et al. 2006). HOS1 has been shown to interact with ICE1 and mediate its ubiquitination and subsequent degradation, which has been suggested to be critical for the desensitization of plants to cold stress (Dong et al., 2006). In the siz1 mutation, on the other hand, the cold-induction of CBFs and their target COR genes is decreased, but the cold induction of MYB15 is increased (Miura et al., 2007). SIZ1 is encoding a SUMO E3 ligase, which is required for the accumulation of SUMO conjugates during cold stress. In contrast to HOS1, which promotes the proteolysis of ICE1, SIZ1 is mediating sumoylation of ICE1 during cold acclimation (Miura et al., 2007). SIZ1 is conjugating SUMO to L393 of ICE1, and the sumoylation is blocked by a K393R substitution. Consequently, transgenic Arabidopsis lines overexpressing ICE1 (K393R) do not show the enhanced cold induction of CBFs and increased freezing tolerance that is seen in lines expressing wild-type ICE1. Similar to the ice1 mutant plants, ICE1 (K393R) overexpressing transgenic plants also showed increased expression of MYB15 during LT treatment and sensitivity to freezing (Miura et al., 2007). It appears that SIZ1-mediated sumoylation increases ICE1 stability against HOS1 mediated ubiquitination to ensure the induction of CBFs and repression of MYB15 expression. Interplay between HOS1 and SIZ1 is then used to fine-tune the transcription of the genes during cold acclimation (Miura et al., 2007). Recently, Doherty et al. (2009) demonstrated that two of the calmodulin-binding transcriptional activators (CAMTAs), CAMTA1 and CAMTA3, were required for full cold acclimation. CAMTA3 was also shown to be required for activation of the CBF1 and CBF2 genes, as their expression was reduced by 40% and 50%, respectively, in the CAMTA3 T-DNA insertion lines (Doherty et al., 2009). The involvement of the members of the CAMTA transcription factor family in the early events in cold acclimation is also providing a bridge between cold responsive gene expression and the rapid Ca+2 influx that occurs when plants are exposed to low temperature. An additional level of regulation of the CBF regulon is emerging from the analysis of the sfr6 mutant of Arabidopsis (Knight et al., 2009). Warren and others (1996) originally isolated a set of mutants impaired in their cold acclimation, called sfr (sensitive to freezing) mutants. In the sfr6 mutant, the accumulation of cold-induced mRNA corresponding to genes containing the CRT element in their promoter regions was markedly reduced, but the level of the expression of CBFs or cold-responsive genes without the CRT element was unaffected, indicating that SFR6 specifically affects a component in the signaling pathway downstream of CBF transcription (Knight et al., 1999; Boyce et al., 2003). Recently, a cDNA corresponding to SFR6 was cloned, and the sequence predicts a plant-specific protein with a molecular weight of 137 kD. However, no function has yet been assigned to the SFR6 protein (Knight et al., 2009). The unaltered level of the CBF transcripts and reduction in the expression of the CBF target genes suggest that the SFR6 mutation interferes with either the translation of the CBF mRNAs or the function of the CBF proteins. Knight and others (2009) demonstrated that the level of the CBF proteins is not affected, indicating that the SFR6 is needed for the transcriptional activator function of the CBFs. In accordance to the predicted level of action, they were showing that the SFR6 protein is predominantly localized in the nucleus (Knight et al., 2009). CBF-independent Regulons The function of the CBF regulon has, to a large extent, relied on isola-

tion of mutants with altered expression of CBF target genes or the CBFs themselves. A significant finding emerging from these studies is that even if several of the isolated mutants exhibit superinduction of CBFs and CBF target genes they do not show improvement in freezing tolerance (Xiong et al., 2001; Xiong et al., 2002). This indicates that the cold acclimation involves additional regulons, whose contribution is needed for development of tolerance. Initial transcript profiling by using the microarray containing ∼8,000 Arabidopsis genes indicated that the CBFs regulon is containing only 12% of the total cold-responsive transcriptome (Fowler and Thomashow, 2002). Out of the

202

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

cold-induced genes that were not part of the CBF regulon, 15 encoded known transcription factors indicated that multiple regulons/induction pathways are involved in cold acclimation (Fowler and Thomashow 2002). Subsequent analysis using the affymetrix gene chip for 24,000 Arabidopsis genes has refined the results, and the current estimate is that ∼28% of the cold responsive genes are in the CBF regulon (Vogel et al., 2005). However, a large portion of cold-responsive genes seems to be regulated by other transcription factors. Vogel and others analyzed the role of five transcription factors, RAV1, MYB73, ZAT10, CZF2, and ZAT12, whose genes were LT responsive, with similar kinetics to the CBF2, in configuration of the LT transcriptome. Analysis of the transcriptome in transgenic lines overproducing these transcription factors failed to assign any genes as being targets for these proteins, with the exception of ZAT12 (Vogel et al., 2005). However, the activity of these transcription factors might require LT-dependent post-translational modifications, or they might act in combination with other factors produced/activated only in low temperature. Therefore, the involvement of these transcription factors in cold acclimation remains to be elucidated. Out of the transcription factors analyzed by Vogel and others (2005), only ZAT12 was clearly having an effect on the transcription of the LT responsive genes. Twenty-four genes were responsive to ZAT12, nine of which were cold induced, and fifteen cold repressed. Seven of the ZAT12 regulon genes were also responsive to CBF2, indicating coordinated activation of these genes (Vogel et al., 2005). ESK1 (ESKIMO1) was recently suggested to be a component of a CBF-independent pathway conferring freezing tolerance (Xin et al., 2007). Xin and Browse (1998) originally identified the eskimo1 (esk1) mutant in Arabidopsis. This mutant was constitutively freezing tolerant and accumulated high levels of proline, indicating that the ESK1 was acting as a negative regulator of freezing tolerance development (Xin and Browse 1998). The ESK1 gene was recently isolated, and it was found to encode a 56.7 kD protein that contains a plant-specific DUF231 domain (domain of unknown function 231). However, no molecular function has been assigned to the protein (Xin et al. 2007). ESK1 gene was found not to be responsive to low temperature. The analysis of the transcriptome in the esk1 mutant relieved that 312 genes showed altered expression, with 173 genes upregulated and 139 genes downregulated as compared with the corresponding wild type. Interestingly, only 12% of the genes upregulated in the esk1 were the same as reported to be upregulated in the CBF2 overexpressing Arabidopsis (Xin et al., 2007). This indicates that the enhanced freezing tolerance in the esk1 mutant is to a large extent determined by factors not regulated by the CBF pathway (Xin et al., 2007). However, the molecular mechanism that leads to activation of these genes remains to be clarified. Bouchabke-Coussa and others (2008) have recently raised a question whether ESK1 is a true negative regulator of tolerance development. They have demonstrated that the esk1 mutant is deficient in water uptake, this leading to a slight constitutive water stress, and speculate that this might lead to the observed alterations in gene expression and tolerance (Bouchabke-Coussa et al., 2008). Members of the WRKY-family of transcription factors are known to be involved in several different processes, including stress responses, particularly in defense against pathogens (Eulgem and Somssich, 2007). In Arabidopsis, the WRKY-family consists of 74 members (Eulgem et al., 2000). Evidence for the involvement of some of the WRKY-type of transcription factors in cold acclimation is slowly emerging. Microarray analysis has demonstrated that some of the WRKY encoding genes are regulated by cold (Karam et al., 2002; Lee et al., 2005); and a cold-inducible WRKYtype of transcription factors have been identified in Solanum dulcamara and barley (Huang and Duman, 2002; Mare et al., 2004). However, whether these proteins are having an active role in cold acclimation remains to be seen. Recently, Zhou and others (2008) identified 64 WRKY-type transcription factor genes in soybean. Several of the genes exhibited responsiveness to abiotic stresses, including eight that were also LT responsive (Zhou et al., 2008). Overexpression of

GENES AND GENE REGULATION FOR LOW-TEMPERATURE TOLERANCE

203

GmWRKY21, one of the cold-responsive WRKYs in Arabidopsis resulted in enhanced tolerance to freezing, but had no effect on salt or osmotic stress tolerance, indicating that WRKY-type of transcription factors are involved in cold acclimation (Zhou et al., 2008). Another large, plant-specific, transcription-factor family, whose members are known to be involved in abiotic stress responses is the NAC-family. NAC-family in Arabidopsis contains more than 100 members, and some of those have been shown to be responsive to low temperature (Tran et al., 2004; Fujita et al., 2004). The role of NAC-proteins in drought and osmotic stress is well established, and they appear to act both negatively and positively to stress responses. The expression of the Arabidopsis NAC-protein encoding gene ATAF1 has been shown to be responsive to dehydration and ABA (Lu et al., 2007). However, analysis of T-DNA insertion mutants of ATAF1 showed that the dehydration tolerance of these mutants was higher than in the corresponding wild type, and the increased tolerance was correlating with increased expression of stress responsive genes (Lu et al., 2007). This indicates that ATAF1 is negatively regulating drought responses in Arabidopsis. On the other hand, overexpression of three Arabidopsis NAC-encoding genes ANAC019, ANAC055, and ANAC072 increases drought tolerance and enhances expression of stress responsive genes (Tran et al., 2004). Stress responsive genes encoding NAC-proteins have also been characterized in rice, and several of those have been shown to confer drought tolerance when overexpressed in transgenic rice (Hu et al., 2006; Nakashima et al., 2007; Zheng et al., 2009). Recently, overexpression of NAC-encoding genes in rice has also been shown to increase cold tolerance (Hu et al., 2008). Genetic analysis of Arabidopsis mutants has also demonstrated that in addition to ICE1, other constitutively expressed transcription factors are mediating the LT responses. HOS9 and HOS10, encoding a homeodomain- and R2R3 MYB-type of transcription factors, respectively, were isolated as mutants with enhanced expression of CBF target genes, but with no alteration in the expression of the CBF genes (Zhu et al., 2004; Zhu et al., 2005). Both hos9 and hos10 mutants were incapable for cold acclimation, and the basal level of freezing tolerance was severely reduced (Zhu et al., 2004; Zhu et al., 2005; Zhu et al., 2007). Microarray analysis has also demonstrated that HOS9, HOS10, and CBFs are affecting different sets of genes during cold acclimation. Interestingly, by using extensive bioinformatics analysis, Benedict and others (2006) have placed HOS9 in the same pathway and downstream from ICE1.

Post-transcriptional Regulation

Recent studies have revealed that post-transcriptional regulation plays a critical role in cold acclimation (Chinnusamy et al., 2007). Post-transcriptional gene expression is regulated by several different strategies including pre-mRNA processing, mRNA transport and stability as well as translation. In a genetic screen using transgenic Arabidopsis harboring the promoter from the abiotic stress and ABA inducible gene RD29A fused to the LUC reporter gene, Lee and others (2006) identified a mutation, where the stability of the normally highly unstable LUC mRNA was enhanced. The mutated gene was identified, and it was shown to encode STABILIZED1 (STA1), a nuclear pre-mRNA splicing factor, and subsequent genetic analysis revealed that STA1 was required for splicing during low temperature. The sta1 mutant was found to be defective in splicing of the cold-induced COR15A pre-mRNA (Lee et al., 2006). Apparently STA1 is regulating both mRNA turnover and splicing during cold acclimation (Lee et al., 2006). Similarly in wheat, two LT responsive genes (encoding a ribokinase and a C3H2C3 RING-finger protein) have been shown to be regulated through alternative splicing under cold stress (Mastrangelo et al., 2005).

204

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

It is a well-known fact that pre-mRNA splicing is coupled with export of mRNAs from the nucleus to the cytoplasm through the nuclear pore complex (NPC). NPCs are composed of several proteins called nucleoporins (NUP). NUPs together with RNA export factors and DEAD-box protein 5 (Dbp5) function in nucleocytoplasmic traffic of nuclear proteins (Cole and Scarcelli, 2006). Recently, the AtNUP160 was found to have a critical role in cold acclimation (Dong et al., 2006). Arabidopsis atnup160-1 mutant plants were not only impaired in the cold induction of CBFs and several other genes involved in cold acclimation, but also hypersensitive to chilling and freezing stresses, indicating a role for the nuclear pore complex (NPC) in chilling and freezing tolerance (Dong et al., 2006). On the other hand, nuclear trafficking not only involves the NUPs, but also karyopherins. By employing a genetic screen, an important β-domain/karyopherin protein SAD2, has been identified (Verslues et al., 2006). SAD2 was found to be involved in nucleocytoplasmic trafficking during ABA treatment, and the sad2-1 mutant plants exhibited hypersensitive response to ABA and enhanced expression of stress in response to ABA, salt, and low temperature (Verslues et al., 2006). Consequently, SAD2 was suggested to function either in the import of a negative regulator or in the export of a positive regulator of ABA responses in Arabidopsis (Verslues et al., 2006). The DEAD-box family of RNA helicases contains proteins that are involved in different aspects of RNA metabolism, including mRNA export (Cole and Scarcelli, 2006). A role for a DEAD-box RNA helicase in mRNA export during abiotic stress was revealed from the analysis of the los4 mutant of Arabidopsis (Gong et al., 2002). The los4-1 mutant showed a reduced expression of the CBF genes, as well as the CBF target genes during cold acclimation. Furthermore, the los4-1 had a chilling-sensitive phenotype, and it was incapable of cold acclimation. The LOS4 gene was cloned, and it was shown to encode a DEAD-box RNA helicase (Gong et al., 2002). Recently, another mutant allele for LOS4 was isolated. This mutant los4-2 (also called cryophyte) exhibits increased chilling and freezing tolerance and enhanced expression CBF2 and CBF target genes (Gong et al., 2005). Interestingly, the los4-2 mutant exhibited reduced heat tolerance (Gong et al., 2005). The function of the LOS4 protein was demonstrated to be in mRNA transport. The los4-1 and los4-2 alleles were defective in mRNA transport, los4-2 being a temperature-sensitive allele of the gene (Gong et al., 2005). Recently, Kim and others (2008) demonstrated that two other Arabidopsis genes, AtRH9 and AtRH25, encoded LT responsive DEAD-box RNA helicases. Furthermore, by analyzing T-DNA mutants of the genes and transgenic Arabidopsis plants, where the genes were overexpressed, they were able to demonstrate that AtRH25, but not AtRH9, contributes to freezing tolerance. These differences in their function also correlate with differences in their nucleic acid binding capacity (Kim et al., 2008). In bacterial cells, the cold tolerance is to a large extent dependent on the production of cold shock proteins (CSP), acting as RNA chaperones with functions in transcriptional antitermination and facilitation of translation (Phadtare et al., 1999). Plants also harbor genes-encoding proteins with high homology to the bacterial CSPs. Whereas in bacteria the CSPs are almost exclusively small proteins, only containing the RNA binding cold shock domain (CSD). In plants the CSD is nearly always connected to auxiliary domains, which contain a glycine-rich region and two or more copies of CCHC-type of Zn2+-fingers (Karlson and Imai, 2003). Cold-responsive CSD-proteins have been identified in several plant species, including wheat (Karlson et al., 2002) and Arabidopsis (Karlson and Imai, 2003). The wheat CSD-protein, WCSD1, was also demonstrated to bind both DNA and RNA (Karlson et al., 2002). An RNA-binding protein, related to the CSD-protein, has also been suggested to be involved in cold acclimation. Kim and others (2005) identified a cold responsive gene, atRZ-1a, encoding a protein with an N-terminal RNA-binding domain, different from the CSD, but similar to a domain found in a cold-responsive RNA-binding protein in cyanobacteria (Maruyama et al., 1999), connected to a glycine-rich region and CCHC-type of zincfingers. Overexpression of atRZ-1a in transgenic Arabidopsis increased the freezing tolerance of the

GENES AND GENE REGULATION FOR LOW-TEMPERATURE TOLERANCE

205

plants, indicating that atRZ-1a is involved in cold acclimation (Kim et al., 2005). Both overexpressors of atRZ-1a and atRZ-1a T-DNA lines did not show any changes in cold-responsive induction of gene expression, indicating that the atRZ-1a is not affecting cold acclimation on the transcriptional level, and consequently, atRZ-1a was suggested to act as an RNA chaperone during LT stress (Kim and Kang, 2006). Small noncoding RNAs known as microRNAs (miRNA) and short interfering RNAs (siRNA) are 20–25 nucleotide-long double-stranded RNA molecules that play a variety of roles in RNAirelated pathways and are ubiquitous repressors of gene expression in animals and plants (Sunkar and Zhu, 2004). Both miRNAs and siRNAs act as repressors by binding to their target mRNAs and guiding them to degradation or inhibiting their translation, depending on the complementarity between the mi/siRNA and the target (Bartel, 2004). MiRNAs that are induced in response to stress can inhibit the expression of genes encoding negative regulators, and inhibition of the expression of miRNAs can lead to the accumulation of their target mRNAs, encoding positive regulators, or determinants of stress tolerance (Sunkar et al., 2007). Several miRNA genes have been shown to be regulated by abiotic stresses (Sunkar and Zhu, 2004; Liu et al., 2008). Regarding low temperature, two current studies have defined 8 miRNA families (Zhou et al., 2008) and 10 miRNAs (Liu et al., 2008) as LT responsive. However, the targets for these miRNAs are currently not well defined. However, one of the cold-responsive miRNAs, miR393, has been shown to target five genes, encoding members of the TIR1 family F-box E3 ubiquitin ligases (Sunkar and Zhu, 2004). Thus, cold-upregulated miR393 might lead to the cleavage of E3 ubiquitin ligase mRNAs, resulting in reduced proteolysis of their target proteins during cold acclimation. Furthermore, the miR393 might be involved in sugar signaling during stress responses as one of its predicted targets, At4g03190, is encoding an F-box protein similar to the yeast glucose repression resistance 1 (GRR1) (Sunkar et al., 2007). Modification of the histone proteins in the nucleosomal core is leading to alterations in chromatin structure, which has long been known to be an important level of gene regulation in eukaryotic cells (reviewed in Pfluger and Wagner, 2007). Acetylation of histones leads to more open chromatin structure, thus facilitating binding of transcription factors and increasing transcription. Conversely, deacetylation of histones leads to more compact chromatin and repression of transcription. Recently, Zhu and others (2008) demonstrated that histone modifications are involved in cold acclimation, by identifying an Arabidopsis gene, HOS15, that encodes a protein involved in deacetylation of chromatin. Hos15 mutant exhibits enhanced induction of CBF target genes, but had no effect on the transcription of the CBFs (Zhu et al., 2008). HOS15 is a WD-repeat protein that specifically binds to histone 4 (H4), and the level of H4 acetylation in the promoter region of the CBF target gene RD29a was increased in the hos15 mutant (Zhu et al., 2008). CBFs have previously been shown to interact with a histone acetyltransferase, AtGCN5, indicating that the CBF regulation of its target genes is including chromatin alterations (Stockinger et al., 2001; Vlachonasios et al., 2003). Interestingly, the hos15 mutant was sensitive to freezing, indicating that repression of HOS15 target genes is essential for development of freezing tolerance (Zhu et al., 2008).

Abscisic Acid (ABA)-dependent Cold Signal Pathway

The phytohormone ABA plays a key role in plant development, regulation of the stomatal aperture, and responses to environmental stresses (Finkelstein et al., 2002). The involvement of ABA in development of drought and salt stresses has been extensively characterized (Zhu, 2002; Chinnusamy et al., 2004; Verslues and Zhu, 2007), but the involvement of ABA in cold acclimation has not been unequivocally demonstrated. However, ABA is clearly an important phytohormone for

206

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

development of freezing tolerance, as indicated by studies demonstrating that both ABA-insensitive (abi) and ABA (aba)-deficient mutants are partially impaired in cold acclimation (Heino et al., 1990; Gilmour and Thomashow, 1991; Mäntylä et al., 1995). In addition, exogenous application of ABA at normal growth temperature leads to increased freezing and chilling tolerance (Chen et al., 1983; Lång et al., 1989; Li et al., 1997, 2003). The level of ABA has also been shown to increase transiently in both herbaceous and woody plants exposed to low temperature (Chen et al., 1983; Li et al., 2002; Mäntylä et al., 2005). However, the increase in the ABA level during cold acclimation is modest, as compared to the increase during drought or salt stresses, and this has raised the question whether this increase is physiologically significant. Recent studies have, however, provided support for the active involvement of ABA also in the cold acclimation process. Cuevas and others (2008) have demonstrated that accumulation of the polyamine putrescine is essential for cold acclimation. The mutants defective in putrescine biosynthesis (adc1 and adc2) exhibited reduced freezing tolerance, and the reduction was caused, at least partially, by the inability of the mutants to increase the ABA level during cold acclimation, due to the lack of induction of the NCED3 gene, encoding 9-cis-epoxycarotenoid dioxygenase, a key enzyme in ABA biosynthesis (Cuevas et al., 2008). The defect in freezing tolerance as well as the reduced activation of ABAresponsive genes during cold acclimation could be complemented by added ABA, indicating that the increase in the level of ABA is essential for development of freezing tolerance (Cuevas et al., 2008). The HOS10 transcription factor (see above) is also affecting the ABA levels, because the hos10 mutant is unable to fully induce the NCED3 gene. The fact that the hos10 mutant has lost the cold acclimation capacity is further indicating a role for ABA in this process. The main regulation of the ABA-responsive genes during abiotic stresses is by basic leucine zipper (bZIP)-transcription factors called ABFs/AREBs, binding to ABA-responsive elements (ABRE) (consensus sequence C/TACGTGGC) in the promoter regions of their target genes (Xiong et al., 2002). In Arabidopsis four ABF genes have been identified and one of them, ABF1, is specifically induced with low temperature and ABA (Choi et al., 2000). A cold and ABA responsive Zn-finger protein SCOF-1, has been characterized in soybean (Kim et al., 2001). Overexpression of SCOF-1 in transgenic Arabidopsis leads to expression of LT-responsive genes at normal growth temperature and constitutive freezing tolerance (Kim et al., 2001). SCOF-1-mediated gene activation was shown to be dependent on its interaction with the bZIP-transcription factor SGBF1, which then is binding to the ABRE and activating gene expression (Kim et al., 2001). Recently, Liao and others (2008) identified three genes (GmbZIP44, GmbZIP62, and GmbZIP78) that encode bZIPtranscription factors negatively regulating ABA responses in soybean by binding to the ABRE. The overexpression of the genes in Arabidopsis lead to decreased ABA sensitivity, but increased the freezing and salt tolerance of the plants, demonstrating the complexity between ABA and stress responses (Liao et al., 2008). The primary source for increased ABA level during stress responses is through de novo synthesis (Cutler and Krochko, 1999). However, plants constantly need to adjust to fluctuating environmental conditions by rapid and often transient responses. It appears that plants also use the glucose-conjugated, inactive ABA (ABA-GE) for fine tuning of the ABA levels (Lee et al., 2006). ABA-GE is hydrolyzed by β-glucosidases; and in Arabidosis, the AtBG1 have been shown to have activity toward ABA-GE (Lee et al., 2006). AtBG1 was initially identified as a salt responsive gene in Arabidopsis, and subsequent analysis revealed that the encoded protein was localized in the endoplasmic reticulum (ER) and was able to hydrolyze ABA-GE. The hydrolytic activity was greatly enhanced by stress-induced polymerization of the enzyme (Lee et al., 2006). A T-DNA insertion line for AtGB1 was shown to have lower ABA levels and to exhibit increased sensitivity to dehydration stress, whereas overexpression of AtBG1 resulted in higher ABA level and increased stress

GENES AND GENE REGULATION FOR LOW-TEMPERATURE TOLERANCE

207

tolerance (Lee et al., 2006). This indicates that release of ABA from ABA-GE is an essential part of ABA-mediated processes in stress signaling (Lee et al., 2006). In leaves, the ABA-GE is localized in the apoplast and vacuoles (Dietz et al., 2000). The localization of the AtBG1 in the ER indicates a currently unknown active transport mechanism for ABA-GE (Lee et al. 2006). The SFR2 protein, which in Arabidosis is required for development of freezing tolerance, was recently identified as a chloroplast outer membrane localized β-glucosidase (Fourrier et al., 2008). Currently, the substrates for SFR2 have not been identified, and it is tempting to speculate that SFR2 could also be involved in hydrolysis of ABA-GA.

Cross Talk between Abiotic and Biotic Stress Responses

Design of new strategies for plants with enhanced tolerance to abiotic stress should include consideration for the consequences that such engineering might have on other plant responses. In nature and in the field, plants are continuously exposed to a variety of abiotic and biotic stresses. Consequently, timely and appropriate responses to different environmental insults are essential for plant survival and are executed through an intricate web of interacting signal transduction cascades. Furthermore, it is important to note that exposure to stress not only triggers protective mechanisms but seems to repress growth-related genes, and growth and stress tolerance appear often inversely correlated due to the high metabolic load of stress adaptation (Heil et al., 2000). Thus, plant adaptation to cold and freezing temperatures is not an isolated phenomenon in plant physiology but part of a network that may also affect other plant responses. There is an increasing body of literature suggesting that plants can shift their stress response priorities between abiotic and biotic response pathways. (For reviews, see Mauch-Mani and Mauch, 2005; Fujita et al., 2006; Asselbergh et al., 2008.) Hormonal signaling is central to this cross talk, and different hormones govern abiotic (ABA) as well as biotic (SA, JA, ET) stress responses. ABA appears as the key phytohormone mediating plant responses to drought, osmotic stress, and low temperature. In contrast to its positive role in abiotic stress signaling, ABA appears as a negative regulator of a number of pathogen responses and disease resistance in plants (Mohr and Cahill, 2003; Andersson et al., 2004; Kariola et al., 2006; de Torres-Zabala et al., 2007) although some exceptions do exist (see Mauch-Mani and Mauch, 2005). For example, EARLY RESPONSE TO DEHYDRATION 15 (ERD15) is a negative regulator of ABA signaling, and silencing of the gene leads to ABA hypersensitivity as well as improved freezing and drought tolerance of transgenic Arabidopsis (Kariola et al., 2006). In contrast, ERD15 overexpressors show ABA insensitivity, impaired freezing tolerance but enhanced induction of SAR marker genes and resistance bacterial pathogens. In accordance, it has been recently demonstrated exogenous ABA can suppress SAR signaling while activation of SAR resulted in suppression of ABA-biosynthetic and ABA-responsive genes (Yasuda et al., 2008). Similarly, our recent work with defense-related WRKY TFs suggests that downregulation of the corresponding genes results in a shift of plant stress response priorities toward more efficient responses to abiotic stress and consequently to enhanced tolerance to freezing and drought (Jing Z Li and E. Tapio Palva, unpublished).

Conclusions and Future Perspectives

Low-temperature and freezing stress are major environmental factors limiting plant growth, distribution, and productivity. Therefore, elucidation of the mechanisms of cold acclimation and

208

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

development of freezing tolerance is of vital importance for design of novel crop varieties that can better adapt to the changing environment and allow use of marginal lands for agricultural production. A concerted effort by using physiological, molecular, and forward and reverse genetic and genomic techniques is required to understand the complex quantitative trait of LT tolerance. Cold acclimation and survival of freezing stress are highly complex processes that involve the combined action of a vast amount of signaling compounds and transcription factors to regulate the network of events ultimately leading to the structural and metabolic alterations essential for tolerance. Systems biology approaches, such as large-scale profiling of the transcriptome and metabolome, have provided us a picture of this network, but more studies are required for mechanistic understanding of the underlying molecular events. For example, LT sensing is still enigmatic, and no actual receptor has been identified, and hence identifying the cold sensor(s) remain as one of the major challenges in cold acclimation research. Experimental evidence indicates that the early steps in cold acclimation involve membrane rigidification, followed by Ca2+-influx. The Ca2+-signal is then decoded by several different types of Ca2+-binding proteins, including CaMs and CLBs, which activate a diverse array of protein kinases, like CDPKs, CIPKs, and MAPKs. These then transmit the signal to transcription factors activating gene expression. A major thrust in this research has been the identification of the key transcription factors and the regulons controlled by these, such as the CBF regulon. Emerging evidence indicates that the regulatory systems and protective mechanisms are surprisingly similar throughout the plant kingdom. For example, the CBF-regulon central for cold acclimation in the model species Arabidopsis is also found in numerous other plant species. The use of transcript profiling and large-scale analysis of proteins and metabolites are providing the first, albeit still incomplete, views of the complex alterations that take place in the transcriptome, proteome, and metabolome during cold acclimation in plants and will define the key genes controlling these processes. Continuation of such studies are likely to provide increasing amounts of information and a combination of the obtained information will hopefully in the near future reveal the causal relationships between gene expression, physiological alterations, and development of tolerance. Extensive cross talk between LT stress and other abiotic and biotic stresses is central to stress adaptation as plants are constantly exposed to a variety of environmental cues and need to adjust their growth and metabolism accordingly. Hence, identifying the specific events required for cold acclimation and freezing tolerance development provides an additional challenge to this research. Furthermore, responses and tolerance to several stresses may share common components and processes. For example, tolerance to freeze-induced dehydration clearly involves mechanisms that are also participating in drought tolerance. Additionally, generation of excess ROS is a consequence of many stresses, both biotic and abiotic, and results in common protective responses. The major future challenge will be to use the obtained information for generation of transgenic plants that exhibit increased tolerance to freezing temperatures without any deleterious side effects. Approaches aiming at genetic improvement of freezing tolerance by transgenic technologies can be divided in two broad categories. The first approach is the introduction or ectopic expression of single genes encoding protective proteins or proteins needed for production of protective metabolites. This approach aims at alterations of a single character and has resulted in some improvement in tolerance as discussed above. However, due to the multitude of different processes involved in development of freezing tolerance, such an approach is of limited value, but could be enhanced by pyramiding several such genes affecting different processes required for tolerance. A more promising approach is to use strategies that aim to simultaneously control several different processes. Regulon engineering through ectopic expression of transcription factors, for example, provides the possibility of simultaneously altering the expression of a number of response genes and consequently affecting several characteristics. Ectopic expression of the CBF transcription factors has

GENES AND GENE REGULATION FOR LOW-TEMPERATURE TOLERANCE

209

been successfully used to enhance freezing tolerance of plants, even those growing at normal temperature. However, the adaptation to adverse environmental conditions is costly and is often combined with a general reduction in normal growth processes. Indeed, the transgenic plants with constitutive overexpression of CBFs exhibit severe growth retardation under normal growth conditions. Recently, this was shown to be due to the effect of CBFs on gibberellic acid (GA) mediated processes (Achard et al., 2008). Apparently CBFs are positively regulating the GA2 oxidase gene, which leads to decreased levels of GA and accumulation of DELLA proteins, which then negatively regulate the growth of the plants (Achard et al., 2008). To avoid such detrimental effects caused by the extensive cross talk between stress adaptation and growth-related processes, for example, it is important to design strategies where the ectopic expression of the transcription factors is tightly regulated, for example, by choosing a promoter that is only active when the response is needed. Hence, priming of the response could offer a strategy that provides sufficient protection to plants exposed to stress but without the detrimental effects on growth and productivity. Such an approach would also minimize the negative effects of enhanced freezing tolerance on disease resistance, for example. So far, the novel strategies have been mainly tested in laboratories, greenhouses, or sometimes experimental fields, but there is a need to develop efficient evaluation methods for the performance of the transgenic plants in field conditions over extended periods to assess their true value for agriculture. However, it is not overly enthusiastic to believe that the combined efforts of molecular biologists and plant breeders will in the future lead to a significant contribution to agricultural productivity.

Acknowledgments

The authors wish to thank all of the members of the laboratory for stimulating discussions. This work was supported by the Academy of Finland Center of Excellence program.

References Aalto, M.A., Heino, P., and Palva, E.T. (2006) Improving low temperature tolerance in plants. In Model plants and crop improvement. (Varshney, R.K. and Koebner, R.K., Eds.) Taylor and Francis Group, Boca Raton. Achard, P., Gong, F., Cheminant, S., Alioua, M., Hedden, P., and Genschik, P. (2008) The cold-inducible CBF1 factor-dependent signaling pathway modulates the accumulation of the growth-repressing DELLA proteins via its effect on gibberillin metabolism. Plant Cell 20:2117–2129. Agarwal, M., Hao, Y., Kapoor, A., Dong, C-H., Fujii, H., Zheng, X., and Zhu, J-K. (2006) A R2R3 type MYB transcription factor is involved in the cold regulation of CBF genes and in acquired freezing tolerance. J Biol Chem 281:37636–37645. Albrecht, V., Weinl, S., Blazevic, D., D’Angelo, C., Batistic, O., Kolukisaoglu, U., Bock, R., Schulz, B., Harter, K., and Kudla, J. (2003) The calcium sensor CBL1 integrates plant responses to abiotic stresses. Plant J 36:457–470. Allen, G.J., Muir, S.R., and Sanders, D. (1995) Release of Ca2+ from individual plant vacuoles by both InsP3 and cyclic ADPribose. Science 268:735–737. Alexandre, J., Lasalle, J.P., and Kado, R.T. (1990) Opening of Ca2+ channels in isolated red beet vacuole membrane by inositol-1, 4, 5-triphosphate. Nature 343:567–570. Alsheikh, M.K., Svensson, J.T., and Randall S.K. (2005) Phosphorylation regulated ion-binding is a property shared by the acidic subclass dehydrins. Plant Cell Environ 28:1114–1122. Anchordoguy, T.J., Rudolph, A.S., Carpenter, J.F., and Crowe J.H. (1987) Modes of interaction of cryoprotectants with membrane phospholipids during freezing. Cryobiology 24:324–331. Anderson, J.P., Badruzsaufari, E., Schenk, P.M., Manners, J.M., Desmond, O.J., Ehlert, C., Maclean, D.J., Ebert, P.R., and Kazan K. (2004) Antagonistic interaction between abscisic acid and jasmonateethylene signaling pathways modulates defense gene expression and disease resistance in Arabidopsis. Plant Cell 16:3460–3479.

210

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

Antikainen, M., Griffith, M., Zhang, J., Hon, W.C., Yang, D.S.C., and Pihakaski-Maunsbach, K. (1996) Immunolocalization of antifreeze proteins in winter rye leaves, crown and roots by tissue printing. Plant Physiol 110:845–857. Asselbergh, B., De Vleesschauwer, D., and Hofte, M. (2008) Global switches and fine-tuning—ABA modulates plant pathogen defense. Molecular Plant Microbe Interactions 21:709–719. Baker, S.S., Wilhelm, K.S., and Thomashow, M.F. (1994) The 5′-region of Arabidopsis thaliana cor15a has cis-acting elements that confer cold-, drought- and ABA-regulated gene expression. Plant Mol Biol 24:701–713. Bartel, D.P. (2004) MicroRNAs: Genomics, Biogenesis, Mechanism, and Function. Cell 116:281–297. Benedict, C., Geisler, M., Trygg, J., Huner, N., and Hurry, V. (2006) Consensus by democracy. Using meta-analyzes of microarray and genomic data to model the cold acclimation signalling pathway in Arabidopsis. Plant Physiol 141:1219–1232. Bouchabke-Coussa, O., Quashie, M-L., Seoane-Redondo, J., Fortabat, M.-N., Gery, C., Yu, A., Linderme, D., Trouverie, J., Granier, F., Téoulé, E., and Durand-Tardif, M. (2008) ESKIMO1 is a key gene involved in water economy as well as cold acclimation and salt tolerance. BMC Plant Biology 8:125. Bouché, N., Scharlat, A., Snedden, W., Bouchez, D., and Fromm, H. (2002) A novel family of calmodulin-binding transcription activators in multicellular organisms. J Biol Chem 277:21851–21861. Boyce, J.M., Knight, H., Deyholos, M., Openshaw, M.R., Galbraith, D.W., Warren, G., and Knight, M.R. (2003) The sfr6 mutant of Arabidopsis is defective in transcriptional activation via CBF/DREB1 and DREB2 and shows sensitivity to osmotic stress. Plant J 34:395–406. Bravo, L.A., Close, T.J., Corcuera, L.J., and Guy, C.L. (1999) Characterization of an 80-kDa dehydrin-like protein in barley responsive to cold acclimation. Physiol Plant 106:177–183. Bravo, L.A., Gallardo, J., Navarrete, A., Olave, N., Martínez, J., Alberdi, M., Close, T.J., and Corcuera, L.J. (2003) Cryoprotective activity of a cold induced dehydrin purified from barley. Physiologia Plantarum 118:262–269. Bray, E.A. (1993) Molecular responses to water-deficit. Plant Physiol 103:1035–1040. Chen, H-H., Li, P.H., and Brenner, M.L. (1983) Involvement of abscisic acid in potato cold acclimation. Plant Physiol 71:362–365. Cheong, Y.H., Kim, K-N., Pandey, G.P., Gupta, R., Grant, J.J., and Luan, S. (2003) CBL1, a calcium sensor that differentially regulates salt, drought, and cold responses in Arabidopsis. The Plant Cell 15:1833–1845. Chinnusamy, V., Ohta, M., Kanrar, S., Lee, B.H., Hong, X., Agarwal, M., and Zhu, J.K. (2003) ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis. Genes Dev 17:1043–1054. Chinnusamy, V., Schumaker, K., and Zhu, J.K. (2004) Molecular genetic perspectives on cross-talk and specificity in abiotic stress signalling in plants. Journal of Experimental Botany 55(395):225–236. Chinnusamy, V., Zhu, J., and Zhu, J.-K. (2007) Cold stress regulation of gene expression in plants. TRENDS in Plant Science 12(10). Choi, D.-W., Rodriguez, E.M., and Close, T.J. (2002) Barley cbf3 gene identification, expression pattern, and map location. Plant Physiology 129:1781–1787. Choi, H.-I., Hong, J.-H., Ha, J.-O., Kang, J.-Y., and Kim, S.Y. (2000) ABFs, a family of ABA-responsive element binding factors. J Biol Chem 275(3):1723–1730. Cole, C.N. and Scarcelli, J.J. (2006) Transport of messenger RNA from the nucleus to the cytoplasm. Curr Opin Cell Biol 18:299–306. Cook, D., Fowler, S., Fiehn, O., and Thomashow, M.F. (2004) A prominent role for the CBF cold response pathway in configuring the low-temperature metabolome of Arabidopsis. PNAS 101(42):15243–15248. Cossins, A.R. (1994) Homeoviscous adaptation of biological membranes and its functional significance, In A.R. Cossins (Ed.), Temperature Adaptation of Biological Membranes. Portland Press, London 63–76. Crowe, J.H., Oliver, A.E., Hoekstra, F.A., and Crowe, L.M. (1997) Stabilization of dry membranes by mixtures of hydroxyethyl starch and glucose: the role of vitrification. Cryobiology 35:20–30. Cuevas, J.C., López-Cobollo, R., Alcázar, R., Zarza, X., Koncz, C., Altabella, T., Salinas, J., Tiburcio, A.F., and Ferrando, A. (2008) Putrescine is involved in Arabidopsis freezing tolerance and cold acclimation by regulating ABA levels in response to low temperature. Plant Physiology Preview. DOI:10.1104/pp. 108. 122945. Cutler, A.J. and Krochko, J.E. (1999). Formation and breakdown of ABA. Trends Plant Sci 4:472–478. D’Angelo, C., Weinl, S., Batistic, O., Pandey, G.K., Cheong, Y.H., Schültke, S., Albrecht, V., Ehlert, B., Schulz, B., Harter, K., Luan, S., Bock, R., and Kudla, J. (2006) Alternative complex formation of the Ca2+-regulated protein kinase CIPK1 controls abscisic acid-dependent and independent stress responses in Arabidopsis. The Plant Journal 48:857–872. Danyluk, J., Perron, A., Houde, M., Limin, A., Fowler, B., Benhamou, N., And Sarhan, F. (1998) Accumulation of an acidic dehydrin in the vicinity of the plasma membrane during cold acclimation in wheat. Plant Cell 10:623–638. de Torres-Zabala, M., Truman, W., Bennett, M.H., Lafforgue, G., Mansfield, J.W., Rlodrigurz Egea, P., Bogre, L., and Grant, M. (2007) Pseudomonas syringae p.v. tomato hijacks the Arabidopsis abscisic acid signaling pathway to cause disease. Embo J 26:1434–1443. Delauney, A.J. and Verma, D.P. (1993) Proline biosynthesis osmoregulation in plants. Plant Journal 4:215–223.

GENES AND GENE REGULATION FOR LOW-TEMPERATURE TOLERANCE

211

Dietz, K.J., Sauter, A., Wichert, K., Messdaghi, D., and Hartung, W. (2000) Extracellular beta-glucosidase activity in barley involved in the hydrolysis of ABA glucose conjugate in leaves. J Exp Bot 51:937–944. Ding, J.P. and Pickard, B.P. (1993) Mechanosensory calcium-selective cation channels in epidermal cells. The Plant Journal 3(1) 8:3–110. Doherty, C.J., Van Buskirk, H.A., Myers, S.J. and Thomashow, M.F. (2009) Roles for Arabidopsis CAMTA Transcription Factors in Cold-Regulated Gene Expression and Freezing Tolerance. The Plant Cell 21:972–984. Dong, C.H., Hu, X., Tang, W., Zheng, X., Kim, Y.S., Le, B.-ha, and Zhu, J.-K. (2006) A putative Arabidopsis nucleoporin AtNUP160 is critical for RNA export and required for plant tolerance to cold stress. Mol Cell Biol 26:9533–9543. Dubouzet, J.G., Sakuma, Y., Ito, Y., Kasuga, M., Dubouzet, E.G., Miura, S., Seki, M., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2003) OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought-, high-salt- and cold-responsive gene expression. Plant J 33:751–763. Dure, III, L., Greenway, S.C., and Galau, G.A. (1981) Developmental biochemistry of cottonseed embryogenesis and germination: changing messenger ribonucleic acid populations as shown by in vitro and in vivo protein synthesis. Biochemistry 20:4162–4168. Dure, III, L., Crouch, M., Harada, J., David, T.H., Mundy, J., Quatrano, R., Thomas, J., and Sung, Z.R. (1989) Common amion acid sequence domains among the LEA proteins of higher plants. Plant Mol Biol 12:475–486. Eulgem, T., Rushton, P.J., Robatzek, S., and Somssich, I.E. (2000) The WRKY superfamily of plant transcription factors. Trends Plant Sci 5:199–206. Eulgem, T. and Somssich, I.E. (2007) Networks of WRKY transcription factors in defense signaling. Curr Opin Plant Biol 10(4):366–371. Finkelstein, R.R., Gampala, S.S.L., and Rock, C.D. (2002) Abscisic Acid Signaling in Seeds and Seedlings. The Plant Cell S15–S45. Fourrier, N., Bédard, J., Lopez-Juez, E., Barbrook, A., Bowyer, J., Jarvis, P., Warren, G., and Thorlby, G. (2008) A role for SENSITIVE TO FREEZING2 in protecting chloroplasts against freeze-induced damage in Arabidopsis. Plant J 55(5):734–745. Fowler, S. and Thomashow, M.F. (2002) Arabidopsis transcriptome profiling indicates multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold-response pathway. Plant Cell 14:1675–1690. Frank, W., Munnik, T., Kerkmann, K., Salamini, F., and Bartles, D. (2000) Water deficit triggers phospholipase D activity in the resurrection plant Craterostigma plantagineum. Plant Cell 12:111–124. Fujita, M., Fujita, Y., Maruyama, K., Seki, M., Hiratsu, K., Ohme-Takagi, M., Tran, L.S.P., Shinozaki, K., and YamaguchiShinozaki, K. (2004) A dehydration-induced NAC protein, RD26 is involved in a novel ABA-dependent stress signaling pathway. Plant J. 39:863–876. Fujita, M., Fujita, Y., Noutoshi, Y., Takahashi, F., Narusaka, Y., Yamaguchi-Shinozaki, K., and Shinozaki, K. (2006) Crosstalk between abiotic and biotic stress responses: a current view from the points of convergence in the stress signaling networks. Curr Opin Plant Biol 9(4):436–442. Fursova, O.V., Pogorelko, G.V., and Tarasov, V.A. (2009) Identification of ICE2, a gene involved in cold acclimation which determines freezing tolerance in Arabidopsis thaliana. Gene 429:98–103. Gardiner, J.C., Harper, J.D.I., Weerakoon, N.D., Collings, D.A., and Ritchi, S. (2001) A 90-kD phospholipase D from tobacco binds to microtubules and the plasma membrane. Plant Cell 13:2143–2158. Gilmour, S.J. and Thomashow, M.F. (1991) Cold acclimation and cold-regulated gene expression in ABA mutants of Arabidopsis thaliana. Plant Mol Biol 17:1233–1240. Gilmour, S.J., Zarka, D.G., Stockinger, E.J., Salazar, M.P., Houghton, J.M., and Thomashow, M.F. (1998) Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold induced COR gene expression. Plant J 16:433–443. Goldstein, G. and Nobel, P.S. (1994) Water Relations and Low-Temperature Acclimation for Cactus Species Varying in Freezing Tolerance. Plant Physiol 104(2):675–681. Gong, Z., Lee, H., Xiong, L., Jagendorf, A., Stevenson, B., and Zhu, J.K. (2002) RNA helicase-like protein as an early regulator of transcription factors for plant chilling and freezing tolerance. Proc Natl Acad Sci 99:11507–11512. Gong, Z.Z., Dong, C.H., Lee, H., Zhu, J.H., Xiong, L.M., Gong, D.M., Stevenson, B., and Zhu, J.K. (2005) A DEAD box RNA helicase is essential for mRNA export and important for development and stress responses in Arabidopsis. Plant Cell 17:256–267. Goyal, K., Walton, L.J., and Tunnacliffe, A. (2005) LEA proteins prevent protein aggregation due to water stress. Biochem J 388:151–157. Griffith, M., Ala, P., Yang, D.S.C., Hon, W.C., and Moffat, B. (1992) Antifreeze protein produced endogenously in winter rye leaves. Plant Physiol 100:593–596. Griffith, M. and Yaish, M.W.F. (2004) Antifreeze proteins in overwintering plants: a tale of two activities. Trends Plant Sci 9:399–405.

212

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

Griffith, M., Lumb, C., Wiseman, S.B., Wisniewski, M., Johnson, R.W., and Marangoni, A.G. (2005) Antifreeze Proteins Modify the Freezing Process In Planta. Plant Physiology 138:330–340. Guinchard, M.P., Robin, C., Grieu, P., and Guckert, A. (1997) Cold acclimation in white clover subjected to chilling and frost: Changes in water and carbohydrates status. European Journal of Agronomy 6:225–233. Guo, Y., Xiong, L., Song, C.P., Gong, D., Halfter, U., and Zhu, J.K. (2002) A calcium sensor and its interacting protein kinase are global regulators of abscisic acid signaling in Arabidopsis. Dev Cell 3:233–244. Guy, C.L. and Li, Q.B. (1998) The organization and evolution of the spinach stress 70 molecular chaperone gene family. Plant Cell 10:539–556. Haake, V., Cook, D., Riechmann, J.L., Pineda, O., Thomashow, M.F., and Zhang, J.Z. (2002) Transcription Factor CBF4 Is a Regulator of Drought Adaptation in Arabidopsis. Plant Physiology 130:639–648. Hannah, M.A., Heyer, A.G., and Hincha D.K. (2005) A global survey of gene regulation during cold acclimation in Arabidopsis thaliana. PLoS Genet 1, e26. Hara, M., Terashima, S., and Kuboi, T. (2001) Characterization and cryoprotective activity of cold-responsive dehydrin from Citrus unshiu. Journal of Plant Physiology 158:1333–1339. Hara, M., Terashima, S., Fukaya, T., and Kuboi, T. (2003) Enhancement of cold tolerance and inhibition of lipid peroxidation by citrus dehydrin in transgenic tobacco. Planta 217:290–298. Heil, M., Hilpert, A., Kaiser, W., and Linsenmair, K.E. (2000). Reduced growth and seed set following chemical induction of pathogen defence: Does systemic acquired resistance (SAR) incur allocation costs? J Ecol 88:645–654. Heino, P., Sandman, G., Lång, V., Nordin, K., and Palva, E.T. (1990) Abscisic acid deficiency prevents development of freezing tolerance in Arabidopsis thaliana (L.) Heynh. Theor Appl Genet 79:801–806. Heino, P. and Palva, T. (2003) Signal transduction in plant cold acclimation. Topics in Current Genetics 4. Heino, P., Nilsson, O., and Palva, E.T. (2009) Photoperiodic control of dormancy and flowering in trees. In Photoperiodism: The biological calendar. (Nelson, R.J., Denlinger, D.L., and Somers, D.E., Eds.) Oxford University Press, New York (in press). Hincha, D.K., Bratt, P.J., and Williams, W.P. (1997a) A cryoprotective lectin reduces the solute permeability and lipid fluidity of thylakoid membranes. Cryobiology 34:193–199. Hincha, D.K., Neukamm, B., Sror, H.A.M., Sieg, F., Weckwarth, W., Rückels, M., Lullien-Pellerin, V., Schröder, W., and Schmitt, J.M. (2001) Cabbage cryoprotectin is a member of the nonspecific plant lipid transfer protein gene family. Plant Physiol 125:835–846. Hincha, D.K., Popova, A.V., and Cacela C. (2006) Effects of sugars on the stability of lipid membranes during drying. In: Advances in planar lipid bilayers and liposomes, Elsevier 3:189–217. Hincha, D.K., Rennecke, P., and Oliver, A.E. (2007) Protection of liposomes against fusion during drying by oligosaccharides is not predicted by the calorimetric glass transition temperatures of the dry sugars. European Biophysics Journal 37:503–508. Holmström, K.O., Somersalo, S., Mandal, A., Palva, E.T., and Welin, B. (2000) Improved tolerance to salinity and low temperature in transgenic tobacco producing glycine betaine. Journal of Experimental Botany 51(343):177–185. Hon, W.C., Griffith, M., Chong, P., and Yang, D.S.C. (1994) Extraction and isolation of antifreeze proteins from winter rye (Secale cereale L.). Plant Physiol 104:971–980. Hong, S.W., Jon, J.H., Kwak, J.M., and Nam, H.G. (1997) Identification of a receptor-like protein kinase gene rapidly induced by abscisic acid, dehydration, high salt, and cold treatments in Arabidopsis thaliana. Plant Physiology 113:1203–1212. Houde, M., Dallaire, S., N’Dong, D., and Sarhan, F. (2004) Overexpression of the acidic dehydrin WCOR410 improves freezing tolerance in transgenic strawberry leaves. Plant Biotechnology Journal 2:381–387. Hsieh, T.-H., Lee, J.-T., Yang, P.-T., Chiu, L.-H., Charng, Y.Y., Wang, Y.-C., and Chan, M.-T. (2002) Heterology expression of the Arabidopsis C-repeat/dehydration response element binding factor 1 gene confers elevated tolerance to chilling and oxidative stresses in transgenic tomato. Plant Physiol 129:1086–1094. Hu, H., Dai, M., Yao, J., Xiao, B., Li, X., Zhang, Q., and Xiong, L. (2006) Overexpressing a NAM, ATAF, and CUC (NAC) transcription factor enhances drought resistance and salt tolerance in rice. Proc Natl Acad Sci 35:12987–12992. Hu, H.H., You, J., Fang, Y.J., Zhu, X.Y., Qi, Z.Y., and Xiong, L.Z. (2008) Characterization of transcription factor gene SNAC2 conferring cold and salt tolerance in rice. Plant Mol Biol 67:169–181. Huang, T. and Duman, J.G. (2002) Cloning and characterization of a thermal hysteresis (antifreeze) protein with DNA-binding activity from winter bittersweet nightshade, Solanum dulcamara. Plant Mol Biol 48:339–350. Hummel, I., Bourdais, G., Gouesbet, G., Couée, I., Malmberg, R.L., and El Amrani, A. (2004) Differential gene expression of ARGININE DECARBOXYLASE ADC1 and ADC2 in Arabidopsis thaliana: characterization of transcriptional regulation during seed germination and seedling development. New Phytologist 163(3):519–531. Hundertmark, M. and Hincha, D.K. (2008) LEA (Late Embryogenesis Abundant) proteins and their encoding genes in Arabidopsis thaliana. BMC Genomics 9:118–139. Hurry, V.M., Malmberg, G., Gardeström, P., and Öquist, G. (1994) Effects of a short-term shift to low temperature and of longterm cold hardening on photosynthesis and ribulose 1,5 biphosphate carboxylase/oxygenase and sucrose phosphate synthase activity in leaves of winter rye (Secale cereale L.). Plant Physiology 106:983–990.

GENES AND GENE REGULATION FOR LOW-TEMPERATURE TOLERANCE

213

Hurry, V.M., Tobieson, M., Kodama, H., Krömer, S., Gardeström, P., and Öquist, G. (1995) Mitochondria contribute to increased photosynthetic capacity of leaves of winter rye (Secale cereale L.) following cold hardening. Plant cell and Environment 18:69–76. Ingram, J. and Bartels, D. (1996). The molecular basis of dehydration tolerance in plants. Annu Rev Plant Physiol Plant Mol Biol 47:377–403. Ito, Y., Katsura, K., Maruyama, K., Taji, T., Kobayashi, M., Seki, M., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2006) Functional analysis of rice DREB1/CBF-type transcription factors involved in cold-responsive gene expression in transgenic rice. Plant Cell Physiol 47:141–153. Jaglo, K.R., Kleff, S., Amundsen, K., Zhang, X., Haake, V., Zhang, J., Deits, T., and Thomashow, M.F. (2001) Components of the Arabidopsis CBF cold-response pathway are conserved in Brassica napus and other plant species. Plant Physiol 127:910–917. Jaglo-Ottosen, K.R., Gilmour, S.J., Zarka, D.G., Schabenberger, O., and Thomashow, M.F. (1998) Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance. Science 280:104–106. Kanaoka, M.M., Pillitteri, L.J., Fujii, H., Yoshida, Y., Bogenschutz, N.L., Takabayashi, J., Zhu, J.-K., and Torii, K.U. (2008) SCREAM/ICE1 and SCREAM2 specify three cell-state transitional steps leading to Arabidopsis stomatal differentiation. Plant Cell 20:1775–1785. Kaplan, F. and Guy, C.L. (2004) Beta-Amylase induction and the protective role of maltose during temperature shock. Plant Physiol 135:1674–1684. Kaplan, F., Kopka, J., Haskell, D.W., Zhao, W., Schiller, K.C., Gatzke, N., Sung, D.Y., and Guy, C.L. (2004) Exploring the temperature-stress metabolome of Arabidopsis. Plant Physiol 136:4159–4168. Kaplan, F., Sungb, D.Y., and Guy, C.L. (2006) Roles of β-amylase and starch breakdown during temperatures stress. Physiologia Plantarum 126:120–128. Karam, B.S., Rhonda, C.F., and Luis O.-S. (2002) Transcription factors in plant defense and stress response. Curr Opin Plant Biol 5:430–436. Kariola, T., Brader, G., Helenius, E., Li, J., Heino, P., and Palva, E.T. (2006) Early Response To Dehydration 15. A negative regulator of ABA responses in Arabidopsis. Plant Physiology 142:1559–1573. Karlson, D., Nakaminami, K., Toyomasu, T., and Imai, R. (2002) A cold-regulated nucleic acid-binding protein of winter wheat shares a domain with bacterial cold shock proteins. J Biol Chem 277:35248–35256. Karlson, D. and Imai, R. (2003) Conservation of the cold shock domain protein family in plants. Plant Physiol 131:12–15. Kasuga, M., Liu, Q., Miura, S., Yamaguchi-Shinozaki, K., and Shinozaki, K. (1999) Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nature Biotech 17:287–291. Kavi Kischor, P.B., Hong, Z., Miao, G.H., Hu, C.A., and Verma D.P. (1995) Overexpression of δ-pyrroline-5-carboxylate synthetase increases proline production and confers osmotolerance in transgenic plants. Plant Physiology 108:1387– 1394. Kawamura, Y. and Uemura, M. (2003) Mass spectrometric approach for identifying putative plasma membrane proteins of Arabidopsis leaves associated with cold acclimation. Plant Journal 36:141–154. Kazuoka, T. and Oeda, K. (1994) Purification and characterization of COR85-oligomeric complex from cold-acclimated spinach. Plant and Cell Physiology 35:601–611. Kim, J.C., Lee, S.H., Cheong, Y.H., Yoo, C-M., Lee, S.I., Chun, H.J., Yun, D.-J., Lee, S.Y., Lim, C.O., and Cho, M.J. (2001) A novel cold-inducible zinc finger protein from soybean, SCOF-1, enhances cold tolerance in transgenic plants. The Plant Journal 25(3):247–259. Kim, J.S, Kim, K.A., Oh, T.R., Park, C.M., and Kang, H. (2008) Functional Characterization of DEAD-Box RNA Helicases in Arabidopsis thaliana under Abiotic Stress Conditions. Plant and Cell Physiology 49(10):1563–1571; doi:10.1093/pcp/ pcn125. Kim, K.-N., Cheong, Y.H., Grant, J.J., Pandey, G.K., and Luan, S. (2003) CIPK3, a calcium sensor-associated protein kinase that regulates abscisic acid and cold signal transduction in Arabidopsis. Plant Cell 15:411–423. Kim, Y.O., Kim, J.S., and Kang, H. (2005) Cold-inducible zinc finger-containing glycine-rich RNA-binding protein contributes to the enhancement of freezing tolerance in Arabidopsis thaliana. Plant J 42:890–900. Kim, Y.O. and Kang, H. (2006) The role of a zinc finger-containing glycinerich RNA-binding protein during the cold adaptation process in Arabidopsis thaliana. Plant Cell Physiol 47:793–798. Klee, C.B., Ren, H., and Wang, X. (1998). Regulation of calmodulin stimulated protein phosphatase, calcineurin. Journal of Biological Chemistry 273:13367–13370. Knight, C.A., Wen, D.Y., and Laursen, R.A. (1995) Nonequilibrium antifreeze peptides and the recrystallization of ice. Cryobiology 32:23–34. Knight, H., Trewavas, A.J., and Knight, M.R. (1996) Cold calcium signalling in Arabidopsis involves two cellular pools and a change in calcium signature after acclimation. Plant Cell 8:489–503. Knight, H., Veale, E.L., Warren, G.J., and Knight, M.R. (1999) The sfr6 mutation in Arabidopsis suppresses low-temperature induction of genes dependent on the CRT/DRE sequence motif. Plant Cell 11:875–886.

214

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

Knight, H., Mugford Nee Garton, S.G., Gao, D., Thorlby, G., and Knight, M.R. (2009) Identification of SFR6, a key component in cold acclimation acting post-translationally on CBF function. The Plant Journal doi: 10.1111/j.1365–313X.2008.03763.x. Koiwa, H., Barb, A.W., Xiong, L., Li, F., McCully, M.G., Lee, B.-ha, Sokolchick, I., Zhu, J., Gong, Z., Reddy, M., Sharkhuu, A., Manabe, Y., Yokoi, S., Zhu, J.-K., Bressan, R.A., and Hasegava, P.M. (2002) C-terminal domain phosphatise-like family members (AtCPLs) differentially regulate Arabidopsis thaliana abiotic stress signalling, growth, and development. Proc Natl Acad Sci 99:10893–10898. Koster, K.L. and Lynch, D.V. (1992) Solute accumulation and compartmentation during the cold acclimation of puma rye. Plant Physiology 98:108–113. Kreps, J.A., Wu, Y., Chang, H.-S., Zhu, T., Wang, X., and Harper, J.F. (2002) Transcriptome Changes for Arabidopsis in Response to Salt, Osmotic, and Cold Stress. Plant Physiol 130:2129–2141. Kruger, C., Berkowitz, O., Stephan, U.W., and Hell, R. (2002) A metalbinding member of the late embryogenesis abundant protein family transports iron in the phloem of Ricinus communis. L. Journal of Biological Chemistry 277:25062–25069. Kubien, D.S., von Caemmerer, S., Furbank, R.T., and Sage, R.F. (2003) C4 photosynthesis at low temperature. A study using transgenic plants with reduced amounts of rubisco. Plant Physiol 132:1577–1585. Kudla, J., Xu, Q., Harter, K., Gruissem, W., and Luan, S. (1999) Genes for calcineurin B-like proteins in Arabidopsis are differentially regulated by stress signals. Proc Natl Acad Sci USA 96:4718–4723. Lång, V., Heino, P., and Palva, E.T. (1989) Low temperature acclimation and treatment with exogenous abscisic acid induce common polypeptides in Arabidopsis thaliana (L.) Heynh. Theor Appl Genet 77:729–734. Lee, H., Xiong, L., Gong, Z., Ishitani, M., Stevenson, B., and Zhu, J.K. (2001) The Arabidopsis HOS1 gene negatively regulates cold signal transduction and encodes a RING finger protein that displays cold-regulated nucleo-cytoplasmic partitioning. Genes Dev 15:912–924. Lee, H., Guo, Y., Ohta, M., Xiong, L., Stevenson, B., and Zhu, J.-K. (2002) LOS2, a genetic locus required for cold responsive gene transcription encodes a bi-functional enolase. EMBO J 21:2692–2702. Lee, B.-H., Henderson, D.A., and Zhu, J.-K. (2005) The Arabidopsis cold-responsive transcriptome and its regulation by ICE1. Plant Cell 17:3155–3175. Lee, B.H., Kapoor, A., Zhu, J., and Zhu, J.-K. (2006) STABILIZED1, a stress-upregulated nuclear protein, is required for premRNA splicing, mRNA turnover, and stress tolerance in Arabidopsis. Plant Cell 18:1736–1749. Levitt, J. (1980) Responses of Plants to Environmental Stresses: Chilling, Freezing and High Temperature Stress, Academic Press, New York. Li, P.H., Chen, W.-P., Jian, L., and Xin, Z. (1997) Abscisic acid–induced chilling tolerance in maize. In Plant Cold Hardiness, molecular biology, biochemistry and physiology (Li, P.H. and Chen, T.H.H., Eds.) 215–224. Plenum Press, New York and London. Li, C., Puhakainen, T., Welling, A., Viherä-Aarnio, A., Ernstsen, A., Junttila, O., Heino, P., and Palva, E.T. (2002) Cold acclimation in silver birch (Betula pendula). Development of freezing tolerance in different tissues and climatic ecotypes. Physiol Plant 116:478–488. Li, C., Junttila, O., Heino, P., and Palva, E.T. (2003) Different responses of northern and southern ecotypes of Betula pendula to exogenous ABA application. Tree Physiol 23:481–487. Liao, Y., Zou, H.-F., Wei, W., Hao, Y.-J., Tian, A.-G., Huang, J., Liu, Y.-F., Zhang, J.-S., and Chen, S.-Y. (2008) Soybean GmbZIP44, GmbZIP62 and GmbZIP78 genes function as negative regulator of ABA signaling and confer salt and freezing tolerance in transgenic Arabidopsis. Planta 228(16):225–240. Liu, J. and Zhu, J.K. (1998) A calcium sensor homolog required for plant salt tolerance. Science 280:1943–1945. Liu Q., Kasuga, M., Sakuma, Y., Abe, H., Miura, S., Kyamaguchi-Shinozaki, K., and Shinozaki, K. (1998) Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain, separate two cellular signal transduction pathways in drought- and low temperature responsive gene expression, respectively, in Arabidopsis. Plant Cell 10:1391–1406. Liu, Z., Sall, A., and Yang, D. (2008) MicroRNA: an Emerging Therapeutic Target and Intervention Tool. Int J Mol Sci 9(6):978–999. Lu, P.L., Chen, N.Z., An, R., Su, Z., Qi, B.S., Ren, F., Chen, J., and Wang, X.C. (2007) A novel drought-inducible gene, ATAF1, encodes a NAC family protein that negatively regulates the expression of stress-responsive genes in Arabidopsis. Plant Mol Biol 63:289–305. Luan, S., Kudla, J., Rodriguez-Concepcion, M., Yalovsky, S., and Gruissem, W. (2002) Calmodulins and calcineurin B-like proteins: calcium sensors for specific signal response coupling in plants. Plant Cell 14:S389–S400. Luan, S. (2008) The CBL–CIPK network in plant calcium Signalling. Trends in Plant Science 14(1). Ludwig, A.A. Saitoh, H., Felix, G., Freymark, G., Miersch, O., Wasternack, C., Boller, T., Jones, J.D.G., Romeis, T. (2005) Ethylene-mediated cross-talk between calcium-dependent protein kinase and MAPK signaling controls stress responses in plants. PNAS 102:10736–10741.

GENES AND GENE REGULATION FOR LOW-TEMPERATURE TOLERANCE

215

Magnan, F., Ranty, B., Charpenteau, M., Sotta, B., Galaud, J.-P., and Aldon, D. (2008) Mutations in AtCML9, a calmodulin-like protein from Arabidopsis thaliana, alter plant responses to abiotic stress and abscisic acid. The Plant Journal 56:575–589. Mahajan, S. and Tuteja, N. (2005) Cold, salinity and drought stresses: an overview. Archives of Biochemistry and Biophysics 444:139–158. Mäntylä, E., Lång, V., and Palva, E.T. (1995) Role of abscisic acid in drought-induced freezing tolerance, cold acclimation, and accumulation of LTI78 and RAB18 proteins in Arabidopsis thaliana. Plant Physiol 107:141–148. Mare, C., Mazzucotelli, E., Crosatti, C., Francia, E., Stanca, A.M., and Cattivelli, L. (2004) Hv-WRKY38: a new transcription factor involved in cold- and drought-response in barley. Plant Mol Biol 55:399–416. Marentes, E., Griffith, M., Mlynarz, A., and Brush, R.E. (1993) Proteins accumulate in the apoplast of winter rye leaves during cold acclimation. Physiol Plant 87:499–507. Maruyama, K., Sato, N., and Ohta, N. (1999) Conservation of structure and cold-regulation of RNA-binding proteins in cyanobacteria: probable convergent evolution with eukaryotic glycine-rich RNA-binding proteins. Nucleic Acids Research 27(9):2029–2036. Maruyama, K., Sakuma, Y., Kasuga, M., Ito, Y., Seki, M., Goda, H., Shimada, Y., Yoshida, S., Shinozaki, K., and YamaguchiShinozaki, K. (2004) Identification of cold-inducible downstream genes of the Arabidopsis DREB1A/CBF3 transcriptional factor using two microarray systems. The Plant Journal 38:982–993. Mastrangelo, A.M., Belloni, S., Barilli, S., Ruperti, B., Di Fonzo, N., Stanca, A.M., and Cattivelli, L. (2005) Low temperature promotes intron retention in two e-cor genes of durum wheat. Planta 221:705–715. Mauch-Mani, B. and Mauch, F. (2005) The role of abscisic acid in plant-pathogen interactions. Curr Opin Plant Biol 8:409–414. McCormack, E. and Braam, J. (2003) Calmodulins and related potential calcium sensors of Arabidopsis. New Phytologist 159:585–598. McCormack, E., Tsai, Y.-C., and Braam, J. (2005) Handling calcium signaling: Arabidopsis CaMs and CMLs. TRENDS in Plant Science 10(8). McKersie, B.D. and Bowley, S.R. (1998) Active oxygen and freezing tolerance in transgenic plants. In Plant Cold Hardiness (Li, P.H. and Chen, T.H.H. (Eds.) Plenum Press, New York. McNeil, P.L. and Kirchhausen, T. (2005) An emergency response team for membrane repair Nat Rev Mol Cell Biol 6: 499–505. Mikami, K., Katagiri, T., Iuchi, S., Yamaguchi-Shinozaki, K., and Shinozaki, K. (1998) A gene encoding phosphatidylinositol4-phosphate 5-kinase is induced by water stress and abscisic acid in Arabidopsis thaliana. Plant J 15:563–568. Mikami, K., Suzuki, I., and Murata, N. (2002) Sensors of abiotic stress in Synechocystis. Topics in Current Genetics: Plant Responses to Abiotic Stress (Hirt, H. and Shinozaki, K., Eds.), Springer 103–119. Mittler, R. (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7:405–410. Mittler, R., Vanderauwera, S., Gollery, M., and Van Breusegem, F. (2004) Reactive oxygen gene network of plants. Trends in Plant Science 9:490–498. Miura, K., Jin, J.O., Lee, J., Yoo, C.Y., Stirm, V., Miura, T., Ashworth, E.N., Bressan, R.A., Yun, D.-J., and Hasegawa, P.M. (2007) SIZ1-mediated sumoylation of ICE1 controls CBF3/DREB1A expression and freezing tolerance in Arabidopsis. Plant Cell 19:1403–1414. Mizoguchi, T., Irie, K., Hirayama, T., Hayashida, N., and Yamaguchi-Shinozaki, K. (1996) A gene encoding a mitogen-activated protein kinase kinase kinase is induced simultaneously with genes for a mitogen-activated protein kinase and an S6 ribosomal protein kinase by touch, cold and water stress in Arabidopsis thaliana. Proc Natl Acad Sci USA 93:765–769. Mohr, P.G. and Cahill, D.M. (2003) Abscisic acid influences the susceptibility of Arabidopsis thaliana to Pseudomonas syringae pv. tomato and Peronospora parasitica. Funct Plant Biol 30:461–469. Monroy, A.F., Sarhan, F., and Dhindsa, R.S. (1993) Cold-induced changes in freezing tolerance, protein phosphorylation, and gene expression: evidence for a role of calcium. Plant Physiol 102:1227–1235. Monroy, A.F. and Dhindsa, R.S. (1995) Low-temperature signal transduction: induction of cold acclimation-specific genes of alfalfa by calcium at 25 °C. Plant Cell 7:321–331. Monroy, A.F., Sangwan, V., and Dhindsa, R.S. (1998) Low temperature signal transduction during cold acclimation: protein phosphatise 2A as an early target for cold-inactivation. Plant J 13:653–660. Munnik, T., Irvine, R.F., and Musgrave, A. (1998) Phospholipid signalling in plants. Biochim Biophys Acta 1389:222–272. Murata, N., Ishizaki-Nishizawa, O., Higashi, S., Hayashi, H., Tasaka, Y., and Nishida, I. (1992) Genetically engineered alteration in the chilling sensitivity of plants. Nature 356:710–713. Murata, N. and Los, D.A. (1997) Membrane Fluidity and Temperature Perception. Plant Physiol 11(5):875–879. Nakashima, K., Tran, L.-S. P., Nguyen, D.V., Fujita, M., Maruyama, K., Todaka, D., Ito, Y., Hayashi, N., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2007) Functional analysis of a NAC-type transcription factor OsNAC6 involved in abiotic and biotic stress-responsive gene expression in rice. Plant J 51:617–630.

216

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

Nakashima, K., Ito, Y., and Yamaguchi-Shinozaki, K. (2009) Transcriptional regulatory networks in response to abiotic stresses in Arabidopsis and grasses. Plant Physiol 149:88–95. Nanjo, T., Kobayashi, M., Yoshiba, Y., Kakubari, Y., Yamaguchi-Shinozaki, K., and Shinozaki, K. (1999) Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis thaliana. FEBS Lett 461:205–210. Newton, S.S. and Duman, J.G. (2000) An osmotin-like cryoprotective protein from the bittersweet nightshade Solanum dulcamara. Plant Mol Biol 44:581–589. Nordin, K., Vakala, T., and Palva, E.T. (1993) Differential expression of two related, low-temperature-induced genes in Arabidopsis thaliana (L.). Heynh. Plant Mol Biol 21:641–653. Novillo, F., Alonso, J.M., Ecker, J.R., and Salinas, J. (2004) CBF2/DREB1C is a negative regulator of CBF1/DREB1B and CBF3/DREB1A expression and plays a central role in stress tolerance in Arabidopsis. Proc Natl Acad Sci USA 16:101(11):3985–3990. Novillo, F., Medina, J., and Salinas, J. (2007) Arabidopsis CBF1 and CBF3 have a different function than CBF2 in cold acclimation and define different gene classes in the CBF regulon. Proc Natl Acad Sci 104(52):21002–21007. Nylander, M., Svensson, J., Palva, E.T., and Welin, B.V. (2001) Stress induced accumulation and tissue-specific localization of dehydrins in Arabidopsis thaliana. Plant Molecular Biology 45:263–279. Ohtake, S., Schebor, C., and de Pablo, J.J. (2006) Effects of trehalose on the phase behavior of DPPC-cholesterol unilamellar vesicles. Biochimica et Biophysica Acta 1758:65–73. Oliver, A.E., Hincha, D.K., and Crowe, J.H. (2002) Looking beyond sugars: the role of amphiphilic solutes in preventing adventitious reactions in anhydrobiotes at low water contents. Comp Biochem Physiol A 131:515–525. Öquist, G., Hurry, V.M., and Huner, N.P.A. (1993) The Temperature-Dependence of the Redox State of Q(a) and Susceptibility of Photosynthesis to Photoinhibition. Plant Physiology and Biochemistry 31:683–691. Örvar, B.L., Sangwan, V., Omann, F., and Dhindsa, R.S. (2000) Early steps in cold sensing by plant cells: the role of actin cytoskeleton and membrane fluidity. Plant J 23:785–794. Pfluger, J. and Wagner, D. (2007) Histone modifications and dynamic regulation of genome accessibility in plants. Current Opinion in Plant Biology 10:645–652. Phadtare, S. and Inouye, M. (1999) Sequence-selective interactions with RNA by CspB, CspC and CspE, members of the CspA family of Escherichia coli. Mol Microbiol 33:1004–1014. Plieth, C., Hansen, U.-P., Knight, H., and Knight, M.R. (1999) Temperature sensing by plants: the primary characteristics of signal perception and calcium response. Plant J 18:491–497. Puhakainen, T., Hess, M.W., Makela, P., Svensson, J., Heino, P., and Palva, E.T. (2004a) Overexpression of multiple dehydrin genes enhances tolerance to freezing stress in Arabidopsis. Plant Mol Biol 54:743–753. Puhakainen, T., Li, C., Boije, M., Kangasjärvi, J., Heino, P., and Palva, E.T. (2004b) Short day photoperiod potentiates low temperature-induced expression of a CBF-controlled gene during cold acclimation in silver birch (Betula pendula Roth.). Plant Physiol 136:4169–4183. Qin, F., Sakuma, Y., Li, J., Liu, Q., Li, Y.-Q., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2004) Cloning and functional analysis of a novel DREB1/CBF transcription factor involved in cold-responsive gene expression in Zea mays L. Plant Cell Physiol. Plant Cell Physiol 45:1042–1052. Rajashekar, C.B., Zhou, H.E., Zhang, Y., Li, W., and Wang, X. (2006) Suppression of phospholipase Dalpha1 induces freezing tolerance in Arabidopsis: response of cold-responsive genes and osmolyte accumulation. J Plant Phys 163:916–926. Robinson, S.J. and Parkin, I.A.P. (2008) Differential SAGE analysis in Arabidopsis uncovers increased transcriptome complexity in response to low temperature. BMC Genomics 9:434. Rorat, T., Irzykowski, W., and Grygorowicz, W.J. (1997) Identification and expression of novel cold induced genes in potato (Solanum sogarandinum). Plant Science 124:69–78. Rorat, T. (2006) Plant dehydrins—tissue location, structure and function. Cell Mol Biol Lett 11:536–556. Rudd, J.J. and Franklin-Tong, V.E. (2001) Unravelling response-specificity in Ca2+ signalling pathways in plant cells. New Phytol 151:7–33. Ruelland, E., Cantrel, C., Gawer, M., Kader, J.C., and Zachowski, A. (2002) Activation of phospholipases C and D is an early response to a cold exposure in Arabidopsis suspension cells. Plant Physiol 130:999–1007. Ruiz, J.M., Sánchez, E., García, P.C., López-Lefebre, L.R., Rivero, R.M., and Romero, L. (2002) Proline metabolism and NAD kinase activity in greenbean plants subjected to cold-shock. Phytochemistry 59:473–478. Sangwan, V., Foulds, I., Singh, J., and Dhindsa, R.S. (2001) Cold-activation of Brassica napus BN 115 promoter is mediated by structural changes in membranes and cytoskeleton, and requires Ca2+ influx. Plant J 27:1–12. Sangwan, V., Orvar, B.L., Beyerly, J., Hirt, H., and Dhindsa, R.S. (2002) Opposite changes in membrane fluidity mimic cold and heat stress activation of distinct plant MAP kinase pathways. The Plant Journal 31:629–638. Santarius, K. (1992) Some Mosses Survive Cryopreservation without Prior Pretreatment. The Bryologists 106(2):270–277.

GENES AND GENE REGULATION FOR LOW-TEMPERATURE TOLERANCE

217

Savoure, A., Hua, X.J., Bertauche, N., Van Montagu, M., and Verbruggen, N. (1997) Abscissic acid-independent and abscissic acid-dependent regulation of proline biosynthesis following cold and osmotic stresses in Arabidopsis thaliana. Molecular and General Genetics 254:104–109. Shi, H., Ishitani, M., Kim, C., and Zhu, J.-K. (2000) The Arabidopsis thaliana salt tolerance gene SOS1 encodes a putative NA+/ H+ antiporter. Proc Natl Acad Sci USA 97:6896–6901. Shinozaki, K.Y. and Shinozaki, K. (2006) Transcriptional Regulatory Networks in Cellular Responses and Tolerance to Dehydration and Cold Stresses. Annu Rev Plant Biol 57:781–803. Slade, L. and Levine, H. (1991) Beyond water activity: recent advances based on an alternative approach to the assessment of food quality and safety. Critical Reviews in Food Science and Technology 30:115–360. Smallwood, M., Worrall, D., Byass, L., Elias, L., Ashford, D., Doucet, C.J., Holt, C., Telford, J., Lillford, P., and Bowles, D.J. (1999) Isolation and characterization of a novel antifreeze protein from carrot (Daucus carota). Biochem J 340:385–391. Soitamo, A.J., Piippo, M., Allahverdiyeva, Y., Battchikova, N., and Aro, E.M. (2008) Light has a specific role in modulating Arabidopsis gene expression at low temperature. BMC Plant Biology 2008, 8:13 doi:10.1186/1471-2229-8-13. Steponkus, P.L., Uemura, M., and Webb, M.S. (1993) Membrane destabilization during freeze-induced dehydration. Curr Topics Plant Physiol 10:37–47. Steponkus, P.L., Uemura, M., Joseph, R.A., Gilmour, S.J., and Thomashow, M.F. (1998) Mode of action of the COR15a gene on the freezing tolerance of Arabidopsis thaliana. Proc Natl Acad Sci USA 95:14570–14575. Stockinger, E.J., Gilmour, S.J., and Tomashow, M.F. (1997) Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcription activator that binds to the C repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc Natl Acad Sci USA 94:1035–1040. Stockinger, E.J., Mao, Y., Regier, M.K., Triezenberg, S.J., and Thomashow, M.F. (2001) Transcriptional adaptor and histone acetyltransferase proteins in Arabidopsis and their interactions with CBF1, a transcriptional activator involved in coldregulated gene expression. Nucleic acids research 29(7):1524–1533. Strauss, G. and Hauser, H. (1986) Stabilization of lipid bilayer vesicles by sucrose during freezing. Proc Natl Acad Sci USA 83:2422–2426. Sunkar, R. and Zhu, J.K. (2004) Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. Plant Cell 16:2001–2019. Sunkar, R., Chinnusamy, V., Zhu, J., and Zhu, J.-K. (2007) Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. Trends Plant Sci 12:301–309. Suzuki, I., Los, D.A., Kanesaki, Y., Mikami, K., and Murata, N. (2000) The pathway for perception and transduction of lowtemperature signals in Synechocystis. EMBO J 19:1327–1334. Tähtiharju, S., Sangwan, V., Monroy, A.F., Dhindsa, R.S., and Borg M. (1997) The induction of kin genes in cold-acclimating Arabidopsis thaliana. Evidence of a role for calcium. Planta 203:442–447. Tähtiharju, S. and Palva, E.T. (2001) Antisense inhibition of protein phosphatase 2C accelerates cold acclimation in Arabidopsis thaliana. Plant J 26:461–470. Teige, M., Scheikl, E., Eulgem, T., Doczi, R., Ichimura, K., Shinozaki, K., Dangl, J.L., and Hirt, H. (2004) The MKK2 pathway mediates cold and salt stress signaling in Arabidopsis. Mol Cell 15:141–152. Thomashow, M.F. (1999) Plant cold acclimation: freezing tolerance genes and regulatory mechanism. Annu Rev Plant Physiol Plant Mol Biol 50:571–599. Thorlby, G., Fourrier, F., and Warren, G. (2004) The SENSITIVE TO FREEZING2 Gene, Required for Freezing Tolerance in Arabidopsis thaliana, Encodes a β-Glucosidase. The Plant Cell 16:2192–2203. Townley, H.E. and Knight, M.R. (2002) Calmodulin as a negative regulator of Arabidopsis COR gene expression. Plant Physiol 128:1169–1172. Tran, L.S., Nakashima, K., Sakuma, Y., Simpson, S.D., Fujita, Y., Maruyama, K., Fujita, M., Seki, M., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2004) Isolation and functional analysis of Arabidopsis stress-inducible NAC transcription factors that bind to a drought-responsive cis-element in the early responsive to dehydration stress 1 promoter. Plant Cell 16:2481–2498. Tran, L.S., Urao, T., Qin, F., Maruyama, K., Kakimoto, T., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2007) Functional analysis of AHK1/ATHK1 and cytokinin receptor histidine kinases in response to abscisic acid, drought, and salt stress in Arabidopsis. Proc Natl Acad Sci USA. 18:104(51):20623–20628. Trewavas, A.J. and Malhó, R. (1997) Signal perception and transduction: The origin of the phenotype. Plant Cell 9:1181–1195. Uemura, M., Joseph, R.A., and Steponkus, P.L. (1995) Cold acclimation of Arabidopsis thaliana. Effect on plasma membrane lipid composition and freeze-induced lesions. Plant Physiol 109:15–30. Uemura, M. and Steponkus, P.L. (1997) Effect of cold acclimation on membrane lipid composition and freeze-induced membrane destabilization. See Ref 68:171–179.

218

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

Uemura, M., Tominaga, Y., Nakagawara, C., Shigematsu, S., Minami, A., and Kawamura, Y. (2006) Responses of the plasma membrane to low temperatures. Physiol Plant 126:81–89. Ulrich, H.D. (2005) Mutual interactions between the SUMO and ubiquitin systems: a plea of no contest. TRENDS in Cell Biology 15(10). Urao, T., Yakubov, B., Satoh, R., Yamaguchi-Shinozaki, K., Seki, M., Hirayama, T., and Shinozaki, K. (1999) A transmembrane hybrid-type histidine kinase in Arabidopsis functions as an osmosensor. Plant Cell 11:1743–1754. Valluru, R. and Van den Ende, W. (2008) Journal of Experimental Botany 59(11):2905–2916. Vaultier, M.N., Cantrel, C., and Vergnolle, C., Justin, A.M., Demandre, C., Benhassaine-Kesri, G., Cicek, D., and Zachowski, A. (2006) Desaturase mutants reveal that membrane rigidification acts as a cold perception mechanism upstream of the diacylglycerol kinase pathway in Arabidopsis cells. FEBS Lett 580:4218–4223. Vereyken, I.J., Albert van Kuik, J., Evers, T.H., Rijken, P.J., and de Kruijff, B. (2003) Structural requirements of the fructan–lipid interaction. Biophysical Journal 6:3759–3766. Verslues, P.E., Guo, Y., Dong, C.H., Ma, W., and Zhu, J.-K. (2006) Mutation of SAD2, an importin beta-domain protein in Arabidopsis, alters abscisic acid sensitivity. Plant J 47:776–787. Verslues, P.E. and Zhu, J.K. (2007) New developments in abscisic acid perception and metabolism. Curr Opin Plant Biol 10(5):447–452. Vlachonasios, K.E., Thomashow, M.F., and Triezenberg, S.J. (2003) Disruption mutations of ADA2b and GCN5 transcriptional adaptor genes dramatically affect Arabidopsis growth, development, and gene expression. Plant Cell 15(3):626–638. Vogel, J.T., Zarka, D.G., Van Buskirk, H.A., Fowler, S.G., and Thomashow, M.F. (2005) Roles of the CBF2 and ZAT12 transcription factors in configuring the low temperature transcriptome of Arabidopsis. Plant J 41:195–211. Wang, X., Li, W., Li, M., and Welti, R. (2006) Profiling lipid changes in plant response to low temperatures. Physiol Plant 126:90–96. Warren, G., McKown, R., Marin, A., and Teutonico, R. (1996) Isolation of mutations affecting the development of freezing tolerance in Arabidopsis thaliana (L.) Heynh. Plant J 17:445–452. Welling, A. and Palva, E.T. (2006) Molecular control of cold acclimation in trees. Physiologia Plantarum 127:167–181. Welling, A. and Palva, E.T. (2008) Involvement of CBF Transcription Factors in Winter Hardiness in Birch. Plant Physiology 147:1199–1211. Welti, R., Li, W., Li, M., Sang, Y., Biesiada, H., Zhou, H.E., Rajashekar, C.B., Williams, T.D., and Wang, X. (2002) Profiling membrane lipids in plant stress responses: Role of phospholipase D alpha in freezing-induced lipid changes in Arabidopsis. J Biol Chem 277:31994–32002. Williams, M.E., Torabinejad, J., Cohick, E., Parker, K., Drake, E.D., Thompson, J.E., Hortter, M., and DeWald, D.B. (2005) Mutations in the Arabidopsis Phosphoinositide Phosphatase Gene SAC9 Lead to Overaccumulation of PtdIns(4,5)P2 and Constitutive Expression of the Stress-Response Pathway. Plant Physiology 138:686–700. Wise, M.J. and Tunnacliffe, A. (2004) POPP the question: What do LEA proteins do? Trends Plant Sci 9:13–17. Xiong, L., Ishitani, M., Lee, H., and Zhu, J.K. (2001) The Arabidopsis LOS5/ ABA3 locus encodes a molybdenum cofactor sulfurase and modulates cold stress- and osmotic stress-responsive gene expression. Plant Cell 13:2063–2083. Xiong, L., Lee, H., Ishitani, M., Tanaka, Y., Stevenson, B., Koiwa, H., Bressan, R.A., Hasegawa, P.M., and Zhu, J.-K. (2002) Repression of stress-responsive genes by FIERY2, a novel transcriptional regulator in Arabidopsis. Proc Natl Acad Sci USA 99:10899–10904. Xin, Z. and Browse, J. (1998) Eskimo1 mutants of Arabidopsis are constitutively freezing-tolerant. Proc Natl Acad Sci 95:7799–7804. Xin, Z. and Browse, J. (2000) Cold comfort farm: the acclimation of plants to freezing temperatures. Plant Cell and Environment 23:893–902. Xin, Z., Mandaokar, A., Chen, J., Last, R.L., and Browse, J. (2007) Arabidopsis ESK1 encodes a novel regulator of freezing tolerance. The Plant Journal 49:786–799. Yamaguchi-Shinozaki, K. and Shinozaki, K. (1994) A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress. Plant Cell 6:251–264. Yamazaki, T., Kawamura, Y., Minami, A., and Uemura, M. (2008) Calcium- dependent freezing tolerance in Arabidopsis involves membrane resealing via synaptotagmin SYT1. Plant Cell 20:3389–3404. Yasuda, M., Ishikawa, A., Jikumaru, Y., Seki, M., Umezawa, T., Asami, T., Maruyama-Nakashita, A., Kudo, T., Shinozaki, K., Yoshida, S., and Nakashita, H. (2008) Antagonistic Interaction between Systemic Acquired Resistance and the Abscisic Acid–Mediated Abiotic Stress Response in Arabidopsis. The Plant Cell 20:1678–1692. Yoshida, S. (1979) Freezing injury and phospholipids degradation in vivo in woody plant cells. Plant Physiol 64:241–246. Young, S.A, Wang, X., and Leach, J.E. (1996) Changes in the plasma membrane distribution of rice phospholipase D during resistant interaction with Xanthomonas oryzae pv. Oryzae. Plant Cell 8:1079–1090.

GENES AND GENE REGULATION FOR LOW-TEMPERATURE TOLERANCE

219

Zarka, D.G., Vogel, J.T., Cook, D., and Thomashow, M.F. (2003) Cold induced expression of Arabidopsis CBF genes involves multiple ICE (inducer of CBF expression) promoter elements and a cold-regulatory circuit that is desensitized by low temperature. Plant Physiol 133:910–918. Zhang, X., Fowler, S.G., Cheng, H., Lou, Y., Rhee, S.Y., Stockinger, E.J., and Thomashow, M.F. (2004) Freezing-sensitive tomato has a functional CBF cold response pathway, but a CBF regulon that differs from that of freezing-tolerant Arabidopsis. Plant J 39:905–919. Zheng, X., Chen, B., Lu, G., and Han, B. (2009) Overexpression of NAC transcription factor enhances rice drought and salt tolerance. Biochem Biophys Res Cummun, doi:10.1016/j.bbrc.2008.12.163. Zhou, Q.-Y., Tian, A.-G., Zou, H.-F., Xie, Z.-M., Lei, G., Huang, J., Wang, C.-M., Wang, H.-W., Zhang, J.-S., and Chen, S.-Y. (2008) Soybean WRKY-type transcription factor genes, GmWRKY13, GmWRKY21, and GmWRKY54, confer differential tolerance to abiotic stresses in transgenic Arabidopsis plants. Plant Biotechnology Journal 6:486–503. Zhu, J.-K. (2002) Salt and drought stress signal transduction in plants. Annu Rev Plant Biol 53:247–273. Zhu, J., Shi, H., Lee, B.H., Damsz, B., Cheng, S., Stirm, V., Zhu, J.K., Hasegawa, P.M., and Bressan, R.A. (2004) An Arabidopsis homeodomain transcription factor gene, HOS9, mediates cold tolerance through a CBF-independent pathway. Proc Natl Acad Sci 101:9873–9878. Zhu, J., Verslues, P.E., Zheng, X., Lee, B.H., Zhan, X., Manabe, Y., Sokolchik, I., Zhu, Y., Dong, C.H., Zhu, J.K., Hasegawa, P.M., and Bressan, R.A. (2005) HOS10 encodes an R2R3-type MYB transcription factor essential for cold acclimation in plants. Proc Natl Acad Sci USA 102:9966–9971. Zhu, P., Zhou, W., Wang, J., Puc, J., Ohgi, K.A., Erdjument-Bromage, H., Tempst, P., Glass, C.K., and Rosenfeld, M.G. (2007) A Histone H2A deubiquitinase complex coordinating histone acetylation and H1 dissociation in transcriptional regulation. Molecular Cell 27:609–621. Zhu, J., Jeong, J.C., Zhu, Y., Sokolchik, I., Miyazaki, S., Zhu, J.-K., Hasegawa, P.M., Bohnert, H.J., Shi, H., Yun, D.-J., and Bressan, R.A. (2008) Involvement of Arabidopsis HOS15 in histone deacetylation and cold tolerance. Proc Natl Acad Sci USA 105:4945–4950. Zien, C.A., Wang, C., Wang, X., and Welti, R. (2001) In vitro substrates and the contribution of the common phospholipase D, PLDα to wound-induced metabolism of lipids in Arabidopsis. Biochim Biophys Acta 1530:236–248.

9

Genetic Approaches toward Improving Heat Tolerance in Plants Mamatha Hanumappa and Henry T. Nguyen

Introduction

Warming of the climate system in recent decades is unequivocal, evident from increases in global average air and ocean temperatures, widespread melting of snow and ice, and rising global sea level. Of the 12 years between 1995 and 2006, 11 rank among the 12 warmest years since 1850. It is very likely that hot extremes and heat waves will continue to become more frequent (IPCC, 2007). Assuming that water and nutrients are optimal, rising temperatures may benefit some agricultural crops by extending geographical distribution and the growing season of crops, which may be useful where shoot mass is of economic value. In contrast, global warming is expected to reduce crop yields in staple cereals by reducing the length of the growing season and hastening senescence (Porter, 2005). With the hydrological cycle closely linked to changes in atmospheric temperature, and due to population growth, the negative impact far outweighs the benefit. Only three weather events have caused damage worth more than $50 billion in the U.S. between 1980 and 2007, one of which was hurricane Katrina, and the other two were a combination of heat and drought. Heat stress is usually accompanied by drought and has caused more than $140 billion worth of damage during this period (Figure 9.1). High-temperature stress is one of the most serious threats to crop production worldwide (Boyer, 1982), an observation recently echoed by Battisti and Naylor (2009).

Thermotolerance

A plant undergoes heat stress when the temperature rises beyond an optimal threshold level for a period of time and causes irreversible damage to growth and development. A transient increase in temperature of about 10–15 °C above ambient is considered heat shock or heat stress. It is a complex interaction of heat intensity, duration, rapidity, and stage of growth. Cool-season crops are more susceptible than warm-season crops. Conversely, heat tolerance is the ability of the plant to overcome this adverse effect and produce economic yields. Although some researchers have reported that the night temperature is more critical (Willits and Peet, 1998), it is generally accepted that diurnal mean temperatures are better indicators of plant response to heat stress. The ability of an organism to cope with high temperatures has two components: inherent and acquired thermotolerance. Inherent or basal thermotolerance is a constitutive component resulting from the evolutionary thermal adaptation of a species. Acquired thermotolerance (AT) is the ability Genes for Plant Abiotic Stress Editors Matthew A. Jenks and Andrew J. Wood © 2010 Blackwell Publishing ISBN: 978-0-813-81502-2

221

Billion Dollar Weather Damage in the US between 1980–2007

160 140

US $ in Billion

120 100 80 60 40 20 0 Drought

Drought+Heat

Flood

Freeze+Hail

Figure 9.1 Costs are normalized to 2007 dollars using GNP Inflation/Wealth Index. Source: National Climatic Data Center (http//www.ncdc.noaa.gov/oa/reports/billionz.html).

Figure 9.2 Acquired thermotolerance in Arabidopsis thaliana. Seedlings were heated to 45 °C for 2 hours directly (left) or after pretreatment at 38 °C, 1.5 hour + 22 °C, 2 hours (right), before moving back to 22 °C. Only the acclimated seedlings on the right survived after 5 days. Photo courtesy of Dr. Elizabeth Vierling, University of Arizona, Tucson, Arizona, USA. For color detail, please see color plate section.

222

GENETIC APPROACHES TOWARD IMPROVING HEAT TOLERANCE IN PLANTS

223

of a plant to survive normally lethal temperatures after an exposure to a mild stress (Figure 9.2). AT relies on the induction of a specific pathway during the acclimation period and a subsequent acquisition of thermotolerance. Intraspecies difference in AT is significant but is small in inherent thermotolerance. Thus, AT measurement is a more useful tool in crop breeding and selection (Klueva et al., 2001).

High Temperature Impact and Plant Response to Heat Stress

The extent of damage caused by any stress depends on the crop species, the stage of growth, and the geographical zone it is adapted to. Seedling emergence, flowering, and seed filling are the most critical stages for all crop species (reviewed by Wahid et al., 2007). However, as plant development is dependent on several environmental conditions, it is difficult to determine a consistent upper threshold temperature (Miller et al., 2001). Several internal factors dictate a plant’s ability for thermotolerance such as antioxidant capacity, accumulation and stability of proteins and enzymes, signaling cascade, membrane composition, and maintenance of transcript function (Klueva et al., 2001). Heat stress can cause damage at the whole plant and cellular level. It can manifest as morphological, physiological, and biochemical responses as discussed below.

Morphological Response

Typically, high-temperature stress results in scorched leaves, flower and young pod abscission, inhibition of shoot and root growth, and fruit and pod discoloration; ultimately, the result is reduction in yield (Figure 9.3) (Hall, 1992). High temperatures during seed germination may lead to poor seedling emergence similar to prolonged effect on developing seed, which may result in loss of vigor, poor germination, and poor seedling establishment. High temperatures during the diurnal cycle can also cause stress. In maize, coleoptile growth reduced and ultimately ceased at 45 °C under diurnally varying temperatures (Weaich et al., 1996). Significant decrease in growth, shoot dry mass, and net assimilation rate were observed due to prolonged heat stress in monocotyledons. Though the tiller number increased, internode length and therefore total biomass were compromised in sugarcane (Ebrahim et al., 1998). Because soil surface temperatures can reach very high levels, carbohydrate translocation to roots due to heat girdling of the shoot base is affected in sorghum, where seedling emergence and survival is also a concern (see Hall, 1992). In wheat, delayed maturity coupled with reduction in grain density and weight led to reduced yield (Ferris et al., 1998; Guilioni et al., 2003). Reductions in starch, protein, and oil content were observed in the maize kernel (Wilhelm et al., 1999). Similarly, anther dehiscence and pollen germination were affected in rice, leading to poor yield (Yoshida et al., 1981). Yield loss due to heat stress is a concern in dicotyledons also. Many aspects of reproduction such as pollen and ovule viability, pollination, fertilization, and postfertilization processes were affected in tomato (Foolad, 2005). Lack of anther dehiscence and excessive style elongation may be major responses to heat stress that limit pollination. Fruit set in tomato and boll set in cotton is also affected by night temperature, the optimal temperature being 15–20 °C. Heat stress combined with long day results in floral bud abortion in cowpea. Similar damage was reported for snap and common beans (reviewed by Hall, 1992) whereas in chickpea, high-temperature stress during pod development is more critical for yield than at any other reproductive stage (Wang et al., 2006).

224

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

Physiological Response

A decrease in leaf water potential as a result of enhanced transpiration during the daytime leads to many physiological perturbations (Figure 9.3) (Tsukaguchi et al., 2003). When moisture is ample, plants tend to maintain a stable tissue water status that is severely impaired by high temperatures (Machado and Paulsen, 2001). High-temperature stress is frequently associated with reduced soil water availability (Simoes-Araujo et al., 2003). High night temperature combined with water stress resulted in smaller leaves with lower water potential, while an increase in leaf trichomes and number of xylem vessels in stem and root was observed in the ornamental Lotus creticus (Bañon et al., 2004). Leaf water potential was affected in sugarcane and tomato plants exposed to heat stress when soil moisture and relative humidity conditions were optimal (Morales et al., 2003; Wahid and Close, 2007), suggesting an effect of heat stress on root hydraulic conductivity. Osmotic Adjustment As an adaptive strategy when exposed to abiotic stresses such as water deficit,

salinity, and extreme temperatures, plants accumulate a variety of organic compounds of low molecular mass in a process generally known as osmotic adjustment. Such compatible osmolytes are thought to enhance stress tolerance. Transgenic studies have shown that enhanced production of the quarternary ammonium compound glycine betaine (GB) improves drought and heat tolerance in maize and several other crops. While some species such as maize, sugarcane, spinach, and barley accumulate high levels, others such as rice, Brassica spp., and tobacco do not produce GB under stress conditions (Sakamoto and Murata, 2002; Quan et al., 2004; Wahid and Close, 2007). Proline is another compound that is known to occur naturally and accumulate to high levels under stress (Kavi Kishore et al., 2005). Proline levels increased in barley plants subjected to heat stress (Georgieva et al., 2003) and cotton cultivars with higher proline content suffered less heat damage (Ashraf et al., 1994). Proline, like GB, is thought to buffer cellular redox potential under stress (Wahid and Close, 2007). Accumulation of soluble sugars appears to be associated with heat tolerance in sugarcane whereas disruption of sugar metabolism and proline transport due to heat stress in reproductive stage resulted in poor fruit set in tomato plants (Sato et al., 2006; Wahid and Close, 2007). Genetically engineering plants to accumulate trehalose, fructans, or mannitol has also been

Figure 9.3 Symptoms of high-temperature stress can vary. Left: Leaf scalding in maize. Photograph courtesy of Robert L. Croissant, deceased (from the collection of Dr. Howard F. Schwartz, Colorado State University, Bugwood Network Image). Right: Leaf rolling in potato. Photograph courtesy of Dr. Howard F. Schwartz, Colorado State University, Bugwood Network Image. For color detail, please see color plate section.

GENETIC APPROACHES TOWARD IMPROVING HEAT TOLERANCE IN PLANTS

225

demonstrated to enhance heat tolerance (Hare et al., 1998). Accumulation of γ-aminobutyric acid (GABA) in response to heat stress has been reported (Shelp et al., 1999; Bouché et al., 2004) while galactinol and raffinose were shown to rescue plants from oxidative damage by protecting salicylate from attack by hydroxyl radicals (Nishizawa et al., 2008). Photosynthesis and Carbon Partitioning The photosynthetic apparatus and associated metabolic processes are heat sensitive, and heat-induced inhibition of photosynthetic CO2 fixation has been reported in many plant species (Berry and Bjorkman, 1980). Though photosystem II (PSII) is thermolabile, it can be acclimated to heat stress (Havaux and Tardy, 1996; Law and CraftsBrandner, 1999), and it may be protected from damage by the action of a chloroplast localized heat shock protein (Hsp) (Heckathorn et al., 1998b). Carbon exchange rate (CER) declined with increasing leaf temperature and was more sensitive in wheat than in cotton. Inhibition of CER was primarily due to a heat-stress-induced decrease in the activation state of Rubisco via inhibition of Rubisco activase (Law and Crafts-Brandner, 1999). Rubisco activation has been documented as the primary site of inhibition by heat stress and is directly related to the ability of Rubisco activase (Weis, 1981a, 1981b; Kobza and Edwards, 1987; Eckardt and Portis, 1997; Feller et al., 1998), which is disrupted by high temperature (Crafts-Brandner et al., 1997). An elevation in carotenoid content in thermotolerant tomato cultivars has been reported, indicating that these compounds are involved in protection from heat stress (Camejo et al., 2005; Wahid and Ghazanfar, 2006). Heat stress may lead to the dissociation of oxygen evolving complex (OEC), disruption of proteins in the PSII reaction center (De Las Rivas and Barber, 1997; Yamane et al., 1998; De Ronde et al., 2004), and reduced activities of sucrose phosphate synthase (Chaitanya et al., 2001). ADP-glucose pyrophosphorylase and invertase (Vu et al., 2001) have been reported. The duration of the developmental phase is also affected by heat stress leading to smaller organs, reduced light perception, and carbon assimilation processes (Stone, 2001). At high atmospheric CO2 concentration, assimilate export is more affected by heat stress than net photosynthesis in some plant species (Leonardos et al., 1996) such as soybean where seed oil accumulation was found to be negatively affected by high temperature (Rotundo and Westgate, 2009). Considerable genotypic variation in assimilate partitioning occurs in crop plants such as wheat (Yang et al., 2002) where high temperature has a direct effect on source-sink activity than on translocation of assimilates (Wardlaw, 1974).

Heat stress denatures membrane proteins and increases unsaturated fatty acids, which makes the lipid bilayer more fluid (Savchenko et al., 2002). The increased solute leakage that occurs as a result of disrupted membrane integrity is an indication of decrease in cell membrane thermostability (CMT) and is used as an indirect measure of heat stress tolerance in many plant species (see Wahid et al., 2007). In Arabidopsis, the ratio of unsaturated to saturated fatty acids in membranes of plants grown in high temperature decreased to onethird of normal levels and total lipid content decreased by about 50% (Somerville and Browse, 1991). Electrolyte leakage is correlated with the organ sampled, developmental stage and age of tissue, growing season, and plant species tested. It varies among plant species and is not a direct measure of the relationship between CMT and yield. However, a direct relationship was observed in sorghum (Sullivan and Ross, 1979), but not in soybean (Shanahan et al., 1990). Cell viability test based on the principle of tetrazolium (triphenyl tetrazolium chloride [TTC]) salt reduction to formazan by dehydrogenase respiratory enzymes evaluates the residual respiratory activity of the stressed tissue (Krishnan et al., 1989). A significant but not absolute correlation was found between CMS and TTC at the seedling stage and flowering stage in wheat (Chen et al., 1982; Fokar et al., 1998).

Cell Membrane Stability and Cell Viability

226

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

Hormones, Antioxidants, and Secondary Metabolites Some of the heat-induced processes are medi-

ated by hormones or can result as a consequence of altered hormonal status by heat stress (Hoffman and Parsons, 1991). The stress hormone abscisic acid (ABA) mediates dehydration and other stress signaling pathways. In the field, heat and drought stresses may occur simultaneously, and ABA induction can be an important component of thermotolerance (Maestri et al., 2002). Many heat shock proteins (Hsp) are ABA-inducible (Pareek et al., 1998; Rojas et al., 1999), and several cisand trans-acting regulatory elements have been recognized by analysis of ABA-responsive promoters (Swamy and Smith, 1999). However, in bentgrass (Agrostis palustris), ABA accumulated during recovery from heat stress, suggesting a role in heat stress recovery rather than signal transduction (Larkindale and Huang, 2005). Ethylene is also known to confer protection against heat and drought stress in several plant species (Larkindale and Knight, 2002). Ethylene signaling mutants such as ethylene response 1 and ethylene insensitive 2 (etr1 and ein2) are sensitive to heat stress (Larkindale et al., 2005). In wheat leaves, ethylene production was inhibited severely at 40 °C whereas it was inhibited only at 45 °C in soybean. Moreover, though its precursor 1-amino-cyclopropane-1carboxylic acid (ACC) accumulated at 40 °C in both species, its conversion into ethylene occurred only in soybean but not in wheat. Ethylene production could be induced by transferring wheat leaves to 18 °C after brief exposure to 40 °C (Tan et al., 1988), indicating a role in recovery similar to its role in creeping bentgrass (Larkindale and Huang, 2005). Inhibition of ethylene production arrests ripening of kiwi fruit at temperatures above 35 °C but not respiration (Antunes and Sfatiotakis, 2000). From similar results obtained in pepper (Piper nigrum), and in sunflower and lettuce seed germination (Corbineau et al., 1989; Huberman et al., 1997; Nascimento et al., 2004), it is clear that ethylene plays a positive role in overcoming heat stress and that the deleterious effect of heat stress is due to the inhibition of conversion of ACC to ethylene. However, airborne ethylene at concentrations found in polluted areas has been reported to have negative effect on holm oak plant responses to heat and drought stress (Munné-Bosche et al., 2004). Salicylic acid (SA), an important signaling component of the defense response system (Kawano et al., 1998), is also suggested to be involved in heat-stress response of plants. It induces long-term thermotolerance in plants where both Ca2+ homeostatis and antioxidant systems are thought to be involved (Wang and Li, 2006b). In cucumber, Shi and co-workers (2006) reported that treatment with sulphosalicylic acid (SSA), a derivative of SA, can effectively detoxify H2O2 through the activity of catalase to increase heat tolerance. Likewise, spraying mustard seedlings with acetyl-SA (Dat et al., 1998) and oak with methyl salicylate resulted in enhanced thermotolerance by increasing the level of ascorbate and α-tocopherol in leaves (Llusià et al., 2005; Wang and Li, 2006a). Jasmonates mediate plant responses to many biotic and abiotic stresses including heat and drought. In water-stressed strawberry and Arabidopsis leaves methyl jasmonate (MeJA) treatment prevented loss of ascorbate (Wang, 1999; Maksymiec and Krupa, 2002). An epimerase that interacts with an Hsp70.3 heat shock protein has been suggested to play an important role in ascorbate homeostatis in stress conditions (Wolucka and Van Montagu, 2003). In Arabidopsis and tobacco suspension cells, it was shown that MeJA treatment increases the de novo synthesis of ascorbic acid that coincides with enhanced transcription of at least two MeJA responsive genes encoding enzymes for vitamin C (L-ascorbic acid) biosynthesis (Wolucka et al., 2005). Tocopherols (vitamin E) have been shown to accumulate in Arabidopsis plants after jasmonate treatment (Sandorf and Hollander-Czytko, 2002). Ascorbic acid is required for the biosynthesis and regeneration of vitamin E (Prescott and John, 1996). Jasmonates thus seem to regulate the biosynthesis of both vitamin C and E that are involved in stress responses (Wolucka et al., 2005). Gibberellins and cytokinins have opposite effects in regulating heat tolerance. A heat-tolerant dwarf mutant of barley, which is impaired in gibberellin biosynthesis, was rendered heat sensitive by the application of gibberellic acid, which

GENETIC APPROACHES TOWARD IMPROVING HEAT TOLERANCE IN PLANTS

227

could be reversed back to heat tolerant by the application of paclobutrazol, a gibberellin antagonist (Vettakkorumakankav et al., 1999). A reduction in kernel filling and yield as a result of a hightemperature-induced decrease in cytokinin content was reported in wheat (Banowetz et al., 1999). From limited reports available, it can be concluded that brassinosteroids also play a role in alleviating heat stress in tomato, oilseed rape (Dhaubhadel et al., 1999), and rice (Wu et al., 2008). In summary, though there are several reports suggesting the role of plant hormones in regulating heat stress, it is unknown yet if they act individually or in concert. It is also unknown if auxins have any role to play in this process, though an elevation in ascorbate biosynthesis was reported in Arabidopsis cell suspension in medium containing exogenously supplied auxin, cytokinin, and MeJA. A depletion in ascorbate was observed when tobacco bright-yellow (BY-2) cells were grown on medium containing only MeJA (Wolucka et al., 2005), suggesting a cross talk between auxin, cytokinin, and MeJA in stress regulation. Antioxidants are scavengers of reactive oxygen species such as H2O2, which are key intermediates in cellular signal transduction (Carcamo et al., 2004). H2O2 is a secondary messenger mediating responses to developmental cues, hormones, and biotic and abiotic stresses (Neill et al., 2002). Vitamin C is an important primary metabolite that functions as an antioxidant and as a cell-signaling modulator in a wide array of crucial physiological processes (Wolucka et al., 2005). Modulation of the ascorbate pool resulted in improved stress tolerance in transgenic tobacco plants (Kwon et al., 2003). Ascorbate is a cofactor of several plant-specific enzymes that catalyze a wide range of reactions in important pathways leading to the biosynthesis of defense-related secondary metabolites such as hormones and stress-induced phenylpropanoids and alkaloids (Prescott and John, 1996; Wolucka et al., 2005). It is also required by dioxygenases responsible for oxidative cleavage of carotenoids in abscisic acid biosynthesis (Seo and Koshiba, 2002), which is involved in plant adaptation to environmental stress. It is a reductant in the xanthophyll cycle that plays an important role in the protection of chloroplasts against photo-oxidative damage (Rockholm and Yamamoto, 1996; Smirnoff, 2000) by preventing reactive oxygen species (ROS) triggered peroxidative damage to membrane lipids (Havaux, 1998; Horton, 2002; Wahid et al., 2007). α-Tocopherol, the major vitamin E compound, is another antioxidant that deactivates photosynthesis-derived ROS and prevents lipid peroxidation by scavenging lipid peroxyl radicals in thylakoid membranes (Munné-Bosch, 2005). Stress-tolerant plants display increased tocopherol levels while the most sensitive species show net tocopherol loss under stress leading to oxidative damage and cell destruction (Munné-Bosch and Alegre, 2003). Drought or warm temperatures during seed maturation were reported to increase α-tocopherol in soybean seed by decreasing δ- and γ-tocopherol (Britz and Kremer, 2002). When ascorbate, but not α-tocopherol availability in chloroplasts is limited as in the Arabidopsis vtc1 mutants, recycling of radicals is affected leading to its irreversible degradation (Munné-Bosch and Alegre, 2002). Flavonoids are stress-induced phenylpropanoids that possess an antioxidant activity and provide protection against microbes, high light, and oxidative stress. The biosynthesis of flavonoids involves four closely related ascorbate-dependent dioxygenases (Nakajima et al., 2001; Martens et al., 2003). Lettuce plants in which the activity of phenyl ammonia lyase (PAL) was inhibited showed increased sensitivity to heat shock treatments suggesting that activation of secondary metabolism as well as the antioxidative metabolism is integral to plant adaptation to stress (Oh et al., 2009). Similar results were reported in watermelon where thermal stress-induced biosynthesis but suppressed oxidation of phenolics, triggered acclimation to heat stress (Rivero et al., 2001). Heat-stressed vegetative tissues of many species including rose and sugarcane leaves show an accumulation of anthocyanins (Wahid and Ghazanfar, 2006). These pigments serve as UV screen and decrease leaf osmotic

228

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

potential, which is linked to increased uptake and reduced transpiration loss of water under environmental stresses (Chalker-Scott, 2002), allowing leaves to respond quickly to changing environmental conditions. Another class of secondary products called isoprenoids is emitted in plants that display better photosynthesis under heat stress (Velikova and Loretto, 2005). This protects the PSII apparatus from damage caused by ROS produced during heat-induced oxygenase action of rubisco, even though the photosynthetic rate approaches zero (Sharkey et al., 2005; Velikova et al., 2005). However, recent studies provide evidence that it is the whole set of antioxidant defenses rather than a single antioxidant that helps plants to withstand environmental stress (Munné-Bosche, 2005).

Molecular Responses Reactive Oxygen Species Once considered toxic by-products of aerobic metabolism, ROS are signaling molecules that control processes such as programmed cell death, abiotic stress responses, pathogen defense, and systemic signaling (Mittler, 2002). Partially reduced forms of oxygen, these intermediates (superoxide, hydrogen peroxide, hydroxyl radical, and singlet oxygen) can react with lipids, proteins, nucleic acids, pigments, and other important cellular components, causing their degradation (Havaux, 2003). They are also generated due to high-temperature-induced cellular injury (Liu and Huang, 2000). Other than the normal source of ROS such as photosynthesis and respiration, there are other sources that are enhanced during abiotic stress such as glycolate oxidase in peroxisomes, NADPH oxidases, amine oxidases, and cell-wall bound peroxidases. Because of this dual source, plants regulate intracellular ROS concentrations by mechanisms involving fine modulation of low levels of ROS for signaling purposes and by ROS scavenging or detoxification during stress. Major ROS-scavenging mechanisms include superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT). Antioxidants such as ascorbic acid and glutathione are crucial for plant defense against oxidative stress as revealed by mutants that are hypersensitive to stress conditions (reviewed in Mittler, 2002). Ascorbate can be a direct scavenger of ROS or an indirect substrate for APX to detoxify hydrogen peroxide (H2O2) in the following reaction:

2ascorbate + H 2 O2 = 2monodehydroascorbate + 2H 2 O APX expression is induced in response to many environmental stresses that result in the accumulation of ROS (Karpinski et al., 1999). The monodehydroascorbate radical undergoes reduction to ascorbate through the action of NAD(P)H-dependent monodehydroascorbate reductase (MDAR) (Hossain and Asada, 1985; De Leonardis et al., 1995) or through nonenzymatic reactions (Miyake and Asada, 1994; Navas et al., 1994). Expression of key enzymes involved in this reaction, monodehydro- and dehydroascorbate reductase (DHAR) is elevated under stress conditions (Mittova et al., 2003; Nishikawa et al., 2003). Additionally, ROS production can be decreased by alternate oxidase enzymes (AOX), which can increase the sensitivity of plants to oxidative stress (Maxwell et al., 1999). Chloroplast AOX is induced in transgenic plants that lack APX and/or CAT, and in normal plants in response to high light (Rizhsky et al., 2002). Several components involved in ROS sensing have recently been identified including mitogen-activated protein kinases AtANP1, NtNPK1, AtMPK3 and 6, and Ntp46MAPK (Kovtun et al., 2000; Samuel et al., 2000), a nucleoside diphosphate kinase (NDPK2) (Moon et al., 2003; Tang et al., 2008) and calmodulin (Harding et al., 1997; Desikan et al., 2001).

GENETIC APPROACHES TOWARD IMPROVING HEAT TOLERANCE IN PLANTS

229

Molecular Chaperones and Heat-induced Proteins Molecular chaperones are defined as a family of

unrelated classes of protein that mediate the correct assembly of other polypeptides but that are not components of the functional assembled structures (Ellis and Hemmingsen, 1989). In addition, chaperones play an important role in resolubilization and/or degradation of proteins partially denatured and/or aggregated by mutation or environmental stresses such as high temperature and oxidative conditions (Ellis, 2005). Many molecular chaperones are stress proteins that are abundant even in the absence of stress. Thus, the stress response can be viewed as an amplification of the basic chaperone function. It is possible that all stress proteins act as molecular chaperones, and many chaperones were originally identified as Hsps. Hsps are the major class involved in acquired thermotolerance (Vierling, 1991), though LEA, dehydrins, and ubiquitins also play a role. Five major families of chaperones are conservatively recognized: chaperonins/Hsp60, Hsp70, Hsp90, Hsp100, and the small Hsp (sHsp) family (see Wang et al., 2004). SHsps are low molecular weight (LMW) proteins of 15–30 kiloDalton (kDa) that can increase as much as 200-fold under stress. All small Hsps in plants are encoded by six nuclear gene families, with each gene family corresponding to proteins found in distinct cellular compartments like cytosol, chloroplast, endoplasmic reticulum (ER), mitochondria, and membranes. Heat shock inhibited respiration to a greater extent in wheat and rye than in maize mitochondria. A single 20 kDa LMW Hsp was found in heat-treated wheat and rye mitochondria whereas maize expressed five LMW proteins, suggesting that this diversity in LMW Hsp expression affects the thermal stability of maize compared to wheat and rye (Korotaeva et al., 2001). Under heat stress, Hsps aggregate into a granular structure, possibly protecting the protein biosynthesis machinery (Miroshnichenko et al., 2005), an ability that may be a prerequisite for survival. Plant sHsps respond to a wide range of environmental stresses, including heat, cold, drought, salinity, and oxidative stress. A chloroplast sHsp26.2 was isolated from a heat-tolerant variant of Agrostis stolonifera grass, demonstrating a clear association (Wang and Luthe, 2003). A dual role in protecting PSII from oxidative damage and in fruit development has been identified for a tomato LMW Hsp21 (Neta-Sharir et al., 2005). Hsp68, a constitutively expressed, mitochondria-localized protein, was elevated in heat-stressed potato, maize, tomato, soybean, and barley (Neumann et al., 1993). Levels of a nuclear localized Hsp101 increased enormously in response to heat shock in developing tassel, ear, silks, endosperm, and embryo and moderately in roots and leaves of maize (Young et al., 2001). A 45 kDa Hsp was also found to be involved in recovery from heat stress (Ristic and Cass, 1992), while the cytosolic accumulation of many nuclear-encoded chloroplast proteins was found to be reversible upon recovery from heat stress in many plants (Heckathorn et al., 1998a). Elongating segments of primary roots of several plant species exhibited a strong ability to induce nucleuslocalized Hsps that have a role in thermotolerance (Nieto-Sotelo et al., 2002). Further, a 45-kDa protein was found to be synthesized in the whole plant whereas 64- and 72-kDa peptides were induced in germinating maize pollen when under heat stress (Ristic et al., 1996). Acquisition of thermotolerance is directly related to the synthesis and accumulation of Hsps (Bowen et al., 2002). In many species, induction of Hsp70 and Hsp101 has been shown to be essential for thermotolerance (Schöffl et al., 1999). Expression profile analysis of the Arabidopsis and spinach Hsp70 genes demonstrated that these proteins are expressed in response to heat, cold, and drought (Wang et al., 2004). It has been hypothesized that Hsp70 participates in ATPdependent protein unfolding or assembly/disassembly reactions and prevents protein denaturation during heat stress (Iba, 2002) through evidence obtained from mutants lacking Hsp70 that are heat sensitive (Burke, 2001). LMW proteins may also play a role in maintaining membrane integrity as evidenced from localization in chloroplast membranes, further suggesting that they protect PSII from adverse effects of high heat (Barua et al., 2003). Under continuous heat stress, Hsp22 remained

230

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

at an elevated level (Lund et al., 1998), and tobacco plants with elevated binding protein (BiP) levels were more tolerant to drought stress and to tunicamycin, a potent activator of the unfolded protein response (UPR) pathway (Alvim et al., 2001). UPR is a mechanism whereby the ERresident BiP keeps the accumulated proteins unfolded to prevent aggregation under stress. Overexpression of BiP proteins in tobacco protoplasts (Kaufman, 1999) prevented the induction of UPR-induced genes and increased cell tolerance to tunicamycin-induced stress (Leborgne-Castel 1999). This suggests that BiP proteins may directly alleviate stress. Overexpression of a soybean BiP (BiPD) conferred drought tolerance and delayed leaf senescence in tobacco and soybean plants. Conversely, antisense tobacco plants showed advanced leaf senescence (Alvim et al., 2001; Valente et al., 2008). Though constitutively expressed, plant Hsp90 genes show a stress response. In Arabidopsis, Hsp90 expression is developmentally regulated and responds to various stress conditions, including heat, cold, and salt. The removal of nonfunctional but potentially harmful polypeptides arising from misfolding, denaturation, and aggregation is important for the maintenance of cellular homeostasis, which is carried out by the Hsp100/Clp family. This mechanism of rescuing proteins from aggregation involves cooperation of the Hsp70 family. The proteins have been reported in many plant species where they are constitutively expressed, developmentally regulated, and induced by heat, cold, dehydration, and salt stress. Another example for cross talk between different chaperone families was provided in pea plants where sHsp18.1 can stably bind heat denatured protein and maintain it in a folding-competent state for further refolding by Hsp70/Hsp100 complex (reviewed by Wang et al., 2004). Heat-induced proteins other than Hsps have been reported in plants such as ubiquitin (Sun and Callis, 1997), cytosolic Cu/Zn-SOD (Herouart and Inze, 1994), and Mn-peroxidase (Brown et al., 1993; Iba, 2002). Ubiquitin synthesis during the first 30 minutes of heat exposure is an important mechanism of heat tolerance in legumes Prosopis chilensis and soybean (Ortiz and Cardemil, 2001). Collectively called Pir proteins, osmotin-like proteins induced by heat and nitrogen stresses were found to be overexpressed in heat-stressed yeast cells (Yun et al., 1997). Late embryogenesis abundant (LEA) proteins prevent aggregation and protect citrate synthase from desiccating conditions like heat and drought stress (Goyal et al., 2005). When exposed to heat and drought, geranium leaves accumulated dehydrin proteins (Arora et al., 1998) similar to heatexposed sugarcane (Wahid and Close, 2007). These proteins are related to protein degradation pathways, minimizing the adverse effects of dehydration and oxidative stress during heat stress (Schöffl et al., 1999).

Mechanism of Heat Tolerance in Plants

Plants adapt to heat stress through long-term evolutionary manifestation of developmental and morphological changes and short-term acclimation mechanisms such as leaf orientation and transpirational cooling. Similar mechanisms may be used to overcome other stresses, overlapping the response ranges and placing emphasis on cellular and physiological strategies that have a broad and over-reaching adaptation mechanism. Early maturation is an escape mechanism that translates to decreased yield in many crop plants (Queitsch et al., 2000; Adams et al., 2001). Root respiratory acclimation to high temperatures associated with root thermotolerance has been reported in Agrostis grass species (Rachmilevitch et al., 2008). Root thermotolerance in A. scabra was associated with both short- and long-term acclimation to temperature changes, leading to reduction in carbon

GENETIC APPROACHES TOWARD IMPROVING HEAT TOLERANCE IN PLANTS

231

expenditure or maintenance costs and resulting in extended root survivability. The first response to heat stress, such as plasma membrane fluidity disruption and osmotic changes, triggers downstream signaling and transcriptional cascade activating a stress-responsive pathway leading to reestablishment of cellular homeostasis and reparation of damaged proteins and membranes (Figure 9.4). Inadequate or delayed response in any step may lead to cell death (Vinocur and Altman, 2005; Bohnert et al., 2006). The acquisition of thermotolerance may reflect a basic mechanism designed to cope with major diurnal fluctuations (Hong et al., 2003), and a mild stress episode is only an acceleration of a normal cellular program rather than an abrupt event linked to stress (Guilioni et al., 1997). The effect of heat stress is not restricted to one level or organelle (Sung et al., 2003), but the initial effects are on the plasmalemma, which becomes more fluid under stress triggering Ca2+ influx and cytoskeletal reorganization, resulting in the upregulation of some mitogen-activated and calcium-dependent kinases. Signaling of these cascades at nuclear level leads to antioxidant production and compatible solute accumulation. Membrane fluidity changes also lead to ROS generation in organelles and signaling in the cytoplasm (Bohnert et al., 2006). The capacity of wheat genotypes to acquire thermotolerance correlated with the activities of CAT and SOD, higher ascorbic acid content, and less oxidative damage (Sairam and Tyagi, 2004). Induction of Hsps and other proteins such as LEA and dehydrins is another mechanism of thermotolerance. These proteins interact with other stress response mechanisms such as production of osmolytes and antioxidants (Diamant et al., 2001; Panchuk et al., 2002). Hsps are involved in stress-signal transduction and gene activation (Nollen and Morimoto, 2002) as well as in the regulation of cellular redox state (Arrigo, 1998). Localization of sHsp with chloroplastic membranes upon heat stress suggests that they play a role in protecting photosynthetic electron transport (Heckathorn et al., 1998b). Detailed analysis of heat shock transcription factors (Hsfs), the terminal components of signal transduction pathway for gene activation in response to heat stress, and a large number of chemical stressors (Zhu et al., 2006; Nover et al., 2001 and references therein) have been reported. The basic modular structure of Hsfs includes a highly conserved DNA-binding domain (DBD), oligomerization domain (OD), nuclear localization signal (NLS), and the least conserved C-terminal activation domain (CTAD). The transcription-activating function of Hsfs is related to the short peptide motifs (AHA motifs) within the CTADs. The palindromic and heat-responsive heat shock elements (HSE) are always present in the promoter regions of heat shock genes. The HSE (5′-AGAAnnTTCT-3′) is the binding target and recognition site for the trans-active Hsfs (Nover and Scharf, 1997; Schöffl et al., 1998). The essential role of HSE for heat-dependent transcriptional activation in plants has been established by promoter deletion analysis in tomato. Hsp activation mechanism by Hsfs is highly conserved and includes the dissociation of a negative regulatory molecule R (repressor) from the Hsfs in monomer form followed by the oligomerization of Hsf molecules. Subsequently, monomeric Hsfs with low affinity for DNA binding are converted into trimers with high affinity for DNA binding. The trimeric Hsfs then bind to HSE of Hsp and activate their transcription. Biosynthesis of Hsps feeds back to the regulation of Hsf expression negatively. Heat stress can also swiftly alter gene expression pattern inducing expression of Hsp components and suppressing others (Yost and Lindquist, 1986; Yang et al., 2006). However, though the sequential cascade in response to heat stress is well known (see Figure 9.5), the mechanisms leading to the specific event and the preferential stress-responsive upregulation and activation of genes and proteins involved, such as Hsps, is yet to be elucidated. It should be noted that transcript expression does not always correlate with protein expression. For example, the Arabidopsis annexin1 (AnnAt1) gene itself is constitutively expressed while the protein expression increased in response to salt stress and was translocated from the cytosol to the membrane (Lee et al., 2004). Investigating the

Heat Stress

Disruption of Cellular Homeostatis Protein and Membrane Damage

Signal Perception and Transduction ROS, Hormones, Kinases, Ca2+, NO

Transcription Factors DREB2A, MBF1c, Hsf, bZIP, NF-X1 Detoxification SOD, APX, CAT

Hips and Chaperones Hsps, sHsps, BiP Gene Regulation Activation of HSR

Osmotic Adjustment proline, trehalose, sugar polyols, glycine betaine

Secondary Metabolites Antioxidants flavonols, ascorbate, tocopherol, isoprenoids

Cell Wall Modification, Dehydrins EXP, LEA, ASR

Protein and Membrane Protection Readjustment of Cellular Homeostatis

Gene Engineering and Translational Genomics Chromosome Engineering and Gene Stacking

Thermotolerance

Plant Introduction and Breeding, MAS, QTL analysis

Profiling/Omics/Bioinformatics, in vitro mutagenesis, TILLING Gene Silencing

Phenotypic Screening and Selection Figure 9.4 Thermotolerance is a complex, multi-component trait and can be enhanced by employing various forward and reverse genetics approaches. Ultimately, appropriate phenotypic and physiological screening is essential to identify heat and multiple stress-tolerant plants. Abbreviations: (APX) ascorbate peroxidase; (ASR) abscisic acid, senescence and ripening related; (BiP) binding protein; (CAT) catalase; (bZIP) basic leucine zipper; (DREB2A) dehydration responsive element binding 2A; (EXP) expansin; (Hsf) heat shock factor; (Hip) heat induced proteins; (Hsp) heat shock proteins; (sHsp) small heat shock proteins; (LEA) late embryogenesis abundant; (MAS) marker assisted selection; (MBF1c) multiprotein bridging factor 1c; (NF-X1) NF-X1-zinc finger; (NO) nitrous oxide; (QTL) quantitative trait locus; (RNAi) RNA inhibition; (ROS) reactive oxygen species; (SOD) superoxide dismutase; (TILLING) targeted-induced lesion IN genome.

232

GENETIC APPROACHES TOWARD IMPROVING HEAT TOLERANCE IN PLANTS

233

Heat Stress

ABA

H2O2

Hsp70/90

MBF1C

DREB2A

SA

Hsf

Ethylene

PR-1

TPS5

BI1, CTL1, TFL2 NF-X1

Trehalose

ETR1, Ein2

sHsp, Hsp101

Osmotic Adjustment

Antioxidants Cell Membrane Stability

Protection of protein homeostatis

Protection from oxidative damage

Thermotolerance Figure 9.5 Skeletal overview of signaling factors involved in thermotolerance. Dashed arrows indicate hypothesized connections. The box represents few of the gene products known to be involved in thermotolerance but whose particular functions are still unknown; DREB2A, MBF1c, CTL1, and NF-X1 are known or speculated regulators of combined stress. Abbreviations: (ABA) abscisic acid; (BI1) bax inhibitor 1; (CTL1) chitinase-like 1; (DREB2A) dehydration responsive element binding 2A; (Ein2) ethylene insensitive 2; (ETR1) ethylene response 1; (H2O2 hydrogen peroxide; (Hsf) heat shock factor; (Hsp) heat shock protein; (MBF1c) multiprotein bridging factor 1c; (NF-X1) NF-X1-zinc finger; (PR-1) pathogen related 1; (SA) salicylic acid; (TFL2) terminal flower 2; (TPS5) trehalose phosphate synthase 5. Source: Simplified and adapted from Kotak et al., 2007 and Suzuki et al., 2008.

effect of heat stress on rice leaf proteome, Lee and co-workers (2007) analyzed at mRNA level four differentially accumulated proteins corresponding to antioxidant enzymes. The results revealed that the transcription levels were not concomitant with translation. Posttranslational modification of proteins plays a critical role in cellular processes. In eukaryotes, one such modification is the attachment of a small polypeptide to a target protein, of which ubiquitin is the best understood. Another class of such molecules that has recently gained interest in plant stress tolerance is the small ubiquitin-like modifier (SUMO) that is related to ubiquitin

234

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

(Geiss-Friedlander and Melchior, 2007). SUMOylation, the process of SUMO maturation and attachment onto substrates, has been shown to play a role in plant abiotic stress regulation (Kurepa et al., 2003; Miura et al., 2005, 2007; Conti et al., 2008). Abiotic stresses including heat shock and H2O2 have been reported to trigger a significant increase in SUMO-protein conjugate levels. Catala and co-workers (2007) reported the involvement of SIZ1, a SUMO ligase, in drought and heat stress. In an earlier study, the siz1 mutants were reported to have reduced basal tolerance to heat shock (Yoo et al., 2006). Whereas a major function of ubiquitin is to mark proteins for intracellular degradation, SUMO modification of proteins leads to a number of biological consequences from antagonism of ubiquitination to regulation of transcription factor activity, alteration of protein subcellular localization and changes in protein-protein interaction (Hochstrasser, 2001; Girdwood et al., 2004; Johnson, 2004; Watts, 2004).

Signal Perception

The primary response to stress is stimulus sensing leading to the key step of setting a signaling cascade in motion to achieve adaptation or tolerance. Some of these stress perception and signaling pathways may be shared while some are specific to a stress (Chinnusamy et al., 2004). In bacteria and yeast, two-component histidine kinases (HK) function as stress sensors. Thermosensors such as the cyanobacterium Hik33 and DesK (from Bacillus subtilis) induce gene expression in response to low temperature. Though such sensors have been reported for other abiotic stresses (reviewed in Gan et al., 2008), no heat sensor has been identified so far. However, change in membrane fluidity plays a critical role in this regard both under high and low temperatures, suggesting that sensors are located in membrane microdomains. Further, these sensors are capable of detecting physical phase transition, eventually leading to conformational changes through cycles of phosphorylation and dephosphorylation due to temperature changes (Plieth, 1999). The sensor may be located on the thylakoid membrane as suggested by the altered heat shock gene expression profiles that ensue when the membrane gains rigidity (Horváth et al., 1998). Owing to the highly unsaturated lipid profile and to the presence of a temperature-sensitive photosystem, thylakoid membrane acting as temperature sensor is physiologically relevant (Sung et al., 2003). Two-component systems typically consist of a membrane localized HK that senses the input signal and a response regulator (see Hwang et al., 2002). The Arabidopsis ATHK1 was identified to be an osmosensor that can also function in yeast. It functions upstream of several stress responsive transcription factors (TF) as a positive regulator of drought and salt-stress responses (Urao et al., 1999; Tran et al., 2007). Similarly, mitogen-activated protein kinase (MAPK) cascades are important for signal transduction (Kaur and Gupta, 2005), and a heat-shock activated MAPK (HAMK) triggered by changes in membrane fluidity coupled with cytoskeletal remodeling has been reported (Sangwan and Dhindsa, 2002). Second messenger molecules such as Ca2+ and ROS are generated once an extracellular stimulus is perceived. A downstream signal cascade that phosphorylates TFs is subsequently activated, which then regulates the expression of a set of genes involved in stress adaptation (Xiong et al., 2002). A sharp rise in cytosolic Ca2+ occurs as a signaling response to temperature stress (Larkindale and Knight, 2002). Ca2+ influx and activation of Ca2+-dependent protein kinases (CDPK) are closely correlated with the expression of Hsps (Sangwan and Dhindsa, 2002). Ca2+ binding activates calmodulin (CaM), which induces a regulatory event cascade including Hsp genes (Liu et al., 2003). Conversely, an elevated cytosolic Ca2+ level under heat stress may alleviate heat injury by increas-

GENETIC APPROACHES TOWARD IMPROVING HEAT TOLERANCE IN PLANTS

235

ing activity of antioxidants, but may not be required for the production of Hsps in plants (Gong et al., 1997). Cytosolic Ca2+ may also influence guard cell turgor maintenance (Webb et al., 1996), and enable plants to survive under heat stress. Other than this, signaling molecules such as SA, ABA, H2O2 and ACC may induce heat tolerance in plants by reducing oxidative damage (Larkindale and Huang, 2004), while nitrous oxide (NO) is emerging as another stress signaling molecule (Dat et al., 2000).

Genetic Approaches to Improve Heat Tolerance in Crops

Although predictable in regions prone to high temperatures, heat stress can cause severe losses in regions that are not normally prone to it due to timing and intensity, even if brief. Additional factors, such as combination or sequential stress and adverse effect on plant-microbe symbiotic association, compound the loss. Application of genetic and biotechnological approaches can significantly reduce such problems, and requires both a sound biological knowledge of the species and the mechanism underlying the tolerance. Though Arabidopsis, rice, Medicago truncatula, and Lotus japonicus continue to serve as model plants, the recent advances in maize and soybean genome sequencing have opened up enormous opportunity for solving stress-induced crop losses. A most recent, giant leap in enhancing stress tolerance of crop plants is the completion of sorghum genome sequencing and its partial analysis revealing several clusters that may be instrumental in the evolution of a cultivated grass species that can withstand extreme drought and heat conditions (Paterson et al., 2009). Cultural practices such as planting time, planting method, and soil and irrigation management have long been in use to minimize stress effects. For example, adequate irrigation during hot periods (Burke, 1990), synchronizing critical growth stage with most favorable weather, and planting shade and barrier trees are some practices adopted around the world based on experience. However, cultural practices alone are not adequate, and yield loss can be minimized further by combining such methods with genetic improvement. Plant breeding has achieved significant increases in total production (Warren, 1998), but genetic improvement for stress tolerance can be an economically viable solution for crop production under stressful environments (Blum, 1988). Summarized below are various strategies available to improve plant heat tolerance and progress to date.

Classical Breeding

Abiotic stress tolerance is a complex trait that is controlled by more than one gene, and is highly influenced by environmental variation (Blum, 1988). Direct selection of resistant cultivars under field conditions is hampered by lack of precise and repeatable trials because it is a developmental stage-specific phenomenon. Screening heat-tolerant breeding progeny in production environments prone to hot temperatures (Ehlers and Hall, 1998) is compounded by the occurrence of other stresses such as drought and pathogen attack. There is a chance of inadvertent selection for traits that may or may not be desirable in commercial production environments. Nevertheless, considerable success was achieved in the 1980s in breeding heat tolerant varieties such as “Pima” cotton, “Saladette” tomato, and cowpea (reviewed by Hall, 1992). Recently, several heat-tolerant tomato hybrid and inbred lines have been released that are also pathogen resistant and have good fruit quality, such as “Neptune,” “Equinox,” and “Solar Fire” (Scott et al., 1995a, 1995b, 2006). The Asian Vegetable

236

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

Research and Development Center (AVRDC) in Taiwan has played a considerable role in improving heat tolerance in vegetable cultivars for the tropics. Growing screening material in greenhouses can be advantageous because it allows precise control over day/night temperatures and timing of heat induction. Choice of container is also important because soil beds may produce more accurate results when testing species where root systems are heat sensitive (Hall, 1992). Proper screening methods and effective selection criteria are essential to a good breeding program. Heat tolerance index (HTI) has been suggested as a good method to screen for differences in coleoptile growth of sorghum seedlings exposed to high temperatures of about 50 °C (Young et al., 2001). In tomato and other species where a strong correlation between heat stress and fruit yield exists, evaluation of germplasm by screening for high fruit set under high temperatures is effective, similar to pollen viability, pollen tube growth and seed viability (Berry and Rafique-Uddin, 1988). Nonsignificant correlation between flower number and yield has been reported in tomato (Lin et al., 2006). For this reason, asexual reproduction may be very useful to develop cultivars suitable for high temperatures. Apomixis could be exploited to indefinitely propagate superior hybrids or specific genotypes bearing complex gene sets (Albertini et al., 2005). However, parthenocarpic tomato fruits are often poor in quality (Hall, 1992). In rice, where high day temperatures prevent dehiscence of anthers, heat resistance could be enhanced by selecting genotypes that initiate anther dehiscence earlier in the morning when diurnal temperatures are minimal (Yoshida et al., 1981). Nonreproductive traits such as photosynthetic efficiency, assimilated translocation, mesophyll resistance, and disorganization of cellular membranes (Chen et al., 1982) are also good selection parameters. In cases where heritability for fruit production is low, several morphological traits conferring heat tolerance can be screened (Hall, 1992). For example, flowering time, absence of floral bud abortion, high number of pods per peduncle for cowpea, and stigma exertion for tomato can be useful traits. Limited genetic variability in crops such as tomato and soybean, in traits such as fruit size (tomato) and grain composition (wheat), is a constraint to traditional breeding (reviewed by Wahid et al., 2007). Classical breeding is more effective when combined with biotechnological approaches to overcome major problems such as lack of natural sources of tolerance and sexual incompatibility (Dita et al., 2006), and seedling screening systems may be more appropriate for genetic engineering strategies because they allow easy manipulation and quick evaluation, particularly for traits such as germination and seedling establishment.

Biotechnology

Different components of tolerance controlled by different sets of genes are critical for heat tolerance at different stages of plant development in different tissues (Howarth, 2005; Bohnert et al., 2006). The use of genetic stocks with different degrees of heat tolerance, correlation and co-segregation analyzes, molecular biology techniques, and molecular markers to identify tolerance quantitative trait loci (QTL) are promising approaches to dissect the genetic basis of thermotolerance (Maestri et al., 2002).

Marker-assisted Selection (MAS)

DNA marker technology derived from molecular genetics and genomic research holds great promise for plant breeding. Owing to genetic linkage, MAS can precisely and efficiently detect allelic

GENETIC APPROACHES TOWARD IMPROVING HEAT TOLERANCE IN PLANTS

237

variation in genes underlying traits. Thus, it has become a component of the new discipline of “molecular breeding” (Collard and Mackill, 2008). Molecular markers are particularly useful when targeting traits controlled by several genes. Mapping different QTL contributing to a trait and identifying linked molecular markers holds possibility for QTL pyramiding to develop an elite cultivar. Numerous marker-assisted techniques such as random amplified polymorphism (RAPD), restricted fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), and simple sequence repeat (SSR) have been reported for both biotic and abiotic stresses (reviewed by Dita et al., 2006). In tomato, Lin and co-workers (2006) reported the use of RAPD markers to identify yield traits under heat stress. Using 43 F7 recombinant inbred lines (RIL) derived from a cross between heat-tolerant CL5915 and heat-sensitive L4422, they identified 14 RAPD markers that were associated with heat tolerance. Four of these markers contributed to high fruit number, high fruit weight, and high yield under heat stress conditions—traits that could be used for MAS breeding using CL5915 as donor parent. In sorghum, stay-green is a drought-resistant trait that enables plants to resist premature senescence during post-flowering stage. Four stay-green QTL located on three linkage groups (LG) were identified using a RFLP map developed from a B35xTX7000 F7 RIL population (Xu et al., 2000). Of these, 2 QTL regions on LG A, Stg1 and Stg2, also contain genes for key photosynthetic enzymes, heat shock proteins, and an ABA responsive gene, suggesting that this LG is important for drought and heat-stress tolerance and yield in sorghum. In maize, 11 QTL-controlling pollen germination and pollen tube growth under heat stress were identified by RFLP mapping (Frova and Sari-Gorla, 1994). As evident from the Stg QTL, stress tolerance mechanisms may be shared. Phenotypic traits such as root depth may be critical in overcoming abiotic stresses such as drought, heat, and heavy metal toxicity. In rice, root length was found to be associated with aluminum tolerance (Nguyen et al., 2003). Further comparison revealed that this QTL located on chromosome 3 of rice was conserved across cereal species. QTL associated with root traits, earliness, and yield are known in rice, maize, and barley (reviewed by Collard and Mackill, 2008). Testing these RILs for heat tolerance alone or in combination with other stresses may confirm the utility of these markers in identifying stress tolerant lines. In addition, MAS can be combined with other technology such as genetic engineering to improve its efficiency.

Somaclonal Variation and in vitro Mutagenesis

Tissue culture generates a wide range of genetic variation in plants, which can be incorporated in plant-breeding programs (Jain, 2001). The ability of somaclonal variation involving callus cultivation and somatic embryogenesis to generate genetic variation is well known (Larkin and Scowcroft, 1981). Though not desirable for genetic transformation and massive micropropagation, such variations can be useful for breeding, especially when combined with mutagenesis (Dita et al., 2006). Induction of point mutations, deletions, or insertions through in vitro mutagenesis strategies such as ethyl methane sulphonate (EMS), fast neutron radiation, and insertional mutagenesis has been useful in breeding for multiple abiotic stress tolerance in cauliflower (Fuller and Eed, 2003) and drought tolerance in wheat (Khan et al., 2001). Further, mutagenesis techniques such as targeted-induced lesion IN genome (TILLING) can be combined with MAS. An Arabidopsis EMS-generated mutant that has lost the ability for acquired thermotolerance, AtTS02, was shown

238

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

to have a reduced level of a 27 kD Hsp (Burke et al., 2000). The major disadvantage of mutagenesis is the large number of individuals that need to be screened to obtain a desired trait. This can be bypassed to some extent through in vitro selection, which has been used to address salinity and zinc tolerance (Flowers, 2004; Zair et al., 2003; Samantaray et al., 1999) and holds promise for heat tolerance. In particular, this method can be coupled with conventional breeding and transgenic approaches where screening is difficult. Another tissue-culture-based method that can be used to produce homozygous lines rapidly is the double haploid (DH) technology where anther cultures are used to obtain haploid embryos. The technique can greatly reduce the time and cost of cultivar development (Liu et al., 2002) and has been used in rice to develop cold tolerant lines (Qian et al., 2000). Somatic hybrids have been generated by fusing heat-tolerant radish (Raphanus sativus) and Brassica oleracea with Brassica nigra (Arumugam, et al., 2002). Reports of such hybrids are available for zinc tolerance (between Thlaspi caerulescens and Brassica napus) (Brewer et al., 1999) and for transfer of salt tolerance from Aeleuropus littoralis sinensis to wheat (Wei et al., 2001). Such wide hybridizations can be used to introgress desirable characters from wild germplasm into economic targets through embryo rescue and protoplast fusion to overcome reproductive barriers.

Genetic Engineering Overexpression and Mutational Analysis Knowledge gained in genetic, molecular, and biochemical aspects of plant-stress tolerance and advances in transformation methods have contributed greatly toward genetic engineering of plants to overexpress a beneficial gene (Figure 9.5; Table 9.1). However, the constraint of multi-genic control of abiotic stress tolerance is still a major limitation in developing heat-tolerant transgenic plants. Alleviation of oxidative damage by overexpressing genes involved in ROS detox, such as glutatathione peroxidase, aldehyde dehydrogenase, and SOD was achieved to obtain drought, salt, and chilling stress (Sunkar et al., 2003; Kirch et al., 2004; Yoshimura et al., 2004). Tobacco plants where a chloroplast desaturase enzyme was silenced to produce more dienoic fatty acids compared to wild type were found to be more heat tolerant (Murakami et al., 2000). The Dnak1 gene that confers salt tolerance in cyanobacterium was transferred into tobacco plants where it conferred heat tolerance (Ono et al., 2001). Enhancing glycinebetaine levels by overexpressing the Betaine Aldehyde Dehydrogenase (BADH) gene has been suggested as a way to achieve heat-stress tolerance (Yang et al., 2005), and tobacco plants with reduced rubisco activase activity were found to be more heat sensitive (Sharkey et al., 2001). A tomato MT-sHsp was demonstrated to induce heat tolerance in tobacco plants (Sanmiya et al., 2004). Queitsch and co-workers (2000) conducted elegant experiments to show that Hsp101 is required for acquired thermotolerance as is evident from defects in Arabidopsis seedling survival and hypocotyl elongation under heat stress. Basal thermotolerance required for seed germination was also abrogated in the Hsp101 mutants. In yet another study, Charng and others (2006) presented evidence to suggest that an sHsp, Hsa32, is required for maintenance rather than for induction of acquired thermotolerance. In the absence of Hsa32, Arabidopsis transgenic plants revealed a faster decaying thermotolerance. Enhanced tolerance to various stresses was achieved in Arabidopsis plants by introducing a Cucurbita ficifolia spermidine synthase gene (Kasukabe et al., 2004). Overexpression of trehalose in rice plants improved tolerance to many abiotic stresses (Garg et al., 2002; Jang et al., 2003; Miranda et al., 2007) resulting in a higher photosynthetic rate and decreased photo-oxidative damage.

Table 9.1 Genetic components implicated and their functions in plant heat tolerance. (ER) endoplasmic reticulum; (Hsp) heat shock protein; (Hsfs) heat shock transcription factor; (MAP) mitogen activated protein; (NAC), NAM, ATAF, and CUC; (ROS) reactive oxygen species; (SUMO) small ubiquitin modifying enzyme; (TF) transcription factor; (UPR) unfolded protein response. Gene

Class and Function

Species

Approach Used

Reference

ABA1, 2, and 3

ABA biosynthesis; basal thermotolerance ABA signaling; acquired thermotolerance Peroxidase; ROS scavenging

Arabidopsis

Mutational analysis

Larkindale et al., 2005

Arabidopsis

Mutational analysis

Larkindale et al., 2005

Arabidopsis

Osmolyte; osmotic adjustment Anti-apoptosis ER-UPR; delayed leaf senescence; drought tolerance TF; regulation of Hsp, heat tolerance Ca2+Binding; ROS sensing, kinase activation

Tobacco Arabidopsis Soybean, Tobacco

Biochemical analysis Mutational analysis Overexpression Mutational analysis Overexpression

Mittler, 2002, Davletova et al., 2005 Yang et al., 2005 Watanabe et al., 2006 Alvim et al., 2001 Valente et al., 2008

Arabidopsis

Mutational analysis

Gao et al., 2008

Arabidopsis Tobacco

Desikan et al., 2001 Harding et al., 1997

ABI1 and 2 APX BADH BI1 BiP

bZIP28 Calmodulin

CAT Choline Kinase Cu/Zn SOD

DREB2A ETR1; EIN2 EXP1 FAD7 GAD1 GBSS HOT2/CTL1 Hsa32 HsfA1

HsfA2 HsfA4a HsfA7a HsfB1

Hsp16, 17, 26 Hsp17.6A

Peroxidase; ROS scavenging Kinase; acquired thermotolerance Peroxidase: ROS scavenging, heat stability of chloroplasts TF; regulation of HsfA3, drought tolerance Ethylene signaling; stress signaling Expansin; restore cell extension Trienoic fatty acid synthesis; temperature acclimation GABA synthesis; heat tolerance Starch synthesis; grain filling Chitinase; salt tolerance and acquired thermotolerance sHsp; maintenance of AT TF; regulate HsfA2 and HsfB1 (tomato), thermotolerance, activation of Hsp70 TF; regulate APX2 TF; ROS sensing, anti-apoptotic TF; acquired thermotolerance

Cucumber Arabidopsis

Microarray Transgenic culture assay Upregulation Mutational analysis

Chenopodium

Enzyme assay

Arabidopsis Arabidopsis

Mutational analysis Transgenic analysis Mutational analysis

Sakuma et al., 2006; Qin et al., 2007 Larkindale et al., 2005

Agrostis

Proteomics

Xu et al., 2007

Tobacco

Gene silencing

Murakami et al., 2000

Arabidopsis

Mutational analysis

Bouché et al., 2004

Rice Arabidopsis

Microarray Mutational analysis

Yamakawa et al., 2007 Kwon et al., 2007

Arabidopsis Tomato Soybean

Mutational analysis Mutational analysis Overexpression

Charng et al., 2006 Mishra et al., 2002; Zhu et al., 2006;

Arabidopsis Arabidopsis, Rice

Expression analysis Mutational analysis Transgenic analysis Mutational analysis

TF; recovery of gene expression, co-regulator of other Hsf sHsp; heat tolerance sHsp; heat, drought and salt tolerance

Arabidopsis, Tomato

Nishizawa et al., 2007 Davletova et al., 2005; Yamanouchi et al., 2002 Larkindale and Vierling, 2008 Baniwal et al., 2004

Arabidopsis

Winter wheat Arabidopsis

Promoter, Mutational and Transgenic analysis Proteomics Overexpression

Shi et al., 2006 Larkindale and Vierling, 2008 Khanna-Chopra and Sabarinath, 2004

Krishnan et al., 1989 Sun et al., 2001

239

Table 9.1 Continued Gene

Class and Function

Species

Approach Used

Reference

Hsp18.1 Hsp20 Hsp21

sHsp; protein folding sHSP; heat tolerance LMW Hsp; PSII oxidative damage protection sHsp; heat tolerance sHSP; heat tolerance

Pea Maize Tomato

Enzyme folding assay Expression analysis Overexpression

Lee and Vierling, 2000 Korataeva et al., 2001 Neta-Sharir et al., 2005

Tomato Agrostis

Iwahashi and Hosoda, 2000 Wang and Luthe, 2003

Hsp; drought and heat tolerance Hsp; heat tolerance Hsp; heat tolerance

Maize

Proteomics Gene expression and proteomics Proteomics

Wu and Laidman, 1997 Neumann et al., 1993

Hsp70

Hsp, protein folding; heat, drought tolerance

Arabidopsis, Spinach, Tobacco

Proteomics Immuno and EST screening Expression profiling

Hsp90

Hsp; heat, cold and salt stress tolerance Hsp; protein disaggregation and unfolding Hsp; basal and acquired thermotolerance

Arabidopsis

Genome analysis

Many

Biochemical and Expression studies Mutational analysis Gene silencing Expression analysis Mutational analysis

Hsp22 Hsp26.2 Hsp45 Hsp55 Hsp68

Hsp100 Hsp101/HOT1

Mung bean Tomato, Potato

Arabidopsis Maize Arabidopsis

Ristic et al., 1991

Schöffl et al., 1999 Burke et al., 2001 Wang et al., 2004; Cho and Hong, 2006 Krishna and Gloor, 2001 Wang et al., 2004 Hong et al., 2003, Queitsh et al., 2000; Young et al., 2001 Larkindale and Vierling, 2008 Arora et al., 1998, Goyal et al., 2005

Hsp110

Hsp; heat tolerance

LEA

Dehydrins; prevent protein aggregation, desiccation and heat tolerance Transcription coactivator; multiple stress regulation

Geranium

Phosphate kinase; heat, cold and salt stress Zinc finger TF; heat and salt stress

Arabidopsis Potato Arabidopsis

MAP Kinase; ROS sensing, heat tolerance Osmolyte; drought and heat stress tolerance

Tobacco

Overexpression

Soybean Arabidopsis

Overexpresssion Mutational analysis

Arabidopsis

Mutant analysis

Rice

Overexpression

Hu et al., 2008

SOD

SUMO ligase; drought and heat stress NAC TF; multiple stress tolerance Peroxidase; ROS scavenging

Simon-Sarkadi et al., 2005 Larkindale and Vierling, 2008 Catala et al., 2007

Tobacco, Agrostis, Wheat

Overexpression Proteomics

Sucrose synthase TFL2 Thaumatin

Sucrose biosynthesis; root heat tolerance Hsp; heat tolerance Pir protein; heat acclimation

Agrostis

Proteomics

Yoshimura et al., 2004; Sairam and Tyagi, 2004; Xu and Huang, 2008 Xu et al., 2007

Arabidopsis Arabidopsis

Mutational analysis Mutational analysis

MBF1c

NDPK2 NF-X1

NPK1 Proline

SIZ1 SNAC2

240

Arabidopsis

Immunoblotting Thermal aggregation assay Overexpression Mutational analysis Mutational analysis Overexpression Mutational analysis

Suzuki et al., 2005, 2008

Moon et al., 2003; Tang et al., 2008 Lisso et al., 2006 Larkindale and Vierling, 2008 Kovtun et al., 2000

Bennett et al., 2005 Larkindale and Vierling, 2008

GENETIC APPROACHES TOWARD IMPROVING HEAT TOLERANCE IN PLANTS

241

Table 9.1 Continued Gene

Class and Function

Species

Approach Used

Reference

Thioredoxin h Trehalose

Redox; heat tolerance Osmolyte; multiple stress tolerance; higher photosynthetic rate; decreased photo oxidative damage sHsp; acquired thermotolerance Proteolysis; mark proteins for degradation Ascorbate synthesis; ROS scavenging

Poplar Rice, Arabidopsis

MALDI-TOF/TOF Overexpression

Arabidopsis

EMS mutagenesis

Ferreira et al., 2006 Hare et al., 1998; Jang et al., 2003; Miranda et al., 2007; Suzuki et al., 2008 Burke et al., 2000

Prosopis, Soybean

Immunoblotting

Ortiz and Cardemil, 2001

Arabidopsis

Mutational analysis

Munné-Bosch and Alegre, 2002

TSO2 Ubiquitin VTC1, 2

In a recent study, the Arabidopsis hot2 was shown to be sensitive to salt stress and have a mutation in the AtCTL1 gene, encoding a chitinase-like protein (Kwon et al., 2007). Interestingly, the hot2 mutant was originally identified based on a defect in acquired thermotolerance of hypocotyl elongation despite wild-type level of induction of Hsp (Hong et al., 2003). Chitin serves a structural role in the cuticles of insects, the shells of crustaceans and the cell walls of many fungi. Chitinaselike genes have been identified in bacteria, viruses, animals, and plants (Cohen-Kupiec and Chet, 1998; Patil et al., 2000), organisms that do not contain chitin. It is assumed that plant chitinases function in plant defense by attacking chitin, which is a common constituent of cell walls in fungi. In the same genetic screen, a mutant associated with Hsp101 (hot1) was identified. It exhibited higher levels of ion leakage under normal conditions and at 45 °C was not protected by a 38 °C pretreatment as wild-type plants where the transcript level remained constant under stress. Other genetic screens have identified mutants affected in chlorophyll accumulation after heat shock and mutants that have reduced thermotolerance (reviewed by Kotak et al., 2007). Mutants such as tu8 in the Terminal Flower 2 (TFL2) gene that has reduced accumulation of Hsp90 (Bennett et al., 2005) and AtBI1 (Bax-Inhibitor 1) (Watanabe and Lam, 2006) have also been identified. In a largescale analysis, 45 Arabidopsis mutants defective in signaling, reactive oxygen, and fatty acid metabolism were tested for basal and acquired thermotolerance at different stages of growth (Larkindale et al., 2005). ABA signaling mutants showed stronger defects in acquired thermotolerance while ethylene signaling and reactive oxygen metabolism mutants were more defective in basal thermotolerance. All mutants tested accumulated wild-type levels of Hsp 101 and sHsps. The data obtained from this comprehensive study indicate that acquired thermotolerance induced by heat shock proteins is distinct from that induced by signaling pathways and that cross talk exists between high light stress and heat stress. Transcription Factors Hsfs are regulatory proteins that control the transcription of Hsp encoding genes (Baniwal et al., 2004), serving as terminal components of signal transduction (Kotak et al., 2007). TFs are attractive candidates for genetic engineering because a single TF can result in multiple stress tolerance (Yamaguchi-Shinozaki and Shinozaki, 2005). Plant Hsfs, consisting of 21 members in Arabidopsis, more than 18 and about 23 in tomato and rice, respectively, comprise three conserved evolutionary classes, A, B, and C (Nover et al., 2001). In tomato, HsfA1a, HsfA2,

242

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

and HsfB1 form a regulatory network (Baniwal et al., 2004), of which HsfA1a is constitutively expressed and regulates the expression of the other two. Therefore, it is defined as the master regulator of heat shock response in tomato (Mishra et al., 2002). Knockout mutant analysis shows that HsfA1a and HsfA1b are important for the initial phase of gene expression, and that expression under prolonged heat shock and recovery is controlled by HsfA2 (Lohmann et al., 2004; Schramm et al., 2006; Nishizawa et al., 2007). In Arabidopsis, heat-shock-induced expression of HsfA2 is not regulated by HsfA1a or HsfA1b unlike in tomato (Busch et al., 2005). Neither single nor double mutants of AtHsfA1a and AtHsfA1b (Lohmann et al., 2004) affected the heat-stress response and the longterm thermotolerance of Arabidopsis. The role of HsfB1 underlines a preprogrammed recovery period for rapid resumption of housekeeping and developmental gene expression (Bharti et al., 2004; Baniwal et al., 2004). HsfA2 is important for abiotic stress tolerance as is evident from its involvement in the regulation of APX2, which is key to oxidative stress regulation (Nishizawa et al., 2007). DREB2A, a TF that regulates dehydration-responsive genes, has been shown to regulate the Arabidopsis HsfA3 (Sakuma et al., 2006). Although HsfA4a and HsfA8 may act as ROS sensors (Davletova et al., 2005), in sunflower, HsfA9 appears to be unique to seed development and not required for stress tolerance (Kotak et al., 2007; Prieto-Dapena et al., 2006). A point mutation in HsfA4a induces spontaneous necrotic lesions in rice leaves suggesting a role as an antiapoptotic factor (Yamanouchi et al., 2002). Overexpression of GmHSFA1 in soybean-enhanced thermotolerance, activated GmHSP70 under normal temperature and enhanced its expression under high temperature (Zhu et al., 2006). NAC (NAM, ATAF and CUC) is a plant-specific TF family with diverse roles in development and stress regulation. Transgenic rice plants overexpressing a stress-responsive NAC gene (SNAC2) displayed multiple stress tolerances (Hu et al., 2008). Microarray analysis revealed upregulation of stress response and adaptation genes such as peroxidase and Hsp, unique to SNAC2 among SNAC genes reported previously. A putative membrane tethered transcription factor called basic leucine zipper (bZIP)28 was shown to be upregulated in response to heat (Gao et al., 2008). The heat-sensitive null mutant shows attenuation of heatinducible expression of the ER chaperone BiP2 and HSP26.5-P. Heat stress releases the TF from the ER membrane and redistributes it to the nucleus. Functional Genomics Suppression of gene expression and function is a powerful tool and can be

achieved by several methods such as anti-sense RNA suppression/RNA interference (RNAi)/posttranscriptional gene silencing (PTGS), T-DNA insertional mutagenesis, and TILLING. Some heat shock response (HSR) genes have been identified by sequence comparison (GmHsfA1) (Zhu et al., 2006), and knockout and insertion mutants have been used extensively. The tomato HsfA1 was silenced by co-suppression and PTGS to compare the dosage effect in wild-type and overexpressing lines, identifying the indispensable master regulator of thermotolerance (Mishra et al., 2002). TILLING populations are available for many species now adding to the repertoire for functional characterization of heat-tolerance genes. The action of specific TFs that bind to conserved cis-acting promoter elements is well documented as a cause of changes in gene expression, particularly those induced by abiotic stress. Promoters of stress-inducible genes contain binding motifs or elements that are activated upon environmental cue, useful to overexpress desired candidate genes. Constitutive promoters put unwanted strain on the plant that is already undergoing stress. Tissuespecific and temporally controlled expression is highly advantageous and rational if the aim is to enhance, for example, heat tolerance in the root or a particular developmental stage. Profiling and “Omics” Expressed sequence tags (EST) are available in public databases for many

plant species whose genome sequence is not yet available. cDNA libraries are generated from

GENETIC APPROACHES TOWARD IMPROVING HEAT TOLERANCE IN PLANTS

243

various stressed tissues, at particular developmental stages, enabling identification of useful candidates. Such libraries are especially important in crops that have a large and complex genome such as wheat, where several abiotic stress-associated pathways have been identified based on ESTs (Houde et al., 2006). Microarray technology is useful to study expression patterns of genes involved in stress regulation. A number of genome-wide microarray datasets have been made publicly available by the AtGenExpress consortium (Schmid et al., 2005). Using such datasets for analysis of transcriptional response profiles of Arabidopsis Hsfs and Hsps, Swindell and co-workers (2007) found evidence for considerable cross talk between heat and nonheat stress regulatory network. Several members of the Hsp20, 70, and 100 families exhibited parallel expression patterns during recovery from heat stress. Effect of high temperature on grain filling during the milky stage of rice was elucidated recently (Yamakawa et al., 2007), showing the upregulation of starch-consuming genes such as α-amylase and the downregulation of several starch synthesis genes such as granule bound starch synthase (GBSS) and starch branching enzyme (SBE). The results corresponded with biochemical defects in starch grains formed under high temperatures such as reduced amylase content and suppression of amylopectin side chain elongation leading to decreased grain size. Using a full-length cDNA library from leaves of poplar subjected to multiple stresses such as heat, dehydration, cold, and salt stress, more than 30,000 ESTs were sequenced out of which about 4,500 were found to be nonredundant (Nanjo et al., 2004). Characterization of an EST dataset for cassava that was enriched for drought-responsive genes identified several Hsfs and Hsps including HSP70 and HsfA3 (Lokko et al., 2007). Advance in molecular and capturing techniques has now made it possible to isolate mRNA from a single cell in order to study the gene expression pattern in isolated cells and zygotes (Brandt et al., 2002; Okamoto et al., 2005) and localized events such as lateral root and root hair development. Proteomic reference maps during grain filling and maturation have been charted for maize (Méchin et al., 2004) and wheat (Vensel et al., 2005) endosperm and for barley grain (Finnie et al., 2002). Studies on the effect of heat stress on wheat grain filling at protein level showed induction of Hsps and downregulation of several proteins involved in starch metabolism (Majoul et al., 2003, 2004). A comparative study of HSR in the two legumes Prosopis chilensis and Glycine max cv. McCall was conducted to evaluate several parameters associated with basal and acquired thermotolerance (Ortiz and Cardemil, 2001). Although no differences in quantum yield of photosynthesis was found between the two species, Prosopis showed higher relative levels of free Hsp70, which increased dramatically after 20 minutes of heat shock. In a similar study in two lines of maize that differ in drought and heat resistance (Ristic et al., 1991), 20-day-old resistant line ZPBL 1304 and the sensitive line ZPL 389, were exposed to 45 °C. The resistant line synthesized a 45-kD Hsp, which was not found in the sensitive line. In the thermal-tolerant winter wheat variety Mustang, unique Hsps of 16, 17, and 26 kDa were observed when heat stressed at 34 °C, but not in the sensitive study (Krishnan et al., 1989). These reports provide a correlation between the synthesis of specific Hsps and the degree of thermal tolerance within a species, and they support the hypothesis that low-molecular-weight Hsps have an adaptive role in acquisition of thermotolerance. Such heat-tolerant lines are valuable as breeding parents. In contrast, no qualitative differences in Hsps were observed between heat resistant and sensitive cotton (Fender and O’Connell, 1989) and sorghum (Ougham and Stoddard, 1986). In the C3 grass Agrostis, an expansin gene AsEXP1 was found to be associated with heat tolerance (Xu et al., 2007). Thermal Agrostis scabra is adapted to high temperatures in geothermal areas and was subjected to 20 °C control temperature and 40 °C heat stress for 7 days. Differential display analysis identified AsEXP1 protein to be strongly upregulated in leaves but not in roots exposed to heat stress. Heat-tolerant ecotypes of the turf grass Agrostis stolonifera were also found to have elevated AsEXP1. Expansins are believed

244

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

to be able to fully restore cell extension in heat denatured cell walls (Wu and Cosgrove, 2000), and this work unveils a molecular marker to select heat-tolerant grass germplasm. Proteomics offers a powerful approach to discover the proteins and pathways that are crucial for stress responsiveness and tolerance (Xu and Huang, 2008). Two-dimensional gel electrophoresis (2-DGE) and mass spectrometry (MS) are core technologies used to dissect the proteomes of several species (reviewed by Agarwal et al., 2005). In heat-shocked (42 °C) mung bean hypocotyls, 10 Hsps with molecular masses ranging from 20 to 85 kDa were separated and visualized of which Hsp55 was the most strongly radiolabeled (Wu and Laidman, 1997). A total of 1,200 proteins were resolved from heat-stressed tomato pericarp with 14 proteins being unique to heat stress, a 22 kDa mitochondrial Hsp, and isoforms of APX among them (Iwahashi and Hosoda, 2000). In heatstressed rice leaves, Hsps and antioxidant enzymes were upregulated while enzymes related to metabolic pathway were differentially accumulated (Lee et al., 2007). Evaluating effects of heat stress on poplar (Populus euphratica), Ferreira and co-workers (2006) profiled protein accumulation in leaves from young plants and found that proteins related to lipid biosynthesis, cytoskeleton structure, and sulfate assimilation were affected. Carbon metabolism was adjusted to achieve photostatis along with a decrease in PSII abundance and an increase in PSI contribution. A putative Hsp90 was also found to be upregulated in stressed tissue. The authors conclude that Thioredoxin h may have a special role in heat tolerance in poplar. Extending their study on Agrostis, Xu and Huang (2008) compared root proteomic responses to heat stress. Thermal A. scabra roots had more upregulated proteins than A. stolonifera roots. Mass spectrometry results suggest that the upregulation of sucrose synthase, glutathione S-transferase, SOD, and stress-inducible protein (Sti) may contribute to the superior root thermotolerance of A. scabra. Further, protein modification via phosphoproteomic analysis revealed that thermal A. scabra had greater phosphorylation of fructosebiphosphate aldolase isoforms than A. stolonifera, suggesting that aldolase phosphorylation might be involved in root thermotolerance.

The Effect of Stress Combination

In the field, the adverse effect of a stress condition can worsen when accompanied by another stress (reviewed by Mittler, 2006). Plant response to a combination of stress may be different from its response to individual stresses (Rizhsky et al., 2004; Mittler, 2006). For example, plants cool their leaves by keeping stomata open for transpiration during heat stress. But when combined with drought stress, the leaf temperature goes up as stomata are kept closed. Also, a combination of drought and heat shock resulted in suppression of photosynthesis and enhanced respiration (Rizhsky et al., 2002). Similarly cold or drought stress combined with high light conditions results in enhanced production of ROS due to the limited availability of CO2 for the dark reaction, leaving oxygen as one of the main reductive products of photosynthesis (Mittler, 2002). From combination stress studies, it is clear that each condition requires a unique acclimation response, tailored to the specific needs of the plant (Mittler, 2006) with synergistic or antagonistic interactions (Rizhsky et al., 2004). Check points between environmental and oxidative stresses have also been suggested (Vranova et al., 2000; Verslues et al., 2007). Using cDNA arrays, Rizhsky and co-workers (2002) found that some transcripts induced during drought and some induced during heat shock were suppressed during a combination of drought and heat shock. In contrast, some transcripts were specifically induced during a combination stress. The Arabidopsis multiprotein bridging factor 1c (MBF1c) has been demonstrated to function upstream of salicylic acid and ethylene during heat stress (Suzuki et al., 2005, 2008). The PR-1 (pathogenesis-related protein 1) transcript expression was not detected

GENETIC APPROACHES TOWARD IMPROVING HEAT TOLERANCE IN PLANTS

245

in mbf1c mutant plants during heat stress, indicating that MBF1c acts upstream. Identified in a screen for virus movement protein (MP) interactors in tobacco, MBF1 may modulate host gene expression upon infection by mosaic virus (Matsushita et al. 2002). Yeast-two hybrid screen performed for MBF1c full-length cDNA isolated trehalose phosphate 5 (AtTPS5) as its interacting partner in regulating heat stress. Further evidence was obtained from two independent TPS5 null mutants that were deficient in basal but not acquired thermotolerance. Taken together the results provide evidence for the existence of a tightly coordinated HSR network involving trehalose, SA and ethylene signaling pathways that is under the control of MBF1c, which enhances tolerance to heat and osmotic stress, separately or in combination. Metabolic profiling of Arabidopsis plants subjected to drought, heat, and a combination of the two revealed that in the latter condition plants accumulated sugars rather than proline, which is known to accumulate under drought. Several reports suggest that plants engineered to overaccumulate proline to enhance abiotic stress tolerance may not withstand field conditions that include a combination of drought and heat stress or heat stress alone (Rizhsky et al., 2004 and references therein). Genetic manipulation of proline accumulation enabled transgenic soybean plants to better withstand simultaneous drought and heat stress, but influenced the concentrations of other amino acids (Simon-Sarkadi et al., 2005), reiterating the value of testing such transgenic plants for chemical compounds important for food quality. Expression of wheat HSP101 mRNAs was induced by heat stress and by a dehydration and ABA treatment but not by chilling or wounding (Campbell et al., 2001), indicating a role for Hsp101 in heat and drought stress tolerance. Similarly, in tobacco transgenic plants overexpressing NtHSP70-1, the level of leaf NtHsp70-1 correlated with maintenance of optimum water content when plants were subjected to progressive drought (Cho and Hong, 2006). Constitutive expression of a maize ZmDREB2A gene resulted in an improved drought and heat stress tolerance in Arabidopsis plants (Qin et al., 2007). Similar results were reported by Sakuma and others (2006) in Arabidopisis where the constitutively active DREB2A induced drought-, salt-, and heat shock-related genes. Thermotolerance was significantly increased in plants overexpressing the constitutively active DREB2A gene and decreased in DREB2A knockout plants. DREB2A has been shown to induce HsfA3 during heat stress by activating its promoter, and HsfA3 in turn activates promoters of several Hsp genes to establish thermotolerance in Arabidopsis (Schramm et al., 2008; Yoshida et al., 2008). Exposure of rice seedlings to a high temperature (42 °C) for 24 hours resulted in a significant increase in drought stress tolerance (Sato and Yokoya, 2008). An sHsp gene, sHSP17.7, which was identified as a probable cause (Murakami et al., 2004), was overexpressed, and transgenic plants were tested for survival after drought stress. Though no significant difference was found in water potential between transgenic and wild-type seedlings, only transgenic seedlings recovered from the stress. An Arabidopsis Hsp17.6A was also shown to induce heat, drought, and salt tolerance when overexpressed (Sun et al., 2001). Using gradual, step-wise, and direct heat stress to study molecular events that dictate acquired thermotolerance, it was found that a gradual increase in temperature led to higher survival of Arabidopsis seedlings (Figure 9.2) (Larkindale and Vierling, 2008). A significant difference in the transcript spectrum was observed in the two acclimation methods, but the core components overlapped. Mutant analysis identified eight new genes involved in heat acclimation, such as cytosolic APX, HsfA7a, NF-X1, SGT1a, Proline oxidase, HSP110, Choline kinase, and Thaumatin. The zinc finger TF NF-X1 is upregulated by a wide range of other abiotic stresses. NF-X1 mutants have reduced growth and survival when salt stressed (Lisso et al., 2006). In summary, several Hsps are induced when plants are heat or water stressed, and some key factors such as Arabidopsis DREB2A, NF-X1, MBF1c, and wheat HSP101 may be involved in regulating multiple stress tolerance. Comparison of heat-tolerant and heat-sensitive genotypes of cool season crops such as cabbage shows that head

246

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

formation at high temperatures is associated with aspects of plant water relations (Hall, 1992). Similarly, high temperature stress can be alleviated by adequate irrigation in wheat and cotton (Burke, 1990). However, as water shortage is already a menace on its own, it is important to integrate available genetic and molecular data to combat heat and heat-induced drought stress in plants.

Evolving Techniques Translational Genomics

The genome-sequencing era is poised to benefit crop improvement more than ever before, with more emphasis on translational genomics and research. In plant sciences, the term translational research implies translating or applying genomics knowledge obtained in one species for improvement of another species. For example, the genomic sequence data obtained from model plants, rice, maize, and soybean can be applied to other plants of economic importance. Similarly MAS can be applied to detect differences between elite breeding lines and to introduce desirable traits. Although translational genomics is not an evolving technique per se, it is a concept that has lately gained impetus as an area of research.

Artificial or Minichromosome

Transgenic crop production has been very successful in alleviating many problems associated with yield. Nevertheless, it has several drawbacks such as the limited number of genes that can be integrated into host chromosomes, extensive screening to identify transgenic plants with desired expression level and copy number, and long breeding programs to obtain varieties that are introgressed with a combination of transgenes. Two approaches have been used to construct maize engineered minichromosomes (MMC) (Yu et al., 2007) or maize minichromosome (Carlson et al., 2007) to stack multiple genes or add unlimited amounts of DNA in a sequential manner that are faithfully transmitted to progeny for several generations. Engineered minichromosomes were generated by modifying natural A or supernumerary B chromosome by telomere-mediated truncation, whereas MMC were constructed by using repetitive DNA found in centromeres, the chromosomal regions needed for inheritance. The minichromosome platform offers several advantages that can facilitate crop biotechnology by combining numerous trait genes in a defined sequence for more consistent expression. Additional genes, multigene complexes, or whole metabolic pathways can be added to a genotype through site-specific recombination cassettes introduced for future manipulations. Breeding programs are required to introgress an integrated transgene into desired germplasm, and while such programs eliminate undesirable linked loci, they stretch over many years. Minichromosomes form an independent linkage group, thus accelerating the breeding process. The two minichromosome approaches offer an unprecedented opportunity to deliver gene stacks and designer chromosomes with multiple desirable traits.

Small RNA

Stress tolerance is achieved by transcriptional and post-transcriptional regulation. Another mode of post-transcriptional regulation by microRNA (miRNA) and small interfering RNA (siRNA) is now known where these small RNAs silence genes by guiding target mRNA for degradation or by

GENETIC APPROACHES TOWARD IMPROVING HEAT TOLERANCE IN PLANTS

247

repressing translation (Mallory and Vaucheret, 2006). MiRNAs are genome-encoded 20–24 nucleotide duplex-structured small RNAs that are evolutionarily conserved. Manipulation of miRNAguided gene regulation can help engineer oxidative stress resistant plants (Sunkar et al., 2006). Under normal conditions, Cu-Zn SOD1 and 2 (CSD1 and 2) mRNAs do not accumulate because of cleavage by miR398. Under oxidative stress, miR398 is transcriptionally downregulated to derepress CSD1 and 2 genes, which then accumulate and scavenge ROS. Transgenic plants that carry miR398-resistant mutations in the CSD2 mRNA were shown to be resistant to diverse abiotic stress conditions compared to the wild type. Genome analyzes and expression profiling have revealed thousands of genes in convergent overlapping pairs that can generate complementary transcripts and widespread antisense transcription in plants (Wang et al., 2006). In Arabidopsis, a natural antisense transcript-derived siRNA (nat-siRNA) derived from a cis-NAT pair of SRO5 and P5CDH genes was demonstrated to play an important role in osmoprotection and oxidative stress management under salt stress (Borsani et al., 2005). The SRO5-P5CDH nat-siRNAs together with the P5CDH and SRO5 proteins form an important regulatory loop controlling proline and ROS production and stress tolerance. Small RNAs were recently cloned from abiotic stressed tissue of poplar (Lu et al., 2008), and 68 putative miRNA sequences were identified. Upon experimental validation, the expression of several miRNAs was found to alter in response to abiotic stress including heat, providing useful information for development of plants and trees with greater stress tolerance.

Conclusion and Perspectives

Thermotolerance is a complex interaction of several components such as mRNA, protein and cell membrane stability, heat shock proteins and molecular chaperones, and antioxidants. Recovery from stress is the critical factor that determines crop yield. Dissection of the underlying molecular mechanisms is vital for effective stress management without compromising other traits. Designing appropriate stress treatments and downstream biochemical and physiological assays in the laboratory is crucial for field success of transgenic plants. For example, a gradual rise in temperature is more reflective of field condition than shifting plants from one temperature to another. In view of the vast evidence for considerable cross talk between various stress response pathways, testing transgenic plants for resistance to an array of stress conditions is essential. Seed pretreatment may be a low cost and high benefit strategy. In grasses, a root or foliar treatment with H2O2 led to heat tolerance (Uchida et al., 2002; Larkindale and Huang, 2004), but its field utility is not known. Plant introduction from regions prone to extremely high temperatures is useful for marker-assisted breeding in crops where limited genetic variation impedes improvement. Such introductions are also good sources for selection of candidates for translational research. Using historic examples and data from 23 global climate models to illustrate the magnitude of damage to food systems, Battisti and Naylor (2009) predict a very grim future where today’s scattered events could become longterm trends without major adaptation investments to develop heat and heat-induced water stress tolerant crop varieties. The need to design strategies to develop crops that can adapt to new and possibly limited growing areas or to climate changes in existing growing areas is more imperative than ever.

References Adams, S.R., Cockshull, K.E., and Cave, C.R.J. (2001) Effect of temperature on the growth and development of tomato fruits. Annals of Botany 88:869–877.

248

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

Agarwal, G.K., Yonekura, M., Iwahashi, Y., Iwahashi, H., and Rakwal, R. (2005) System, trends and perspectives of proteomics in dicot plants Part III. Unraveling the proteomes influenced by the environment, and at the levels of function and genetic relationships. Journal of Chromatography B 815:137–145. Albertini, E., Marconi, G., Reale, L., Barcaccia, G., Porceddu, A., Ferranti, F., and Falcinelli M. (2005) SERK and APOSTART: candidate genes for apomixis in Poa pratensis. Plant Physiology 138:2185–2199. Alvim, F.C., Carolino, S.M.B., Cascardo, J.C.M., Nunes, C.C., Martinez, C.A., Otoni, W.C., and Fontes, E.P.B. (2001) Enhanced accumulation of BiP in transgenic plants confers tolerance to water stress. Plant Physiology 126(3): 1042–1054. Antunes, M.D.C. and Sfatiotakis, E.M. (2000) Effect of high temperature stress on ethylene biosynthesis, respiration and ripening of “Hayward” kiwifruit. Postharvest Biology and Technology 20:251–259. Arora, R., Pitchay, D.S., and Bearce, B.C. (1998) Water-stress-induced heat tolerance in geranium leaf tissues: A possible linkage through stress proteins? Physiologia Plantarum 103:24–34. Arrigo, A.P. (1998) Small stress proteins: chaperones that act as regulators of intracellular redox state and programmed cell death. Biological Chemistry 379:19–26. Arumugam, N., Mukhopadhyay, A., Gupta, V., Sodhi, Y.S., Verma, J.K., Pental, D., and Pradhan, A.K. (2002) Synthesis of somatic hybrids (RCBB) by fusing heat-tolerant Raphanus sativus (RR) and Brassica oleracea (CC) with Brassica nigra (BB). Plant Breeding 121:168–170. Ashraf, M., Saeed, M.M., and Qureshi, M.J. (1994) Tolerance to high temperature in cotton (Gossypium hirsutum L.) at initial growth stages. Environmental and Experimental Botany 34:275–283. Baniwal, S.K., Bharti, K., Chan, Y.K., Fauth, M., Ganguli, A., Kotak, S., Mishra, S.K., Nover, L., Port, M., Scharf, K.-D., Tripp, J., Weber, C., Zielinski, D., and von Koskull-Döring, P. (2004) Heat stress response in plants: a complex game with chaperones and more than twenty heat stress transcription factors. Journal of Bioscience 29(4):471–487. Bañon, S., Fernandez, J.A., Franco, J.A., Torrecillas, A., Alarcon, J.J., and Sanchez-Blanco, M.J. (2004) Effects of water stress and night temperature preconditioning on water relations and morphological and anatomical changes of Lotus creticus plants. Scientia Horticulturae 101:333–342. Banowetz, G.M., Ammar, K., and Chen, D.D. (1999) Temperature effects on cytokinin accumulation and kernel mass in dwarf wheat. Annals of Botany 83:303–307. Barua, D., Downs, C.A., and Heckathorn, S.A. (2003) Variation in chloroplast small heat-shock protein function is a major determinant of variation in thermotolerance of photosynthetic electron transport among ecotypes of Chenopodium album. Functional Plant Biology 30:1071–1079. Battisti, D.S. and Naylor, R.L. (2009) Historical warnings of future food insecurity with unprecedented seasonal heat. Science 323:240–244. Bennett, R.N., Wenke, T., Freudenberg, B., Mellon, F.A., and Ludwig-Müller, J. (2005) The tu8 mutation of Arabidopsis thaliana encoding a heterochromatin protein 1 homolog causes defects in the induction of secondary metabolite biosynthesis. Plant Biology 7:348–357. Berry, J.A. and Bjorkman. O. (1980) Photosynthetic response and adaptation to temperature in higher plants. Annual Review of Plant Physiology 31:491–543. Berry, S.Z. and Rafique-Uddin, M. (1988) Effect of high temperature on fruit set in tomato cultivars and selected germplasm. HortScience 23:606–608. Bharti, K., von Koskull-Döring, P., Bharti, S., Kumar, P., Tintschl-Korbitzer, A., Treuter, E., and Nover, L. (2004) Tomato heat stress transcription factor HsfB1 represents a novel type of general transcription coactivator with a histone-like motif interacting with HAC1/CBP. Plant Cell 16:1521–1535. Blum, A. (1988) Plant breeding for stress environments. p. 223. CRC Press Inc. Boca Raton, Florida. Bohnert, H.J., Gong, Q., Li, P., and Ma, S. (2006) Unraveling abiotic stress tolerance mechanisms—getting genomics going. Current Opinion in Plant Biology 9:180–188. Borsani, O., Zhu, J., Verslues, P.E., Sunkar, R., and Zhu, J.K. (2005) Endogenous siRNAs derived from a pair of natural cisantisense transcripts regulate salt tolerance in Arabidopsis. Cell 123:1279–1291. Bouché, N., Eait, A., Zik, M., and Fromm, H. (2004) The root-specific glutamate decarboxylase (GAD1) is essential for sustaining GABA levels in Arabidopsis. Plant Molecular Biology 55:315–325. Bowen, J., Michael, L.-Y., Plummer, K., and Ferguson, I. (2002) The heat shock response is involved in thermotolerance in suspension-cultured apple fruit cells. Journal of Plant Physiology 159:599–606. Boyer, J.S. (1982) Crop productivity and environment. Science 218:443–448. Brandt, S., Kloska, S., Altmann, T., and Kehr, J. (2002) Using array hybridization to monitor gene expression at the single cell level. Journal of Experimental Botany 53:2315–2323. Brewer, E.P., Saunders, J.A., Angle, J.S., Chaney, R.L., and McIntosh, M,S. (1999) Somatic hybridization between the zinc accumulator Thlaspi caerulescens and Brassica napus. Theoretical and Applied Genetics 99:761–771.

GENETIC APPROACHES TOWARD IMPROVING HEAT TOLERANCE IN PLANTS

249

Britz, S.J. and Kremer, D.F. (2002) Warm temperatures or drought during seed maturation increase free α-tocopherol in seeds of soybean (Glycine max [L.] Merr.) Journal of Agricultural and Food Chemistry 50:6058–6063. Brown, J.A., Li, D., and Ic, M. (1993) Heat shock induction of manganese peroxidase gene transcription in Phanerochaete chryosporium. Applied and Environmental Microbiology 59:4295–4299. Burke, J.J. (1990) “High Temperature Stress and Adaptations in Crops.” In Stress Responses in Plants: Adaptation and Acclimation Mechanisms, edited by Alscher RG and Cumming JR, pp. 295–308. Wiley-Liss, Inc. New York. Burke, J.J. (2001) Identification of genetic diversity and mutations in higher plant acquired thermotolerance. Physiologia Plantarum 112:167–170. Burke, J.J., O’Mahony, P.J., and Oliver, M.J. (2000) Isolation of Arabidopsis mutants lacking components of acquired thermotolerance. Plant Physiology 123(2):575–588. Busch, W., Wunderlich, M., and Schöffl, F. (2005) Identification of novel heat shock factor-dependent genes and biochemical pathways in Arabidopsis thaliana. Plant Journal 41:1–14. Camejo, D., Rodriguez, P., Marales, M.S., Dell’amico, J.M., Torrecillas, A., and Alarcón, J.J. (2005) High temperature effects on photosynthetic activity of two tomato cultivars with different heat susceptibility. Journal of Plant Physiology 162:281–289. Campbell, J.L., Klueva, N.Y., Zheng, H.-G., Nieto-Sotelo, J., Ho, T.-H.D., and Nguyen, H.T. (2001) Cloning of new members of heat shock protein HSP101 gene family in wheat (Triticum aestivum (L.) Moench) inducible by heat, dehydration, and ABA. Biochimica et Biophysica Acta 1517:270–277. Carcamo, J.M., Pedraza, A., Borquez–Ojeda, O., Zhang, B., Sanchez, R., and Golde, D.W. (2004) Vitamin C is a kinase inhibitor: dehydroascorbic acid inhibits IκBα kinase beta. Molecular and Cell Biology 24:6645–6652. Carlson, S.R., Rudgers, G.W., Zieler, H., Mach, J.M., Luo, S., Grunden, E., Krol, C., Copenhaver, G.P., and Preuss, D. (2007) Meiotic transmission of an in vitro-assembled autonomous maize minichromosome. PLoS Genetics 3(10):1965–1974. Catala, R., Ouyang, J., Abreu, I.A., Hu, Y., Seo, H., Zhang, X., and Chua, N.-H. (2007) The Arabidopsis E3 SUMO ligase SIZ1 regulates plant growth and drought responses. Plant Cell 19:2952–2966. Chaitanya, K.V., Sundar, D., and Reddy, A.R. (2001) Mulberry leaf metabolism under high temperature stress. Biologia Plantarum 44:379–384. Chalker-Scott, L. (2002) Do anthocyaninins function as osmoregulators in leaf tissue? Advances in Botanical Research 37:103–106. Charng, Y.-Y., Liu, H.-C., Liu, N.-Y., Hsu, F.-C., and Ko, S.-S. (2006) Arabidopsis Hsa32, a novel heat shock protein, is essential for acquired thermotolerance during long recovery after acclimation. Plant Physiology 140(4):1297–1305. Chen, T.H.H., Shen, Z.Y., and Lee, P.H. (1982) Adaptability of crop plants to high temperature stress. Crop Science 223:719–725. Chinnusamy, V., Schumaker, K., and Zhu, J.K. (2004) Molecular genetic perspectives on cross-talk and specificity in abiotic stress signaling in plants. Journal of Experimental Botany 55:225–236. Cho, E.K. and Hong, C.B. (2006) Over-expression of tobacco NtHSP70-1 contributes to drought-stress tolerance in plants. Plant Cell Reporter 25:349–358. Cohen-Kupiec, R. and Chet, I. (1998) The molecular biology of chitin digestion. Current Opinion in Biotechnology 9(3):270–277. Collard, B.C.Y. and Mackill, D.J. (2008) Marker-assisted selection: an approach for precision plant breeding in the twenty-first century. Philosophical Transactions of the Royal Society B 363:557–572. Conti, L., Price, G., O’Donnell, E., Schwessinger, B., Dominy, P., and Sadanandom, A. (2008) Small ubiquitin-like modifier proteases OVERLY TOLERANT TO SALT1 and -2 regulate salt stress responses in Arabidopsis. Plant Cell 20:2894–2908. Corbineau, F., Rudnicki, R.M., and Come, D. (1989) ACC conversion to ethylene by sunflower seeds in relation to maturation, germination and thermodormancy. Plant Growth Regulator 8:105–115. Crafts-Brandner, S.J., van de Loo, F.J., and Salvucci, M.E. (1997) The two forms of ribulose-1, 5-bisphosphate carboxylase/ oxygenase activase differ in sensitivity to elevated temperature. Plant Physiology 114:439–444. Dat, J., Vandenbeelé, S., Vranova, E., Van Montagu, M., Inzé, D., and Van Breusegem, F. (2000) Dual action of the active oxygen species during plant stress responses. Cell and Molecular Life Sciences 57:779–795. Dat, J.F., Foyer, C.H., and Scott, I.M. (1998) Changes in salicylic acid and antioxidants during induction of thermo tolerance in mustard seedlings. Plant Physiology 118:1455–1461. Davletova, S., Rhizsky, L., Liang, H., Shengqiang, Z., Oliver, D.J., Coutu, J., Shuleev, V., Schlauch, K., and Mittler, R. (2005) Cytosolic ascorbate peroxidase 1 is a central component of the reactive oxygen gene network of Arabidopsis. Plant Cell 17:268–281. De Las Rivas, J. and Barber, J. (1997) Structure and thermal stability of photosystem II reaction centers studied by infrared spectroscopy. Biochemistry 36:8897–8903.

250

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

De Leonardis, S., De Lorenzo, G., Borraccino, G., and Dipierro, S. (1995) A specific ascorbate free radical reductase isozyme participates in the regeneration of ascorbate for scavenging toxic oxygen species in potato tuber mitochondria. Plant Physiology 109:847–851. De Ronde, J.A., Cress, W.A., Kruger, G.H., Strasser, R.J., and Van Staden, J. (2004) Photosynthetic response of transgenic soybean plants, containing an Arabidopsis P5CR gene, during heat and drought stress. Journal of Plant Physiology 161(11):1211–1224. Desikan, R., Mackerness, S.A.-H., Hancock, J.T., and Neill, S.J. (2001) Regulation of the Arabidopsis transcriptome by oxidative stress. Plant Physiology 127:159–172. Dhaubhadel, S., Chaudhary, S., Dobinson, K.F., and Krishna, P. (1999) Treatment with 24-epibrassinolide, a brassinosteroid, increases the basic thermotolerance of Brassica napus and tomato seedlings. Plant Molecular Biology 40:333–342. Diamant, S., Eliahu, N., Rosenthal, D., and Goloubinoff, P. (2001) Chemical chaperones regulate molecular chaperones in vitro and in cells under combined salt and heat stresses. Journal of Biological Chemistry 276:39586–39591. Dita, M.A., Rispail, N., Prats, E., Rubiales, D., and Singh, K.B. (2006) Biotechnology approaches to overcome biotic and abiotic stress constraints in legumes. Euphytica 147:1–24. Ebrahim, M.K., Zingsheim, O., El-Shourbagy, M.N., Moore, P.H., and Komor, E. (1998) Growth and sugar storage in sugarcane grown at temperatures below and above optimum. Journal of Plant Physiology 153:593–602. Eckardt, N.A. and Portis, A.R., Jr. (1997) Heat denaturation profiles of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase and the inability of Rubisco activase to restore activity of heat-denatured Rubisco. Plant Physiology 113:243–248. Ehlers, J.D. and Hall, A.E. (1998) Heat tolerance of contrasting cowpea lines in short and long days. Field Crops Research 55:11–21. Ellis, R.J. and Hemmingsen, S.M. (1989) Molecular chaperones: proteins essential for the biogenesis of some macromolecular structures. Trends in Biochemical Sciences 14(8):339–42. Ellis, R.J. (2005) “Chaperone Function: The Orthodox View.” In Molecular chaperones and cell signaling, edited by Henderson, B. and Pockley, G.A., pp 3–21. Cambridge University Press, Cambridge, UK. Feller, U., Crafts-Brandner, S.J., and Salvucci, M.E. (1998) Moderately high temperatures inhibit ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase-mediated activation of Rubisco. Plant Physiology 116:539–546. Fender, S.E. and O’Connell, M.A. (1989) Heat shock protein expression in thermotolerant and thermosensitive lines of cotton. Plant Cell Reporter 8:37–40. Ferreira, S., Hjernø, K., Larsen, M., Wingsle, G., Larsen, P., Fey, S., Roepstorff, P., and Pais, M.S. (2006) Proteome profiling of Populus euphratica Oliv. upon heat stress. Annals of Botany 98:361–377. Ferris, R., Ellis, R.H., Wheeler, T.R., and Hadley, P. (1998) Effect of high temperature stress at anthesis on grain yield and biomass of field grown crops of wheat. Plant Cell Environment 34:67–78. Finnie, C., Melchior, S., Roepstorff, P., and Svensson, B. (2002) Proteome analysis of grain filling and seed maturation in barley. Plant Physiology 129:1308–1319. Flowers, T.J. (2004) Improving crop salt tolerance. Journal of Experimental Botany 55:307–319. Fokar, M., Nguyen, H.T., and Blum, A. (1998) Heat tolerance in spring wheat. I. estimating cellular thermotolerance and its heritability. Euphytica 104:1–8. Foolad, M.R. (2005) “Breeding for abiotic stress tolerances in tomato.” In Abiotic Stresses: Plant Resistance Through Breeding and Molecular Approaches, pp 613–684. The Haworth Press Inc., New York, USA. Frova, C. and Sari-Gorla, M. (1994) Quantitative trait loci (QTLs) for pollen thermotolerance detected in maize. Molecular Genetics and Genomics 245:424–430. Fuller, M.P. and Eed, M.H.I. (2003) The development of multiple stress-resistant cauliflower using mutagenesis in conjunction with a microshoot tissue culture technique. In XXVI. International Horticultural Congress: Environmental Stress and Horticultural Crops, Toronto, Canada. (2003) Acta Horticulturae 618:71–76. Gan, J.-P., Chao, D.-Y., and Lin, H.-X. (2008) Toward understanding molecular mechanisms of abiotic stress responses in rice. Rice 1:36–51. Gao, H., Brandizzi, F., Benning, C., and Larkin, R.M. (2008) A membrane-tethered transcription factor defines a branch of the heat stress response in Arabidopsis thaliana. Proceedings of the National Academy of Sciences USA 105(42):16398–16403. Garg, A., Kin, J.K., Owens, T.F., Ranwala, A.P., Choi, Y.D., Kochian, L.V., and Wu, R.J. (2002) Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses. Proceedings of the National Academy of Sciences USA 99:25898–15903. Geiss-Friedlander, R. and Melchior, F. (2007) Concepts in sumoylation: A decade on. Nature Review Molecular Cell Biology 8:947–956. Georgieva, K., Fedina, I., Maslenkova, L., and Peeva, V. (2003) Response of chlorina barley mutants to heat stress under low and high light. Functional Plant Biology 30:515–524.

GENETIC APPROACHES TOWARD IMPROVING HEAT TOLERANCE IN PLANTS

251

Girdwood, D.W., Tatham, M.H., and Hay, R.T. (2004) SUMO and transcriptional regulation. Seminars in Cell and Developmental Biology 15:201–210. Gong, M., Chen, S.N., Song, Y.Q., and Li, Z.G. (1997) Effect of calcium and calmodulin on intrinsic heat tolerance in relation to antioxidant systems in maize seedlings. Australian Journal of Plant Physiology 24:371–379. Goyal, K., Walto, L.J., and Tunnacliffe, A. (2005) LEA proteins prevent protein aggregation due to water stress. The Biochemical Journal 388:151–157. Guilioni, L., Wery, J., and Tardieu, F. (1997) Heat stress-induced abortion of buds and flowers in pea: is sensitivity linked to organ age or to relations between reproductive organs? Annals of Botany 80:159–168. Guilioni, L., Wery, J., and Lecoeur, J. (2003) High temperature and water deficit may reduce seed number in field pea purely by decreasing plant growth rate. Functional Plant Biology 30:1151–1164. Hall, A.E. (1992) Breeding for heat tolerance. Plant Breeding Reviews 10:129–168. Harding, S.A., Oh, S.-H., and Roberts, D.M. (1997) Transgenic tobacco expressing a foreign calmodulin gene shows an enhanced production of active oxygen species. EMBO Journal 16(6):1137–1144. Hare, P.D., Cress, W.A., and Staden, J.V. (1998) Dissecting the roles of osmolyte accumulation during stress. Plant, Cell and Environment 21:535–553. Havaux, M. and Tardy, F. (1996) Temperature-dependent adjustment of the thermal stability of photosystem II in vivo: possible involvement of xanthophyll-cycle pigments. Planta 198:324–333. Havaux, M. (1998) Carotenoids as membrane stabilizers in chloroplasts. Trends in Plant Science 3:147–151. Havaux, M. (2003) Spontaneous and thermoinduced photon emission: new methods to detect and quantify oxidative stress in plants. Trends in Plant Science 8(9):409–413. Heckathorn, S.A., Downs, C.A., and Coleman, J.S. (1998a) Nuclear-encoded chloroplast proteins accumulated in the cytosol during severe heat stress. International Journal of Plant Science 159:39–45. Heckathorn, S.A., Downs, C.A., Sharkey, T.D., and Coleman, J.S. (1998b) The small, methionine-rich chloroplast heat-shock protein protects photosystem II electron transport during heat stress. Plant Physiology 116:439–444. Herouart, D., Van Montagu, M., and Inzé, D. (1994) Developmental and environmental regulation of the Nicotiana plumbaginifolia cytosolic Cu/Zn-superoxide dismutase promoter in transgenic tobacco. Plant Physiology 104:873–880. Hochstrasser, M. (2001) SP-RING for SUMO: New functions bloom for a ubiquitin-like protein. Cell 107:5–8. Hoffman, A.A. and Parsons, P.A. (1991) Evolutionary genetics and environmental stress. Oxford University Press, Oxford. Hong, S.-W., Lee, U., and Vierling, E. (2003) Arabidopsis hot mutants define multiple functions required for acclimation to high temperatures. Plant Physiology 132:757–767. Horton, P. (2002) Crop improvement through alteration in the photosynthetic membrane. ISB News Report. Virginia Tech. Blacksburg, VA. Horváth, G., Arellano, J.B., Droppa, M., and Baron, M. (1998) Alterations in Photosystem II electron transport as revealed by thermoluminescence of Cu-poisoned chloroplasts. Photosynthesis Research 57:175–181. Hossain, M.A. and Asada, K. (1985) Monodehydroascorbate reductase from cucumber is a flavin adenine dinucleotide enzyme. Journal of Biological Chemistry 260:12920–12926. Houde, M., Belcaid, M., Ouellet, F., Danyluk, J., Monroy, A.F., Dryanova, A., Gulick, P., Bergeron, A., Laroce, A., Links, M.G., MacCarthy, L., Crosby, W.L., and Sarhan, F. (2006) Wheat EST resources for functional genomics of abiotic stress. BMC Genomics 7:149. Howarth, C.J. (2005) “Genetic improvements of tolerance to high temperature.” In Abiotic stresses: Plant resistance through breeding and molecular approaches, edited by Ashraf M and Harris PJC. Howarth Press Inc. New York. Hu, H., You, J., Fang, Y., Zhu, X., Qi, Z., and Xiong, L. (2008) Characterization of transcription factor gene SNAC2 conferring cold and salt tolerance in rice. Plant Molecular Biology 67:169–181. Huberman, M., Riov, J., Aloni, B., and Goren, R. (1997) Role of ethylene biosynthesis and auxin content and transport in high temperature-induced abscission of pepper reproductive organs. Journal of Plant Growth Regulator 16:129–135. Hwang, I., Chen, H.-C., and Sheen, J. (2002) Two–component signal transduction pathways in Arabidopsis. Plant Physiology 129:500–515. Iba, K. (2002) Acclimative response to temperature stress in higher plants: approaches of gene engineering for temperature tolerance. Annual Review of Plant Biology 53:225–245. IPCC. (2007) “Summary for Policymakers.” In Climate Change 2007: The Physical Science Bases. Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change, edited by Solomon, S., Qin, D., Manning, M., Chen, Z., Marquis, M., Averyt, K.B., Tignor, M., and Miller, H.L. Cambridge University Press, Cambridge, United Kingdom and New York, USA. Iwahashi, Y. and Hosoda, H. (2000) Effect of heat stress on tomato fruit protein expression. Electrophoresis 21(9):1766–1771. Jain, S.M. (2001) Tissue culture-derived variation in crop improvement. Euphytica 118:153–166. Jang, I.-C., Oh, S.-J., Seo, J.-S., Choi, W.-B., Song, S.I., Kim, C.H., Kim, Y.S., Seo, H.S., Choi, Y.D., Nahn, B.H., and Kim, J.-K. (2003) Expression of a bifunctional fusion of the Escherichia coli genes for trehalose-6-phosphate phosphatase in

252

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

transgenic rice plants increases trehalose accumulation and abiotic stress tolerance without stunting growth. Plant Physiology 131:516–524. Johnson, E.S. (2004) Protein modification by SUMO. Annual Review of Biochemistry 73:355–382. Karpinski, S., Reynolds, H., Karpinska, B., Wingsle, G., Creissen, G., and Mullineaux, P. (1999) Systemic signaling and acclimation in response to excess excitation energy in Arabidopsis. Science 284:654–657. Kasukabe, Y., He, L., Nada, K., Misawa, S., Ihara, I., and Tachibana, S. (2004) Overexpression of spermidine synthase enhances tolerance of multiple environmental stresses and up-regulates the expression of various stress-regulated genes in transgenic Arabidopsis thaliana. Plant and Cell Physiology 45:712–722. Kaufman, R.J. (1999) Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes and Development 13:1211–1233. Kaur, N. and Gupta, A.K. (2005) Signal transduction pathways under abiotic stresses in plants. Current Science 88: 1771–1780. Kavi Kishore, P.B., Sangam, S., Amrutha, R.N., Laxmi, P.S., Naidu, K.R., Rao, K.R.S.S., Rao, S., Reddy, K.J., Theriappan, P., and Sreenivasulu, N. (2005) Regulation of proline biosynthesis, degradation, uptake and transport in higher plants: its implications in plant growth and abiotic stress tolerance. Current Science 88:424–438. Kawano, T., Sahashi, N., Takahashi, K., Uozumi, N., and Muto, S. (1998) Salicylic acid induces extracellular superoxide generation followed by an increase in cytosolic calcium ion in tobacco suspension culture: the earliest events in salicylic acid signal transduction. Plant and Cell Physiology 39:721–730. Khan, A.J., Hassan, S., Tariq, M., and Khan, T. (2001) Haploidy breeding and mutagenesis for drought tolerance in wheat. Euphytica 120: 409–414. Khanna-Chopra, R. and Sabarinath, S. (2004) Heat-stable chloroplastic Cu/Zn superoxide dismutase in Chenopodium murale. Biochemical and Biophysical Research Communications 320:1187–1192. Kirch, J.J., Bartels, D., Wei, Y., Schnable, P., and Wood, A. (2004) The aldehyde dehydrogenase gene superfamily of Arabidopsis thaliana. Trends in Plant Science 9:371–377. Klueva, N.Y., Maestri, E., Marimoli, N., and Nguyen, H.T. (2001) “Mechanisms of Thermotolerance in Crops.” In Crop Responses and Adaptations to Temperature Stress, edited by A.S. Basra, pp 177–217, Food Products Press. New York. Kobza, J. and Edwards, G.E. (1987) Influences of leaf temperature on photosynthetic carbon metabolism in wheat. Plant Physiology 83:69–74. Korotaeva, N.E., Antipina, A.I., Grabelynch, O.K., Varakina, N.N., Borovskii, G.B., and Voinikov, V.K. (2001) Mitochondrial low-molecular-weight heat-shock proteins and the tolerance of cereal mitochondria to hyperthermia. Russian Journal of Plant Physiology 48(6):798–803. Kotak, S., Larkindale, J., Lee, U., von Koskull-Döring, P., Vierling, E., and Scharf, K.-D. (2007) Complexity of the heat stress response in plants. Current Opinion in Plant Biology 10:310–316. Kotak, S., Vierling, E., Baumlein, H., and von Koskull-Döring, P. (2007) A novel transcriptional cascade regulating expression of heat stress proteins during seed development of Arabidopsis. Plant Cell 19:182–195. Kovtun, Y., Chiu, W.-L., Tena, G., and Sheen, J. (2000) Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants. Proceedings of the National Academy of Sciences USA. 97(6):2940–2945. Krishna, P. and Gloor, G. (2001) The Hsp90 family of proteins in Arabidopsis thalinana. Cell Stress Chaperones 6:238–246. Krishnan, M., Nguyen, H.T., and Burke, J.J. (1989) Heat shock protein synthesis and thermal tolerance in wheat. Plant Physiology 90:140–145. Kurepa, J., Walker, J.M., Smalle, J., Gosink, M.M., Davis, S.J., Durham, T.L., Sung, D.-Y., and Vierstra, R.D. (2003) The small ubiquitin-like modifier (SUMO) protein modification system in Arabidopsis. Accumulation of SUMO1 and -2 conjugates is increased by stress. Journal of Biological Chemistry 278:6862–6872. Kwon, S.Y., Choi, S.M., Ahn, Y.O., Lee, H.S., Lee, H.B., Park, Y.M., and Kwak, S.S. (2003) Enhanced stress-tolerance of transgenic tobacco plants expressing a human dehydroascorbate reductase gene. Journal of Plant Physiology 160:347–353. Kwon, Y., Kim, S.H., Jung, M.S., Kim, M.S., Oh, J.E., Ju, H.W., Kang, K., Vierling, E., Lee, H., and Hong, S.W. (2007) Arabidopsis hot2 encodes an endochitinase-like protein that is essential for tolerance to heat, salt and drought stresses. Plant Journal 49:184–193. Larkin, P.J. and Scowcroft, W.R. (1981) Somaclonal variation—a novel source of variability from cell-cultures for plant improvement. Theoretical and Applied Genetics 60:197–214. Larkindale, J. and Knight, M.R. (2002) Protection against heat stress-induced oxidative damage in Arabidopsis involves calcium, abscisic acid, ethylene and salicylic acid. Plant Physiology 128:682–695. Larkindale, J. and Huang, B. (2004) Thermotolerance and antioxidant system in Agrostis stolonifera: involvement of salicylic acid, abscisic acid, calcium, hydrogen peroxide, and ethylene. Journal of Plant Physiology 161:405–413. Larkindale, J., Hall, J.D., Knight, M.R., and Vierling, E. (2005) Heat Stress phenotypes of Arabidopsis mutants implicate multiple signaling pathways in the acquisition of thermotolerance. Plant Physiology 138:882–897.

GENETIC APPROACHES TOWARD IMPROVING HEAT TOLERANCE IN PLANTS

253

Larkindale, J. and Huang, B. (2005) Effects of abscissic acid, salicylic acid, ethylene and hydrogen peroxide in thermotolerance and recovery for creeping bentgrass. Plant Growth Regulator 47:17–28. Larkindale, J. and Vierling, E. (2008) Core genome responses involved in acclimation to high temperature. Plant Physiology 146(2):748–761. Law, R.D. and Crafts-Brandner, S.J. (1999) Inhibition and acclimation of photosynthesis to heat stress is closely correlated with activation of ribulose-1,5-bisphosphate Carboxylase/Oxygenase. Plant Physiology 120(1):173–182. Leborgne-Castel, N., Jelitto-Van Dooren, E.P.W.M., Crofts, A.J., and Denecke, J. (1999) Overexpression of BiP in tobacco alleviates endoplasmic reticulum stress. Plant Cell 11:459–470. Lee, G. and Vierling, E. (2000) A small heat shock protein cooperates with heat shock protein 70 systems to reactivate a heatdenatured protein. Plant Physiology 122(1):189–198. Lee, D.G., Ahsan, N., Lee, S.H., Kang, K.Y., Bahk, J.D., Lee, I.J., and Lee, B.H. (2007) A proteomic approach in analyzing heat-responsive proteins in rice leaves. Proteomics 7(18):3369–3383. Lee, S., Lee, E.J., Yang, E.J., Lee, J.E., Park, A.R., Song, W.H., and Park, O.K. (2004) Proteomic identification of annexins, calcium-dependent membrane binding proteins that mediate osmotic stress and abscisic acid signal transduction in Arabidopsis. Plant Cell 16:1378–1391. Leonardos, E.D., Tsujita, M.J., and Grodzinski, B. (1996) The effect of source or sink temperature on photosynthesis and 14C partitioning in and export from a source leaf of Alstroemeria. Physiologia Plantarum 97:563–575. Lin, H.-K., Lo, H.-F., Lee, S.-P., Kuo, G.C., Chen, J.-T., and Yeh, W.-L. (2006) RAPD markers for the identification of yield traits in tomatoes under heat stress via bulked segregant analysis. Hereditas 143:142–154. Lisso, J., Altmann, T., and Mussig, C. (2006) The AtNFXL1 gene encodes a NF-X1 type zinc finger protein required for growth under salt stress. FEBS Letters 580:4851–4856. Liu, H.-T., Li, B., Shang, Z.-L., Li, X.-Z., Mu, R.-L., Sun, D.-Y., and Zhou, R.-G. (2003) Calmodulin is involved in heat shock signal transduction in wheat. Plant Physiology 132:1186–1195. Liu, W., Zheng, M.Y., Polle, E.A., and Konzak, C.F. (2002) Highly efficient doubled-haploid production in wheat (Triticum aestivum L.) via induced microspore embryogenesis. Crop Science 42:686–692. Liu, X. and Hwang, B. (2000) Heat stress injury in relation to membrane lipid peroxidation in creeping bent grass. Crop Science 40:503–510. Llusià, J., Peñuelas, J., and Munné-Bosch, S. (2005) Sustained accumulation of methyl salicylate alters antioxidant protection and reduces tolerance of holm oak to heat stress. Physiologia Plantarum 124:353–361. Lohmann, C., Eggers-Schumacher, G., Wunderlich, M., and Schöffl, F. (2004) Two different heat shock transcription factors regulate immediate early expression of stress genes in Arabidopsis. Molecular Genetics and Genomics 271:11–21. Lokko, Y., Anderson, J.V., Rudd, S., Raji, A., Horvath, D., Mikel, M.A., Kim, R., Liu, L., Hernandez, A., Dixon, A.G.O., and Ingelbrecht, I.L. (2007) Characterization of an 18,166 EST dataset for cassava (Mannihot esculenta Crantz) enriched for drought-responsive genes. Plant Cell Reports 26(9):1605–1618. Lu, S., Sun, Y.-H., and Chiang, V. (2008) Stress-responsive microRNAs in Populus. The Plant Journal 55:131–151. Lund, A.A., Blum, P.H., Bhattramakki, D., and Elthon, T.E. (1998) Heat-stress response of maize mitochondria. International Journal of Plant Science 159:39–45. Machado, S. and Paulsen, G.M. (2001) Combined effects of drought and high temperature on water relations of wheat and sorghum. Plant and Soil 233:179–187. Maestri, E., Klueva, N., Perrotta, C., Gulli, M., Nguyen, H.T., and Marimoli, N. (2002) Molecular genetics of heat tolerance and heat shock proteins in cereals. Plant Molecular Biology 48:667–681. Majoul, T., Bancel, E., Triboi, E., Hamida, B., and Branlard, G. (2003) Proteomic analysis of the effect of heat stress on hexaploid wheat grain: characterization of heat-responsive proteins from total endosperm. Proteomics 3:175–183. Majoul, T., Bancel, E., Triboi, E., Hamida, B., and Branlard, G. (2004) Proteomic analysis of the effect of heat stress on hexaploid wheat grain: characterization of heat-responsive proteins from non-prolamins fraction. Proteomics 4:505–513. Maksymiec, W. and Krupa, Z. (2002) The in vivo and in vitro influence of methyl jasmonate on oxidative processes in Arabidopsis thaliana leaves. Acta Physiologia Plantarum 24:351–357. Mallory, A.C. and Vaucheret, H. (2006) Functions of microRNAs and related small RNAs in plants. Nature Genetics 38:S31–S36. Martens, S., Forkmann, G., Britsch, L., Wellman, F., Matern, U., and Lukacin, R. (2003) Divergent evolution of flavonoid 2-oxoglutarate-dependent dioxygenases in parsley. FEBS Letters 544:93–98. Matsushita, Y., Miyakawa, R., Deguchi, M., Nishiguchi, M., and Nyunoya, H. (2002) Cloning of a tobacco cDNA coding for a putative transcriptional coactivator MBF1c that interacts with the tomato mosaic virus movement protein. Journal of Experimental Botany 53(373):1531–1532. Maxwell, D.P., Wang, Y., and McIntosh, L. (1999) The alternative oxidase lowers mitochondrial reactive oxygen production in plant cells. Proceedings of the National Academy of Sciences USA 96(14):8271–8276.

254

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

Méchin, V., Balliau, T., Chateau-Joubert, S., Davanture, M., Langella, O., Negroni, L., Prioul, J., Thevenot, C., Zivy, M., and Damerval, C. (2004) A two-dimensional proteome map of maize endosperm. Phytochemistry 65:1609–1618. Miller, P., Lanier, W., and Brandt, S. (2001) Using growing degree days to predict plant stages. Ag/Extension Communications Coordinator, Communications Services, Montana State University-Bozeman, Bozeman, MT. Miranda, J., Avonce, N., Suarez, R., Thevelein, J.M., Van Dijck, P., and Iturriaga, G. (2007) A bifunctional TPS-TPP enzyme from yeast confers tolerance to multiple and extreme abiotic-stress conditions in transgenic Arabidopsis. Planta 226(6):1411–1421. Miroshnichenko, S., Tripp, J., Nieden, U., Neumann, D., Conrad, U., and Manteuffel, R. (2005) Immunomodulation of function of small heat shock proteins prevents their assembly into heat stress granules and results in cell death at sublethal temperatures. Plant Journal 41:269–281. Mishra, S.K., Tripp, J., Winkelhaus, S., Tschiersch, B., Theres, K., Nover, L., and Scharf, K.D. (2002) In the complex family of heat stress transcription factors, HsfA1 has a unique role as master regulator of thermotolerance in tomato. Genes and Development 6:177–189. Mittler, R. (2002) Oxidative stress, antioxidants and stress tolerance. Trends in Plant Science 7(9):405–410. Mittler, R. (2006) Abiotic stress, the field environment and stress combination. Trends in Plant Science 11(1):15–19. Mittova, V., Tal, M., Volokita, M., and Guy, M. (2003) Up-regulation of the leaf mitochondrial and peroxisomal antioxidative systems in response to salt-induced oxidative stress in the wild-salt tolerant tomato species Lycopersicon pennellii. Plant, Cell and Environment 26:845–856. Miura, K., Jin, J.B., Lee, J., Yoo, C.Y., Stirm, V., Miura, T., Ashworth, E.N., Bressan, R.A., Yun, D.-J., and Hasegawa, P.M. (2007) SIZ1-mediated sumoylation of ICE1 controls CBF3/DREB1A expression and freezing tolerance in Arabidopsis. Plant Cell 19:1403–1414. Miura, K., Rus, A., Sharkhuu, A., Yokoi, S., Karthikeyan, A.S., Raghothama, K.G., Baek, D., Koo, Y.D., Jin, J.B., Bressan, R.A., Yun, D.-J., and Hasegawa, P.M. (2005) The Arabidoposis SUMO E3 ligase SIZ1 controls phosphate deficiency responses. Proceedings of the National Academy of Sciences USA 102:7760–7765. Miyake, C. and Asada, K. (1994) Ferredoxin-dependent photoreduction of the monodehydroascorbate radical in spinach thylakoids. Plant and Cell Physiology 35:539–549. Moon, H., Lee, B., Choi, G., Shin, D., Prasad, D.T., Lee, O., Kwak, S.-S., Kim, D.H., Nam, J., Bahk, J., Hong, J.C., Lee, S.Y., Cho, M.J., Lim, C.O., and Yun, D.-J. (2003) NDP kinase 2 interacts with two oxidative stress-activated MAPKs to regulate cellular redox state and enhances multiple stress tolerance in transgenic plants. Proceedings of the National Academy of Sciences USA 100(1):358–363. Morales, D., Rodriguez, P., Dell’amico, J., Nicolas, E., Torrecillas, A., and Sanchez-Blanco, M.J. (2003) High-temperature preconditioning and thermal shock imposition affects water relations, gas exchange and root hydraulic conductivity in tomato. Biologia Plantarum 47:203–208. Munné-Bosch, S. and Alegre, L. (2002) Interplay between ascorbic acid and lipophilic antioxidant defences in chloroplasts of water-stressed Arabidopsis plants. FEBS Letters 524:145–148. Munné-Bosch, S. and Alegre, L. (2003) Drought-induced changes in the redox state of α-tocopherol, ascorbate and the diterpene carnosic acid in chloroplasts of Labiatae species differing in carnosic acid contents. Plant Physiology 131:1816–1825. Munné-Bosch, S., Peñuelas, J., Asensio, D., and Llusià, J. (2004) Airborne ethylene may alter antioxidant protection and reduce tolerance of holm oak to heat and drought stress. Plant Physiology 136(2):2937–2947. Munné-Bosch, S. (2005) The role of α-tocopherol in plant stress tolerance. Journal of Plant Physiology 162:743–748. Murakami, T., Matsuba, S., Funatsuki, H., Kawaguchi, K., Saruyama, H., Tanida, M., and Sato, Y. (2004) Over-expression of a small heat shock protein, sHSP17.7, confers both heat tolerance and UV-B resistance to rice plants. Molecular Breeding 13:165–175. Murakami, Y., Tsuyama, M., Kobayashi, Y., Kodama, H., and Iba, K. (2000) Trienoic fatty acids and plant tolerance of high temperature. Science 287:476–479. Nakajima, J., Tanaka, Y., Yamazaki, M., and Saito, K. (2001) Reaction mechanism from leucoanthocyanidin to anthocyaniding 3-glucoside, a key reaction for coloring in anthocyanin biosynthesis. Journal of Biological Chemistry 276:25797–25803. Nanjo, T., Futamura, N., Nishiguchi, M., Igasaki, T., Shinozaki, K., and Shinohara, K. (2004) Characterization of full-length enriched expressed sequence tags of stress treated poplar leaves. Plant and Cell Physiology 45(12):1738–1748. Nascimento, W.M., Cantliffe, D.J., and Huber, D.J. (2004) Ethylene evolution and endo-b-mannanase activity during lettuce seed germination at high temperature. Scientia Agricola 61:156–163. Navas, P., Villalba, J.M., and Cordoba, F. (1994) Ascorbate function at the plasma membrane. Biochimica et Biophysica Acta 1197:1–13. Neill, S.J., Desikan, R., Clarke, A., Hurst, R.D., and Hancock, J.T. (2002) Hydrogen peroxide and nitric oxide as signaling molecules in plants. Journal of Experimental Botany 53:1237–1247.

GENETIC APPROACHES TOWARD IMPROVING HEAT TOLERANCE IN PLANTS

255

Neta-Sharir, I., Isaacson, T., Lurie, S., and Weiss, D. (2005) Dual role for tomato heat shock protein 21: protecting photosystem II from oxidative stress and promoting color changes during fruit maturation. Plant Cell 17:1829–1838. Neumann, D.M., Emmermann, M., Thierfelder, J.M., Zur Nieden, U., Clericus, M., Braun, H.P., Nover, L., and Schmitz, U.K. (1993) HSP68-a DNAK-like heat-stress protein of plant mitochondria. Planta 190:32–43. Nguyen, B.D., Brar, D.S., Bui, B.C., Nguyen, T.V., Pham, L.N., and Nguyen, H.T. (2003) Identification and mapping of the QTL for aluminum tolerance introgressed from the new source, ORYZA RUFIPOGON Griff., into indica rice (Oryza sativa L.). Theoretical and Applied Genetics 106:583–593. Nieto-Sotelo, J., Martinez, L.M., Ponce, G., Cassab, G.I., Alagón, A., Meeley, R.B., Ribaut, J.-M., and Yang, R. (2002) Maize HSP101 plays important roles in both induced and basal thermotolerance and primary root growth. Plant Cell 14:1621–1633. Nishikawa, F., Kato, M., Hyodo, H., Ikoma, Y., Sugiura, M., and Yano, M. (2003) Ascorbate metabolism in harvested broccoli. Journal of Experimental Botany 54:2439–2448. Nishizawa, A., Yabuta, Y., and Shigeoka, S. (2008) Galactinol and Raffinose constitute a novel function to protect plants from oxidative damage. Plant Physiology 147:1251–1263. Nishizawa, A., Yabuta, Y., Yoshida, E., Maruta, T., Yoshimura, K., and Shigeoka, S. (2007) Arabidopsis heat shock transcription factor A2 as a key regulator in response to several types of environmental stress. Plant Journal 48:535–547. Nollen, E.A.A. and Morimoto, R.I. (2002) Chaperoning signaling pathways: molecular chaperones as stress-sensing “heat shock” proteins. Journal of Cell Science 115:2809–2816. Nover, L. and Scharf, K.-D. (1997) Heat stress proteins and transcription factors. Cell and Molecular Life Science 53:80– 103. Nover, L., Bharti, K., Döring, P., Mishra, S.K., Ganguli, A., and Scharf, K.-D. (2001) Arabidopsis and the heat stress transcription factor world: how many heat stress transcription factors do we need? Cell Stress and Chaperones 6(3):177–189. Oh, M.-M., Trick, H.N., and Rajashekar, C.B. (2009) Secondary metabolism and antioxidants are involved in environmental adaptation and stress tolerance in lettuce. Journal of Plant Physiology 166(2):180–191. Okamoto, T., Scholten, S., Lorz, H., and Kranz, E. (2005) Identification of genes that are up- or down-regulated in the apical or basal cell of maize two-celled embryos and monitoring their expression during zygote development by a cell manipulationand PCR-based approach. Plant and Cell Physiology 46:332–338. Ono, K., Hibono, T., Kohinata, T., Suzuki, S., Tanaka, Y., Nakamura, T., Takabe, T., and Takabe, T. (2001) Overexpression of DnaK from a halotolerant cyanobacterium Aphanothece halophytica enhances the high-temperature tolerance of tobacco during germination and early growth. Plant Science 160:455–461. Ortiz, C. and Cardemil, L. (2001) Heat-shock responses in two leguminous plants: a comparative study. Journal of Experimental Botany 52(361):1711–1719. Ougham, H.J. and Stoddard, J.L. (1986) Synthesis of heat-shock protein and acquisition of thermotolerance in high-temperature tolerant and high-temperature susceptible lines of Sorghum. Plant Science 44:163–167. Panchuk, I.I., Volkov, R.A., and Schöffl, F. (2002) Heat stress- and heat shock transcription factor-dependent expression and activity of ascorbate peroxidase in Arabidopsis. Plant Physiology 129: 838–853. Pareek, A., Singla, S.L., and Grover, A. (1998) Protein alterations associated with salinity, desiccation, high and low temperature stresses and abscisic acid application in seedlings of Pusa 169, a high-yielding rice (Oryza sativa L.) cultivar. Current Science 75:1023–1035. Paterson, A.H., Bowers, J.E., Bruggmann, R., Dubchak, I., Grimwood, J., Gundlach, H., Haberer, G., Hellsten, U., Mitros, T., Poliakov, A., Schmutz, J., Spannagl, M., Tang, H., Wang, X., Wicker, T., Bharti, K.A., Chapman, J., Feltus, A.F., Gowik, U., Grigoriev, I.V., Lyons, E., Maher, C.A., Martis, M., Narechania, A., Otillar, R.P., Penning, B.W., Salamov, A.A., Wang, Y., Zhang, L., Carpita, N.C., Freeling, M., Gingle, A.R., Hash, T.C., Keller, B., Klein, P., Kresovich, S., McCann, M.C., Ming, R., Peterson, D.G., ur-Rehman, M., Ware, D., Westhoff, P., Mayer, K.F.X., Messing, J., and Rokhsar, D.S. 2009. The Sorghum bicolor genome and the diversification of grasses. Nature 457:551–556. Patil, R.S., Ghormade, V.V., and Deshpande, M.V. (2000) Chitinolytic enzymes: an exploration. Enzyme and Microbial Technology 26(7):473–483. Plieth, C. (1999) Temperature sensing by plants: calcium-permeable channels as primary sensors—a model. Journal of Membrane Biology 172:121–127. Porter, J.R. (2005) Rising temperatures are likely to reduce crop yields. Nature 436:174. Presott, A.G. and John, P. (1996) DIOXYGENASES: molecular structure and role in plant metabolism. Annual Review of Plant Physiology and Plant Molecular Biology 47:245–271. Prieto-Dapena, P., Castano, R., Almoguera, C., and Jordano, J. (2006) Improved resistance to controlled deterioration in transgenic seeds. Plant Physiology 142:1102–1112. Qian, Q., Zeng, D., He, P., Zheng, X., Chen, Y., and Zhu, L. (2000) QTL analysis of the rice seedling cold tolerance in a double haploid population derived from anther culture of a hybrid between indica and japonica rice. Chinese Science Bulletin 45:448–453.

256

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

Qin, F., Kakimoto, M., Sakuma, Y., Maruyama, K., Osakabe, Y., Tran, L.-S., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2007) Regulation and functional analysis of ZmDREB2A in response to drought and heat stresses in Zea mays L. The Plant Journal 50(1):54–69. Quan, R., Shang, M., Zhang, H., Zhao, Y., and Zhang, J. (2004) Engineering of enhanced glycine betaine synthesis improves drought tolerance in maize. Plant Biotechnology Journal 2:477–486. Queitsch, C., Hong, S.-W., Vierling, E., and Lindquist, S. (2000) Heat shock protein 101 plays a crucial role in thermotolerance in Arabidopsis. Plant Cell 12:479–492. Rachmilevitch, S., Lambers, H., and Huang, B. (2008) Short-term and long-term root respiratory acclimation to elevated temperatures associated with root thermotolerance for two Agrostis grass species. Journal of Experimental Botany 59(14):3803–3809. Ristic, Z. and Cass, D.D. (1992) Chloroplast structure after water and high temperature stress in two lines of maize that differ in endogenous levels of abscisic acid. International Journal of Plant Science 153:186–196. Ristic, Z., Gifford, D.J., and Cass, D.D. (1991) Heat shock proteins in two lines of Zea mays L. that differ in drought and heat resistance. Plant Physiology 97:1430–1434. Ristic, Z., Williams, G., Yang, G., Martin, G., and Fullerton, S. (1996) Dehydration, damage to cellular membranes, and heatshock proteins in maize hybrids from different climates. Journal of Plant Physiology 149:424–432. Rivero, R.M., Ruiz, J.M., Garcia, P.C., Lopez-Lefebre, L.R., Sanchez, E., and Romero, L. (2001) Resistance to cold and heat stress: accumulation of phenolic compounds in tomato and watermelon plants. Plant Science 160:315–321. Rizhsky, L., Hallak-Herr, E., Breusegem, F.V., Rachmilevitch, S., Barr, J.E., Rodermel, S., Inze, D., and Mittler, R. (2002) Double antisense plants lacking ascorbate peroxidase and catalase are less sensitive to oxidative stress than single antisense plants lacking ascorbate peroxidase or catalase. The Plant Journal 34:329–342. Rizhsky, L., Hongjian, L., and Mittler, R. (2002) The combined effect of drought stress and heat shock on gene expression in tobacco. Plant Physiology 130:1143–1151. Rizhsky, L., Hongjian, L., Shuman, J., Shulaev, V., Davletova, S., and Mittler, R. (2004) When defense pathways collide. The response of Arabidopsis to a combination of drought and heat stress. Plant Physiology 134:1683–1696. Rockholm, D.C. and Yamamoto, H.Y. (1996) Violaxanthin de-epoxidase. Plant Physiology 110:697–703. Rojas, A., Almoguera, C., and Jordano, J. (1999) Transcriptional activation of a heat shock gene promoter in sunflower embryos: synergism between ABI3 and heat shock factors. Plant Journal 20:601–610. Rotundo, J.L. and Westgate, M.E. (2009) Meta-analysis of environmental effects on soybean seed composition. Field Crops Research 110:147–156. Sairam, R.K. and Tyagi, A. (2004) Physiology and molecular biology of salinity stress tolerance in plants. Current Science 86:407–421. Sakamoto, A. and Murata, N. (2002) The role of glycine betaine in the protection of plants from stress: clues from transgenic plants. Plant Cell Environment 25:163–171. Sakuma, Y., Maruyama, K., Qin, F., Osakabe, Y., and Shinozaki, K. (2006) Dual function of an Arabidopsis transcription factor DREB2A in water-stress-responsive and heat-stress-responsive gene expression. Proceedings of the National Academy of Sciences USA 103(49):18822–18827. Samantaray, S., Rout, G.R., and Das, P. (1999) In vitro selection and regeneration of zinc tolerant calli from Setaria italica L. Plant Science 143:201–209. Samuel, M.A., Miles, G.P., and Ellis, B. (2000) Ozone treatment rapidly activates MAP kinase signaling in plants. Plant Journal 22(4):367–376. Sandorf, I. and Holländer-Czytko, H. (2002) Jasmonate is involved in the induction of tyrosine aminotransferase and tocopherol biosynthesis in Arabidopsis thaliana. Planta 216:173–179. Sangwan, V. and Dhindsa, R.S. (2002) In vivo and in vitro activation of temperature-responsive plant MAP kinases. FEBS Letters 531:561–564. Sanmiya, K., Suzuki, K., Egawa, Y., and Shono, M. (2004) Mitochondrial small heat shock protein enhances thermotolerance in tobacco plants. FEBS Letters 557:265–268. Sato, S., Kamiyama, M., Iwata, T., Makita, N., Furukawa, H., and Ikeda, H. (2006) Moderate increase of mean daily temperature adversely affects fruit set of Lycopersicon esculentum by disrupting specific physiological processes in male reproductive development. Annals of Botany 97:731–738. Sato, Y. and Yokoya, S. (2008) Enhanced tolerance to drought stress in transgenic rice plants overexpressing a small heat-shock protein, sHSP17.7. Plant Cell Reports 27(2):329–334. Savchenko, G.E., Klyuchareva, E.A., Abrabchik, L.M., and Serdyuchenko, EV. (2002) Effect of periodic heat shock on the membrane system of etioplasts. Russian Journal of Plant Physiology 49:349–359. Schmid, M., Davison, T.S., Henz, S.R., Pape, U.J., Demar, M., Vingron, M., Scholkopf, B., Weigel, D., and Lohmann, J.U. (2005) A gene expression map of Arabidopsis thaliana development. Nature Genetics 37:501–506.

GENETIC APPROACHES TOWARD IMPROVING HEAT TOLERANCE IN PLANTS

257

Schöffl, F., Prändl, R., and Reindl, A. (1998) Regulation of heat stress response. Plant Physiology 117:1135–1141. Schöffl, F., Prändl, R., and Reindl, A. (1999) “Molecular responses to heat stress.” In Molecular Responses to Cold, Drought, Heat and Salt Stress in Higher Plants, edited by Shinozaki, K. and Yamaguchi-Shinozaki, K., pp 81–98, RG Landes Co, Austin, Texas. Schramm, F., Ganguli, A., Kiehlmann, E., Englich, G., Walch, D., and von Koskull-Döring, P. (2006) The heat stress transcription factor HsfA2 serves as a regulatory amplifier of a subset of genes in the heat stress response in Arabidopsis. Plant Molecular Biology 60:759–772. Schramm, F., Larkindale, J., Kiehlmann, E., Ganguli, A., Englich, G., Vierling, E., and von Koskull-Döring, P. (2008) A cascade of transcription factor DREB2A and heat stress transcription factor HsfA3 regulates the heat stress response of Arabidopsis. The Plant Journal 53:264–274. Scott, J.W., Jones, J.B., Somodi, G.C., Chellemi, D.O., and Olson, S.M. (1995a) “Neptune,” a heat-tolerant, bacterial-wilt-tolerant tomato. HortScience 30:641–642. Scott, J.W., Olson, S.M., Bryan, H.H., Bartz, J.A., Maynard, D.N., and Stofella, P.J. (2006) “Solar Fire” hybrid tomato: Fla. 7766 tomato breeding line. HortScience 41:1504–1505. Scott, J.W., Olson, S.M., Howe, T.K., Stofella, P.J., Bartz, J.A., and Bryan, H.H. (1995b) “Equinox,” heat-tolerant hybrid tomato. HortScience 30:647–648. Seo, M. and Koshiba, T. (2002) Complex regulation of ABA biosynthesis in plants. Trends in Plant Science 7:41–48. Shanahan, J.F., Edwards, I.B., Quick, J.S., and Fenwick, J.R. (1990) Membrane thermostability and heat tolerance of spring wheat. Crop Science 30:247–251. Sharkey, T.D., Badger, M.R., Von-Caemmerer, S., and Andrews, T.J. (2001) Increased heat sensitivity of photosynthesis in tobacco plants with reduced Rubisco activase. Photosynthesis Research 67:147–156. Sharkey, T.D. (2005) Effects of moderate heat stress on photosynthesis: importance of thylakoid reactions, rubisco deactivation, reactive oxygen species, and thermotolerance provided by isoprene. Plant Cell and Environment 28:269–277. Shelp, B.J., Bown, A.W., and McLean, M.D. (1999) Metabolism and functions of gamma-aminobutyric acid. Trends in Plant Science 4(11):446–452. Shi, Q., Bao, Z., Zhu, Z., Ying, Q., and Qian, Q. (2006) Effects of different treatments of salicylic acid on heat tolerance, chlorophyll fluorescence, and antioxidant enzyme activity in seedlings of Cucumis sativa L. Plant Growth Regulator 48:127–135. Simoes-Araujo, J.L., Rumjanek, N.G., and Margis-Pinheiro, M. (2003) Small heat shock protein genes are differentially expressed in distinct varieties of common bean. Brazilian Journal of Plant Physiology 15:33–41. Simon-Sarkadi, L., Kocsy, G., Varhegyi, A., Galiba, G., and De Ronde, J.A. (2005) Genetic manipulation of proline accumulation influences the concentrations of other amino acids in soybean subjected to simultaneous drought and heat stress. Journal of Agricultural Food Chemistry 53:7512–7517. Smirnoff, N. (2000) Ascorbate biosynthesis and function in photoprotection. Philosophical Transactions of the Royal Society London B Biological Sciences 355:1455–1464. Somerville, C. and Browse, J. (1991) Plant lipids, metabolism and membranes. Science 252:80–87. Stone, P. (2001) “The effects of heat stress on cereal yield and quality.” In Crop Responses and Adaptation to Temperature Stress, edited by Basra AS, pp 243–291, Food Products Press, Binghamton, New York. Sullivan, C.Y. and Ross, W.M. (1979) “Selecting for drought and heat resistance in grain sorghum.” In Stress physiology in crop plants, edited by Mussell, H. and Staple, R., pp 263–281, Wiley, New York. Sun, C.W. and Callis, J. (1997) Independent modulation of Arabidopsis thaliana polyubiquitin mRNAs in different organs of and in response to environmental changes. Plant Journal 11:1017–1027. Sun, W., Bernard, C., van de Cotte, B., Van Montagu, M., and Verbruggen, N. (2001) At-HSP17.6A, encoding a small heat-shock protein in Arabidopsis, can enhance osmotolerance upon overexpression. The Plant Journal 27(5):407–415. Sung, D.-Y., Kaplan, F., Lee, K.-J., and Guy, C.L. (2003) Acquired tolerance to temperature extremes. Trends in Plant Science 8:179–187. Sunkar, R., Bartels, D., and Kirch, H.H. (2003) Overexpression of a stress-inducible aldehyde dehydrogenase gene from Arabidopsis thaliana in transgenic plants improves stress tolerance. Plant Journal 35:452–464. Sunkar, R., Kapoor, A., and Zhu, K. (2006) Posttranscriptional induction of two Cu/Zn superoxide dismutase genes in Arabidopsis is mediated by downregulation of miR398 and important for oxidative stress tolerance. Plant Cell 18:2051–2065. Suzuki, N., Bajad, S., Shuman, J., Shulaev, V., and Mittler, R. (2008) The transcriptional co-activator MBF1c is a key regulator of thermotolerance in Arabidopsis thaliana. Journal of Biological Chemistry 283(14):9269–9275. Suzuki, N., Rizhsky, L., Liang, H., Shuman, J., Shulaev, V., and Mittler, R. (2005) Enhanced tolerance to environmental stress in transgenic plants expressing the transcription coactivator Multiprotein Bridging Factor 1c. Plant Physiology 139:1313–1322.

258

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

Swamy, P.M. and Smith, B.N. (1999) Role of abscissic acid in plant stress tolerance. Current Science 76:1220–1227. Swindell, W.R., Huebner, M., and Weber, A.P. (2007) Transcriptional profiling of Arabidopsis heat shock proteins and transcription factors reveals extensive overlap between heat and non-heat stress response pathways. BMC Genomics 8:125. Tan, C., Yu, Z.W., Yang, H.D., and Yu, S.W. (1988) Effect of high temperature on ethylene production in two plant tissues. Acta Phytophysiologica Sinica 14:373–379. Tang, L., Kim, M.D., Yang, K.S., Kwon, S.Y., Kim, S.H., Kim, J.S., Yun, D.J., Kwak, S.S., and Lee, H.S. (2008) Enhanced tolerance of transgenic potato plants overexpressing nucleoside diphosphate kinase 2 against multiple environmental stresses. Transgenic Research 17(4):705–715. Tran, L.-S., Urao, T., Qin, F., Maruyama, K., Kakimoto, T., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2007) Functional analysis of AHK1/ATHK1 and cytokinin receptor histidine kinases in response to abscisic acid, drought, and salt stress in Arabidopsis. Proceedings of the National Academy of Sciences USA 104:20623–20628. Tsukaguchi, T., Kawamitsu, Y., Takeda, H., Suzuki, K., and Egawa, Y. (2003) Water status of flower buds and leaves as affected by high temperature in heat tolerant and heat-sensitive cultivars of snap bean (Phaseolus vulgaris L.). Plant Production Science 6:4–27. Uchida, A., Jagendorf, A.T., Hibino, T., Takabe, T., and Takabe, T. (2002) Effects of hydrogen peroxide and nitric oxide on both salt and heat stress tolerance in rice. Plant Science 163:515–523. Urao, T., Yakubov, B., Satoh, R., Yamaguchi-Shinozaki, K., Seki, M., Hirayama, T., and Shinozaki, K. (1999) A transmembrane hybrid-type histidine kinase in Arabidopsis functions as an osmosensor. Plant Cell 11:1743–1754. Valente, M.A., Faria, J.A., Soares-Ramos, J.R., Reis, P.A., Pinheiro, G.L., Piovesan, N.D., Morais, A.T., Menezes, C.C., Cano, M.A., Fietto, L.G., Loureiro, M.E., Aragäo, F.J., and Fontes, EP. (2008) The ER luminal binding protein (BiP) mediates an increase in drought tolerance in soybean and delays drought-induced leaf senescence in soybean and tobacco. Journal of Experimental Botany. 3 Dec 2008 Epub. Velikova, V. and Loretto, F. (2005) On the relationship between isoprene emission and thermotolerance in Phragmites australis leaves exposed to high temperatures and during the recovery from a heat stress. Plant Cell and Environment 28:318–327. Velikova, V., Edreva, A., and Loretto, F. (2005) Endogenous isoprene protects Phragmites australis leaves against singlet oxygen. Plant Cell and Environment 28:318–327. Vensel, W.H., Tanaka, C.K., Cai, N., Wong, J.H., Buchanan, B.B., and Hurkman, W.J. (2005) Developmental changes in the metabolic protein profiles of wheat endosperm. Proteomics 5:1594–1611. Verslues, P.E., Batelli, G., Grillo, S., Agius, F., Kim, Y.-S., Zhu, J., Agarwal, M., Katiyar-Agarwal, S., and Zhu, J.-K. (2007) Interaction of SOS2 with Nucleoside Diphosphate Kinase 2 and Catalases reveals a point of connection between salt stress and H2O2 signaling in Arabiodpsis thaliana. Molecular and Cellular Biology 27(22):7771–7780. Vettakkorumakankav, N.N., Falk, D., Saxena, P., and Fletcher, R.A. (1999) A crucial role for gibberellins in stress protection of plants. Plant and Cell Physiology 40(5):542–548. Vierling, E. (1991) The roles of heat-shock proteins in plants. Annual Review of Plant Biology 42:579–620. Vinocur, B. and Altman, A. (2005) Recent advances in engineering plant tolerance to abiotic stress: achievements and limitations. Current Opinion in Biotechnology 16:123–132. Vranova, E., Langebartels, C., Van Montagu, M., Inzé, D., and Van Camp, W. (2000) Oxidative stress, heat shock and drought differentially affect expression of a tobacco protein phosphatase 2C. Journal of Experimental Botany 51(351):1763–1764. Vu, J.C.V., Gesch, R.W., Pennanen, A.H., Allen, L.H.J., Boote, K.J., and Bowes, G. (2001) Soybean photosynthesis, Rubisco and carbohydrate enzymes function at supra-optimal temperatures in elevated CO2. Journal of Plant Physiology 158:295–307. Wahid, A. and Close, T.J. (2007) Expression of dehydrins under heat stress and their relationship with water relations of sugarcane leaves. Biologia Plantarum 51:104–109. Wahid, A. and Ghazanfar, A. (2006) Possible involvement of some secondary metabolites in salt tolerance of sugarcane. Journal of Plant Physiology 163:723–730. Wahid, A., Gelani, S., Ashraf, M., and Foolad, M.R. (2007) Heat tolerance in plants: An overview. Environmental and Experimental Botany 61:199–233. Wang, D. and Luthe, D.S. (2003) Heat sensitivity in a bentgrass variant. Failure to accumulate a chloroplast heat shock protein isoform implicated in heat tolerance. Plant Physiology 133:319–327. Wang, H., Chua, N.-H., and Wang, X.-J. (2006) Prediction of trans-antisense transcripts in Arabidopsis thaliana. Genome Biology 7(10):R92. Wang, J., Gan, Y.T., Clarke, F., and McDonald, C.L. (2006) Response of chickpea yield to high temperature stress during reproductive development. Crop Science 46:2171–2178. Wang, J.B. and Li, R.Q. (1999) Changes in Ca2+ distribution in mesophyll cells of pepper under heat stress. Acta Horticulturae Sinica 26:57–58.

GENETIC APPROACHES TOWARD IMPROVING HEAT TOLERANCE IN PLANTS

259

Wang, L.-J. and Li, S.-L. (2006a) Thermotolerance and related antioxidant enzyme activities induced by heat acclimation and salicylic acid in grape (Vitis vinifera L.) leaves. Plant Growth Regulator 48:137–144. Wang, L.-J. and Li, S.-L. (2006b) Salicylic acid-induced heat or cold tolerance in relation to Ca2+ homeostatis and antioxidant systems in young grape plants. Plant Science 170:685–694. Wang, W., Vinocur, B., Shoseyov, O., and Altman, A. (2004) Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends in Plant Science 9(5):1360–1385. Wardlaw, I.F. (1974) “Temperature control of translocation.” In Mechanism of regulation of plant growth, edited by Bielske, R.L., Ferguson, A.R., and Cresswell, M.M., pp 533–538, Bulletin of the Royal Society of New Zealand, Wellington. Warren, G.F. (1998) Spectacular increases in crop yields in the twentieth century. Weed Technology 12:752–760. Watanabe, N. and Lam, E. (2006) Arabidopsis Bax inhibitor-1 functions as an attenuator of biotic and abiotic types of cell death. Plant Journal 45(6):884–894. Watts, F.Z. (2004) SUMO modification of proteins other than transcription factors. Seminars in Cell and Developmental Biology 15:211–220. Weaich, K., Briston, K.L., and Cass, A. (1996) Modeling preemergent maize shoot growth II. High temperature stress conditions. Agricultural Journal 88:398–403. Webb, A.A.R., McAinsh, M.R., Taylor, J.E., and Hetherington, A.M. (1996) Calcium ions as intercellular second messengers in higher plants. Advances in Botanical Research 22:45–96. Wei, Y., Guangmin, X., Daying, Z., and Huimin, C. (2001) Transfer of salt tolerance from Aeleuropus littoralis sinensis to wheat (Triticum aestivum L.) via asymmetric somatic hybridization. Plant Science 161(2):259–266. Weis, E. (1981a) The temperature sensitivity of dark-inactivation and light-activation of the ribulose-1,5-bisphosphate carboxylase in spinach chloroplasts. FEBS Letters 129:197–200. Weis, E. (1981b) Reversible heat-inactivation of the Calvin cycle: a possible mechanism of the temperature regulation of photosynthesis. Planta 151:33–39. Wilhelm, E.P., Mullen, R.E., Keeling, P.L., and Singletary, G.W. (1999) Heat stress during grain filling in maize: effects of kernel growth and metabolism. Crop Science 39:1733–1741. Willits, D.H. and Peet, M.M. (1998) The effect of night temperature on greenhouse grown tomato yields in warm climates. Agricultural and Forest Meteorology 92(3):191–202. Wolucka, B.A. and Van Montagu, M. (2003) GDP-mannose 3′,5′-epimerase forms GDP-L-gulose, a putative intermediate for the de novo biosynthesis of vitamin C in plants. Journal of Biological Chemistry 278:47483–47490. Wolucka, B.A., Goossens, A., and Inzé, D. (2005) Methyl jasmonate stimulates the de novo biosynthesis of vitamin C in plant cell suspensions. Journal of Experimental Botany 56(419):2527–2538. Wu, C.-Y., Trieu, A., Radhakrishnan, P., Kwok, S.F., Harris, S., Zhang, K., Wang, J., Wan, J., Zhai, H., Takatsuto, S., Matsumoto, S., Fujioka, S., Feldmann, K.A., and Pennell, R.I. (2008) Brassinosteroids regulate grain filling in rice. The Plant Cell 20:2130–2145. Wu, D.H. and Laidman, D.L. (1997) Isolation of six low molecular weight heat shock proteins and partial characterization of heat shock protein 29 from mung bean hypocotyl. Phytochemistry 44(6):985–989. Wu, Y. and Cosgrove, D.J. (2000) Adaptation of roots to low water potentials by changes in cell wall extensibility and cell wall proteins. Journal of Experimental Botany 51:1543–1553. Xiong, L., Schumaker, K.S., and Zhu, J.K. (2002) Cell signaling during cold, drought, and salt stress. Plant Cell 14:S165–183. Xu, C. and Huang, B. (2008) Root proteomic responses to heat stress in two Agrostis grass species contrasting in heat tolerance. Journal of Experimental Botany 59(15):4183–4194. Xu, J., Tian, J., Belanger, F.C., and Huang, B. (2007) Identification and characterization of an expansin gene AsEXP1 associated with heat tolerance in C3 Agrostis grass species. Journal of Experimental Botany 58(13):3789–3796. Xu, S., Subudhi, P., Crasta, O.R., Rosenow, D.T., Mullet, J.E., and Nguyen, H.T. (2000) Molecular mapping of QTLs conferring stay-green in grain sorghum (Sorghum bicolor L. Moench). Genome 43:461–469. Yamaguchi-Shinozaki, K. and Shinozaki, K. (2005) Organization of cis-acting regulatory elements in osmotic- and cold-stressresponsive promoters. Trends in Plant Science 10:88–94. Yamakawa, J., Hirose, T., Kuroda, M., and Yamaguchi, T. (2007) Comprehensive expression profiling of rice grain filling-related genes under high temperature using DNA microarray. Plant Physiology 144:258–277. Yamane, Y., Kashino, Y., Koike, H., and Satoh, K. (1998) Effects of high temperatures on the photosynthetic systems in spinach: oxygen-evolving activities, fluorescence characteristics and the denaturation process. Photosynthetic Research 57:51–59. Yamanouchi, U., Yano, M., Lin, H., Ashikari, M., and Yamada, K. (2002) A rice spotted leaf gene, Spl7, encodes a heat stress transcription factor protein. Proceedings of the National Academy of Sciences USA 99:7530–7535. Yang, J., Sears, R.G., Gill, B.S., and Paulsen, G.M. (2002) Genotypic differences in utilization of assimilate sources during maturation of wheat under chronic heat and heat shock stresses. Euphytica 125:179–188.

260

GENES FOR PLANT TOLERANCE TO TEMPERATURE EXTREMES

Yang, K.A., Lim, C.J., Hong, J.K., Park, C.Y., Cheong, Y.H., Chung, W.S., Lee, K.O., Lee, S.Y., Cho, M.J., and Lim, C.O. (2006) Identification of cell wall genes modified by a permissive high temperature in Chinese cabbage. Plant Science 171:175–182. Yang, X., Liang, Z., and Lu, C. (2005) Genetic engineering of the biosynthesis of glycinebetaine enhances photosynthesis against high temperature stress in transgenic tobacco plants. Plant Physiology 138:2299–2309. Yoo, C.Y., Miura, K., Jin, J.B., Lee, J., Park, H.C., Salt, D.E., Yun, D.-J., Bressan, R.A., and Hasegawa, P.M. (2006) SIZ1 small ubiquitin-like modifier E3 ligase facilitates basal thermotolerance in Arabidopsis independent of salicylic acid. Plant Physiology 142:1548–1558. Yoshida, S., Satake, T., and MacKill, D.J. (1981) High-temperature stress in rice. IRRI Research Paper Series 67. International Rice Research Institute, Manila, Philippines. Yoshida, T., Sakuma, Y., Todaka, D., Maruyama, K., Qin, F., Mizoi, J., Kidoro, S., Fujita, Y., Shinozaki, K., and YamaguchiShinozaki, K. (2008) Functional analysis of an Arabidopsis heat-shock transcription factor HsfA3 in the transcriptional cascade downstream of the DREB2A stress-regulatory system. Biochemical and Biophysical Research Communications 368:515–521. Yoshimura, K., Miyao, K., Gaber, A., Takeda, T., Kanaboshi, H., and Shigeoka, S. (2004) Enhancement of stress tolerance in transgenic tobacco plants overexpressing Chlamydomonas glutathione peroxidase in chloroplasts or cytosol. Plant Journal 37:21–33. Yost, H.J. and Lindquist, S. (1986) RNA splicing is interrupted by heat shock and is rescued by heat shock protein synthesis. Cell 45:185–193. Young, T.E., Ling, J., Geisler-Lee, C.J., Tanguay, R.L., Caldwell, C., and Gallie, D.R. (2001) Developmental and thermal regulation of maize heat shock protein, HSP101. Plant Physiology 127:777–791. Yu, W., Han, F., Gao, Z., Vega, J.M., and Birchler, J.A. (2007) Construction and behavior of engineered minichromosomes in maize. Proceedings of the National Academy of Sciences USA 104(21):8924–8929. Yun, D.-J., Zhao, Y., Pardo, J.M., Narasimhan, M.L., Damsz, B., Lee, H., Abad, L.R., D’urzo, M.P., Hasegawa, P.M., and Bressan, R.A. (1997) Stress proteins on the yeast cell surface determine resistance to osmotin, a plant antifungal protein. Proceeding of the National Academy of Sciences USA 94:7082–7087. Zair, I., Chlyah, A., Sabounji, K., Tittahsen, M., and Chlyah, H. (2003) Salt tolerance improvement in some wheat cultivars after application of in vitro selection pressure. Plant Cell, Tissue and Organ Culture 73:237–244. Zhu, B., Ye, C., Lü, H., Chen, X., Chai, G., Chen, J., and Wang, C. (2006) Identification and characterization of a novel heat shock transcription factor gene, GmHsfA1, in soybeans (Glycine max). Journal of Plant Research 119:247–256.

Section 4 Integrating Plant Abiotic Stress Responses

Genes for Plant Abiotic Stress Editors Matthew A. Jenks and Andrew J. Wood © 2010 Blackwell Publishing ISBN: 978-0-813-81502-2

10

Genetic Networks Underlying Plant Abiotic Stress Responses Arjun Krishnan, Madana M.R. Ambavaram, Amal Harb, Utlwang Batlang, Peter E. Wittich, and Andy Pereira

Introduction

Plants are sessile organisms that have evolved a wide spectrum of adaptations to cope with the challenges of various environmental stresses during their life cycle. Factors that can cause stress to plants and affect plant productivity can be classified into seven major classes (Elstner and Oswald, 1994): • • • • • • •

light (high and low) radiation (UV-B, UV-A, etc.) temperature (high, low, chilling, and freezing) hydration (drought and flooding) chemical factors (salt, heavy metals, pH, etc.) mechanical factors biological influence

Abiotic stresses, especially drought, salinity, and cold, are recognized as the primary causes of crop loss worldwide (Boyer, 1982). In the face of a global scarcity of water resources and erratic rainfall, the improvement of stress tolerance is especially important. The progress of conventional breeding for improvement of stress-tolerant crops has been rather slow, mainly due to the complex nature of the stress response and tolerance traits. Therefore, a systems-level understanding of the genetic and molecular basis of abiotic stress tolerance would aid in crop improvement for stress tolerance. Plant adaptation to environmental stresses is dependent upon the activation of cascades of molecular networks involved in stress perception, signal transduction, and the expression of specific stress-related genes and metabolites. The present review starts with short descriptions of individual abiotic stress components and their interaction to plant processes, and then deals with the genetic networks underlying plant abiotic stress responses. These include stress responsive “global transcriptome analysis” and gene interaction networks that play an important role in plant adaptation to abiotic stress.

Abiotic Stresses

Drought or water deficit can be the consequence of a shortage in rainfall, coarse-textured soils that retain little water in the root zone, or drying winds (Swindale and Bidinger, 1981). Plant adaptation Genes for Plant Abiotic Stress Editors Matthew A. Jenks and Andrew J. Wood © 2010 Blackwell Publishing ISBN: 978-0-813-81502-2

263

264

INTEGRATING PLANT ABIOTIC STRESS RESPONSES

to drought stress can involve avoidance mechanisms with reduced shoot growth and increased root development resulting in increased water absorption and reduced transpiration, thereby maintaining plant tissue water status. Cold temperature stress can reduce the fluidity of membranes causing them to be rigid. In addition, freeze-induced membrane damage results primarily from severe dehydration associated with freezing (Steponkus, 1984). Salinity stress is due to the presence of excessive sodium (Na+) in soils causing osmotic stress, which produces a suite of responses identical to water stress due to drought. In later stages, excessive salts enter the plant, eventually becoming toxic to older leaves, causing senescence and reducing assimilate transport to young leaves. Salinity may result in Ca2+ deficiency (Munns, 2002), or disruption in ion distribution leading to growth arrest or death (Zhu, 2001). Heat stress causes growth inhibition, leaf senescence, and death of shoots and roots. Heat stress inhibits photosynthesis, limits carbohydrate accumulation (Huang and Gao, 1999), and damages cell membranes leading to cell death (Abernethy et al., 1989). Nutrient deficiency can be caused by limitation of the 14 essential nutrients of which nitrogen (N), phosphorus (P), potassium (K), and iron (Fe) are the most important to agricultural production worldwide (Wang et al., 2002). Flooding stress of the soil and deeper submergence impose stress on plants through inadequate supply of oxygen (anoxia). This causes blocks in oxidative phosphorylation of mitochondria and tricarboxylic acid cycle, leading to anaerobic fermentation for ATP generation (Liao and Ling, 2001), and reduction of root and vegetative growth. Other stresses such as by ozone (O3), sulfur dioxide (SO2), and ultraviolet B (UV-B) radiation have increased due to anthropogenic activities in industrialized parts of the world, and may reach levels that are toxic to plants. Exposure of plants to O3, SO2, and UV-B has been found to elicit responses to oxidative stress (Willeken et al., 1994).

Plant Responses to Environmental Stresses

In order to escape or avoid stress, plants have evolved a myriad of strategies and mechanisms with behavioral, physiological, or morphological adjustments to adapt to a variety of stresses. These include stress avoidance, escape, and tolerance responses. Experimental evidence established that plants adapt to the surrounding environmental conditions on a daily, or even on an hourly basis. The early events by which plants respond and adapt to environmental stresses include sensing of stress and subsequent signal transduction events that activate various physiological and metabolic responses. Such responses are mediated via numerous physiological alterations involving numerous genes and regulatory circuits resulting in changes such as alteration of plasma membrane composition, changes in phytohormone levels, and changes in relative water content in the leaves (Vinocur and Altman, 2005), all of which eventually lead to phenotypic changes. These adaptive changes are indeed correlated with a range of adaptive strategies in plants that are by and large tolerant to such stresses.

Abiotic Stress and Plant Development Root Responses Plant roots are capable of growing toward higher water availability and away from

high osmolarity (hydrotropism) (Eapen et al., 2005). In some cases, root growth is stimulated by

GENETIC NETWORKS UNDERLYING PLANT ABIOTIC STRESS RESPONSES

265

soil water deficit to reach deeper water sources by lateral root formation or root elongation. However, more often root development is inhibited in dry soil, and lateral root primordial emergence is repressed (Munns and Sharp, 1993; Xiong et al., 2006). Vegetative Responses Leaf rolling and wilting are responses of the plant to very low water availability. Mild water deficits may cause an adaptive response of inhibition in shoot growth and leaf area development and is often more severe than the root growth response on water deficit (Munns and Sharp, 1993). This growth inhibition results in a reduction of water evaporation, lower photosynthetic activity (reduced sugar production), and root mineral ion uptake. The remaining available resources are allocated toward osmotic adjustment processes for turgor maintenance and cell water retention (Neumann, 2008). Leaf senescence as a result of low water availability may be a mechanism to transfer nutrients from leaves to growing seeds in order to guarantee survival of the next generation (Neumann, 2008). In tobacco, an isopentenyltransferase gene linked to a stress-activated promoter showed delayed leaf senescence and drought tolerance (Rivero et al., 2007). Although ABA has been implicated as the primary signaling molecule from the root to the shoot for soil water deficit, there is increasing evidence that hydraulic signals are involved in root-to-shoot signaling when plants become more water stressed, also leading to stomatal closure and leaf growth inhibition. Other factors like ABA conjugates, cytokinins, and pH may be involved in a chemical communication between roots and shoots (Neumann, 2008; Schachtman and Goodger, 2008). Meristem Activity Abiotic stress signaling molecules inhibit growth by stimulating cell cycle checkpoints, resulting in an impaired G1-to-S transition, slowing down of DNA replication, and/ or delayed entry into mitosis. A decrease in root mitotic activity as a result of water stress has been reported (Schuppler et al., 1998). In wheat and maize, when mitotic activity in the meristematic zone of leaves is reduced by water stress, the meristematic zone becomes smaller. Reduced activity is correlated with an increased proportion of deactivated 34-kD Cdc2-like kinase (Granier et al., 2000). A direct link between stress perception and cell cycle machinery is found in rice, where EL 2 (encoding a plant CDK inhibitor) expression is induced by drought, cold, and propionic acid (Peres et al., 2007).

A short growth cycle is a way of avoidance to an anticipated long, terminal drought period. On the other hand, flower induction can be inhibited as a response to water deficit during the vegetative growth stage. Day length, light, temperature, vernalization, nutritional status, and plant age are factors that contribute to the transition from vegetative to inflorescence meristem development. Interestingly, flowering time is also found to be genetically correlated to Δ13C, which is correlated to water use efficiency (WUE). Arabidopsis mutants that show late flowering also result in less negative Δ13C and thus probably higher WUE. Differences in the levels of the transcription factor Flowering Locus (FLC) may cause the effect on Δ13C (Mckay et al., 2003). Water deficit after flower induction can have reversible or irreversible effects on meiosis, pollen, and ovule development, anthesis, fertilization, and endosperm or embryo development (Boyer and McLaughlin, 2007). In rice, anther dehiscence and panicle exsertion are well known events sensitive to drought, respectively showing irreversible and partly reversible disruption upon stress (Liu et al., 2006). In rice and wheat, a shortage of photosynthetic components due to water shortage can also prevent pollen from storing starch, resulting in low pollen vigor (Boyer and McLaughlin, 2007). In maize, an irreversible effect of drought stress is asynchronous anthesis and silking, resulting in absence of fertilization of the ovules (Liu et al., 2006). Water deficit shortly after fertilization Reproduction

266

INTEGRATING PLANT ABIOTIC STRESS RESPONSES

can cause ovary abortion: low sugar signaling downregulates sucrose processing genes invertases (Incw2 and Ivr2) and upregulates putative senescence genes (RIP2 and PLD1). Reversion of the phenotype was possible by feeding sucrose, suggesting a key role for Incw2 (Boyer and McLaughlin, 2007). Possible regulatory mechanisms of invertase activity are described in detail (Huang et al., 2007). Drought stress during Arabidopsis flowering and seed set is not well studied yet, but application of osmotic stress resulted in reactive oxygen species (ROS) accumulation preceding ovule abortion within 12 hours (Hauser et al., 2006).

Metabolic and Proteomic Responses to Abiotic Stress

This section is an overview of abiotic stress responses at the biochemical and protein levels, by considering the effect of environmental stresses on functional genes that encode enzymes associated with the synthesis of osmotically active compounds, transporters, chaperones, and ROS scavengers. Signal Transduction Pathways under Abiotic Stresses Plants respond and acclimate or adapt to variable environmental conditions with a wide range of cellular and metabolic changes that are triggered by signaling and regulatory pathways. Recently, new insights into signaling networks involved in abiotic stress adaptation have been gained by transcriptome analyzes that suggest the existence of both specific signaling and of cross talk between signal transduction pathways in response to environmental changes. The stress response associated gene products are thought to protect, either directly or indirectly, against a variety of environmental stresses. For example, drought, high salinity, and low temperature, three common abiotic stresses, all cause an accumulation of compatible solutes and antioxidants (Hasegawa et al., 2000). Identification and utilization of specific genes with a role in drought, high salinity, and low temperature responsive functions will help in developing crops with improved tolerance to different stresses mentioned above (Cushman and Bohnert, 2000). However, the success of these approaches has generally been limited by a lack of understanding of genes controlling metabolic flux, compartmentation, and response function. The products of these abiotic stress responsive genes are thought to function not only in stress tolerance but also in the regulation of gene expression and in signal transduction in the stress response. These gene products can be classified into two groups. The first group includes the proteins that are directly involved in stress tolerance. These include genes regulating osmotic adjustments inside a cell to prevent water loss. Basically these are genes involved in the synthesis and accumulation of osmolytes without disturbing the cellular functions. They encode enzymes required for the biosynthesis of various osmoprotectant molecules (sugars, proline, glycinebetaine, etc.), and proteins that protect macromolecules and membranes (LEAproteins, osmolytes, mRNA-binding proteins, etc.). Of the stress-related osmoprotectant compounds, the three most extensively studied are glycinebetaine, alcohol mannitol, and trehalose, a sugar known to play a role in drought resistance of many organisms including the “resurrection” plant. Other genes in this category encode GSTs, catalases, superoxide dismutase, ascorbate peroxidase, and a few others. Chaperones and proteinases that may destroy inactive proteins and enzymes involved in ATP production pathways also fall into this category. In plants, the inevitable production of ROS under any stress leads to singlet oxygen, superoxide, H2O2 and hydroxyl radicals. The mechanisms through which ROS detoxification occurs are enzymatic and nonenzymatic. Stress increases ROS levels followed by upregulation or downregulation of mRNA transcripts and protein levels and presumably leads to an accelerated turnover of components of detoxification

GENETIC NETWORKS UNDERLYING PLANT ABIOTIC STRESS RESPONSES

267

systems, which in turn exert a positive effect on plant performance (Miller et al., 2008). The genes in these groups usually encode enzymes involved in removing toxic-free radicals or proteins that directly mediate detoxification of toxic substances. The second group of abiotic stress responsive gene products consists of proteins involved in signal transduction and regulation of stress responsive gene expression. These include transcriptional factors, protein kinases, protein phosphatases and enzymes involved in phosphoinositide metabolism, and other signaling molecules such as calmodulin-binding proteins. The transcripts of genes encoding several of these proteins are shown to accumulate and lead to complex changes in gene expression resulting in plant adaptation to abiotic stress conditions. The role of these gene products has been reviewed extensively (Shinozaki and Yamaguchi-Shinozaki, 2000; Xiong et al., 2002). Signaling in Stress Responses A generic signal transduction pathway starts with signal perception,

followed by the generation of second messengers such as inositol phosphates (InsP) and ROS. Second messengers can modulate intracellular Ca2+ levels, often initiating a protein phosphorylation cascade that finally targets proteins directly involved in cellular protection, or transcription factors controlling specific sets of stress-regulated genes (Figure 10.1). The products of these genes may be involved in the synthesis of regulatory molecules like plant hormones abscisic acid (ABA), gibberellic acid (GA), cytokinin, jasmonic acid (JA), brassinosteroid (BR), and salicylic acid (SA). These regulatory molecules can, in turn, initiate a second round of signaling that may follow the same generic pathway, although different components are often involved. ROS Signaling Most types of abiotic stresses such as drought, salinity, flooding, and heat and cold

stresses disrupt the metabolic balance of cells. One characteristic cellular feature activated by abiotic stresses is the high production of ROS in the chloroplasts, mitochondria, or in peroxisomes, causing irreversible cellular and tissue damage (Miller et al., 2008). To circumvent this effect, the cellular internal antioxidant mechanism will be elevated, including the antioxidant enzymes of higher plants such as superoxide dismutase, catalase, glutathione transferase, and ascorbate peroxidase. Antioxidant enzymes either catalyze reactions where antioxidant molecules are able to quench ROS without being transformed into destructive radicals, or process ROS directly. Each of the antioxidant enzymes is comprised of several isoforms. Although ROS are associated with many different biotic or abiotic stress conditions, different genes of the ROS gene network of Arabidopsis were found to respond differently to different stress treatments. Plants with high levels of antioxidants, either constitutive or induced, are reported to have a relatively greater resistance to oxidative stress damage (Hasegawa et al., 2000). There are several reports on antioxidant enzymes and their products in relation to the stress tolerance in crop plants, including the use of transgenic plants carrying antioxidant genes that exhibit improved stress tolerance (Vinocur and Altman, 2005; Miller et al., 2008) and antioxidant potential (Reddy et al., 2007). Since the antioxidant enzymes are reported to modulate gene expression through the generation of appropriate signal molecules and the destruction of unnecessary signal molecules, the ROS are also considered as important signaling molecules in crop plants. Calcium-dependent Signaling In abiotic stress signaling, Ca2+-dependent protein kinases (CDPK)

and the Salt Overly Sensitive 3 (SOS3) family of Ca2+ sensors are involved in coupling of this calcium signal to specific protein phosphorylation cascades (Kaur and Gupta, 2005). The important feature of Ca2+ signaling is the presence of repetitive transient bursts. The first round of transient Ca2+ generation leads to generation of secondary signaling molecules like ABA and ROS, which stimulate a second round of Ca2+ increase. These multiple rounds of transient bursts from various

268

Figure 10.1 Major regulatory events underlying response to abiotic stress. Signaling cascades are shown in ellipses, transcription factors (TF) are shown in rectangular boxes with directed arrows, and cis-regulatory elements (CRE) bound by TFs are shown in boxes with flanking lines. Other regulatory arms, phosphoprotein cascades, and small RNAs are shown in rounded rectangles; and gene expression events are shown as dotted lines.

GENETIC NETWORKS UNDERLYING PLANT ABIOTIC STRESS RESPONSES

269

sources will have different signaling consequences and therefore, physiological meaning. A number of studies have shown that the CDPKs are induced or activated under abiotic stresses (Knight and Knight, 2001). Overexpression of OsCDPK7 resulted in increased cold and osmotic stress tolerance in rice. Further, the role of CDPKs and SOS3 in the activation of transcription factors of LEA-type genes and ion homeostasis, respectively, has been extensively reviewed (Xiong et al., 2002; Kaur and Gupta, 2005). Phosphoprotein Signaling In addition to Ca2+–regulated protein kinase pathways, plants also use

other kinds of phosphoproteins for abiotic stress signal transduction such as protein kinases and phosphatases, which respectively catalyze phosphorylation and dephosphorylation of specific substrates. Several mitogen-activated protein kinase (MAPK) modules (i.e., MAPKKK-MAPKKMAPK) that may be involved in abiotic stress signaling are identified in alfalfa and tobacco. Recently, many studies have focused on protein kinases, which catalyze phosphorylation, as a key mechanism to translate external stimuli into cellular responses in both eukaryotic and prokaryotic cells. For example, identification of phosphoproteome targets in Arabidopsis sucrose nonfermenting-like kinase (SnRK2.8) reveals the connection between plant growth and metabolic processes (Shin et al., 2007). A common observation both in plants and in other organisms is that a MAPK module can be used for the transmission of multiple signals (Xiong et al., 2002). However, analysis of the in vivo function of the various MAPK components and their interrelationships will be essential for constructing signaling pathways. A combined phosphoproteomic approach of mass spectrometry and microarray technology could enhance the construction of dynamic plant signaling networks that underlie plant biology (de la Fuente van Bentem and Hirt, 2007). Plant Hormone Responses to Abiotic Stress Recent studies on the effects of the plant hormones on gene transcription have been done using microarrays by the AtGenExpress Consortium (http://web. uni-franfurt.de/fb15/botanik/mcb/AFGN/atgenex.htm and the responses characterized for each hormone (Nemhauser et al., 2006). The analyzes showed that many growth-regulating plant hormones affect the same biological processes by eliciting different and unique transcriptional targets (Nemhauser et al., 2006). The interaction between abiotic stresses and plant hormones has recently been described (Bajguz and Hayat, 2009). ABA is well known as a stress hormone, because its level is elevated under stress conditions, and was suggested to act as a growth inhibitor under stress. Recent studies showed that the increased level of ABA under drought conditions is needed to inhibit ethylene production, and to maintain shoot and root growth (Spollen et al., 2000). On the other hand, leaf elongation of maize is not significantly affected by the level of ABA or the level of ethylene under drought conditions (Voisin et al., 2006). At least four signaling pathways were discovered under stress conditions. Two of the pathways are ABA-dependent, and the other two are ABA-independent pathways (Shinozaki and YamaguchiShinozaki, 1997). Comparison of a set of drought-responsive genes under progressive drought to that of plant hormone-responsive genes showed that 67% of drought-responsive genes were regulated by ABA. Promoter motif analysis of the remaining “drought-specific” ABA-independent genes showed that the expression of these genes is linked to ABA-dependent pathways. These results revealed a vital role for ABA signaling in drought response (Huang et al., 2008). However, gene ontology (GO) analysis of drought responsive genes revealed that many drought upregulated and downregulated genes were responsive to more than one hormone (Huang et al., 2008). The comparison of other hormone-responsive genes to drought-responsive genes showed different percentages of common genes between drought-responsive and hormone-responsive

270

INTEGRATING PLANT ABIOTIC STRESS RESPONSES

genes. From 641 hormone-responsive genes, 491, 197, 95, 85, 60, 46, and 13 were responsive to drought and ABA, methyl JA, auxin, BR, ethylene, cytokinin, and GA, respectively (Huang et al., 2008). Exogenous application of 24-epibrassinolide (EBR), a BR, led to improvement of plant tolerance to many abiotic stresses: drought, cold, high salinity, and thermal stress in Arabidopsis and Brassica napus (Kagale et al., 2007). It was suggested that BRs confer resistance to the different environmental stresses through cross talk with other plant hormones (Krishna, 2003). The possible mechanisms of BR enhancement of plant resistance to various types of environmental stresses have been described (Bajguz and Hayat, 2009), but more investigations are needed to reveal the molecular basis. One study on pea plants using Brassinosteriod (BR)-deficient and BR-insensitive mutants showed that changes in endogenous BR concentrations do not have any significant role in plant response to drought (Jager et al., 2008). Many physiological parameters such as net photosynthesis, conductance, transpiration rate, and water-use efficiency are significantly affected by drought stress. Exogenous BAP, a cytokinin, was found to reduce the negative effect of drought on these physiological parameters in cotton plants (Pandey et al., 2003). In winter wheat, it was found that the changes in starch and protein yield in the grain under postanthesis drought conditions are highly influenced by the changes in the concentration of endogenous plant hormones (Xie et al., 2003). Comparison of the expression profiles of barley plants exposed to salinity stress and plants treated with JA revealed a significant overlap between the genes regulated by the two treatments (Walia et al., 2007). In addition, it was shown that preexposure of barley plants to JA enhanced their tolerance to salinity (Walia et al., 2007). The several studies showing the interaction of plant hormones to plant responses to different stresses are only the beginning of research in this area. Detailed multiple-factor experiments of hormones and stress factors will give us a better understanding of stress system biology.

Transcriptome Analysis of Abiotic Stress Responses

Complete genome sequence information of Arabidopsis (The Arabidopsis Initiative, 2000) and rice (Goff et al., 2002) has provided a valuable resource for gene discovery. Though the genome sequence annotation provides an approximate gene index, it does not throw light on the functional role of all genes and the tissue-specific and environment-influenced regulation. Hence, genomewide expression profiling is a valuable tool for determining the functions of genes and their spatial and temporal expression patterns, as well as elucidating the genetic networks in which they participate. Microarrays offer a powerful platform to find out the expression of genes during different stress conditions/stages at a global level not only in Arabidopsis and rice but also in various crop plants and tree species. Microarray studies have been used successfully to address diverse questions, and are rapidly becoming an essential tool for identifying and analyzing genes involved in, or controlling, various biological processes ranging from development to responses to environmental cues (Gorantla, 2005). A number of stress-inducible genes have been identified using microarray analysis in various plant species, such as Arabidopsis (Seki et al., 2002) and rice (Rabbani et al., 2003). Among the genes identified, several were classified as regulatory genes, such as protein kinases and transcription factors, in addition to functional genes. Now, analyzing the functions of these genes is critical to furthering our understanding of the molecular mechanisms governing plant stress response and tolerance, ultimately leading to enhancement of stress tolerance in crops.

GENETIC NETWORKS UNDERLYING PLANT ABIOTIC STRESS RESPONSES

271

Transcriptional Regulatory Programs Underlying Abiotic Stress Response

The regulatory logic that drives stress response is governed by the combination of signaling regulators, transcription factors (TF), their binding site in the regulatory regions of target genes (cisregulatory elements [CRE]), and other regulatory molecules (e.g., chromatin modifiers and small RNAs). TFs are master regulators that control the expression of many target genes through specific binding of the TF to the corresponding CRE in the promoters of respective target genes. In Arabidopsis, dehydration-responsive element-binding protein 1 (DREB1)/C-repeat binding factor (CBF) and DREB2 function in ABA-independent gene expression, whereas the ABA-responsive element (ABRE) binding protein (AREB)/ABRE binding factor (ABF) functions in ABA-dependent gene expression (Figure 10.1). DREB1/CBF, DREB2, AREB/ABF, and NAC (another class of TFs) have important roles in response to abiotic stresses in rice. Over and above the TFs, the CREs are sites of high rates of evolutionary diversity driving the progressively complex gene expression correlating with organismal complexity. These elements in association with their TFs are key in determining the cellular transcriptional response diversity, which is the spectrum of gene expression states elicited by different environmental conditions including stress. The AtGenExpress consortium has generated extensive data on Arabidopsis root and shoot organs under nine abiotic stress conditions (cold, osmotic stress, salt, drought, genotoxic stress, ultraviolet light, oxidative stress, wounding, and high temperature) at six time points of stress exposure (0.5, 1, 3, 6, 12, and 24 hour) using the same technology platform (affymetrix gene chip— AGI) and reference conditions (Kilian et al., 2007). The regulatory code for transcriptional response across different stresses was recently delineated using the AtGenExpress stress time-series data (Walther et al., 2007). If the series of regulation and gene expression events are thought of as a transcriptional regulatory signaling cascade, the early responders in the cascade are observed to have evolved a greater capacity to respond to many different TFs and then channel the response to common effector genes, which is matched by an association with a greater number of CREs. Most likely to house the many CREs, the early-response genes also have, on average, 30% longer intergenic upstream regions than do late-response genes. These genes are more likely to contain TATA-boxes, implicated in the regulation by nucleosomal mechanisms for chromatin remodeling. Genes involved in the regulation of transcription, signaling, and stress response genes are overrepresented among early-response genes, whereas ribosomal genes and genes generally involved in protein biosynthesis, metabolism, and other nonsignaling processes are more represented among the late-response genes, consistent with the notion of a cascade. Since several loci remain undiscovered and unannotated, not featured in a microarray, and since many active intergenic regions play a role in fine regulation of response, Arabidopsis Affymetrix tiling arrays were used in a study to characterize the whole genome transcriptome under drought, cold, and high-salinity (Matsui et al., 2008). The assay brought to light 7,719 non-AGI transcriptional units (TU) in the unannotated “intergenic” regions of Arabidopsis genome, out of which 1,275 and 181 non-AGI TUs, most of which are nonprotein-coding RNAs belonging to pairs of the fully overlapping sense-antisense transcripts, are upregulated and downregulated, respectively, by one of the stresses. Further analysis showed a significant linear correlation between the expression ratios of the sense and antisense transcripts, indicating that many nonprotein-coding antisense transcripts do not control the accumulation of the sense transcripts negatively. This study directly suggests novel roles of the antisense RNAs in plants.

272

INTEGRATING PLANT ABIOTIC STRESS RESPONSES

Small noncoding RNAs are emerging as important regulators of both transcriptional and posttranscriptional gene silencing. miRNAs, in particular, are being implicated in playing crucial regulatory roles in response to abiotic or biotic stress as well as to diverse physiological processes (Shukla et al., 2008). These are endogenous, ∼22 nucleotide, small noncoding regulatory RNAs that target mRNAs for cleavage or translational repression. Stress-induced miRNAs target transcription factors involved in further regulation of gene expression and signal transduction that probably function in stress responses. Moreover, the miRNA genes contain several known stressresponsive CREs, such as the ABRE, anaerobic induction elements (ARE), MYB binding site involved in drought-inducibility (MBS), heat-stress-responsive elements (HSE), low-temperatureresponsive elements (LTR), and defense- and stress-responsive elements (TC-rich repeats) (Liu et al., 2008). An independent study confirmed that 19 miRNA genes of 11 miRNA families in Arabidopsis are upregulated by cold stress, which also contains many known stress-related CREs in their promoters. The role of another key player—cell identity—in determining stress response was vindicated by an extensive transcriptome analysis of carefully dissected Arabidopsis roots (Dinneny et al., 2008). The transcriptional state of a cell was inferred to be a function of environmental conditions and regulated by a smaller core set of “cell identity” genes. From the study, it is clear that known stress pathways commonly regulate cell-type-specific biological processes, whereas responses occurring in all cells are required to be fine tuned according to the specific stimuli. Although canonical stressrelated CREs were found to be present in genes in all cells, cell-type-specific responses were distinguishable at the promoter level, indicating the role of other cell-type-specific CREs.

Association between Abiotic Stress Responses

Comparison of responses to different abiotic stresses at any level of detail—genes, processes or regulatory elements—points to cross talk between pathways and a significant overlap in the molecular response logic and machinery. Such comparative analyzes have been carried out in Arabidopsis using the AtGenExpress stress series data, and many insights into shared and distinct stress responses have been unearthed, along with ideas about the broad similarities between different stresses. Using a combination of microarray data from various stress experiments followed by analysis of differential expression, a set of 984 genes containing 289 regulatory genes were identified in a study as being responsive to multiple abiotic stresses, and were experimentally validated (Kant et al., 2008). Another study used a probabilistic clustering approach; several modules of genes that comprise the generic and specific responses to stress were recovered (Supper et al., 2007). Extensive cross talk between drought and high-salinity has also been observed in the tiling array experiment (∼1,800 common genes) (Matsui et al., 2008). In total, 423 and 168 genes were observed to be upregulated and downregulated, respectively, during all three stresses. A detailed analysis of the associations between the nine abiotic stress responses, organs, and time-courses was made using genome-wide correlations as well as gene-specific expression responses (Swindell, 2006). The highest average correlation exists between salt and osmotic stress in both organs (a correlation coefficient of 0.642 in roots and 0.565 in shoots), followed by genotoxic and oxidative stress (0.509 in roots, 0.486 in shoots). The smallest correlation was between cold and heat stress (0.101) in roots. The drought stress treatment correlation in shoots was highest to genotoxic (0.455) followed by osmotic and oxidative (0.425) stresses and the lowest to cold (0.196) stress. In the root, drought was most highly correlated to osmotic (0.353) and least to cold (0.149)

GENETIC NETWORKS UNDERLYING PLANT ABIOTIC STRESS RESPONSES

273

stress. Among individual genes, 67 showed a general stress expression with 41 in shoots and 26 in roots. Among these general stress genes, the functional categories overrepresented were associated with cell rescue/defense/virulence, energy, and metabolism. The temporal pattern of the abiotic stress transcriptional response progresses from general-to-specific responses with the responses being more strongly associated at early to middle time points of stress exposures. These analyzes also support multi-stress tolerance to be related to general metabolic processes and allocation of energetic resources. The stress series data was reinterpreted using “genomic signatures,” or sets of genes already known to be responsive to a specific condition (Weston et al., 2008). The analyzes illustrated the complexity of the stress response that is characteristic of cross-talk pathways and multiple secondary effects from prolonged treatments. For example, the early cold stress query signatures (3 hour [h] and 6 h) showed very high similarity to cold signatures with only weak similarity scores to other signature phenotypes. Alternatively, the 24 h cold query showed similarity to cold signatures as well as drought and osmotic signatures. This result likely reflects the secondary effects of the prolonged (3 h and 6 h versus 24 h) cold treatment, and is in contrast with the results reported by Swindell (2006). Modules of genes participating in multiple stress responsive pathways were identified and were enriched for conserved metabolic pathways like photosynthesis and starch/sucrose metabolism. A common abiotic stress responsive module was also identified that was enriched with genes that respond to all treatments, mostly containing genes known to participate in calcium and calmodulin signaling pathways, which have been shown to participate in a multitude of cellular functions including cell death. A large compendium of Arabidopsis stress gene expression datasets were integrated and analyzed to elucidate clusters of genes characterizing the response to individual and a combination of all stresses (Ma and Bohnert, 2007). A cluster downregulated by almost all stresses was defined as a “universal stress response transcriptome” containing 197 genes encompassing diverse processes including known abiotic stress-responses, response to hormones, and signaling pathways related to MAPK, calcium, ROS, phospholipids, apoptosis, and protein degradation. These genes were also induced by ABA treatment. Another group of clusters contained genes that were induced in multiple abiotic stresses and were enriched for the ABRE and DRE motifs. Yet another group of clusters comprised of genes repressed in multiple abiotic stresses, related to general gene expression functions, protein synthesis, metabolism, cell organization, cell cycle progression, and cell division. These clusters were enriched with the MYCATERD1, SORLRP3AT, UP1ATMSD, and E2Frelated motifs. Generally, genes that are differentially expressed in response to a large number of different external stimuli are expected to contain more distinct CREs in their upstream regions than are genes that respond to only a few environmental cues. This has been verified in Arabidopsis where multiple stress response genes have been shown to be shorter, contain an increased absolute CRE count, CRE density, and have more paralogs, which might be important for the capacity of the cell for a greater dynamic expression range, to respond with similar gene products in reaction to varied stimuli, and to use the regulatory segments and gene sequence of paralogs as inventory for novel gene expression (Walther et al., 2007). Finally, several miRNA genes have been identified to respond to multiple stresses (Sunkar and Zhu, 2004). miR393 was found to be strongly upregulated by cold, dehydration, NaCl, and ABA treatments. miR397b and miR402 were slightly upregulated by all the stress treatments. miR389a.1 was observed to be downregulated by all of the stress treatments. Fourteen stress-inducible miRNAs were identified using microarray data under the effect of three abiotic stresses in Arabidopsis (Liu et al., 2008). Among them, 10 high-salinity regulated, 4 drought-regulated, and 10 cold-regulated

274

INTEGRATING PLANT ABIOTIC STRESS RESPONSES

miRNAs were detected, respectively. miR168, miR171, and miR396 responded to all of the stresses.

Gene Network of Universal Abiotic Stress Response

The large number of regulatory genes involved in the response to different abiotic stresses, as well as their overlapping expression profiles, demonstrates the complexity of transcriptional regulation underlying stress response. Genes differentially expressed during different stress treatments have been detected in microarray experiments and have been used for limited molecular genetic studies. Even analysis based on comparison of gene lists has shown that extensive cross talk and overlap among multiple signaling pathways exists. Moreover, the coexistence of multiple CREs within a cluster of genes and their known associations with stress response also indicate the complexity of gene regulatory networks. Hence, it is important for understanding the universal stress mechanisms to apply an integrative molecular network approach and the knowledge to identify tolerance mechanisms against an array of stresses. To elucidate the stress-responsive gene regulatory networks, the components of the transcription regulatory systems need to be identified, including genes encoding transcription factors and genes encoding downstream effector products. A good starting point for this is large-scale gene expression data that can be used to infer the regulatory interactions based on expression correlations, CRE content, and functional annotation. For example, a simple pipeline was used to infer cold acclimation regulatory pathways by combining CRE composition and (experimentally determined) TF–CRE correspondence with time-course gene expression profiles (Chawade et al., 2007). Other more rigorous methods for reconstruction of regulatory networks from gene expression data take into account all pair-wise gene correlations (based on different similarity measures) and process the data into a network after removing all “predicted” indirect interactions. In Arabidopsis, the closest to a genome-scale network reconstruction comes from an analysis of transcriptome data based on a regularized graphical Gaussian model (GGM) (Ma et al., 2007). Through partial correlation, GGM infers coregulation patterns between gene pairs conditional on the behavior of other genes, which is very suitable for inference of transcriptional regulatory interactions where a single is usually dependent on more than one TF. A network of 18,625 interactions (high confidence) among 6,760 genes was inferred. The network contains many coherent subnetworks corresponding to important processes including stress. To make use of this network, all of the genes within modules or clusters declared as “multiple,” “common,” “generic,” or “universal” in all of the abovementioned studies were gathered, which amount to 5,953 genes. Genes reported by Matsui and others (2008) using the tiling arrays seem to encompass many genes from other studies, while genes from Swindell (2006) have the least representation among genes reported in any other study. Of these 5,953 genes, 1,193 were identified in 2 or more studies (955 in 2, 194 in 3, 42 in 4, and 2 in 5 of the studies), and were used further to fish for interacting neighbors. A total of 686 out of 1,193 genes were present in the GGM network, which were found to be interacting with 1,300 genes. A total of 2,917 interactions between this total set of 1,986 genes (containing ∼250 TFs) were recovered to set up the “universal abiotic stress response” network, which is enriched with genes known to respond to abiotic and biotic stress, participate in carbohydrate, lipid, amino acid, and secondary metabolism, and transport (Figure 10.2). This network may contain several novel candidates for stress tolerance that can be tested by high throughput mutation analysis.

GENETIC NETWORKS UNDERLYING PLANT ABIOTIC STRESS RESPONSES

275

Figure 10.2 Universal abiotic stress response network. 2,917 interactions connect 1,986 genes representing many known stressassociated processes including canonical abiotic and biotic stress-response pathways, and carbohydrate, lipid, amino acid, and secondary metabolism.

The next steps are clear: this network needs to be integrated with networks comprising other types of molecular interactions including protein–protein interactions taking place during stress response to arrive at an all-encompassing systems-level model of abiotic stress. A study in this direction used a combination of Y2H assay, gene expression profiling, localization to genetic loci, plant mutations, and bioinformatics analysis to characterize the interactions between more than 200

276

INTEGRATING PLANT ABIOTIC STRESS RESPONSES

rice genes associated with stress response (Cooper et al., 2003). Further large-scale assays for protein-DNA and protein–protein interactions need to be performed, in addition to DNA modification and metabolic profiling specific to different abiotic stresses. The compendium of stress-related transcriptomes needs to be refined to get the subset for abiotic stress, and rigorous regulatory network reconstruction algorithms need to be used to recover a comprehensive gene network for abiotic stress that will pave the way for understanding this complex phenomenon better.

Conclusions

A systems-level approach to abiotic stress response will first make possible an elegant integration of diverse genome-scale data that are snapshots of what goes inside the cell across time points, conditions, and/or tissue types, by treating all of the regulatory and regulated components as interacting entities. Second, this perspective will point out the gaps in the framework that need to be filled, which will warrant the right experiments. Third, with less gene-level conservation across plants, complicated by duplication and divergence, conservation of response at the level of pathways and processes might be more significant, which will be made tractable in a network approach. Fourth, in the field condition, there is always a co-occurrence of abiotic stresses rather than a single stress, necessitating a consideration of the effects of simultaneous stresses and universal stress response, which can be most intuitively handled using an integrative, holistic outlook. Under this unified view, abiotic stress responses in plants can, hence, be seen in relation to other processes to obtain an extensive grasp of the effects of environmental stress, and acclimation and adaptive changes that protect plants. Effects of specific genes or processes can be predicted using such network models, and new ones can be assessed as a function of their association with known essential genes and processes paving the way for discovery of novel candidate points of control in the system that can be modified to improve crop stress tolerance.

References Abernethy, R.H., Thiel, D.S., Peterson, N., and Helm, K. (1989) Thermotolerance is developmentally dependent in germinating wheat seed. Plant Physiology 89(2):569–576. Bajguz, A. and Hayat, S. (2009) Effects of brassinosteroids on the plant responses to environmental stresses. Plant Physiology and Biochemistry 47(1):1–8. Boyer, J.S. 1982. Plant productivity and environment. Science 218(4571):443–448. Boyer, J.S. and McLaughlin, J.E. (2007) Functional reversion to identify controlling genes in multigenic responses: analysis of floral abortion. Journal of Experimental Botany 58:267–277. Chawade, A., Brautigam, M., Lindlof, A., Olsson, O., and Olsson, B. (2007) Putative cold acclimation pathways in Arabidopsis thaliana identified by a combined analysis of mRNA co-expression patterns, promoter motifs and transcription factors. BMC Genomics 8:304. Cooper, B., Clarke, J.D., Budworth, P., Kreps, J., et al. (2003) A network of rice genes associated with stress response and seed development. Proceedings of the National Academy of Sciences USA 100(8):4945–4950. Cushman, J.C. and Bohnert, H.J. (2000) Genomic approaches to plant stress tolerance. Current Opinion in Plant Biology 3(2):117–124. de la Fuente van Bentem, S. and Hirt, H. (2007) Using phosphoproteomics to reveal signalling dynamics in plants. Trends Plant Sci 12(9):404–411. Dinneny, J.R., Long, T.A., Wang, J.Y., Jung, J.W. et al. (2008) Cell identity mediates the response of Arabidopsis roots to abiotic stress. Science 320(5878):942–945. Eapen, D., Barroso, M.L., Ponce, G. Campos, M.E., and Cassab, G.I. (2005) Hydrotropism: root growth responses to water. Trends in Plant Science 10(1):44–50.

GENETIC NETWORKS UNDERLYING PLANT ABIOTIC STRESS RESPONSES

277

Elstner, E.F. and Oswald, W. (1994) Mechanisms of oxygen activation during plant stress. Proceedings of Royal Soc Edinburgh 102B:131–154. Goff, S.A., Ricke, D., Lan, T.-H., et al. (2002) A draft sequence of the rice genome (Oryza sativa L. ssp. japonica). Science 296(5565):92–100. Gorantla, M. (2005) Functional Genomics of Drought Tolerance: Large Scale EST Generation, Annotation, Physical Mapping, and Gene Expression Profiling in Rice (Oryza sativa sub sp., indica cv. Nagina 22). Ph. D. diss., University of Hyderabad, India. Granier, C., Dirk, I., Francois, and T. (2000) Spatial distribution of cell division rate can be deduced from that of p34(cdc2) kinase activity in maize leaves grown at contrasting temperatures and soil water conditions. Plant Physiology 124(3):1393–1402. Hasegawa, P.M., Bressan, R.A., Zhu, J.-K., and Bohnert, H.J. (2000) Plant cellular and molecular responses to high salinity. Annual Review of Plant Physiology and Plant Molecular Biology 51:463–499. Hauser, B.A., Sun, K., Oppenheimer, D.G., and Sage, T.L. (2006) Changes in mitochondrial membrane potential and accumulation of reactive oxygen species precede ultrastructural changes during ovule abortion. Planta 223(3):492–499. Huang, B. and Gao, H. (2000) Growth and carbohydrate metabolism of creeping bent grass cultivars in response to increasing temperature. Crop Science 40(4):1115–1120. Huang, D., Wu, W., Abrams, S.R., and Cutler, A. (2008) The relationship of drought-related gene expression in Arabidopsis thaliana to hormonal and environmental factors. Journal Experimental Botany 59(11):2991–3007. Huang, L.-F., Bocock, P.N., Davis, J.M., and Koch, K.E. (2007) Regulation of invertase: a suite of transcriptional and posttranscriptional mechanisms. Functional Plant Biology 34(6):499–507. Jager, C.E., Symons, G.M., Ross, J.J., and Reid, J.B. (2008) Do brassinosteroids mediate the water stress response? Physiologia Plantarum 133(2):417–425. Kagale, S., Divi, U.K., Krochko, J.E., Keller, WA., and Krishna, P. (2007) Brassinosteroid confers tolerance in Arabidopsis thaliana and Brassica napus to a range of abiotic stresses. Planta 225(2):353–364. Kant, P., Gordon, M., Kant, S., Zolla, G., Davydov, O., Heimer, Y.M., Chalifa-Caspi, V., Shaked, R., and Barak, S. (2008) Functional-genomics-based identification of genes that regulate Arabidopsis responses to multiple abiotic stresses. Plant, Cell & Environment 31(6):697–714. Kaur, N., Gupta and A.K. (2005) Signal transduction pathways under abiotic stresses in plants. Current Science 88(11):1771–1780. Kilian, J., Whitehead, D., Horak, J., Wanke, D., Weinl, S., Batistic, O., D’Angelo, C., Bornberg-Bauer, E., Kudla, J., and Harter, K. (2007) The AtGenExpress global stress expression data set: protocols, evaluation and model data analysis of UV-B light, drought and cold stress responses. The Plant Journal 50(2):347–363. Knight, H. and Knight, MR. (2001) Abiotic stress signalling pathways: specificity and cross-talk. Trends in Plant Science 6(6):262–267. Krishna, P. (2003) Brassinosteroid-mediated stress responses. Journal of Plant Growth regulation 22(4):289–297. Liao, C.-T. and Ling, C.-H. (2001) Physiological adaptation of crop plants to flooding stress. Proceedings of Natural Science Council (ROC) 25(3):148–158. Liu, H.-H., Tian, X., Li, Y-J., Wu, C.-A., and Zheng, C.-C. (2008) Microarray-based analysis of stress-regulated microRNAs in Arabidopsis thaliana. RNA 14(5):836–843. Liu, J.X., Liao, D.Q., Oane, R., Estenor, L., Yang, X.E., Li, Z.C., and Bennett, J. (2006) Genetic variation in the sensitivity of anther dehiscence to drought stress in rice. Field Crops Research 97(1):87–100. Ma, S. and Bohnert, H.J. (2007) Integration of Arabidopsis thaliana stress-related transcript profiles, promoter structures, and cell-specific expression. Genome Biology 8(4):R49. Ma, S., Gong, Q., and Bohnert, H.J. (2007) An Arabidopsis gene network based on the graphical Gaussian model. Genome Research 17(11):1614–1625. Matsui, A., Ishida, J., Morosawa, T., Mochizuki, Y. et al. (2008) Arabidopsis transcriptome analysis under drought, cold, highsalinity and ABA treatment conditions using a tiling array. Plant and Cell Physiology 49(8):1135–1149. Mckay, J.K., Richards, J.H., and Mitchell-Olds, T. (2003) Genetics of drought adaptation in Arabidopsis thaliana: I. Pleiotropy contributes to genetic correlations among ecological traits. Molecular Ecology 12(5):1137–1151. Miller, G., Shulaev, V., and Mittler, R. (2008) Reactive oxygen signaling and abiotic stress. Physiologia Plantarum 133(3):481–489. Munns, R. (2002) Comaparative physiology of salt and water stress. Plant, Cell and Environment 25(2):239–250. Munns, R. and Sharp, R.E. (1993) Involvement of abscisic-acid in controlling plant-growth in soils of low water potential. Australian Journal of Plant Physiology 20(5):425–437. Nemhauser, J.L., Hong, F., and Chory, J. (2006) Different plant hormones regulate similar processes through largely nonoverlapping transcription responses. Cell 126(3):467–475.

278

INTEGRATING PLANT ABIOTIC STRESS RESPONSES

Neumann, P.M. (2008) Coping Mechanisms for Crop Plants in Drought-prone Environments. Annals of Botany 101(7):901–907. Pandey, D.M., Goswami, C.L., and Kumar, B. (2003) Physiological effects of plant hormones in cotton under drought. Biologia Plantarum 47(4):535–540. Peres, A., Churchman, M.L., Hariharan, S., Himanen, K., et al. (2007) Novel plant-specific cyclin-dependent kinase inhibitors induced by biotic and abiotic stresses. Journal of Biological Chemistry 282(35):25588–25596. Rabbani, A.M., Maruyama, K., Abe, H., Khan, A.M., et al. (2003) Monitoring expression profiles of rice genes under cold, drought, and high-salinity stresses and abscisic acid application using cDNA microarray and RNA gel-blot analyzes. Plant Physiology 133(4):1755–1767. Reddy, A.M., Reddy, V.S., Scheffler, B.E., Wienand, U., and Reddy, A.R. (2007) Novel transgenic rice overexpressing anthocyanidin synthase accumulates a mixture of flavonoids leading to an increased antioxidant potential. Metabolic Engineering 9(1):95–111. Rivero, R.M., Kojima, M., Gepstein, A., Sakakibara, H., Mittler, R., Gepstein, S., Blumwald, E. (2007) Delayed leaf senescence induces extreme drought tolerance in a flowering plant. Proceedings of the National Academy of Sciences USA 104(49):19631–19636. Schachtman, D.P. and Goodger, J.Q.D. (2008) Chemical root to shoot signaling under drought. Trends in Plant Science 13(6):281–287. Schuppler, U., He, P.-H., John, P.C.L., and Munns, R. (1998) Effect of water stress on cell division and cell-division-cycle 2-like cell-cycle kinase activity in wheat leaves. Plant Physiology 117(2):667–678. Seki, M., Ishida, J., Narusaka, M., Fujita, M., et al. (2002) Monitoring the expression pattern of around 7000 Arabidopsis genes under ABA treatments using a full-length cDNA microarray. Functional Integrative Genomics 2(6):282–291. Shin, R., Alvarez, S., Burch, A.Y., Jez, J.M., and Schachtman, D.P. (2007) Phosphoproteomic identification of targets of the Arabidopsis sucrose nonfermenting-like kinase SnRK2.8 reveals a connection to metabolic processes. Proceedings of the National Academy of Sciences USA 104(15):6460–6465. Shinozaki, K. and Yamaguchi-Shinozaki, K. (2000) Molecular responses to dehydration and low temperature: differences and cross-talk between two stress signaling pathways. Current Opinion in Plant Biology 3(3):217–223. Shinozaki, K. and Yamaguchi-Shinozaki, K. (1997) Gene expression and signal transduction in water-stress response. Plant Physiology 115(2):327–334. Shukla, L.I., Chinnusamy, V., and Sunkar, R. (2008) The role of microRNAs and other endogenous small RNAs in plant stress responses. Biochimica et Biophysica Acta 1779(11):743–748. Spollen, WG., LeNoble, M.E., Samuels, TD., Bernstein, N., and Sharp, R.E. (2000) Abscisic acid accumulation maintains maize primary root elongation at low water potentials by restricting ethylene production. Plant Physiology 122(3):967–976. Steponkus, P.L. (1984) Role of plasma membrane in freezing injury and cold acclimation. Annual Review of Plant Physiology and Plant Molecular Biology 35:543–584. Sunkar, R. and Zhu, J.-K. 2004. Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. The Plant Cell 16(8):2001–2019. Supper, J., Martin, S., Wanke, D., Harter, K., and Zell, A. (2007) EDISA: extracting biclusters from multiple time-series of gene expression profiles. BMC Bioinformatics 8:334. Swindale, L.D. and Bidinger, F.R. (1981) The human consequences of drought and crop research priorities for their alleviation. p 2–13. In Paleg, L.G. and Aspinal, D. (Eds). The physiology and biochemistry of drought resistance in plants, Academic Press. Sydney. Swindell, W.R. (2006) The association among gene expression responses to nine abiotic stress treatments in Arabidopsis thaliana. Genetics 174(4):1811–24. The Arabidopsis Initiative. (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408(6814):796–815. Vinocur, B. and Altman, A. (2005) Recent advances in engineering plant tolerance to abiotic stress: achievements and limitations. Current Opinion in Plant Biology 16(2):123–132. Voisin, A.-S., Reidy, B., Parent, B., Rolland, G., Redondo, E., Gerentes, D., Tardieu, F., and Muller, B. (2006) Are ABA, ethylene or their interaction involved in the response of leaf growth to soil water deficit? An analysis using naturally occurring variation or genetic transformation of ABA production in maize. Plant, Cell, and Environment 29(9):1829–1840. Walia, H., Wilson, C., Condamine, P., Liu, X., Ismail, A.M., Close, T.J. (2007) Large-scale expression profiling and physiological characterization of jasmonic acid-mediated adaptation of barley to salinity stress. Plant, Cell and Environment 30(4):410–421. Walther, D., Brunnemann, R., and Selbig, J. (2007) The regulatory code for transcriptional response diversity and its relation to genome structural properties in A. thaliana. PLoS Genetics 3(2):e11.

GENETIC NETWORKS UNDERLYING PLANT ABIOTIC STRESS RESPONSES

279

Wang, Y.-W., Garvin, D.F., and Kochian, L.V. (2002) Rapid induction of regulatory and transporter genes in response to phosphorus, potassium, and iron deficiency in tomato roots. Evidence for cross talk in root rhizosphere-mediated signals. Plant Physiology 130(3):1361–1370. Weston, D.J., Gunter, L.E., Rogers, A., and Wullschleger, S.D. (2008) Connecting genes, coexpression modules, and molecular signatures to environmental stress phenotypes in plants. BMC Systems Biology 2:16. Willeken, H., Van Camp, W., Van Montangu, M., Inze, D., Langebartel, C., and Sandermann, H. (1994) Ozone, sulphur dioxide, and ultraviolet B radiation have similar effects on mRNA accumulation of antioxidant genes in Nicotiana plumbaginifolia L. Plant Physiology 106(3):1007–1014. Xie, Z., Jiang, D., Cao, W., Dai, T., and Jing, Q. (2003) Relationships of endogenous plant hormones to accumulation of grain protein and starch in winter wheat under different post-anthesis soil water statuses. Plant Growth Regulation 41(2):117–127. Xiong, L., Schumaker, K.S., and Zhu, J.-K. (2002) Cell Signaling during Cold, Drought, and Salt Stress. Plant Cell 14(1):S165–S183. Xiong, L., Wang, R.-G., Mao, G., and Koczan, J.M. (2006) Identification of Drought Tolerance Determinants by Genetic Analysis of Root Response to Drought Stress and Abscisic Acid. Plant Physiology 142(3):1065–1074. Zhu, J.-K. (2001) Plant salt tolerance. Trends in Plant Science. 6(2):66–71.

11

Discovering Genes for Abiotic Stress Tolerance in Crop Plants Michael Popelka, Mitchell Tuinstra, and Clifford F. Weil

Introduction

The ability of food crops to become more productive in stressful climates and growth conditions has never been more important. Yields have risen steadily from year to year, but the current rate of production will not be enough to support the 39% increase in population predicted by 2050 (Population Reference Bureau, 2008), and forecasts of decreased rates of production due to abiotic stress are now a major concern. Abiotic stresses are already common on a large percentage of existing, arable lands, amplified by worldwide climate change, and as use of more marginal lands becomes necessary, developing crops with enhanced stress tolerance is of vital concern. Conventional breeding strategies and marker-assisted selection to increase crop yields across drought and nondrought environments have been successful (Witcombe et al., 2008), but some of the traits selected for stressful climates may be genetically exhausted in some crops (Duvick, 2005). With the sequencing of several crop genomes now completed, a number of others in progress, tools for comparing genomes and evaluating transcriptome response to abiotic stress, are available. It is now possible to consider comprehensive inventories of abiotic stress genes. Gene discovery methods and plant transformation have helped increase the effectiveness of physiological and cellular mechanisms involved with stress tolerance, have aided in moving tolerance genes between species, and have been useful in devising new, useful combinations of genes and alleles. In addition, traits like stress tolerance that are multigenic can now be examined and manipulated as systems rather than only one gene at a time. These methods hold tremendous potential for modifying and combining genes to meet the stress-tolerant crop needs of the future (reviewed in Cushman and Bohnert, 2000; Umezawa et al., 2006). Recently, several general routes have been followed in discovering genes associated with abiotic stress. Using sequences from stress-related genes in model species such as Arabidopsis (covered extensively elsewhere in this volume), similar, potentially homologous genes have been identified in a wide range of crop species using bioinformatics. Comparative genomics has extended from Arabidopsis all the way up to tree species, where the poplar genome serves as the model (Rios et al., 2008). More directly, where it has been possible to do so (for example, rice, barley, maize, tomato, tobacco, etc.), examining changes in the transcriptome of stressed versus unstressed plants reveals a variety of genes, many previously undescribed (Kore-eda et al., 2004; Agarwal and Grover, 2005; Mantri et al., 2007; Poroyko et al., 2007). For example, gene discovery for drought stress in barley has taken advantage for several years of an affymetrix array (the Barley1 Gene Chip (Close et al., 2004) that represents ∼21,400 genes, derived from extensive mining of a dataset containing ∼400,000 ESTs and 84 cDNA libraries. More recently, profiling studies have moved to even greater coverage using Digital Gene Expression (DGE) or other NextGen sequencing-based methods, which reveal additional genes not found on commercial arrays (∼25% of unique SAGE Genes for Plant Abiotic Stress Editors Matthew A. Jenks and Andrew J. Wood © 2010 Blackwell Publishing ISBN: 978-0-813-81502-2

281

282

INTEGRATING PLANT ABIOTIC STRESS RESPONSES

or DGE tags are not represented on the affymetrix chip) (Ibrahim et al., 2005). Interestingly, the improved methods show that genes induced or repressed specifically under drought or dehydration make up nearly half of the stress response genes in barley (Tommasini et al., 2008). Studies of drought and other stress responses have also taken a more direct approach, identifying cDNAs isolated from stressed seedlings to identify those genes that are upregulated, and then overexpressing them (Jia et al., 2006; Sakurai et al., 2007; Luhua et al., 2008). A recent, innovative twist on this approach likely to see more use in the near future is the use of inducible gene expression libraries followed by analysis of induction-dependent stress tolerance (Papdi et al., 2008). Related, comparative approaches have also profiled differences in gene expression under drought stress due to natural variation of inbreds and in reciprocal hybrids (Kollipara et al., 2002). Finally, an entire area of gene “rediscovery” has been identified in which many genes involved in disease and insect resistance prove to be part of abiotic stress responses as well. Genes upregulated by stress can then be overexpressed in an effort to improve stress tolerance, both in the species from which the gene was identified and when expressed as transgenes in other species (Oh et al., 2005; Oh et al., 2007; Wei et al., 2008). This approach will be made even more powerful as we better understand the physiology of stress tolerance to inform where, when, and in what tissues the transcriptome is examined, allowing predictions of what genes may be the best targets for overexpression. Yet another avenue has been identifying binding sites for stressresponsive transcription factors, revealing additional new genes (Suprunova et al., 2007; Ling et al., 2000; Ogo et al., 2008). Identification of stress-tolerant mutants using both natural and induced variations (Rothan and Causse, 2007) and more traditional QTL analysis remain potent ways to identify new and sometimes unexpected stress-tolerance genes (for example, Price et al., 2002; Foolad et al., 2003; Lanceras et al., 2004); this is especially true in those crop species where transgenics and extensive genomic resources are still under development. In maize, extensive use of transposons harbored by the plant, such as Activator/Dissociation (Ac/Ds) and Mutator, has led to large collections of mutants that are publicly available for screening (Lunde et al., 2003; May et al., 2003; Kolkman et al., 2005; Bai et al., 2007; Settles et al., 2007). Rice collections using transgenic Ac/Ds or Tos17 are also available, as are attempts to use T-DNA introduced by agroinfection (An et al., 2005; Hsing et al., 2007; Qu et al., 2008). An effort is under way to generate Ac/Ds mutant populations in barley (Cooper et al., 2004; Singh et al., 2006; Zhao et al., 2006) and tomato (Knapp et al., 1994; Briza et al., 1995; Meissner et al., 2000). T-DNAs have been used to generate large mutant populations in other crops as well (e.g., various Brassica species). Chemically mutagenized populations, both for forward genetic screening and reverse genetic TILLING, a means of detecting point mutations in specific genes, have been made for maize (Till et al., 2004), rice (Till et al., 2007; Suzuki et al., 2008), wheat (Slade et al., 2005), barley (Caldwell et al., 2004), tomato (Menda et al., 2004), soybean (Cooper et al., 2008), canola (Wang et al., 2008b), Brassica oleracea (Himelblau et al., 2009), and sorghum (Xin et al., 2008), and are being developed for a wide range of other crops. TILLING for natural variation (EcoTILLING) (Comai et al., 2004) has also proven effective in wild populations of poplar (Gilchrist et al., 2006). Although this has not yet been applied directly for screens of stress tolerance, this should be an informative route to pursue. As high throughput DNA sequencing costs continue to come down, these methodologies are being replaced by resequencing, currently for targeted parts of the gene space, but it is now very likely that resequencing of an entire genome to analyze genetic variation will be inexpensive and straightforward in the near future. Genes involved in abiotic stress break into the same three classes (i.e., structural, regulatory, and signaling genes) that have been described for many other processes. Recent data also suggest that

DISCOVERING GENES FOR ABIOTIC STRESS TOLERANCE IN CROP PLANTS

283

the roles of miRNAs and gene silencing in regulating gene expression extend to stress tolerance as well, and this exciting new direction in gene discovery and approach to developing stress-tolerant varieties is only just being tapped (Zhao et al., 2007; Kapoor et al., 2008). In addition, developmental and morphological traits that render plants more resistant to abiotic stresses have been identified in wild relatives of crops (for example, John and Spangenberg, 2005; Sahi et al., 2006; Proite et al., 2007; Myouga et al., 2008; Ergen and Budak, 2009); and, although these are often multigenic traits, there are efforts to move some of them from wild species into their domesticated cousins. As a result gene discovery among wild relatives is also an expanding field. Some genes expressed in response to abiotic stress are generalized stress response genes while others are specific to dealing with one or another stress. A better understanding of both types, how they interact with one another, and the biochemistry behind their effects is increasingly important. Significant effort has been devoted to identifying genes for which expression changes, either in response to abiotic stress or during recovery from stress. While this is an important first step, it is important to remember that not all stress-response genes or their products will confer stress tolerance (defined as retaining yield even in stress conditions) when overexpressed. Gene discovery, at least as it applies to stress tolerance, thus has two components to it: gene identification and efficacy of expressing (or overexpressing) that gene.

Drought Tolerance

Genomics approaches have greatly advanced our ability to identify hundreds of genes that might contribute to improvements in drought tolerance (Ramalingam et al., 2006; Tuberosa and Salvi, 2006; Gorantla et al., 2007; Shinozaki and Yamaguchi-Shinozaki, 2007; Zhou et al., 2007; Degenkolbe et al., 2009). As many as 600 genes held in common among rice, maize, barley, and Arabidopsis show changes in transcription level in response to drought. As we better understand the functions and expression patterns of these genes, they can be harnessed to increase stress tolerance. In addition, proteome-level comparisons have provided another means of comparing potentially useful genes in stressed versus unstressed plants (Hajheidari et al., 2005; Plomion et al., 2006). Proteins have also driven gene discovery efforts through identification of membrane proteins and protein complexes, such as those associated with lipid rafts, in the plasma membrane that are important for abiotic stress response (Luthje et al., 2008; Komatsu, 2008). Improved understanding of stress physiology and fine-tuning gene expression in the biochemical pathways of stress response have provided effective means of discovering which of these genes are favorable candidates for improving drought tolerance (reviewed in Chaves, 2003). A great deal of effort has focused on the signaling process, particularly the hormone signals. For example, in maize blocking, the first step in ethylene biosynthesis prevents senescence of leaves and improves yield under drought conditions (Young et al., 2004). Similarly, abscisic acid (ABA) also accumulates during stress conditions, inducing stomatal closure, and is involved with several signaling pathways for stress-related gene expression. A gene involved with ABA biosynthesis, 9-cisepoxycartenoid dioxygenase (NCED), is activated in maize under drought stress (Burbidge et al., 1997). Overexpression of the Arabidopsis NCED gene in Arabidopsis leads to an increase in ABA levels, a decrease in transpiration rate, and greater tolerance to drought stress than untransformed controls (Iuchi et al., 2001). A similar study using the NCED gene of the tropical pasture legume Stylosanthes guianensis in tobacco has shown comparable results, suggesting the approach can be generalized to other crops (Zhang et al., 2008). An interesting added benefit of overexpressing NCED has been increased antioxidant activity. This increase triggers a different suite of genes,

284

INTEGRATING PLANT ABIOTIC STRESS RESPONSES

possibly improving stress tolerance still further. Consistent with this idea, genes controlling ascorbic acid redox state can be manipulated to allow plants better control over stomatal closure and less evaporative loss under drought stress (Chen and Gallie, 2004). Likewise, nitric oxide synthesis induced by ABA is linked to stomatal closure (Neill et al., 2008). Thus, the downstream changes in gene expression that result from overexpressing transgenes are, themselves, useful avenues of stress tolerance gene discovery (Seong et al., 2007). All of these are studies focused on individual hormones; however, the complex interaction of these hormone pathways is also important in response to drought and other abiotic stresses (reviewed in Davies et al., 2005). Further research will be important in understanding the cross talk between hormone-response pathways and the genes and gene products that mediate them. From the standpoint of discovering new genes, targeting hormone-controlled genes rather than hormone synthesis itself (e.g., overexpressing genes that are ethylene-repressed or inhibiting those that are ethylene-induced), presents an additional, important approach to drought tolerance (Tang et al., 2005; Xu et al., 2007). In addition to signaling pathways of drought response, the structural genes of naturally droughtresistant plants and the wild-type alleles of naturally drought-sensitive mutants and genes that are upregulated during normal periods of dessication such as seed development have all been valuable gene discovery tools. Characteristics of the cuticle, and the structural and regulatory genes that control them are good examples (Kunst and Samuels, 2003; Shepherd and Wynne Griffiths, 2006; Samuels et al., 2008). Even when stomata close, water can still diffuse through the cuticle of the plant. In maize, the genes involved with wax biosynthesis are known as the GLOSSY (GL) loci (Neuffer et al., 1997). Predicted functions for three of these genes (GL1, GL2, and GL8) have been suggested by analyzing gl mutant alleles (Tacke et al., 1995; Sturaro et al., 2005; Xu et al., 2008). Increasing expression of genes in the peroxygenase pathway, which contributes to the formation of cutin, may also be a useful means of increasing drought tolerance (Lequeu et al., 2003). Similarly, the SHINE clade of transcription factors in Arabidopsis affects cuticle metabolism and/or epidermal surface structure; overexpression of these transcription factors induced tolerance to drought conditions and improved recovery time (Aharoni et al., 2004). The detailed mechanism of cuticular wax deposition is still relatively unknown, but lipid transfer proteins (LTP) are possible wax transporters. LTPs have the ability to transfer lipids across different membranes, and some LTP genes are induced under drought stress (Kader, 1996; Colmenero-Flores et al., 1997; Colmenero-Flores et al., 1999; Jang et al., 2004; Cameron et al., 2006). In addition, loss of epicuticular wax is associated with increased water loss. Whether simply increasing the amount of epicuticular wax improves drought tolerance is not yet clear, but it presents a possible approach. Augmenting the expression of gene products that normally increase as seeds dry down is another useful approach. For example, rice transformed with the LEA protein gene OsLEA3-1 under expression by both a CaMV 35S promoter and a promoter strongly induced by drought exhibited an increase in both salt and drought tolerance under water-stress field conditions. No yield penalty was observed in the transgenic rice under optimal conditions (Xiao et al., 2007). The LEA proteins known from a wide variety of crop species could be amenable to similar approaches. In addition, seed dry-down specific transcription factors and their targets also suggest potential drought tolerance mechanisms (for example, Prieto-Dapena et al., 2008). From the standpoint of cellular structure, genes involved in synthesis of membrane lipids regulate individual cells’ abilities to respond to water stress (Knight and Knight, 2001). Identifying and manipulating the genes that control biosynthesis and deposition of these lipids can help improve drought tolerance. For example, changing the fluidity of the membrane and altering protein-lipid interactions can alter the activity of ion channels and proteins, such as aquaporins controlling cell

DISCOVERING GENES FOR ABIOTIC STRESS TOLERANCE IN CROP PLANTS

285

turgor pressure (van der Heide and Poolman, 2000; Lian et al., 2004). This reorganization of proteins in the membrane may activate an additional water-stress-sensing mechanism that includes phospholipases C and D as well as phosphatidyl-4,5-phosphate 5-kinase (Sang et al., 2001; Yamaguchi-Shinozaki and Shinozaki, 2001; Ueda et al., 2004; Bargmann and Munnik, 2006; Drame et al., 2007; Wang et al., 2008a; Bargmann et al., 2009). Interaction among these systems and the genes involved are not well understood to date, and further study is needed; however, they present another route to improving drought tolerance, either alone or in combination with other approaches. Apart from controlling the flow and loss of water within the plant, another set of genes that can impart drought tolerance is those involved in water-use efficiency, defined as biomass or crop yield in relation to the amount of water lost through transpiration. Gene discovery in this area is of primary interest to both industry and public research efforts. Under stress environments such as drought or cold, where water is less available, transpiration is decreased by stomatal closure, osmoprotectant accumulation, etc. Laporte and others (2002) demonstrated that stomatal conductance is decreased by constitutively overexpressing the NADP-malic enzyme from maize in tobacco, altering malate metabolism in the guard cells. The transgenic plants gained more fresh weight per unit of water even though there was no change in morphology or growth rate, decreased soil moisture more slowly, and wilted later than control plants. Similarly, knockout mutants of the AtMRP4 transporter involved with ion flux in the plasma membranes of Arabidopsis guard cells show increased transpiration and decreased drought tolerance (Klein et al., 2004). Plant responses to drought stress extend beyond the stress period itself—there is a recovery process once water again becomes available—and gene discovery efforts in the period following a drought stress have provided novel information about drought tolerance. For example, Zhou and others (2007) report that novel promoter motifs occur in the promoters of genes controlling rehydration after drought stress, suggesting that an important part of drought tolerance in rice may not be occurring during the stress at all but as a coordinated, enhanced ability to recover once water is reintroduced. Another area of gene discovery has been the secondary effects that accrue during drought response, with drought tolerance achieved by preventing these effects. For example, profiling longterm transcript changes under drought stress confirms that closed stomata also decrease carbon assimilation due to decreased CO2 influx (Cornic, 2000; Flexas and Medrano, 2002; Chaves and Oliveira, 2004; Ramachandra Reddy et al., 2004; Ghannoum, 2009). The combination of lowcarbon assimilation with high-light intensity can lead to photoinhibition and ensuing oxidative stress (Ramachandra Reddy et al., 2004), governed by yet another set of gene responses. “Functional stay-green” describes the maintenance of photosynthetically active leaves under various forms of abiotic stress after other varieties have become senescent (Fischer and Turner, 1978; Thomas and Howarth, 2000; Yan et al., 2004; Park et al., 2007; Gay et al., 2008). Leaf senescence is, overall, a form of programmed cell death involving cell degradation and relocation of their contents within the plant. Suppressing inducible senescence has thus been a rich source for identifying the genes that control senescence, and for conferring resistance to oxidative stress caused by other environmental factors such as heat and UV light (del Rio et al., 1998). The regulation of drought-induced early senescence involves hormones, as well as sugar signaling (Wingler and Roitsch, 2008). For instance, drought stress increases the activity of ACC synthase and thus the amount of ethylene production (Apelbaum and Yang, 1981), while intact cotton plants (Morgan et al., 1990) and knockout mutants of two ACC synthase genes in maize show decreased levels of ethylene synthesis in leaves and improved drought tolerance (Young et al., 2004). At 20 days after pollination, a point at which older leaves typically begin to senesce, mutant leaves have

286

INTEGRATING PLANT ABIOTIC STRESS RESPONSES

higher chlorophyll and protein levels (particularly RuBisCO), higher stomatal conductance, transpiration, and CO2 assimilation than wild-type leaves. For other plant hormones, timing of hormone synthesis can have dramatic effects on whether it confers useful drought tolerance. As an example, expression of isopentenyltransferase (IPT), known to increase synthesis of cytokinins, inhibits leaf senescence when expression is placed under the control of the PSAG12 promoter, induced during leaf senescence (Rivero et al., 2007). However, the transformed plants also show nutrient deficiency in young leaves, delayed anthesis, and reduced seedling establishment. IPT expressed in tobacco plants using the PSARK promoter instead, normally induced during late maturation rather than at leaf senescence, did not show these side effects. After 40 days of optimal growth conditions, followed by 15 days of drought stress, transgenic plants showed only 8 and 14% reductions in biomass and seed and no foliar senescence compared with wild-type plants that wilted dramatically, showing a 57% reduction in biomass and 60% reduction in seed yield and senescent leaves. The transgenic plants also had increased levels of antioxidants such as ascorbate and glutathione, revealing an as-yet unexplored link between cytokinin and activation of reactive oxygen scavenging. The most recent additions in gene discovery for drought tolerance are drought-related microRNAs (MiRNAs) and the genes they target (Phillips et al., 2007; Sunkar et al., 2007; Zhao et al., 2007; Sunkar et al., 2008). The promoters of genes for these miRNAs indicate that at least some of them are controlled by CBF/DREBs, the proteins that bind Dehydration Responsive Elements (DRE). MiRNAs are thus likely to be involved in both early and later steps in the drought response.

Salt Stress

In addition to decreasing water availability for crop production, the quality of the water that is available is a major issue (Lexer et al., 2004; Munns et al., 2006). Salinity in soils is increasing in many areas, and tolerance of these conditions is also a focus of gene discovery efforts in crop species and their soil symbionts (reviewed in Sahi et al., 2006). Mining the genes of halophytic species such as Spartina (Jithesh et al., 2006; Baisakh et al., 2008), which normally grow in brackish conditions, has been an important resource in this area as have screens for salt–tolerant mutants directly in crop species and model systems (Quesada et al., 2000; Borsani et al., 2001; Rai et al., 2003; Cao et al., 2007). Numerous studies have shown that plants accumulate molecular ions, sugars, and amino acids under salt stress. This form of salt tolerance is generally referred to as osmotic adjustment. Ogawa and Yamauchi (2006) observed greater increases in these osmolytes in root elongation and elongated zones than in leaves when each was exposed to differing levels of osmotic stress. The increase of osmolytes may cause lower water potential in cells, allowing cells to retain the turgidity needed for cell expansion. There is further speculation that some osmolytes may help prevent oxidative damage, maintain membranes, and prevent denaturation of proteins during abiotic stress conditions. Molecular ions, most notably potassium, lower water potential, are easily transported throughout the plant, and are easily taken up in roots. However, an imbalance of potassium ions can interfere with other cell processes, and potassium overaccumulation is often only a limited solution to salt stress. Proline, an important amino acid in osmotic adjustment, is more metabolically neutral than potassium, but has a higher molecular weight and thus takes more energy to transport (Liu and Zhu, 1997; Verbruggen and Hermans, 2008). Genes involved in proline biosynthesis are relatively well known. Therefore, the focus for its use in salt tolerance has been primarily on the genes of the signaling pathways involved (reviewed in Tuteja, 2007). Numerous studies have also been

DISCOVERING GENES FOR ABIOTIC STRESS TOLERANCE IN CROP PLANTS

287

conducted illustrating the role of glycine betaine (GB), an amino acid derivative thought to stabilize membrane and protein structure under salt and other stresses (Saneoka et al., 1995; Holmstrom et al., 2000; Sakamoto and Murata, 2002; Park et al., 2004), although this has not proven to be true in all cases (Omokawa and Aonuma, 2002). GB has proven to be a fruitful transgenic tool as well. For example, five maize lines engineered for increased activity of a bacterial choline dehydrogenase, an enzyme in GB synthesis, had greater levels of sugars and amino acids than wild types, decreased osmotic potential, increased root biomass, increased total biomass, and increased grain weight per plant when compared to wild-type controls (Quan et al., 2004). Gene discovery in this area now needs to concentrate on those genes governing these responses. Reducing sugars have also been found to concentrate in roots undergoing osmotic stress. These sugars provide carbon for extended root growth at the same time they alter water potential. Interestingly, these compounds may be synthesized where needed by the plant as a secondary response to salt stress and then not relocated; under osmotic stress, maize roots do not accumulate higher levels of sugars until well after other osmolytes had already increased (Ogawa and Yamauchi, 2006). It should be noted that there has been less effort to understand the genes involved in this mode of stress tolerance because there is still debate over how effective osmolyte accumulation is (for example, Munns, 2005; Serraj and Sinclair, 2002). Critics suggest that the benefits of osmolyte accumulation do not become evident under severe drought stress until the point at which yield is already too compromised to be valuable.

Heat Stress

Plants are unequipped to regulate their internal temperatures, and heat stress can damage a variety of tissue types and reduce or completely hinder their ability to maintain homeostasis. Still, although plants are sessile, they are not without mechanisms of natural heat tolerance, and these have been the targets of both natural and artificial selection. Further improvement remains important, however, as sudden or sustained heat stress can overcome these defenses. Gene discovery for heat tolerance has taken similar routes to those described above for drought: profiling transcription changes in response to and during recovery from heat stress, profiling heat tolerant variants, comparing stressed with unstressed transcriptomes (Sanchez-Ballesta et al., 2003), and experimenting with transgenics. Emphasis has been on understanding the systems impacted by heat stress and modifications to those that are more thermotolerant. For example, heat stress impacts membrane fluidity (Sung et al., 2003). Changes in the fluidity or rigidity of membranes, especially the thylakoid membrane, may be a first step in the signal transduction of heat shock factors (HSF), which generate thermotolerance through various mechanisms. Changes in membrane fluidity can, in turn, lead to a change in calcium influx, which may act as a second messenger in thermotolerance (Larkindale and Huang, 2004). During heat stress, Arabidopsis calcium levels peak at the beginning of the heat recovery period, suggesting either that its accumulation during stress is important or that it may be involved with recovery from heat stress rather than the direct heat stress response. A class of transcription factors with the conserved WRKYGQK motif followed by a zinc finger, the so-called WRKY proteins, have proven to be central players in the response to both abiotic and biotic stresses (reviewed in Wu et al., 2005). Improved heat and drought tolerance accompany overexpression of these factors (Wu et al., 2009), and focused efforts to identify all of the WRKY proteins as well as their target genes are under way. Similarly, genes encoding RIP-domain proteins also confer tolerance to both biotic and abiotic stresses (Jiang et al., 2008).

288

INTEGRATING PLANT ABIOTIC STRESS RESPONSES

The well-known effect of heat shock on protein denaturation and chaperonins has been documented for several decades, and represents one of the earliest gene discovery approaches using abiotic stress. Most of the recent attention has been paid to HSFs that induce the expression of chaperonins (HSPs), and overexpression of these transcription factors (Karaba et al., 2007; Xu et al., 2007; Papdi et al., 2008); however, thermotolerance has been most often been reported as overexpression of specific HSPs (Harndahl et al., 1999; Malik et al., 1999; Park and C.B., 2002; Murakami et al., 2004; Sanmiya et al., 2004; Salvucci, 2008). The evolutionary conservation of HSPs has also proven to be a valuable feature allowing expression of even prokaryotic HSPs to improve broad stress tolerance in crop plants (Castiglioni et al., 2008). Extensive data from the overexpression in maize of CspA and CspB cloned from Escherichia coli and Bacillus subtilis, respectively, shows increased tolerance to drought in both vegetative and reproductive growth, leading to higher yields. Rice exhibited tolerance to heat, cold and water deficit. Several other stresses (e.g., calcium, reactive oxygen species, salicylic acid [SA], abscisic acid [ABA], etc.) can induce HSFs in plants (Larkindale and Knight, 2002;, Suzuki and Mittler, 2006; Swindell et al., 2007; Timperio et al., 2008), and NADPH oxidases produce reactive oxygen species in response to increased temperatures. This wide range of inducers has been useful in understanding what the generalized stress response genes are as well as providing candidates for engineering broad-range stress tolerance. Identifying targets of some of these early HSFs has helped delineate some of the stress-specific response genes as well (Rouhier et al., 2005). Evidence also suggest that HSPs can increase tolerance to oxidative stress by acting as antioxidants (Hamilton III and Heckathorn, 2001).

Oxidative Stress

The response to reactive oxygen species (ROS) has become increasingly central to abiotic stress tolerance (Overmyer et al., 2003; Apel and Hirt, 2004; Pitzschke et al., 2006; Shao et al., 2008) and even to the cross talk between abiotic and biotic stress response (Kacperska, 2004; Abuqamar et al., 2008). Gene discovery in this area also impacts overall plant health, even in nonstress situations, because ROS are natural by-products of aerobic metabolism and a normal part of senescence and the hypersensitive response in plant disease resistance. Plant production of reactive oxygen species (ROS) occurs predominantly in the chloroplast, mitochondria, and peroxisomes. ROS scavengers such as antioxidants exist to remove these ROS, because they can have deleterious effects on other cellular components. Abiotic stresses can cause an imbalance between the normal production and removal of ROS, which can lead to oxidative stress damage and programmed cell death (PCD) (Fukao and Bailey-Serres, 2004). Because of the constitutive and inescapable nature of this stress, plants have evolved a wide range of ROS scavenging systems involving low molecular weight antioxidants, the activity of antioxidant enzymes, hormones, sugar sensing (Couee et al., 2006), and a collection of superoxide dismutases (reviewed in Ślesak, et al., 2007). Interestingly, particularly from a gene discovery perspective, the emphasis in developing tolerance to oxidative stress has dwelt far more on the effects of overexpressing single genes or small families of genes of interest than on large-scale gene discovery. It remains possible that too much tolerance to oxidative stress would not be in the best overall interests of the plant, in any case (Allen, 1995; Van Breusegem et al., 1999; Wang et al., 1999); for example, constitutive overexpression of antioxidant enzymes might prevent normal senescence or cause an inability of the plant to defend itself from microbial pathogen attack. Still, successful examples of increasing oxidative stress tolerance have been described, so at least some

DISCOVERING GENES FOR ABIOTIC STRESS TOLERANCE IN CROP PLANTS

289

level of increase must be possible without negative effects (Eltayeb et al., 2007). In Arabidopsis, the 14-3-3 regulatory protein, GF14λ, interacts with ascorbate peroxidase, involved with ROS scavenging (Narendra, 2005). Constitutive expression of Arabidopsis GF14λ in cotton produced a stay-green phenotype conferring enhanced drought tolerance and increased photosynthesis rates under slow progressive water stress when compared to wild-type and nonexpressing plants (Yan et al., 2004). More recent research has focused on using various stress-specific promoters to direct overexpression of ROS degrading enzymes and enzymes that synthesize antioxidants specifically to the times and places they are needed. For example, an MnSOD enzyme from pea was overexpressed in rice to increase tolerance using the SWPA2 promoter, which is induced by oxidative stress (Wang et al., 2005a). Transgenic plants also displayed improved tolerances to drought without slowing growth under optimal conditions. Similar, broader range tolerances to drought and to salt stress in addition to oxidative damage, have been reported by expressing glutathione-S-transferase (GST) (Zhao and Zhang, 2006), S-adenosylmethionine decarboxylase (SAMDC) (Wi et al., 2006), dehydroascorbate reductase (DHAR), cytosolic ascorbate peroxidase (Wang et al., 2005b), and monodehydroascorbate reductase (MDAR) (Eltayeb et al., 2007).

Nutrient/Mineral Stress

Gene discovery as it relates to performance under nutrient stress and to tolerance or hyperaccumulation of toxic minerals has become a major research driver. Nutrient stresses often accompany other factors such as drought, which further decreases nutrient mobility. At the same time, industry is keenly interested in developing ways to reduce the need for fertilizer inputs and to improve nutrient use efficiency without decreasing yield. The responses to nutrient stress, both in the plants and in mycorrhizal symbionts, allow plants to increase the range of their nutrient acquisition beyond the depletion zone immediately surrounding the root. These efforts are directed at understanding genes of crop plant species, as well as the metagenomics of microbial flora in the soil around them. For example, maize arbuscular mycorrhizae (AM) supply roots with immobile nutrients in exchange for reduced carbon. AM symbiosis in maize thus increases tolerance of nutrient stress as well as drought and other abiotic stresses (Subramanian and Charest, 1999; Sheng et al., 2008; Boomsma and Vyn, 2008); similar results are seen in tomato (Aroca et al., 2008) and other species (Daei et al., 2008). AM may affect a combination of physiological processes within the plant including hydraulic conductivity, water uptake, rooting depth, nutrient acquisition, and plant metabolism (Augé, 2001). In any case, discovering the genes that impact formation of these symbioses are, at least indirectly, discovery of genes involved in stress tolerance. The symbiosis is under genetic control in the plant, as demonstrated by the fact that the effects of AM symbiosis on maize vary based on the genotype of the plant (Kaeppler et al., 2000). Details of which plant genes are involved in AM-mediated stress tolerance come from more closely examining the physiological responses involved. For example, in addition to increasing drought tolerance, AM symbiosis in maize improves tolerance to salt stress by increasing the accumulation of soluble sugars in the roots, which feeds the symbiont and changes osmotic pressure within the root system (Feng et al., 2002). It has also been hypothesized that ROS are produced during the process of establishing the mycorrhizae, inducing a mild oxidative stress response in the plant that can contribute to better oxidative stress tolerance (Fester et al., 2002; Fester and Hause, 2005; Walter et al., 2007). Thus, in addition to genes governing hormone responses in AM

290

INTEGRATING PLANT ABIOTIC STRESS RESPONSES

colonized plants (for example, Fester and Hause, 2007), genes that mediate signaling between the plant and its symbionts, and that control the physiological processes altered by the symbiosis are all potential targets for improving stress tolerance. Gene discovery in this instance may also have a “value-added” aspect in that AM symbiosis often either has no effect under nonstress conditions, or can even inhibit growth due to the increased root respiration supporting the symbiosis. There may be opportunities to improve the benefits of symbiosis in the absence of stress. Nutrient stress tolerance is most easily studied using hydroponic growth, and this has proven to be valuable for gene discovery. A recent example of this approach comes from soybean and a study of tolerance to low iron levels and the chlorosis that low iron can induce. Near isogenic lines selected for either efficient or inefficient response to low iron were compared by microarray after growth in low iron was followed by return to iron-sufficient medium (O’Rourke et al., 2007). Four cDNAs remained underexpressed in the inefficient line, even after return to normal levels of iron, confirmed by real-time PCR measurements. These genes are now targets of study for improving tolerance to and recovery from iron deficiency. Very recently, use of inductively coupled plasma mass spectrometry (ICP-MS) to analyze mineral content in plant tissues has provided new assays for gene discovery for mineral stress tolerance (reviewed in Salt et al., 2008). This high throughput analytical method has allowed screening of both natural and induced variation in the ability to accumulate unusually high or low levels of minerals such as potassium, arsenic, iron, nickel, and a dozen others. Using ICP-MS to precisely quantify mineral content, QTL analysis for this variation has revealed several important genes that control mineral tolerance in maize (I. Baxter, D. Salt, unpublished). This assay method promises to be a powerful tool in a wide range of other crop plants in the near future.

Plant Architecture and Morphology

Nutrients becoming unavailable to the plant is a major part of the overall challenge involved with dry soils and soils with high salt content. This stress, as well as drought and mineral toxicity, can be dealt with through changes in root architecture and function. Roots rely heavily on mass flow and diffusion of water to receive nutrients such as nitrogen, phosphorus, and potassium among many others. Plants can therefore either increase efficiency of root absorption in a given root zone or extend their root systems further under osmotic and drought stress in search of more beneficial soil environments; however, any increased investment in root growth needs to be balanced against potential negative effect on yields. Changes in root architecture under different stresses have been the focal point of many studies. Genetic variation for root structure and its response specifically in stress tolerance has been described in rice (Zheng et al., 2000) and in maize, where alleles have been described with many pleiotropic and linkage effects (Hochholdinger et al., 2004; Giuliani et al., 2005; Liu et al., 2006; Hochholdinger and Tuberosa, 2009). Most plants under drought stress have decreased lateral root growth contrasted by an increase in primary root growth associated with abscisic acid, probably in search of moisture located deeper within the soil (Deak and Malamy, 2005; Aloni et al., 2006). In contrast, a plant’s ability to explore its root zone laterally is enhanced by the formation and expansion of seminal and adventitious roots. Hormone signaling, and the genes that synthesize and are affected by these hormones, all play important roles in formation, repression, growth, and differentiation of both primary and lateral roots (Sharp, 2002; Sharp et al., 2004). These responses thus facilitate annotation of root growth genes. In the drought response, there are two key questions:

DISCOVERING GENES FOR ABIOTIC STRESS TOLERANCE IN CROP PLANTS

• •

291

What root architecture is most efficient under drought conditions? What combination of genes is involved in that determination?

Transgenic studies to address this problem have not made a great deal of headway, and much remains unknown. For example, overexpression of the tomato H+-pyrophosphatase (H+-PPase) gene AVP1 conferred larger root biomass and, consequently, faster recovery from drought conditions, although the mechanisms remain unclear (Park et al., 2005). Increased lateral root formation could improve nutrient and water uptake and increase standability (Mullen and Hangarter, 2003); however, the tradeoff between increased root growth and decreased yield has already been mentioned, and the reverse, a decrease in root mass to boost investment in the shoot, can increase vulnerability to pathogens or reduce soil penetration (Lynch, 2007). Transcriptional control of root architecture is a more recent approach to these questions but has not yet produced results. For drought in the field, where extending roots laterally into new areas of soil (as opposed to deeper) does not alleviate the problem, the answer may not be increasing root mass so much as increasing the efficiency with which roots retrieve soil moisture. Gene discovery efforts focus on genes that increase surface area and transport efficiency rather than on root extension. Substantial genetic variation for root hair length and density is seen in maize, controlled by several QTL (Zhu et al., 2005). The gibberellin-DELLA signal pathway has also been shown to effect root hair morphology (Jiang et al., 2007). An advantage of modifying root hairs to increase surface area is their relatively low metabolic cost. Genetically induced, morphological changes in response to stress are not limited to the roots. Yu and others (2008) isolated HDG11, an Arabidopsis homeodomain (HD)-START transcription factor, and found that its expression in tobacco produced transgenic plants with reduced leaf stomata density in addition to deeper and more lateral roots.

Evolutionary Conservation and Gene Discovery

One of the more interesting features of gene discovery and transgenic trials in abiotic stress tolerance has been the degree to which overexpressing heterologous genes has been successful at improving plant performance under stress. This is particularly true for overexpressed transcription factors, where the transcription factor as well as the target genes and the transcription factor binding sites associated with those genes all have to be conserved for the experiment to succeed. While the strong conservation of heat-shock proteins, with their fundamental role as protein chaperones, is perhaps not surprising, there is no a priori reason to expect conservation of so many other, multipart, stress response systems. An example of this point (and by no means the only example) has been the ethylene-responsive factor (ERF) class of transcription factors. ERFs are responsible for, among other things, several of the physiological processes in abiotic stress response. The dehydration-responsive elementbinding proteins (DREB) are a subgroup of the ERF family, and DREB1 and DREB2 are involved with cold and dehydration stress, respectively. Since the initial description of the DREB1A and DREB2B proteins in Arabidopsis over a decade ago (Liu et al., 1998), DREB genes have been discovered in a wide variety of plants suggesting that their manipulation and reintroduction into crop species can be a valuable means of gaining stress tolerance (Dubouzet et al., 2003; Shen et al., 2003; Gutterson and Reuber, 2004; Li et al., 2005; Agarwal et al., 2006; Zhuang et al., 2008; Xu et al., 2009). Interestingly, manipulation of the Arabidopsis DREB genes has been very effective in engineering drought tolerance in other plants. Wheat transformed with the Arabidopsis

292

INTEGRATING PLANT ABIOTIC STRESS RESPONSES

DREB1A gene under the control of a stress-induced promoter showed a 10-day delay in wilting under water stress when compared to checks (Pellegrineschi et al., 2004). Similar studies have been conducted with Arabidopsis DREB1A and DREB2A expressed in maize, where plants also showed improved tolerance to heat, cold, drought, and salinity stress (Al-Abed et al., 2005; Qin et al., 2007) and with a sugarcane ERF expressed in tobacco (Trujillo et al., 2008). In the last decade, zinc-finger proteins in plants have shown the ability to regulate development, programmed cell death, and a wide range of other processes. The ability now to design zinc-finger combinations to bind to any sequence of choice has also raised a great deal of interest in the use of these proteins to improve stress tolerance (Holmes-Davis et al., 2005). An aspect of these factors that are at the same time intriguing and daunting is that the effects of binding a zinc-finger protein in front of a gene in one system may have differing effects in other systems. As one of the largest families of transcription factors in plants, several zinc-finger proteins control abiotic stress acclimation (Ciftci-Yilmaz and Mittler, 2008). The overexpression of a zinc-finger protein in rice has demonstrated the ability to increase tolerance to salt and drought stress with a correlated increase in proline and soluble sugars (Xu et al., 2008). However, zinc finger protein expression in tobacco negatively regulated some stress-induced genes and caused transgenic plants to become more susceptible to drought and salt stresses than wild types (Huang et al., 2008). Research indicates that zinc-finger proteins may play a large role in the future for the genetic engineering agricultural crops. Increasingly, novel transcription factors are being discovered, and some of these impact stress tolerance. For example, plant versions of the multimeric nuclear factor Y (NF-Y) differ from their animal and fungal counterparts both in structure and in the number of gene copies present (Albani and Robert, 1995; Edwards et al., 1998). A subclass of NF-Y from maize, ZmNF-YB2, when overexpressed in maize, conferred increased drought tolerance. The transgenic lines showed delayed senescence and also greater recovery after the drought treatment without yield penalty than did controls. Microarray analysis showed that genes regulated by the NF-Y transcription factor did not overlap with DRE-binding transcription factors or ABA-treated wild-type plants, suggesting that NF-Y is a novel transcription factor (Nelson et al., 2007). Conclusion

Selection for yield under unfavorable environments in conventional breeding has proven successful for methods to increase abiotic stress tolerance in food crops. However, with the inclusion of new technologies, the ability for breeders and plant scientists to multiply their gain in stress resistance may exist. QTL mapping combined with marker-assisted selection allows breeders to rapidly pyramid traits affecting the acclimation and yield improvement of plants experiencing these stressful conditions. At the same time, the discovery and isolation of novel genetic mechanisms and the creation of transgenic plants bode well for the role of biotechnology in crop improvement. Although we have emphasized the different methods that exist for finding and testing genes that improve the ability of crops to withstand abiotic stress, it is important to remember that the essential goals of these efforts will be maintaining or increasing overall yield on the farm while facing increasingly harsher climates and the need to use increasingly marginal lands. References Abuqamar, S., Luo, H., Laluk, K., Mickelbart, M.V., and Mengiste, T. (2008) Cross-talk between biotic and abiotic stress responses in tomato is mediated by the AIM1 transcription factor. Plant J.

DISCOVERING GENES FOR ABIOTIC STRESS TOLERANCE IN CROP PLANTS

293

Agarwal, P.K., Agarwal, P., Reddy, M.K., and Sopory, S.K. (2006) Role of DREB transcription factors in abiotic and biotic stress tolerance in plants. Plant Cell Rep 25, 1263–1274. Agarwal, S. and Grover, A. (2005) Isolation and transcription profiling of low-O2 stress-associated cDNA clones from the flooding-stress-tolerant FR13A rice genotype. Ann Bot (Lond) 96, 831–844. Aharoni, A., Dixit, S., Jetter, R., Thoenes, E., van Arkel, G., and Pereira, A. (2004) The SHINE clade of AP2 domain transcription factors activates wax biosynthesis, alters cuticle properties, and confers drought tolerance when overexpressed in Arabidopsis. Plant Cell 16, 2463–2480. Al-Abed, D., Madasamy, P., Talla, R., Goldman, S., and Rudrabhatla, S. (2005) Genetic Engineering of Maize with the Arabidopsis DREB1A/CBF3 Gene Using Split-Seed Explants. Crop Science 2390–2402. Albani, D. and Robert, L.S. (1995) Cloning and characterization of a Brassica napus gene encoding a homologue of the B subunit of a heteromeric CCAAT-binding factor. Gene 167, 209–213. Allen, R.D. (1995) Dissection of oxidative stress tolerance using transgenic plants. Plant Physiol 107, 1049–1054. Aloni, R., Aloni, E., Langhans, M., and Ullrich, C.I. (2006) Role of cytokinin and auxin in shaping root architecture: regulating vascular differentiation, lateral root initiation, root apical dominance and root gravitropism. Ann Bot (Lond) 97, 883–893. An, G., Jeong, D.H., Jung, K.H., and Lee, S. (2005) Reverse genetic approaches for functional genomics of rice. Plant Mol Biol 59, 111–123. Apel, K. and Hirt, H. (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55, 373–399. Apelbaum, A. and Yang, S.F. (1981) Biosynthesis of Stress Ethylene Induced by Water Deficit. Plant Physiol 68, 594–596. Aroca, R., Del Mar Alguacil, M., Vernieri, P., and Ruiz-Lozano, J.M. (2008) Plant responses to drought stress and exogenous ABA application are modulated differently by mycorrhization in tomato and an ABA-deficient mutant (sitiens). Microb Ecol 56, 704–719. Augé, R.M. (2001) Water relations, drought and vesicular-arbuscular mycorrhizal symbiosis. Mycorrhiza 11, 3–42. Bai, L., Singh, M., Pitt, L., Sweeney, M., and Brutnell, T.P. (2007) Generating novel allelic variation through Activator insertional mutagenesis in maize. Genetics 175, 981–992. Baisakh, N., Subudhi, P.K., and Varadwaj, P. (2008) Primary responses to salt stress in a halophyte, smooth cordgrass (Spartina alterniflora Loisel.). Funct Integr Genomics 8, 287–300. Bargmann, B.O. and Munnik, T. (2006) The role of phospholipase D in plant stress responses. Curr Opin Plant Biol 9, 515–522. Bargmann, B.O., Laxalt, A.M., ter Riet, B., van Schooten, B., Merquiol, E., Testerink, C., Haring, M.A., Bartels, D., and Munnik, T. (2009) Multiple PLDs required for high salinity and water deficit tolerance in plants. Plant Cell Physiol 50, 78–89. Boomsma, C.R. and Vyn, T.J. (2008). Maize drought tolerance: Potential improvements through arbuscular mycorrhizal symbiosis? Field Crops Res 108, 14–31. Borsani, O., Cuartero, J., Fernandez, J.A., Valpuesta, V., and Botella, M.A. (2001) Identification of two loci in tomato reveals distinct mechanisms for salt tolerance. Plant Cell 13, 873–887. Briza, J., Carroll, B.J., Klimyuk, V.I., Thomas, C.M., Jones, D.A., and Jones, J.D. (1995) Distribution of unlinked transpositions of a Ds element from a T-DNA locus on tomato chromosome 4. Genetics 141, 383–390. Burbidge, A., Grieve, T.M., Jackson, A., Thompson, A., and Taylor, I.B. (1997) Structure and expression of a cDNA encoding a putative neoxanthin cleavage enzyme (NCE) isolated from a wilt related tomato (Lycopersicon esculentum Mill.) library. J. Exp. Bot. 47, 2111–2112. Caldwell, D.G., McCallum, N., Shaw, P., Muehlbauer, G.J., Marshall, D.F., and Waugh, R. (2004) A structured mutant population for forward and reverse genetics in Barley (Hordeum vulgare L.). Plant J 40, 143–150. Cameron, K.D., Teece, M.A., and Smart, L.B. (2006) Increased accumulation of cuticular wax and expression of lipid transfer protein in response to periodic drying events in leaves of tree tobacco. Plant Physiol 140, 176–183. Cao, W.H., Liu, J., He, X.J., Mu, R.L., Zhou, H.L., Chen, S.Y., and Zhang, J.S. (2007) Modulation of ethylene responses affects plant salt-stress responses. Plant Physiol 143, 707–719. Castiglioni, P., Warner, D., Bensen, R.J., Anstrom, D.C., Harrison, J., Stoecker, M., Abad, M., Kumar, G., Salvador, S., D’Ordine, R., Navarro, S., Back, S., Fernandes, M., Targolli, J., Dasgupta, S., Bonin, C., Luethy, M.H., and Heard, J.E. (2008) Bacterial RNA chaperones confer abiotic stress tolerance in plants and improved grain yield in maize under water-limited conditions. Plant Physiol 147, 446–455. Chaves, M.M. (2003) Understanding plant responses to drought—from genes to the whole plant. Functional Plant Biology 30, 239–264. Chaves, M.M. and Oliveira, M.M. (2004) Mechanisms underlying plant resilience to water deficits: prospects for water-saving agriculture. J Exp Bot 55, 2365–2384. Chen, Z. and Gallie, D.R. (2004) The ascorbic acid redox state controls guard cell signaling and stomatal movement. Plant Cell 16, 1143–1162.

294

INTEGRATING PLANT ABIOTIC STRESS RESPONSES

Ciftci-Yilmaz, S., and Mittler, R. (2008). The zinc finger network of plants. Cell Mol Life Sci 65, 1150–1160. Close, T.J., Wanamaker, S.I., Caldo, R.A., Turner, S.M., Ashlock, D.A., Dickerson, J.A., Wing, R.A., Muehlbauer, G.J., Kleinhofs, A., and Wise, R.P. (2004) A new resource for cereal genomics: 22K barley GeneChip comes of age. Plant Physiol 134, 960–968. Colmenero-Flores, J.M., Campos, F., Garciarrubio, A., and Covarrubias, A.A. (1997) Characterization of Phaseolus vulgaris cDNA clones responsive to water deficit: identification of a novel late embryogenesis abundant-like protein. Pl Mol Biol 35, 93–405. Colmenero-Flores, J.M., Moreno, L.P., Smith, C.E., and Covarrubias, A.A. (1999) Pvlea-18, a member of a new late-embryogenesis-abundant protein family that accumulates during water stress and in the growing regions of well-irrigated bean seedlings. Plant Physiol 120, 93–104. Comai, L., Young, K., Till, B.J., Reynolds, S.H., Greene, E.A., Codomo, C.A., Enns, L.C., Johnson, J.E., Burtner, C., Odden, A.R., and Henikoff, S. (2004) Efficient discovery of DNA polymorphisms in natural populations by Ecotilling. Plant J 37, 778–786. Cooper, J.L., Till, B.J., Laport, R.G., Darlow, M.C., Kleffner, J.M., Jamai, A., El-Mellouki, T., Liu, S., Ritchie, R., Nielsen, N., Bilyeu, K.D., Meksem, K., Comai, L., and Henikoff, S. (2008) TILLING to detect induced mutations in soybean. BMC Plant Biol 8, 9. Cooper, L.D., Marquez-Cedillo, L., Singh, J., Sturbaum, A.K., Zhang, S., Edwards, V., Johnson, K., Kleinhofs, A., Rangel, S., Carollo, V., Bregitzer, P., Lemaux, P.G., and Hayes, P.M. (2004) Mapping Ds insertions in barley using a sequence-based approach. Mol Genet Genomics 272, 181–193. Cornic, G. (2000) Drought stress inhibits photosynthesis by decreasing stomatal aperture—not by affecting ATP synthesis. Trends in Plant Sci 5, 187–188. Couee, I., Sulmon, C., Gouesbet, G., and El Amrani, A. (2006). Involvement of soluble sugars in reactive oxygen species balance and responses to oxidative stress in plants. J Exp Bot 57, 449–459. Cushman, J.C. and Bohnert, H.J. (2000) Genomic approaches to plant stress tolerance. Curr Opin Plant Biol 3, 117–124. Daei, G., Ardekani, M.R., Rejali, F., Teimuri, S., and Miransari, M. (2008) Alleviation of salinity stress on wheat yield, yield components, and nutrient uptake using arbuscular mycorrhizal fungi under field conditions. J Plant Physiol. Davies, W.J., Kudoyarova, G., and Hartung, W. (2005) Long distance ABA signaling and its relation to other signaling pathways in the detection of soil drying and the mediation of the plant’s response to drought. J. Plant Growth Regulation 24, 285–295. Deak, K. and Malamy, J. (2005) Osmotic regulation of root system architecture. Plant J. 43, 17–28. Degenkolbe, T., Do, P.T., Zuther, E., Repsilber, D., Walther, D., Hincha, D.K., and Kohl, K.I. (2009) Expression profiling of rice cultivars differing in their tolerance to long-term drought stress. Plant Mol Biol 69, 133–153. del Rio, L.A., Pastori, G.M., Palma, J.M., Sandalio, L.M., Sevilla, F., Corpas, F.J., Jimenez, A., Lopez-Huertas, E., and Hernandez, J.A. (1998) The activated oxygen role of peroxisomes in senescence. Plant Physiol 116, 1195–1200. Drame, K.N., Clavel, D., Repellin, A., Passaquet, C., and Zuily-Fodil, Y. (2007) Water deficit induces variation in expression of stress-responsive genes in two peanut (Arachis hypogaea L.) cultivars with different tolerance to drought. Plant Physiol Biochem 45, 236–243. Dubouzet, J.G., Sakuma, Y., Ito, Y., Kasuga, M., Dubouzet, E.G., Miura, S., Seki, M., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2003) OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought-, high-salt- and cold-responsive gene expression. Plant J 33, 751–763. Duvick, D.N. (2005) The contribution of breeding to yield advances in maize (Zea mays L.). Adv Agron 86, 83–145. Edwards, D., Murray, J.A., and Smith, A.G. (1998) Multiple genes encoding the conserved CCAAT-box transcription factor complex are expressed in Arabidopsis. Plant Physiol 117, 1015–1022. Eltayeb, A.E., Kawano, N., Badawi, G.H., Kaminaka, H., Sanekata, T., Shibahara, T., Inanaga, S., and Tanaka, K. (2007) Overexpression of monodehydroascorbate reductase in transgenic tobacco confers enhanced tolerance to ozone, salt and polyethylene glycol stresses. Planta 225, 1255–1264. Ergen, N.Z. and Budak, H. (2009) Sequencing over 13,000 expressed sequence tags from six subtractive cDNA libraries of wild and modern wheats following slow drought stress. Plant Cell Environ 32, 220–236. Feng, G., Zhang, F.S., Li, X.L., Tian, C.Y., Tang, C., and Rengel, Z. (2002) Improved tolerance of maize plants to salt stress by arbuscular mycorrhiza is related to higher accumulation of soluble sugars in roots. Mycorrhiza 12, 185–190. Fester, T. and Hause, G. (2005) Accumulation of reactive oxygen species in arbuscular mycorrhizal roots. Mycorrhiza 15, 373–379. Fester, T. and Hause, B. (2007) Drought and symbiosis—why is abscisic acid necessary for arbuscular mycorrhiza? New Phytologist 175, 383–386. Fester, T., Schmidt, D., Lohse, S., Walter, M.H., Giuliano, G., Bramley, P.M., Fraser, P.D., Hause, B., and Strack, D. (2002) Stimulation of carotenoid metabolism in arbuscular mycorrhizal roots. Planta 216, 148–154.

DISCOVERING GENES FOR ABIOTIC STRESS TOLERANCE IN CROP PLANTS

295

Fischer, R.A. and Turner, N.C. (1978) Plant productivity in the arid and semiarid zones. Ann. Rev. of Plant Physiol 29, 277–317. Flexas, J. and Medrano, H. (2002) Drought-inhibition of photosynthesis in C3 plants: stomatal and non-stomatal limitations revisited. Ann Bot (Lond) 89, 183–189. Foolad, M.R., Zhang, L.P., and Subbiah, P. (2003) Genetics of drought tolerance during seed germination in tomato: inheritance and QTL mapping. Genome 46, 536–545. Fukao, T. and Bailey-Serres, J. (2004) Plant responses to hypoxia—is survival a balancing act? Trends Plant Sci 9, 449–456. Gay, A., Thomas, H., Roca, M., James, C., Taylor, J., Rowland, J., and Ougham, H. (2008) Nondestructive analysis of senescence in mesophyll cells by spectral resolution of protein synthesis-dependent pigment metabolism. New Phytol 179, 663–674. Ghannoum, O. (2009) C4 photosynthesis and water stress. Ann Bot (Lond) 103, 635–644. Gilchrist, E.J., Haughn, G.W., Ying, C.C., Otto, S.P., Zhuang, J., Cheung, D., Hamberger, B., Aboutorabi, F., Kalynyak, T., Johnson, L., Bohlmann, J., Ellis, B.E., Douglas, C.J., and Cronk, Q.C. (2006) Use of Ecotilling as an efficient SNP discovery tool to survey genetic variation in wild populations of Populus trichocarpa. Mol Ecol 15, 1367–1378. Giuliani, S., Sanguineti, M.C., Tuberosa, R., Bellotti, M., Salvi, S., and Landi, P. (2005) Root-ABA1, a major constitutive QTL, affects maize root architecture and leaf ABA concentration at different water regimes. J Exp Bot 56, 3061–3070. Gorantla, M., Babu, P.R., Lachagari, V.B., Reddy, A.M., Wusirika, R., Bennetzen, J.L., and Reddy, A.R. (2007) Identification of stress-responsive genes in an indica rice (Oryza sativa L.) using ESTs generated from drought-stressed seedlings. J Exp Bot 58, 253–265. Gutterson, N. and Reuber, T.L. (2004) Regulation of disease resistance pathways by AP2/ERF transcription factors. Curr Opin Plant Biol 7, 465–471. Hajheidari, M., Abdollahian-Noghabi, M., Askari, H., Heidari, M., Sadeghian, S.Y., Ober, E.S., and Salekdeh, G.H. (2005) Proteome analysis of sugar beet leaves under drought stress. Proteomics 5, 950–960. Hamilton III, E.W. and Heckathorn, S.A. (2001) Mitochondrial adaptations to NaCl. Complex I Is protected by anti-oxidants and small heat shock proteins, whereas complex II is protected by proline and betaine. Plant Physol 126, 1266–1274. Harndahl, U., Hall, R.B., Osteryoung, K.W., Vierling, E., Bornman, J.F., and Sundby, C. (1999) The chloroplast small heat shock protein undergoes oxidation-dependent conformational changes and may protect plants from oxidative stress. Cell Stress Chaperones 4, 129–138. Himelblau, E., Gilchrist, E.J., Buono, K., Bizzell, C., Mentzer, L., Vogelzang, R., Osborn, T., Amasino, R.M., Parkin, I.A., and Haughn, G.W. (2009) Forward and reverse genetics of rapid-cycling Brassica oleracea. Theor Appl Genet. Hochholdinger, F. and Tuberosa, R. (2009) Genetic and genomic dissection of maize root development and architecture. Curr Opin Plant Biol. Hochholdinger, F., Woll, K., Sauer, M., and Dembinsky, D. (2004) Genetic dissection of root formation in maize (Zea mays) reveals root-type specific developmental programmes. Ann Bot (Lond) 93, 359–368. Holmes-Davis, R., Li, G., Jamieson, A.C., Rebar, E.J., Liu, Q., Kong, Y., Case, C.C., and Gregory, P.D. (2005) Gene regulation in planta by plant-derived engineered zinc finger protein transcription factors. Plant Mol Biol 57, 411–423. Holmstrom, K.O., Somersalo, S., Mandal, A., Palva, T.E., and Welin, B. (2000) Improved tolerance to salinity and low temperature in transgenic tobacco producing glycine betaine. J Exp Bot 51, 177–185. Hsing, Y.I., Chern, C.G., Fan, M.J., Lu, P.C., Chen, K.T., Lo, S.F., Sun, P.K., Ho, S.L., Lee, K.W., Wang, Y.C., Huang, W.L., Ko, S.S., Chen, S., Chen, J.L., Chung, C.I., Lin, Y.C., Hour, A.L., Wang, Y.W., Chang, Y.C., Tsai, M.W., Lin, Y.S., Chen, Y.C., Yen, H.M., Li, C.P., Wey, C.K., Tseng, C.S., Lai, M.H., Huang, S.C., Chen, L.J., and Yu, S.M. (2007) A rice gene activation/knockout mutant resource for high throughput functional genomics. Plant Mol Biol 63, 351–364. Huang, J., Wang, M.M., Jiang, Y., Bao, Y.M., Huang, X., Sun, H., Xu, D.Q., Lan, H.X., and Zhang, H.S. (2008) Expression analysis of rice A20/AN1-type zinc finger genes and characterization of ZFP177 that contributes to temperature stress tolerance. Gene 420, 135–144. Ibrahim, A.F., Hedley, P.E., Cardle, L., Kruger, W., Marshall, D.F., Muehlbauer, G.J., and Waugh, R. (2005) A comparative analysis of transcript abundance using SAGE and Affymetrix arrays. Funct Integr Genomics 5, 163–174. Iuchi, S., Kobayashi, M., Taji, T., Naramoto, M., Seki, M., Kato, T., Tabata, S., Kakubari, Y., Yamaguchi-Shinozaki, K., and Shinozaki, K. (2001) Regulation of drought tolerance by gene manipulation of 9-cis-epoxycarotenoid dioxygenase, a key enzyme in abscisic acid biosynthesis in Arabidopsis. Plant J 27, 325–333. Jang, C.S., Lee, H.J., Chang, S.J., and Seo, Y.W. (2004) Expression and promoter analysis of the TaLTP1 gene induced by drought and salt stress in wheat (Triticum aestivum L.). Plant Sci 167, 995–1001. Jia, J., Fu, J., Zheng, J., Zhou, X., Huai, J., Wang, J., Wang, M., Zhang, Y., Chen, X., Zhang, J., Zhao, J., Su, Z., Lv, Y., and Wang, G. (2006) Annotation and expression profile analysis of 2073 full-length cDNAs from stress-induced maize (Zea mays L.) seedlings. Plant J 48, 710–727.

296

INTEGRATING PLANT ABIOTIC STRESS RESPONSES

Jiang, C., Gao, X., Liao, L., Harberd, N.P., and Fu, X. (2007) Phosphate starvation root architecture and anthocyanin accumulation responses are modulated by the gibberellin-DELLA signaling pathway in Arabidopsis. Plant Physiol 145, 1460–1470. Jiang, S.Y., Ramamoorthy, R., Bhalla, R., Luan, H.F., Venkatesh, P.N., Cai, M., and Ramachandran, S. (2008) Genome-wide survey of the RIP domain family in Oryza sativa and their expression profiles under various abiotic and biotic stresses. Plant Mol Biol 67, 603–614. Jithesh, M.N., Prashanth, S.R., Sivaprakash, K.R., and Parida, A.K. (2006) Antioxidative response mechanisms in halophytes: their role in stress defence. J Genet 85, 237–254. John, U.P. and Spangenberg, G.C. (2005). Xenogenomics: Genomic Bioprospecting in Indigenous and Exotic Plants Through EST Discovery, cDNA Microarray-Based Expression Profiling and Functional Genomics. Comp Funct Genomics 6, 230–235. Kacperska, A. (2004) Sensor types in signal transduction pathways in plant cells responding to abiotic stressors: do they depend on stress intensity? Physiol Plant 122, 159–168. Kader, J.C. (1996) Lipid-Transfer Proteins in Plants. Annu Rev Plant Physiol Plant Mol Biol 47, 627–654. Kaeppler, S.M., Parke, J.L., Mueller, S.M., Senior, L., Stuber, C., and Tracy, W.F. (2000) Variation among maize inbred lines and detection of quantitative trait loci for growth at low phosphorus and responsiveness to arbuscular mycorrhizal fungi. Crop Sci 40, 358–364. Kapoor, M., Arora, R., Lama, T., Nijhawan, A., Khurana, J.P., Tyagi, A.K., and Kapoor, S. (2008) Genome-wide identification, organization and phylogenetic analysis of Dicer-like, Argonaute and RNA-dependent RNA Polymerase gene families and their expression analysis during reproductive development and stress in rice. BMC Genomics 9, 451. Karaba, A., Dixit, S., Greco, R., Aharoni, A., Trijatmiko, K.R., Marsch-Martinez, N., Krishnan, A., Nataraja, K.N., Udayakumar, M., and Pereira, A. (2007) Improvement of water use efficiency in rice by expression of HARDY, an Arabidopsis drought and salt tolerance gene. Proc Natl Acad Sci U S A 104, 15270–15275. Klein, M., Geisler, M., Suh, S.J., Kolukisaoglu, H.U., Azevedo, L., Plaza, S., Curtis, M.D., Richter, A., Weder, B., Schulz, B., and Martinoia, E. (2004) Disruption of AtMRP4, a guard cell plasma membrane ABCC-type ABC transporter, leads to deregulation of stomatal opening and increased drought susceptibility. Plant J 39, 219–236. Knapp, S., Larondelle, Y., Rossberg, M., Furtek, D., and Theres, K. (1994) Transgenic tomato lines containing Ds elements at defined genomic positions as tools for targeted transposon tagging. Mol Gen Genet 243, 666–673. Knight, H. and Knight, M.R. (2001) Abiotic stress signalling pathways: specificity and cross-talk. Trends Plant Sci 6, 262–267. Kolkman, J.M., Conrad, L.J., Farmer, P.R., Hardeman, K., Ahern, K.R., Lewis, P.E., Sawers, R.J., Lebejko, S., Chomet, P., and Brutnell, T.P. (2005) Distribution of Activator (Ac) throughout the maize genome for use in regional mutagenesis. Genetics 169, 981–995. Kollipara, K.P., Saab, I.N., Wych, R.D., Lauer, M.J., and Singletary, G.W. (2002) Expression profiling of reciprocal maize hybrids divergent for cold germination and desiccation tolerance. Plant Physiol 129, 974–992. Komatsu, S. (2008) Plasma membrane proteome in Arabidopsis and rice. Proteomics 8, 4137–4145. Kore-eda, S., Cushman, M.A., Akselrod, I., Bufford, D., Fredrickson, M., Clark, E., and Cushman, J.C. (2004) Transcript profiling of salinity stress responses by large-scale expressed sequence tag analysis in Mesembryanthemum crystallinum. Gene 341, 83–92. Kunst, L. and Samuels, A.L. (2003) Biosynthesis and secretion of plant cuticular wax. Prog Lipid Res 42, 51–80. Lanceras, J.C., Pantuwan, G., Jongdee, B., and Toojinda, T. (2004) Quantitative trait loci associated with drought tolerance at reproductive stage in rice. Plant Physiol 135, 384–399. Laporte, M.M., Shen, B., and Tarczynski, M.C. (2002) Engineering for drought avoidance: expression of maize NADP-malic enzyme in tobacco results in altered stomatal function. J Exp Bot 53, 699–705. Larkindale, J. and Knight, M.R. (2002) Protection against heat stress induced oxidative damage in Arabidopsis involves calcium, abscisic acid, ethylene, and salicylic acid. Plant Physiol 128, 682–695. Larkindale, J. and Huang, B. (2004) Thermotolerance and antioxidant systems in Agrostis stolonifera: involvement of salicylic acid, abscisic acid, calcium, hydrogen peroxide, and ethylene. J Plant Physiol 161, 405–413. Lequeu, J., Fauconnier, M.L., Chammai, A., Bronner, R., and Blee, E. (2003) Formation of plant cuticle: evidence for the occurrence of the peroxygenase pathway. Plant J 36, 155–164. Lexer, C., Lai, Z., and Rieseberg, L.H. (2004) Candidate gene polymorphisms associated with salt tolerance in wild sunflower hybrids: implications for the origin of Helianthus paradoxus, a diploid hybrid species. New Phytol 161, 225–233. Li, X.P., Tian, A.G., Luo, G.Z., Gong, Z.Z., Zhang, J.S., and Chen, S.Y. (2005) Soybean DRE-binding transcription factors that are responsive to abiotic stresses. Theor Appl Genet 110, 1355–1362. Lian, H.L., Yu, X., Ye, Q., Ding, X., Kitagawa, Y., Kwak, S.S., Su, W.A., and Tang, Z.C. (2004) The role of aquaporin RWC3 in drought avoidance in rice. Plant Cell Physiol 45, 481–489.

DISCOVERING GENES FOR ABIOTIC STRESS TOLERANCE IN CROP PLANTS

297

Ling, J., Wells, D.R., Tanguay, R.L., Dickey, L.F., Thompson, W.F., and Gallie, D.R. (2000) Heat shock protein HSP101 binds to the Fed-1 internal light regulator y element and mediates its high translational activity. Plant Cell 12, 1213– 1227. Liu, J. and Zhu, J.K. (1997) Proline accumulation and salt-stress-induced gene expression in a salt-hypersensitive mutant of Arabidopsis. Plant Physiol 114, 591–596. Liu, Q., Kasuga, M., Sakuma, Y., Abe, H., Miura, S., Yamaguchi-Shinozaki, K., and Shinozaki, K. (1998) Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell 10, 1391– 1406. Liu, Y., Lamkemeyer, T., Jakob, A., Mi, G., Zhang, F., Nordheim, A., and Hochholdinger, F. (2006) Comparative proteome analyzes of maize (Zea mays L.) primary roots prior to lateral root initiation reveal differential protein expression in the lateral root initiation mutant rum1. Proteomics 6, 4300–4308. Luhua, S., Ciftci-Yilmaz, S., Harper, J., Cushman, J., and Mittler, R. (2008) Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants expressing proteins of unknown function. Plant Physiol 148, 280–292. Lunde, C.F., Morrow, D.J., Roy, L.M., and Walbot, V. (2003) Progress in maize gene discovery: a project update. Funct Integr Genomics 3, 25–32. Luthje, S., Hopff, D., Schmitt, A., Meisrimler, C.N., and Menckhoff, L. (2008) Hunting for low abundant redox proteins in plant plasma membranes. J Proteomics. Lynch, J.P. (2007) Roots of the second Green Revolution. Austr J Bot 55, 493–512. Malik, M.K., Slovin, J.P., Hwang, C.H., and Zimmerman, J.L. (1999) Modified expression of a carrot small heat shock protein gene, hsp17. 7, results in increased or decreased thermotolerance double dagger. Plant J 20, 89–99. Mantri, N.L., Ford, R., Coram, T.E., and Pang, E.C. (2007) Transcriptional profiling of chickpea genes differentially regulated in response to high-salinity, cold and drought. BMC Genomics 8, 303. May, B.P., Liu, H., Vollbrecht, E., Senior, L., Rabinowicz, P.D., Roh, D., Pan, X., Stein, L., Freeling, M., Alexander, D., and Martienssen, R. (2003) Maize-targeted mutagenesis: A knockout resource for maize. Proc Natl Acad Sci USA 100, 11541–11546. Meissner, R., Chague, V., Zhu, Q., Emmanuel, E., Elkind, Y., and Levy, A.A. (2000) Technical advance: a high throughput system for transposon tagging and promoter trapping in tomato. Plant J 22, 265–274. Menda, N., Semel, Y., Peled, D., Eshed, Y., and Zamir, D. (2004) In silico screening of a saturated mutation library of tomato. Plant J 38, 861–872. Morgan, P.W., He, C.J., De Greef, J.A., and De Proft, M.P. (1990) Does Water Deficit Stress Promote Ethylene Synthesis by Intact Plants? Plant Physiol 94, 1616–1624. Mullen, J.L. and Hangarter, R.P. (2003) Genetic analysis of the gravitropic set-point angle in lateral roots of Arabidopsis. Adv Space Res 31, 2229–2236. Munns, R. (2005) Genes and salt tolerance: bringing them together. New Phytol 167, 645–663. Munns, R., James, R.A., and Lauchli, A. (2006) Approaches to increasing the salt tolerance of wheat and other cereals. J Exp Bot 57, 1025–1043. Murakami, T., Matsuba, S., Funatsuki, H., Kawaguchi, K., Saruyama, H., Tanida, M., and Sato, Y. (2004) Over-expression of a small heat shock protein, sHSP17.7, confers both heat tolerance and UV-B resistance to rice plants. Molec Breeding 13, 165–175. Myouga, F., Hosoda, C., Umezawa, T., Iizumi, H., Kuromori, T., Motohashi, R., Shono, Y., Nagata, N., Ikeuchi, M., and Shinozaki, K. (2008) A Heterocomplex of Iron Superoxide Dismutases Defends Chloroplast Nucleoids against Oxidative Stress and Is Essential for Chloroplast Development in Arabidopsis. Plant Cell 20, 3148–3162. Narendra, S. (2005) The subcellular localization and function of APX3 in Arabidopsis. In Biology (Lubbock, TX: Texas Tech University), pp. 151. Neill, S., Barros, R., Bright, J., Desikan, R., Hancock, J., Harrison, J., Morris, P., Ribeiro, D., and Wilson, I. (2008) Nitric oxide, stomatal closure, and abiotic stress. J Exp Bot 59, 165–176. Nelson, D.E., Repetti, P.P., Adams, T.R., Creelman, R.A., Wu, J., Warner, D.C., Anstrom, D.C., Bensen, R.J., Castiglioni, P.P., Donnarummo, M.G., Hinchey, B.S., Kumimoto, R.W., Maszle, D.R., Canales, R.D., Krolikowski, K.A., Dotson, S.B., Gutterson, N., Ratcliffe, O.J., and Heard, J.E. (2007) Plant nuclear factor Y (NF-Y) B subunits confer drought tolerance and lead to improved corn yields on water-limited acres. Proc Natl Acad Sci USA 104, 16450–16455. Neuffer, M.G., Coe, E.H., and Wessler, S.R. (1997) Mutants of maize. (Cold Spring Harbor: Cold Spring Harbor Press). O’Rourke, J.A., Graham, M.A., Vodkin, L., Gonzalez, D.O., Cianzio, S.R., and Shoemaker, R.C. (2007) Recovering from iron deficiency chlorosis in near-isogenic soybeans: a microarray study. Plant Physiol Biochem 45, 287–292. Ogawa, A. and Yamauchi, C. (2006) Root osmotic adjustment under osmotic stress in maize seedlings. 2. Mode of accumulation of several solutes for osmotic adjustment in roots. Plant Prod Sci 9, 39–46.

298

INTEGRATING PLANT ABIOTIC STRESS RESPONSES

Ogo, Y., Kobayashi, T., Nakanishi Itai, R., Nakanishi, H., Kakei, Y., Takahashi, M., Toki, S., Mori, S., and Nishizawa, N.K. (2008) A novel NAC transcription factor, IDEF2, that recognizes the iron deficiency-responsive element 2 regulates the genes involved in iron homeostasis in plants. J Biol Chem 283, 13407–13417. Oh, S.J., Kwon, C.W., Choi, D.W., Song, S.I., and Kim, J.K. (2007) Expression of barley HvCBF4 enhances tolerance to abiotic stress in transgenic rice. Plant Biotechnol J 5, 646–656. Oh, S.J., Song, S.I., Kim, Y.S., Jang, H.J., Kim, S.Y., Kim, M., Kim, Y.K., Nahm, B.H., and Kim, J.K. (2005) Arabidopsis CBF3/ DREB1A and ABF3 in transgenic rice increased tolerance to abiotic stress without stunting growth. Plant Physiol 138, 341–351. Omokawa, H. and Aonuma, S. (2002) Amelioration of the salt-stressed root growth of rice and normalization of the Na+ distribution between the shoot and root by (S)-alpha-methylbenzyl-2-fluoro-4-methylphenylurea. Biosci Biotechnol Biochem 66, 336–343. Overmyer, K., Brosche, M., and Kangasjarvi, J. (2003) Reactive oxygen species and hormonal control of cell death. Trends Plant Sci 8, 335–342. Papdi, C., Abraham, E., Joseph, M.P., Popescu, C., Koncz, C., and Szabados, L. (2008) Functional identification of Arabidopsis stress regulatory genes using the controlled cDNA overexpression system. Plant Physiol 147, 528–542. Park, E.J., Jeknic, Z., Sakamoto, A., DeNoma, J., Yuwansiri, R., Murata, N., and Chen, T.H. (2004) Genetic engineering of glycinebetaine synthesis in tomato protects seeds, plants, and flowers from chilling damage. Plant J 40, 474–487. Park, S., Li, J., Pittman, J.K., Berkowitz, G.A., Yang, H., Undurraga, S., Morris, J., Hirschi, K.D., and Gaxiola, R.A. (2005) Up-regulation of a H+-pyrophosphatase (H+-PPase) as a strategy to engineer drought-resistant crop plants. Proc Natl Acad Sci USA 102, 18830–18835. Park, S.M. and C.B., H. (2002) Class I small heat-shock protein gives thermotolerance in tobacco. J Plant Physiol 159, 25–30. Park, S.Y., Yu, J.W., Park, J.S., Li, J., Yoo, S.C., Lee, N.Y., Lee, S.K., Jeong, S.W., Seo, H.S., Koh, H.J., Jeon, J.S., Park, Y.I., and Paek, N.C. (2007) The senescence-induced staygreen protein regulates chlorophyll degradation. Plant Cell 19, 1649–1664. Pellegrineschi, A., Reynolds, M., Pacheco, M., Brito, R.M., Almeraya, R., Yamaguchi-Shinozaki, K., and Hoisington, D. (2004) Stress-induced expression in wheat of the Arabidopsis thaliana DREB1A gene delays water stress symptoms under greenhouse conditions. Genome 47, 493–500. Phillips, J.R., Dalmay, T., and Bartels, D. (2007) The role of small RNAs in abiotic stress. FEBS Lett 581, 3592–3597. Pitzschke, A., Forzani, C., and Hirt, H. (2006) Reactive oxygen species signaling in plants. Antioxid Redox Signal 8, 1757–1764. Plomion, C., Lalanne, C., Claverol, S., Meddour, H., Kohler, A., Bogeat-Triboulot, M.B., Barre, A., Le Provost, G., Dumazet, H., Jacob, D., Bastien, C., Dreyer, E., de Daruvar, A., Guehl, J.M., Schmitter, J.M., Martin, F., and Bonneu, M. (2006) Mapping the proteome of poplar and application to the discovery of drought-stress responsive proteins. Proteomics 6, 6509–6527. Poroyko, V., Spollen, W.G., Hejlek, L.G., Hernandez, A.G., LeNoble, M.E., Davis, G., Nguyen, H.T., Springer, G.K., Sharp, R.E., and Bohnert, H.J. (2007) Comparing regional transcript profiles from maize primary roots under well-watered and low water potential conditions. J Exp Bot 58, 279–289. Price, A.H., Townend, J., Jones, M.P., Audebert, A., and Courtois, B. (2002) Mapping QTLs associated with drought avoidance in upland rice grown in the Philippines and West Africa. Plant Mol Biol 48, 683–695. Prieto-Dapena, P., Castano, R., Almoguera, C., and Jordano, J. (2008) The ectopic overexpression of a seed-specific transcription factor, HaHSFA9, confers tolerance to severe dehydration in vegetative organs. Plant J 54, 1004–1014. Proite, K., Leal-Bertioli, S.C., Bertioli, D.J., Moretzsohn, M.C., da Silva, F.R., Martins, N.F., and Guimaraes, P.M. (2007) ESTs from a wild Arachis species for gene discovery and marker development. BMC Plant Biol 7, 7. Qin, F., Kakimoto, M., Sakuma, Y., Maruyama, K., Osakabe, Y., Tran, L.S., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2007) Regulation and functional analysis of ZmDREB2A in response to drought and heat stresses in Zea mays L. Plant J 50, 54–69. Qu, S., Desai, A., Wing, R., and Sundaresan, V. (2008) A versatile transposon-based activation tag vector system for functional genomics in cereals and other monocot plants. Plant Physiol 146, 189–199. Quan, R., Shang, M., Zhang, H., Zhao, Y., and Zhang, J. (2004) Engineering of enhanced glycine betaine synthesis improves drought tolerance in maize. Plant Biotechnol J 2, 477–486. Quesada, V., Ponce, M.R., and Micol, J.L. (2000) Genetic analysis of salt-tolerant mutants in Arabidopsis thaliana. Genetics 154, 421–436. Rai, S.P., Luthra, R., and Kumar, S. (2003) Salt-tolerant mutants in glycophytic salinity response (GSR) genes in Catharanthus roseus. Theor Appl Genet 106, 221–230.

DISCOVERING GENES FOR ABIOTIC STRESS TOLERANCE IN CROP PLANTS

299

Ramachandra Reddy, A., Chaitanya, K.V., and Vivekanandan, M. (2004) Drought-induced responses of photosynthesis and antioxidant metabolism in higher plants. J Plant Physiol 161, 1189–1202. Ramalingam, J., Pathan, M.S., Feril, O., Ross, K., Ma, X.F., Mahmoud, A.A., Layton, J., Rodriguez-Milla, M.A., Chikmawati, T., Valliyodan, B., Skinner, R., Matthews, D.E., Gustafson, J.P., and Nguyen, H.T. (2006) Structural and functional analyzes of the wheat genomes based on expressed sequence tags (ESTs) related to abiotic stresses. Genome 49, 1324–1340. Rios, G., Naranjo, M.A., Iglesias, D.J., Ruiz-Rivero, O., Geraud, M., Usach, A., and Talon, M. (2008) Characterization of hemizygous deletions in citrus using array-comparative genomic hybridization and microsynteny comparisons with the poplar genome. BMC Genomics 9, 381. Rivero, R.M., Kojima, M., Gepstein, A., Sakakibara, H., Mittler, R., Gepstein, S., and Blumwald, E. (2007) Delayed leaf senescence induces extreme drought tolerance in a flowering plant. Proc Natl Acad Sci USA 104, 19631–19636. Rothan, C. and Causse, M. (2007) Natural and artificially induced genetic variability in crop and model plant species for plant systems biology. Exs 97, 21–53. Rouhier, N., Villarejo, A., Srivastava, M., Gelhaye, E., Keech, O., Droux, M., Finkemeier, I., Samuelsson, G., Dietz, K.J., Jacquot, J.P., and Wingsle, G. (2005) Identification of plant glutaredoxin targets. Antioxid Redox Signal 7, 919–929. Sahi, C., Singh, A., Kumar, K., Blumwald, E., and Grover, A. (2006) Salt stress response in rice: genetics, molecular biology, and comparative genomics. Funct Integr Genomics 6, 263–284. Sakamoto, A. and Murata, N. (2002) The role of glycine betaine in the protection of plants from stress: clues from transgenic plants. Plant Cell Environ 25, 163–171. Sakurai, T., Plata, G., Rodriguez-Zapata, F., Seki, M., Salcedo, A., Toyoda, A., Ishiwata, A., Tohme, J., Sakaki, Y., Shinozaki, K., and Ishitani, M. (2007) Sequencing analysis of 20,000 full-length cDNA clones from cassava reveals lineage specific expansions in gene families related to stress response. BMC Plant Biol 7, 66. Salt, D.E., Baxter, I., and Lahner, B. (2008) Ionomics and the study of the plant ionome. Annu Rev Plant Biol 59, 709–733. Salvucci, M.E. (2008) Association of Rubisco activase with chaperonin-60beta: a possible mechanism for protecting photosynthesis during heat stress. J Exp Bot 59, 1923–1933. Samuels, L., Kunst, L., and Jetter, R. (2008) Sealing plant surfaces: cuticular wax formation by epidermal cells. Annu Rev Plant Biol 59, 683–707. Sanchez-Ballesta, M.T., Lluch, Y., Gosalbes, M.J., Zacarias, L., Granell, A., and Lafuente, M.T. (2003) A survey of genes differentially expressed during long-term heat-induced chilling tolerance in citrus fruit. Planta 218, 65–70. Saneoka, H., Nagasaka, C., Hahn, D.T., Yang, W.J., Premachandra, G.S., Joly, R.J., and Rhodes, D. (1995). Salt Tolerance of Glycinebetaine-Deficient and -Containing Maize Lines. Plant Physiol 107, 631–638. Sang, Y., Zheng, S., Li, W., Huang, B., and Wang, X. (2001). Regulation of plant water loss by manipulating the expression of phospholipase Dalpha. Plant J 28, 135–144. Sanmiya, K., Suzuki, K., Egawa, Y., and Shono, M. (2004). Mitochondrial small heat-shock protein enhances thermotolerance in tobacco plants. FEBS Lett 557, 265–268. Seong, E.S., Guo, J., Kim, Y.H., Cho, J.H., Lim, C.K., Hyun Hur, J., and Wang, M.H. (2007) Regulations of marker genes involved in biotic and abiotic stress by overexpression of the AtNDPK2 gene in rice. Biochem Biophys Res Commun 363, 126–132. Serraj, R. and Sinclair, T.R. (2002) Osmolyte accumulation: can it really help increase crop yield under drought conditions? Plant, Cell and Env 25, 333–341. Settles, A.M., Holding, D.R., Tan, B.C., Latshaw, S.P., Liu, J., Suzuki, M., Li, L., O’Brien, B.A., Fajardo, D.S., Wroclawska, E., Tseung, C.W., Lai, J., Hunter, C.T., 3rd, Avigne, W.T., Baier, J., Messing, J., Hannah, L.C., Koch, K.E., Becraft, P.W., Larkins, B.A., and McCarty, D.R. (2007) Sequence-indexed mutations in maize using the Uniform Mu transposon-tagging population. BMC Genomics 8, 116. Shao, H.B., Chu, L.Y., Shao, M.A., Jaleel, C.A., and Mi, H.M. (2008) Higher plant antioxidants and redox signaling under environmental stresses. C R Biol 331, 433–441. Sharp, R.E. (2002) Interaction with ethylene: changing views on the role of abscisic acid in root and shoot growth responses to water stress. Plant Cell Environ 25, 211–222. Sharp, R.E., Poroyko, V., Hejlek, L.G., Spollen, W.G., Springer, G.K., Bohnert, H.J., and Nguyen, H.T. (2004) Root growth maintenance during water deficits: physiology to functional genomics. J Exp Bot 55, 2343–2351. Shen, Y.G., Zhang, W.K., He, S.J., Zhang, J.S., Liu, Q., and Chen, S.Y. (2003) An EREBP/AP2-type protein in Triticum aestivum was a DRE-binding transcription factor induced by cold, dehydration and ABA stress. Theor Appl Genet 106, 923–930. Sheng, M., Tang, M., Chen, H., Yang, B., Zhang, F., and Huang, Y. (2008) Influence of arbuscular mycorrhizae on photosynthesis and water status of maize plants under salt stress. Mycorrhiza 18, 287–296. Shepherd, T. and Wynne Griffiths, D. (2006) The effects of stress on plant cuticular waxes. New Phytol 171, 469–499. Shinozaki, K. and Yamaguchi-Shinozaki, K. (2007) Gene networks involved in drought stress response and tolerance. J Exp Bot 58, 221–227.

300

INTEGRATING PLANT ABIOTIC STRESS RESPONSES

Singh, J., Zhang, S., Chen, C., Cooper, L., Bregitzer, P., Sturbaum, A., Hayes, P.M., and Lemaux, P.G. (2006) High-frequency Ds remobilization over multiple generations in barley facilitates gene tagging in large genome cereals. Plant Mol Biol 62, 937–950. Slade, A.J., Fuerstenberg, S.I., Loeffler, D., Steine, M.N., and Facciotti, D. (2005) A reverse genetic, nontransgenic approach to wheat crop improvement by TILLING. Nat Biotechnol 23, 75–81. S´lesak, I., Libik, M., Karpinska, B., and Miszalski, Z. (2007) The role of hydrogen peroxide in regulation of plant metabolism and cellular signalling in response to environmental stresses. Acta Biochimica Polonica 1, 39–50. Sturaro, M., Hartings, H., Schmelzer, E., Velasco, R., Salamini, F., and Motto, M. (2005) Cloning and characterization of GLOSSY1, a maize gene involved in cuticle membrane and wax production. Plant Physiol 138, 478–489. Subramanian, K.S. and Charest, C. (1999) Acquisition of N by external hyphae of an arbuscular mycorrhizal fungus and its impact on physiological responses in maize under drought-stressed and well-watered conditions. Mycorrhiza 9, 69–75. Sung, D.Y., Kaplan, F., Lee, K.J., and Guy, C.L. (2003) Acquired tolerance to temperature extremes. Trends Plant Sci 8, 179–187. Sunkar, R., Chinnusamy, V., Zhu, J., and Zhu, J.K. (2007) Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. Trends Plant Sci 12, 301–309. Sunkar, R., Zhou, X., Zheng, Y., Zhang, W., and Zhu, J.K. (2008) Identification of novel and candidate miRNAs in rice by high throughput sequencing. BMC Plant Biol 8, 25. Suprunova, T., Krugman, T., Distelfeld, A., Fahima, T., Nevo, E., and Korol, A. (2007) Identification of a novel gene (Hsdr4) involved in water-stress tolerance in wild barley. Plant Mol Biol 64, 17–34. Suzuki, N. and Mittler, R. (2006) Reactive oxygen species and temperature stresses: a delicate balance between signaling and destruction. Physiologia Plantarum 126, 45–51. Suzuki, T., Eiguchi, M., Kumamaru, T., Satoh, H., Matsusaka, H., Moriguchi, K., Nagato, Y., and Kurata, N. (2008) MNU-induced mutant pools and high performance TILLING enable finding of any gene mutation in rice. Mol Genet Genomics 279, 213–223. Swindell, W.R., Huebner, M., and Weber, A.P. (2007) Transcriptional profiling of Arabidopsis heat shock proteins and transcription factors reveals extensive overlap between heat and non-heat stress response pathways. BMC Genomics 8, 125. Tacke, E., Korfhage, C., Michel, D., Maddaloni, M., Motto, M., Lanzini, S., Salamini, F., and Doring, H.P. (1995) Transposon tagging of the maize Glossy2 locus with the transposable element En/Spm. Plant J 8, 907–917. Tang, D., Christiansen, K.M., and Innes, R.W. (2005) Regulation of plant disease resistance, stress responses, cell death, and ethylene signaling in Arabidopsis by the EDR1 protein kinase. Plant Physiol 138, 1018–1026. Thomas, H. and Howarth, C.J. (2000) Five ways to stay green. J Exp Bot 51 Spec No, 329–337. Till, B.J., Cooper, J., Tai, T.H., Colowit, P., Greene, E.A., Henikoff, S., and Comai, L. (2007) Discovery of chemically induced mutations in rice by TILLING. BMC Plant Biol 7, 19. Till, B.J., Reynolds, S.H., Weil, C., Springer, N., Burtner, C., Young, K., Bowers, E., Codomo, C.A., Enns, L.C., Odden, A.R., Greene, E.A., Comai, L., and Henikoff, S. (2004) Discovery of induced point mutations in maize genes by TILLING. BMC Plant Biol 4, 12. Timperio, A.M., Egidi, M.G., and Zolla, L. (2008) Proteomics applied on plant abiotic stresses: role of heat shock proteins (HSP). J Proteomics 71, 391–411. Tommasini, L., Svensson, J.T., Rodriguez, E.M., Wahid, A., Malatrasi, M., Kato, K., Wanamaker, S., Resnik, J., and Close, T.J. (2008) Dehydrin gene expression provides an indicator of low temperature and drought stress: transcriptome-based analysis of barley (Hordeum vulgare L.). Funct Integr Genomics 8, 387–405. Trujillo, L.E., Sotolongo, M., Menendez, C., Ochogavia, M.E., Coll, Y., Hernandez, I., Borras-Hidalgo, O., Thomma, B.P., Vera, P., and Hernandez, L. (2008) SodERF3, a novel sugarcane ethylene responsive factor (ERF), enhances salt and drought tolerance when overexpressed in tobacco plants. Plant Cell Physiol 49, 512–525. Tuberosa, R. and Salvi, S. (2006) Genomics-based approaches to improve drought tolerance of crops. Trends Plant Sci 11, 405–412. Tuteja, N. (2007) Mechanisms of high salinity tolerance in plants. Methods Enzymol 428, 419–438. Ueda, A., Kathiresan, A., Inada, M., Narita, Y., Nakamura, T., Shi, W., Takabe, T., and Bennett, J. (2004) Osmotic stress in barley regulates expression of a different set of genes than salt stress does. J Exp Bot 55, 2213–2218. Umezawa, T., Fujita, M., Fujita, Y., Yamaguchi-Shinozaki, K., and Shinozaki, K. (2006) Engineering drought tolerance in plants: discovering and tailoring genes to unlock the future. Curr Opin Biotechnol 17, 113–122. Van Breusegem, F., Slooten, L., Stassart, J.M., Moens, T., Botterman, J., Van Montagu, M., and Inze, D. (1999) Overproduction of Arabidopsis thaliana FeSOD confers oxidative stress tolerance to transgenic maize. Plant Cell Physiol 40, 515–523. van der Heide, T. and Poolman, B. (2000) Osmoregulated ABC-transport system of Lactococcus lactis senses water stress via changes in the physical state of the membrane. Proc Natl Acad Sci USA 97, 7102–7106. Verbruggen, N. and Hermans, C. (2008) Proline accumulation in plants: a review. Amino Acids 35, 753–759.

DISCOVERING GENES FOR ABIOTIC STRESS TOLERANCE IN CROP PLANTS

301

Walter, M.H., Floss, D.S., Hans, J., Fester, T., and Strack, D. (2007) Apocarotenoid biosynthesis in arbuscular mycorrhizal roots: contributions from methylerythritol phosphate pathway isogenes and tools for its manipulation. Phytochemistry 68, 130–138. Wang, C.R., Yang, A.F., Yue, G.D., Gao, Q., Yin, H.Y., and Zhang, J.R. (2008a) Enhanced expression of phospholipase C 1 (ZmPLC1) improves drought tolerance in transgenic maize. Planta 227, 1127–1140. Wang, F.Z., Wang, Q.B., Kwon, S.Y., Kwak, S.S., and Su, W.A. (2005a) Enhanced drought tolerance of transgenic rice plants expressing a pea manganese superoxide dismutase. J Plant Physiol 162, 465–472. Wang, J., Zhang, H., and Allen, R.D. (1999) Overexpression of an Arabidopsis peroxisomal ascorbate peroxidase gene in tobacco increases protection against oxidative stress. Plant Cell Physiol 40, 725–732. Wang, N., Wang, Y., Tian, F., King, G.J., Zhang, C., Long, Y., Shi, L., and Meng, J. (2008b). A functional genomics resource for Brassica napus: development of an EMS mutagenized population and discovery of FAE1 point mutations by TILLING. New Phytol 180, 751–765. Wang, Y., Wisniewski, M., Meilan, R., Cui, M., Webb, R., and Fuchigami, L. (2005b) Overexpression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling and salt stress. J Am Soc Hort Sci 130, 167–173. Wei, W., Zhang, Y., Han, L., Guan, Z., and Chai, T. (2008) A novel WRKY transcriptional factor from Thlaspi caerulescens negatively regulates the osmotic stress tolerance of transgenic tobacco. Plant Cell Rep 27, 795–803. Wi, S.J., Kim, W.T., and Park, K.Y. (2006) Overexpression of carnation S-adenosylmethionine decarboxylase gene generates a broad-spectrum tolerance to abiotic stresses in transgenic tobacco plants. Plant Cell Rep 25, 1111–1121. Wingler, A. and Roitsch, T. (2008) Metabolic regulation of leaf senescence: interactions of sugar signalling with biotic and abiotic stress responses. Plant Biol (Stuttg) 10 Suppl 1, 50–62. Witcombe, J.R., Hollington, P.A., Howarth, C.J., Reader, S., and Steele, K.A. (2008) Breeding for abiotic stresses for sustainable agriculture. Philos Trans R Soc Lond B Biol Sci 363, 703–716. Wu, K.L., Guo, Z.J., Wang, H.H., and Li, J. (2005) The WRKY family of transcription factors in rice and Arabidopsis and their origins. DNA Res 12, 9–26. Wu, X., Shiroto, Y., Kishitani, S., Ito, Y., and Toriyama, K. (2009) Enhanced heat and drought tolerance in transgenic rice seedlings overexpressing OsWRKY11 under the control of HSP101 promoter. Plant Cell Rep 28, 21–30. Xiao, B., Huang, Y., Tang, N., and Xiong, L. (2007) Over-expression of a LEA gene in rice improves drought resistance under the field conditions. Theor Appl Genet 115, 35–46. Xin, Z., Wang, M.L., Barkley, N.A., Burow, G., Franks, C., Pederson, G., and Burke, J. (2008) Applying genotyping (TILLING) and phenotyping analyzes to elucidate gene function in a chemically induced sorghum mutant population. BMC Plant Biol 8, 103. Xu, D.Q., Huang, J., Guo, S.Q., Yang, X., Bao, Y.M., Tang, H.J., and Zhang, H.S. (2008) Overexpression of a TFIIIA-type zinc finger protein gene ZFP252 enhances drought and salt tolerance in rice (Oryza sativa L.). FEBS Lett 582, 1037–1043. Xu, Z.S., Ni, Z.Y., Li, Z.Y., Li, L.C., Chen, M., Gao, D.Y., Yu, X.D., Liu, P., and Ma, Y.Z. (2009) Isolation and functional characterization of HvDREB1-a gene encoding a dehydration-responsive element binding protein in Hordeum vulgare. J Plant Res 122, 121–130. Xu, Z.S., Xia, L.Q., Chen, M., Cheng, X.G., Zhang, R.Y., Li, L.C., Zhao, Y.X., Lu, Y., Ni, Z.Y., Liu, L., Qiu, Z.G., and Ma, Y.Z. (2007) Isolation and molecular characterization of the Triticum aestivum L. ethylene-responsive factor 1 (TaERF1) that increases multiple stress tolerance. Plant Mol Biol 65, 719–732. Yamaguchi-Shinozaki, K. and Shinozaki, K. (2001) Improving plant drought, salt and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Novartis Found Symp 236, 176–186; discussion 186–179. Yan, J., He, C., Wang, J., Mao, Z., Holaday, S.A., Allen, R.D., and Zhang, H. (2004) Overexpression of the Arabidopsis 14-3-3 protein GF14 lambda in cotton leads to a “stay-green” phenotype and improves stress tolerance under moderate drought conditions. Plant Cell Physiol 45, 1007–1014. Young, T.E., Meeley, R.B., and Gallie, D.R. (2004) ACC synthase expression regulates leaf performance and drought tolerance in maize. Plant J 40, 813–825. Yu, H., Chen, X., Hong, Y.Y., Wang, Y., Xu, P., Ke, S.D., Liu, H.Y., Zhu, J.K., Oliver, D.J., and Xiang, C.B. (2008) Activated expression of an Arabidopsis HD-START protein confers drought tolerance with improved root system and reduced stomatal density. Plant Cell 20, 1134–1151. Zhang, Y., Jang, Y., Lu, S., Chai, J., and Guo, Z. (2008) Overexpressing SgNCED1 in tobacco increases ABA level, antioxidant enzyme activities, and stress tolerance. J Plant Growth Regul 27, 151–158. Zhao, B., Liang, R., Ge, L., Li, W., Xiao, H., Lin, H., Ruan, K., and Jin, Y. (2007) Identification of drought-induced microRNAs in rice. Biochem Biophys Res Commun 354, 585–590. Zhao, F. and Zhang, H. (2006) Expression of Suaeda salsa glutathione S-transferase in transgenic rice resulted in a different level of abiotic stress resistance. J Agricultural Sci 144, 547–554.

302

INTEGRATING PLANT ABIOTIC STRESS RESPONSES

Zhao, T., Palotta, M., Langridge, P., Prasad, M., Graner, A., Schulze-Lefert, P., and Koprek, T. (2006) Mapped Ds/T-DNA launch pads for functional genomics in barley. Plant J 47, 811–826. Zheng, H.G., Babu, R.C., Pathan, M.S., Ali, L., Huang, N., Courtois, B., and Nguyen, H.T. (2000) Quantitative trait loci for root-penetration ability and root thickness in rice: comparison of genetic backgrounds. Genome 43, 53–61. Zhou, J., Wang, X., Jiao, Y., Qin, Y., Liu, X., He, K., Chen, C., Ma, L., Wang, J., Xiong, L., Zhang, Q., Fan, L., and Deng, X.W. (2007) Global genome expression analysis of rice in response to drought and high-salinity stresses in shoot, flag leaf, and panicle. Plant Mol Biol 63, 591–608. Zhu, J., Kaeppler, S.M., and Lynch, J.P. (2005) Mapping of QTLs for lateral root branching and length in maize (Zea mays L.) under differential phosphorus supply. Theor Appl Genet 111, 688–695. Zhuang, J., Cai, B., Peng, R.H., Zhu, B., Jin, X.F., Xue, Y., Gao, F., Fu, X.Y., Tian, Y.S., Zhao, W., Qiao, Y.S., Zhang, Z., Xiong, A.S., and Yao, Q.H. (2008) Genome-wide analysis of the AP2/ERF gene family in Populus trichocarpa. Biochem Biophys Res Commun 371, 468–474.

Index

ABA. See Abscisic acid ABA-responsive element (ABRE), 271 ABC transporters, 123, 126–28, 127f, CP8 ABFs/AREBs, 206 abh1 mutants, 22 Abiotic stress responses CRE in, 271, 273–74 crop loss and, 263 cross talk with biotic stress responses, 207–9 introduction to, 263 major regulatory events in, 268f metabolic and proteomic hormones in, 269–70 signaling in, 267–69, 268f signal transduction pathways under stress, 266–67 plant development and meristem activity, 265 reproduction, 265–66 root responses, 264–65 vegetative responses, 265 stress factors and, 263–64 systems-level approach to, 276 transcriptome analysis of association between responses, 272–74 transcriptional regulatory programs and, 271–72 value of, 270 universal, gene network of, 274–76, 275f Abiotic stress tolerance, discovering genes for in barley, 281–82 components of, 283 drought tolerance, 283–86 evolutionary conservation and, 291–92 goals of, 292 heat tolerance, 287–88 introduction to, 281–83 in maize, 287–89 nutrient/mineral stress, 289–90 oxidative stress, 288–89 plant architecture and morphology in, 290–91 QTL in, 282 in rice, 282 salinity, 286–87 in wild relatives of crops, 283 ABRE. See ABA-responsive element

Abscisic acid (ABA) biosynthesis and catabolism of, 9 circadian clock influencing, 65 cold acclimation and, 187, 196 cross talk between abiotic and biotic stress responses and, 207–8 as drought hormone, 7–10, 8f, 24, 56, 265, 269 heat tolerance and, 226 low-temperature tolerance, gene expression regulation and, 205–7 molecular details of, 5, 6t role of, 71 signaling and, 11–14, 12f, 71–72, 205–7, 283 Acid soils, 113 Acquired heat tolerance, 221–23, 222f, 229, CP15 AFLP. See Amplified fragment length polymorphism AFP. See Antifreeze proteins Agricultural plant sciences, goals of, 144 AHK1, 56, 193, 233 Alfalfa, 195 ALMT1, 118–19 ALS1, 126–28, 127f, CP8 ALS3 aluminum tolerance and, 122–26, 124f, 130–32, 131f, CP7 in Arabidopsis, 124f, 130–32, 131f, CP7 in phloem, 123, 124f, CP7 plasma membrane and, 125 Alternate oxidase enzymes (AOX), 228 alt mutants, 130–37, 131f, 135f, CP9 Aluminum cytoskeleton targeted by, 115–16 DNA damage from, 116, 132–34, 137 exclusion, 117 lipids interacting with, 114 membrane targeted by, 114–15 oxidative stress response and, 116 resistance, 117–20, 128–29 Aluminum-dependent root growth inhibition aluminum exclusion and, 117 Arabidopsis and, 115, 119–22, 124f, 127f, CP7–CP8 aluminum-sensitive mutants, 121–22, 129–37, 131f, 135f, CP9 factors required for resistance to, 128–29

Genes for Plant Abiotic Stress Editors Matthew A. Jenks and Andrew J. Wood © 2010 Blackwell Publishing ISBN: 978-0-813-81502-2

303

304

INDEX

Aluminum-dependent root growth inhibition (continued) cell cycle progression and, 134, 135f, CP9 hyperaccumulation and, 120 introduction to, 113–14 resistance mechanisms of, 117–20, 128–29 root tip and, 114, 119, 123, 128, 136 ROS and, 137 simplicity of, 138 tolerance mechanisms for, 120–21, 128–29 ALS1 and, 126–28, 127f, CP8 ALS3 and, 122–26, 124f, 130–32, 131f, CP7 toxicity mechanisms of, 114–17 AM. See Arbuscular mycorrhizae Ammonium transport, 173 Amplified fragment length polymorphism (AFLP), 236 Anion channels, 13 Anoxia, 93 Anthocyanins, 227–28 Antiapoptotic factor, 242 Antifreeze proteins (AFP), 191 Antioxidants ascorbate, 46 in high-temperature response, 226–28 ROS scavenged by, 227–28 AOX. See Alternate oxidase enzymes Apoplastic leakage, 90 APX. See Ascorbate peroxidase Arabidopsis ALS1 localization analysis in, 127f, CP8 ALS3 expressed in, 124f, 130–32, 131f, CP7 aluminum-dependent root growth inhibition and, 115, 119–22, 124f, 127f, CP7–CP8 aluminum-sensitive mutants, 121–22, 129–37, 131f, 135f, CP9 factors required for resistance to, 128–29 BRs and, 227 cell identity in, 272 cell wall-based osmotic stress tolerance of cellulose biosynthesis and, 36–37 core oligosaccharide assembly and, 40, 41f core oligosaccharide processing and, 42 dolichol biosynthesis and, 38–39, 38f genes influencing, 35–36 GPI anchor biosynthesis and, 46–47, 47f importance of, 48 introduction to, 35 microtubules and, 47–48 N-glycan modifications in, 38, 44–45 N-glycan re-glycosylation, ERAD and, 44 N-glycosylation modifications in, 38 OST and, 40–42 sugar-nucleotide biosynthesis and, 39–40, 39f UPR, osmotic stress signaling and, 42–44, 43f circadian clock influencing, 65 constitutive overexpression of vacuolar transporters in, 94–95 drought response regulatory network in, 58, 61f–62f, 63–66, 71, 74, CP2–CP3

evolutionary conservation and, 291–92 genome sequence of, 7, 270 heat tolerance of, 222f, 225–27, 229, 231, 233–34, 236–37, 241–45, 247, CP15 low-temperature tolerance of, 187–88, 190–94, 197–99, 200f, 201–4, 206–7, CP14 metabolic profiling of, 245 NUE in, 169, 173, 173f phosphate use and, 147–48 phosphoprotein signaling in, 269 PUE in, 147, 151, 153–55 reproduction of, 266 stomatal function of, 5, 6t, 7, 24–25 ABA biosynthesis in, 9 calcium in, 13–14 cell signaling mutants in, 15–22 genome of, 7 hormones in, 10 ion channels in, 11–13 ROS and NO in, 14 stress series data of, 272–73 Arabidopsis Genome Expression project (AtGen Express), 63, 271–72 Arbuscular mycorrhizae (AM), 289–90 Ascorbate, 46, 227 Ascorbate peroxidase (APX), 228 Asian Vegetable Research and Development Center (AVRDC), 235 Asparagine-rich protein (NRP), 44 Assimilate partitioning, 225 AtATR, 133–34, 136–37 Ataxia-telangiectasia Mutated and Rad3-related Kinase (ATR), 132–33 Ataxia-telangiectasia Mutated (ATM) function, 132–33 AtGen Express. See Arabidopsis Genome Expression project AtHK1, 56, 193, 233 ATM function. See Ataxia-telangiectasia Mutated function AtPP2C-HA, 22 ATR. See Ataxia-telangiectasia Mutated and Rad3-related Kinase AtRAC1, 21 Atrichoblast cells, 67 Auto-regulating enzyme, 72 AVP1, 291 AVRDC. See Asian Vegetable Research and Development Center Barley, 119, 169 gene discovery for, 281–82 heat tolerance and, 224, 226, 229 hormone responses in, 270 proteomic reference maps for, 243 Bentgrass, 226 Biofuels, 178 Bioinformatic approaches, 56–63, 58f, 60f–62f, CP1, CP3 Biological stress factors, 263

INDEX

Biosynthesis of ABA, 9 cellulose, 36–37 dolichol, 38–39, 38f GPI anchor, 46–47, 47f organic acid, 118 putrescine, 206 solute, 189–90 sugar-nucleotide, 39–40, 39f Biotic stress responses, 207–9 Brassinosteroids (BRs), 227 Breeding classical, 234–35 conventional, 281 molecular, 236 BRs. See Brassinosteroids Buckwheat, 120–21 Bypass flow, 90 Ca. See Calcium Ca2+-dependent protein kinases (CDPK), 16–17, 233, 267–69 Ca2+-dependent signaling, 267–69 Ca2+-insensitive influx, 90 Ca2+-sensitive influx, 89–90 Cabbage, 245 Calcineurin B-like (CBL) proteins, 194–95 Calcium (Ca) in Arabidopsis stomatal function, 13–14 cytosolic activity of, 98–99 as second messenger, 194–95, 233 Calmodulin (CaM), 194–95 Calmodulin-like (CML) proteins, 195 CaM. See Calmodulin Carbon exchange rate (CER), 225 Carbon partitioning, 225 Carrot, 191 Cassava, 243 Catalase (CAT), 228 Cauliflower, 236 CBF, 198, 204–5, 208–9 CBF-independent regulons, 201–3 CBF regulon, 60f–61f, 66–70, 68t, 197–201, 200f, 271, 286, CP1–CP2, CP14 CBL-interacting protein kinases (CIPK), 16–17, 195 CBL proteins. See Calcineurin B-like proteins CDPK. See Ca2+-dependent protein kinases CDPK and CIPK network, 16–17 Cell identity, 272 Cell membrane thermostability (CMT), 225 Cell signaling mutants with altered stomatal responses in Arabidopsis, 15–22 farnesyl transferase, 21 genes related to humidity sensing, 21–22 G proteins, 19–21 phosphatases, 17–19 protein kinases, 15–17 Cell-specific signaling, 100

305

Cellular dehydration, 185 Cellulose biosynthesis, 36–37 Cellulose synthase (CesA family), 37 Cell viability, 225 Cell wall-based osmotic stress tolerance cellulose biosynthesis and, 36–37 core oligosaccharide assembly and, 40, 41f core oligosaccharide processing and, 42 dolichol biosynthesis and, 38–39, 38f genes influencing, 35–36 GPI anchor biosynthesis and, 46–47, 47f importance of, 48 introduction to, 35 microtubules and, 47–48 N-glycan modifications in, 38, 44–45 N-glycan re-glycosylation, ERAD and, 44 N-glycosylation modifications in, 38 OST and, 40–42 sugar-nucleotide biosynthesis and, 39–40, 39f UPR, osmotic stress signaling and, 42–44, 43f CER. See Carbon exchange rate CesA family. See Cellulose synthase Chaperonins, 288 Chemical stress factors, 263. See also Salinity Chickpea, 223 Chilling-sensitive plants, 185 Chilling-tolerant-but-freezing-sensitive plants, 185, 187 Chitin, 237–38 Chlorophyll accumulation, 241 CIPK. See CBL-interacting protein kinases Circadian clock, 65 9-cis-epoxycartenoid dioxygenase (NCED), 283 Cis-regulatory elements (CRE), 271, 273–74 Cis-regulatory fingerprint, 67, 68f Cis-violaxanthin, 9 Cis-xanthophylls, 9 Citrate export, 117, 119 Citric acid cycle, 117 Classical breeding, 234–35 Climate change, 221, 222f CML proteins. See Calmodulin-like proteins CMT. See Cell membrane thermostability CNGC2/DND1. See CYCLIC NUCLEOTIDE GATED CHANNEL2 Cobra mutants, 37 Co-expressed genes, 59 Co-expression map, 58 Cold acclimation ABA and, 187, 196 complexity of, 208 integration of, 196 process of, 186f, 187, 200f, CP13–CP14 protective mechanisms induced during membrane structure alterations, 188–89 protective proteins production, 190–92 solute biosynthesis, 189–90 Cold shock proteins (CSP), 204 Conventional breeding, 281

306

INDEX

Cotton, 191 heat tolerance and, 224, 234, 243 hormones in, 270 Cowpea, 234 CRE. See Cis-regulatory elements CRHK. See Cytokinin-receptor histidine kinases Crops for biofuels, 178 fertilizer for, 143–45, 167, 176 loss of, 263 NUE in in Arabidopsis, 169, 173, 173f drought tolerance and, 178 genetic engineering to improve, 175 introduction to, 167, 168f, CP11 maize and, 174 metabolite changes related to, 167, 168f, CP11 metanomic tools for extending functional genomics and, 174–75 methylamine and, 169, 173, 173f microarrays and, 173 microbial activity in, 176, 177f, CP12 mutants for isolating plant genes and, 169–73, 170t–172t, 173f mycorrhizal effects in, 178 nitrogen partitioning regulation as, 169 nodule effects in, 178 rice and, 175 transcript analysis in, 174 transgenics lacking a priori evidence for, 175–176, 176t understanding of, 178 water effects in, 178 world food security influenced by, 178 yield influenced by, 169 PUE in in Arabidopsis, 147, 151, 153–55 definition of, 150 genetic determinants for phosphate acquisition in, 151–53 genetic determinants for phosphate utilization efficiency in, 155–56 genetic engineering to improve, 156–58, 157t improved nutrition and, 143–44 introduction to, 143 NUE and, 150 phosphate starvation and, 146–48, 149f, CP10 plant metabolism and, 145–46 root growth and, 148 root system architecture modulated in, 153–55 sensing and signaling networks in, 146–48, 149f, CP10 world food security influenced by, 143–44 yield influenced by, 144–45, 158 tilling programs for, 169 wild relatives of, 283

Crop tolerance. See also Heat tolerance; Low-temperature tolerance to drought, 25, 178, 283–86 to salinity components of, 85–86 future research on, 100 genomics, signaling, and, 97–100 halophytic crops and, 85 osmotic tolerance and, 96–97 sodium exclusion and, 86–94, 87f–88f, CP5–CP6 tissue tolerance and, 94–96 Cross talk, between abiotic and biotic stress responses, 207–9 Cryoprotective proteins, 191 CSP. See Cold shock proteins Cuticle, characteristics of, 284 CYCLIC NUCLEOTIDE GATED CHANNEL2 (CNGC2/ DND1), 13 CYP707A, 9 Cytokinin-receptor histidine kinases (CRHK), 10 Cytokinins as hormones in drought responses, 10 as negative regulators of root growth, 148 Cytoplasmic Na+ tolerance, 95–96 Cytoskeleton aluminum targeting, 115–16 changes in, 189 Cytosolic calcium activity, 98–99 Damage response, tissue tolerance to salinity and, 96 Dehydration-responsive element (DRE), 66–67, 68t, 286 Dehydration-responsive element-binding protein. See DREB/CBF regulon Dehydrins, 192 Dense overlapping region (DOR), 71 Dephosphorylation, of protein, 99 DGE. See Digital Gene Expression DGL1, 41–42 DH technology. See Double haploid technology Digital Gene Expression (DGE), 281 Direct phosphate uptake pathway, 151–52 DNA aluminum damaging, 116, 132–34, 137 marker technology, 235–36 nutrition and, 146 Dolichol biosynthesis, 38–39, 38f DOR. See Dense overlapping region Double haploid (DH) technology, 237 DRE. See Dehydration-responsive element DREB/CBF regulon, 60f–61f, 66–70, 68t, 271, 286, CP1–CP2 Drought cost of, 221, 222f effects of, 263–64 gene discovery for, 283–86 hormones and, 7–11, 8f

INDEX

ABA, 7–10, 8f, 24, 56, 265, 269 cytokinins, 10 ethylene, 10 JA, 10 maize and, 178 NUE and, 178 plant architecture and morphology in, 290–91 rice influenced by, 55 secondary effects of, 285 signaling and, 11–14, 12f, 55–56, 71–72, 283 stress as major stress, 5, 55, 263–64 perception of, 55–56, 57t, CP4 transcriptomic studies of, 57t, 63–66, CP4 tobacco and, 178 tolerance, 25, 178, 283–86 wheat and, 236 Drought response regulatory network. See also Stomatal function, drought response of ABA signaling and, 71–72 in Arabidopsis, 58, 61f–62f, 63–66, 71, 74, CP2–CP3 DREB/CBF regulon and, 60f–61f, 66–70, 68t, CP1–CP2 drought stress perception and, 55–56, 57t, CP4 integration of, 72–73 introduction to, 55 known pathways, protein interactions and, 74–75 reactive oxygen signaling and, 72 systems biology approaches to, 56–63, 58f, 60f–62f, CP1, CP3 transcriptomic studies of drought stress and, 57t, 63–66, CP4 EBC. See Epidermal bladder cell Efflux, to soil, 90–91 Endoplasmic reticulum (ER), 38 core oligosaccharide processing in, 42 in ER-associated protein degradation, 44 in heat tolerance, 239t–241t Enhanced-release fertilizer, 176 Enzymes, 167, 168f, CP11 EPF1. See EPIDERMAL PATTERNING FACTOR 1 Epidermal bladder cell (EBC), 94 EPIDERMAL PATTERNING FACTOR 1 (EPF1), 23–24 ER. See Endoplasmic reticulum ERA1, 21 ERAD. See ER-associated degradation ER-associated degradation (ERAD), 44 ERECTA, 16 ERF. See Ethylene-responsive factor ESK1, 202 EST. See Expressed sequence tags Ethylene as hormone in drought responses, 10 production, 226 Ethylene-responsive factor (ERF), 291 Exogenous BAP, 270

307

Expansion-induced lysis, 188 Expressed sequence tags (EST), 242–43 Extracellular freezing, 185, 191 Farnesyl diphosphate (FPP), 38 Farnesyl transferase, 21 Fertilizer, 143–45, 167, 176 Flavonoids, 227–28 FLC. See Flowering Locus Flooding stress factors, 263–64 Flowering Locus (FLC), 265 Food security, 143–44, 178, 185, 263, 281 FPP. See Farnesyl diphosphate Freezing-tolerant plants, 185, 187 Fructan, 189–90 Functional genomics, for heat tolerance, 242 Gas chromatography (GC), 174 GB. See Glycine betaine GC. See Gas chromatography GDH. See Glutamate dehydrogenase Gene(s). See also specific genes cell identity, 272 in cellulose biosynthesis, 36–37 cell wall and plant stress tolerance influenced by, 35–36 co-expressed, 59 discovery, for abiotic stress tolerance in barley, 281–82 components of, 283 drought tolerance, 283–86 evolutionary conservation and, 291–92 goals of, 292 heat tolerance, 287–88 introduction to, 281–83 in maize, 287–89 nutrient/mineral stress, 289–90 oxidative stress, 288–89 plant architecture and morphology in, 290–91 QTL in, 282 in rice, 282 ROS, 288–89 salinity, 286–87 in wild relatives of crops, 283 expression regulation, low-temperature tolerance and ABA-dependent cold signal pathway and, 205–7 post-transcriptional regulation and, 203–5 signal transduction in, 192–96 transcriptional regulation and, 196–203, 200f, CP14 function assignment of, 64 hormone-responsive, 269–70 humidity related, 21–22 mutants for isolating, 169–73, 170t–172t, 173f network, of universal abiotic stress responses, 274–76, 275f starch synthesis, 243

308

INDEX

Gene ontology (GO), 269 Genetic engineering for heat tolerance, 224–25 functional genomics, 242 overexpression, mutational analysis and, 237–41, 238f, 239t–241t profiling and “omics,” 242–44 TF and, 239t–241t, 241–42 NUE improved by, 175 PUE improved by, 156–58, 157t Genome, of Arabidopsis, 7, 270 GGM. See Graphical Gaussian model GL loci. See GLOSSY loci Global warming, 221, 222f GLOSSY (GL) loci, 284 Glucose, 189 Glutamate dehydrogenase (GDH), 168f, 174, CP11 Glycine betaine (GB), 224, 287 Glycosylphosphatidylinositol (GPI), 46–47, 47f GO. See Gene ontology Golgi apparatus core oligosaccharide processing in, 42 N-glycan modification in, 44–45 GORK, 11 GPCR. See G protein coupled receptors GPI. See Glycosylphosphatidylinositol G protein coupled receptors (GPCR), 19–20 G proteins, 19–21 Graphical Gaussian model (GGM), 274 Groundwater contamination, 167 Growth maintenance, tissue tolerance to salinity and, 96 gsr1, 169, 173 Guard cells role of, 5, 9, 24 signaling events in, 11–14, 12f TGG1 in, 14 Half-type ABC transporter, 126–28, 127f, CP8 Halophytic crops, 85, 90 HAT1, 22 HDG11, 291 Heat-induced proteins, 229–30 Heat shock elements (HSE), 231 Heat shock proteins (Hsps), 229, 234, 239t–241t, 243–45, 288 Heat shock transcription factors (Hsfs), 231, 239t–241t, 241–42, 288 Heat tolerance ABA and, 226 acquired, 221–23, 222f, 229, CP15 of Arabidopsis, 222f, 225–27, 229, 231, 233–34, 236– 37, 241–45, 247, CP15 barley and, 224, 226, 229 cotton and, 224, 234, 243 ER in, 239t–241t evolving techniques for artificial or minichromosome, 246

small RNA, 246–47 translational genomics, 246 future of, 247 gene discovery for, 287–88 genetic approaches to, 224–25 biotechnology, 235 classical breeding, 234–35 MAS, 235–36 somaclonal variation and in vitro mutagenesis, 236–37 genetic engineering for, 224–25 functional genomics, 242 overexpression, mutational analysis and, 237–41, 238f, 239t–241t profiling and “omics,” 242–44 TF and, 239t–241t, 241–42 global warming and, 221, 222f high temperatures in antioxidants and, 226–28 carbon partitioning and, 225 cell membrane stability, cell viability and, 225 cost of, 221, 222f hormones and, 226–28 impact of, 223 molecular response to, 228–30 morphological response to, 223, 224f, CP15 osmotic adjustment to, 224–25 photosynthesis and, 225 physiological response to, 224, 224f, CP15 secondary metabolites and, 226–28 transpiration and, 5 inherent, 221–23 lettuce and, 226–27 maize and, 223, 224f, 229, 234, 245, CP15 mechanism of complexity of, 231, 232f Hsfs and, 231 inadequate, 230 roots and, 230 signal perception, 233–34 MeJA and, 226 potato and, 224f, 229, CP15 radish and, 237 rice and, 223, 234, 237, 245 ROS and, 227–28, 239t–241t signaling factors in, 238f sorghum and, 234, 243 stress combination effect and, 244–46 sugarcane and, 223–24, 227, 230 tobacco and, 224, 237, 245 tomato and, 224, 234 wheat and, 223, 225–26, 229, 245 Herbicides, 173, 178 Heterotrimeric G proteins, 19–20 High temperatures antioxidants and, 226–28 carbon partitioning and, 225 cell membrane stability, cell viability and, 225

INDEX

cost of, 221, 222f hormones and, 226–28 impact of, 223, 264 molecular response to, 228–30 morphological response to, 223, 224f, CP15 osmotic adjustment to, 224–25 photosynthesis and, 225 physiological response to, 224, 224f, CP15 secondary metabolites and, 226–28 transpiration and, 5 High throughput research, 75 Histidine kinases, 10, 56, 193, 233 Histone proteins, 205 Holm oak plant, 226 Hormone-responsive genes, 269–70 Hormones in Arabidopsis, 10 in barley, 270 in cotton, 270 drought stress and, 7–11, 8f ABA, 7–10, 8f, 24, 265, 269 cytokinins, 10 ethylene, 10 JA, 10 in high-temperature response, 226–28 in metabolic and proteomic responses to abiotic stress, 269–70 root system architecture, PUE and, 154–55 in wheat, 270 HSE. See Heat shock elements Hsfs. See Heat shock transcription factors Hsps. See Heat shock proteins Humidity, 21–22 Hurricane Katrina, 221 Hydrangea, 120 Hydration stress factors, 263. See also Drought Hydrogen peroxide, 72 Hydrological cycle, 221 Hydroponic growth, 290 ICE gene, 67, 69, 198–201 Ice nucleating proteins (INP), 191 ICP-MS. See Inductively coupled plasma mass spectrometry Inductively coupled plasma mass spectrometry (ICP-MS), 290 Inherent heat tolerance, 221–23 INP. See Ice nucleating proteins In silico mutagenesis, 67 Integrators, 73 In vitro mutagenesis, 236–37 Ion channels, 11–13, 12f IPP. See Isopentenyl diphosphate IPT, 286 IRE1, 42 Irrigation, 83, 234 Isopentenyl diphosphate (IPP), 38 Isoprenoids, 228

309

Jasmonates (JA) circadian clock influencing, 65 as hormones in drought responses, 10 KAT1, 11 Kiwi fruit, 226 Large-scale assays, 276 Late embryogenesis abundant (LEA) proteins, 191–92, 284 LCBK, 22 Leaf elongation, inhibition of, 84 Leaf senescence, 265 Leaf wilting 2 (LEW2), 36, 39 Lettuce, 226–27 LEW2. See Leaf wilting 2 Light stress factors, 263 Lime, soil pH influenced by, 113 Lipids, aluminum interacting with, 114 Lipid transfer proteins (LTP), 284 Low phosphate availability, 143 Low-temperature tolerance of Arabidopsis, 187–88, 190–94, 197–99, 200f, 201–4, 206–7, CP14 cold acclimation and ABA and, 187, 196 complexity of, 208 integration of, 196 process of, 186f, 187, 200f, CP13–CP14 protective mechanisms induced during, 188–92 cross talk between abiotic and biotic stress responses and, 207–9 development of, 186f, CP13 future perspectives of, 207–9 gene expression regulation and ABA-dependent cold signal pathway and, 205–7 post-transcriptional regulation and, 203–5 signal transduction in, 192–96 transcriptional regulation and, 196–203, 200f, CP14 introduction to, 185–87, 186f, CP13 optimal temperature ranges and, 185 ROS and, 187, 192 signaling and, 187, 192–96 LRR receptor kinases, 15–16 LTP. See Lipid transfer proteins Maize, 118 as chilling-sensitive plant, 185 consumption of, 144 direct phosphate uptake pathway in, 151 drought and, 178 gene discovery in, 287–89 heat tolerance and, 223, 224f, 229, 234, 245, CP15 meristem activity of, 265 NUE and, 174 proteomic reference maps for, 243 reproduction of, 265 root system architecture of, 154

310

INDEX

Maltose accumulation, 190 MAPK. See Mitogen-activated protein kinase MAP kinases cascade, 17 humidity and, 21–22 Marker-assisted selection (MAS), 235–36, 281 Mass spectrometry (MS), 174, 244, 290 Mechanical stress factors, 263 MeJA. See Methyl jasmonate Membrane. See Plasma membrane Membrane transport-related proteins, 11–13, 12f Meristem activity, 265 Metabolic responses, to abiotic stress hormones in, 269–70 signaling in, 267–69, 268f signal transduction pathways under stress, 266–67 Metabolism, phosphorus and, 145–46 Metabolites NUE and, 167, 168f, CP11 secondary, in high-temperature response, 226–28 Metanomic tools, for extending functional genomics, 174–75 Methylamine, 169, 173, 173f Methylene urea (MU), 176 Methyl jasmonate (MeJA), 10 circadian clock influencing, 65 heat tolerance and, 226 Microarray NUE and, 173 studies, 70 usefulness of, 243, 270 Microbial activity, in NUE, 176, 177f, CP12 MicroRNA (miRNA), 205, 246–47, 272–73, 286 Microtubules, 47–48 Mineral stress, gene discovery for, 289–90 Minichromosome (MMC), 246 Mir399/PHO2 pathway role, 156 miRNA. See MicroRNA Mitogen-activated protein kinase (MAPK), 195–96, 233, 269 MMC. See Minichromosome Molecular breeding, 236 Molecular chaperones, 229–30 Movement protein (MP), 245 MS. See Mass spectrometry MU. See Methylene urea Mustard, 226 Mutational analysis, in genetic engineering for heat tolerance, 237–41, 238f, 239t–241t Mycorrhizal effects, in NUE, 178 NAC-family, of transcription factors, 203, 242 Na+ tolerance, cytoplasmic, 95–96 NCED. See 9-cis-epoxycartenoid dioxygenase Negative transcriptional regulation, 152 Network reconstruction algorithms, 276 N-glycan

modifications, 38, 44–45 re-glycosylation, 44 N-glycosylation ascorbate as interface with, 46 modifications, 38 Nitrate use efficiency (NUE) in Arabidopsis, 169, 173, 173f drought and, 178 genetic engineering to improve, 175 introduction to, 167, 168f, CP11 maize and, 174 metabolite changes related to, 167, 168f, CP11 metanomic tools for extending functional genomics and, 174–75 methylamine and, 169, 173, 173f microarrays and, 173 microbial activity in, 176, 177f, CP12 mutants for isolating plant genes and, 169–73, 170t–172t, 173f mycorrhizal effects in, 178 nitrogen partitioning regulation as, 169 nodule effects in, 178 rice and, 175 transcript analysis in, 174 transgenics lacking a priori evidence for, 175–76, 176t understanding of, 178 water effects in, 178 world food security influenced by, 178 yield influenced by, 169 Nitric oxide (NO), 11, 14 Nitrogen methylene urea as, 176 partitioning regulation, 169 role of, 167 NO. See Nitric oxide Nodule effects, in NUE, 178 NPC. See Nuclear pore complex NRP. See Asparagine-rich protein Nuclear pore complex (NPC), 204 Nucleoporins (NUPs), 204 NUE. See Nitrate use efficiency NUPs. See Nucleoporins Nutrient deficiency, 264 Nutrient stress, gene discovery for, 289–90. See also Nitrate use efficiency; Phosphorus use efficiency Nutrition DNA, RNA and, 146 improved, 143–44 O3. See Ozone Oligosaccharides, 38 assembly of, 40, 41f processing of, 42 Oligosaccharyltransferase (OST), 15, 40–42 Operational window, 70 Optimal temperature ranges, 185 Organic acid biosynthesis, 118

INDEX

Orthophosphates (Pi), 145. See also Phosphorus use efficiency Osmoprotectant synthesis, 96–97 Osmotic adjustment to high temperatures, 224–25 to salinity, 286 Osmotic stress signaling, 42–44, 43f Osmotic tolerance. See also Cell wall-based osmotic stress tolerance osmoprotectant synthesis and, 96–97 stomatal closure and, 97 OST. See Oligosaccharyltransferase Overexpression, in genetic engineering for heat tolerance, 237–41, 238f, 239t–241t Oxidative stress gene discovery for, 288–89 response aluminum and, 116 ascorbate as interface with, 46 Ozone (O3), 264 PA. See Phosphatidic acid Papaya, 118 PCD. See Programmed cell death Pepper, 226 Peroxide, 72 Phi cell, 90 Phloem ALS3 in, 123, 124f, CP7 recirculation in, 93 PHO1, 155–56 Phosphatases, 17–19 Phosphate starvation, 146–48, 149f, 154–58, CP10 Phosphatidic acid (PA), 11 Phospholipase C (PLC), 14 Phospholipase D (PLD), 20 Phospholipids, 188–89 Phosphoprotein signaling, 269 Phosphorus acquisition of, 153–55 metabolism and, 145–46 regulatory and structural functions of, 145–46 starvation and, 146–48, 149f, CP10 yield influenced by, 144–45, 158 Phosphorus use efficiency (PUE) in Arabidopsis, 147, 151, 153–55 definition of, 150 genetic determinants for phosphate acquisition in, 150–53 genetic determinants for phosphate utilization efficiency in, 155–56 genetic engineering to improve, 156–58, 157t improved nutrition and, 143–44 introduction to, 143 phosphate starvation and, 146–48, 149f, CP10 plant metabolism and, 145–46 QTL and, 154

311

root growth and, 148 root system architecture modulated in, 153–55 sensing and signaling networks in, 146–48, 149f, CP10 world food security influenced by, 143–44 yield influenced by, 144–45, 158 Phosphorylation, of protein, 99 Photosynthesis in high-temperature response, 225 water in, 5 PHR1, 147–48, 153 Pht families, 151–52 Pi. See Orthophosphates Plasma membrane ALS3 and, 125 aluminum targeting, 114–15 during freeze/thaw cycle, 192 hyperpolarization of, 11 rigidification, 193, 200f, CP14 stability of, 225 structure, cold acclimation altering, 188–89 PLC. See Phospholipase C PLD. See Phospholipase D Polysaccharides, 189 Poplar, 244 Population growth, 143–44, 178, 185, 281 Post-transcriptional regulation, low-temperature tolerance and, 203–5 Potato, 185, 224f, 229, CP15 PP2A, 19 PP2C, 18–19 Primary root length (PRL), 154 Primary salinity, 83 PRL. See Primary root length Profiling, in genetic engineering for heat tolerance, 242–44 Programmed cell death (PCD), 288 Proline, 224 Protective proteins, production of, 190–92 Protein(s) antifreeze, 191 ATM, 132–33 CBL, 194–95 in cellulose biosynthesis, 36–37 CML, 195 CSP, 204 dephosphorylation of, 99 in drought response regulatory network, 74–75 ER-associated degradation of, 44 G, 19–21 heat-induced, 229–30 histone, 205 Hsps, 229, 234, 239t–241t, 243–45, 288 INP, 191 interaction map of, 59–63, 60f–62f, CP1, CP3 interaction predictions and, 74–75 IRE1, 42 LEA, 191–92, 284

312 Protein(s) (continued) LTP, 284 membrane transport-related, 11–13, 12f MP, 245 NRP, 44 patenting of, 173 phosphorylation of, 99 posttranslational modification of, 233 protective, 190–92 in signal transduction, 267 transmembrane, 125 UPR and, 42–44, 43f, 230, 239t–241t zinc-finger, 292 Protein kinases, 15–17, 194–96 Protein phosphatases, 17–19, 196 Protein tyrosine phosphatase (PTP), 19 Proteomic responses, to abiotic stress hormones in, 269–70 signaling in, 267–69, 268f signal transduction pathways under stress, 266–67 Proteomics, 243–44 PTP. See Protein tyrosine phosphatase PUE. See Phosphorus use efficiency Putrescine biosynthesis, 206 QTL. See Quantitative Trait Locus Quantitative Trait Locus (QTL), 128–29, 153, 235–36 ERECTA gene and, 16 in gene discovery, 282 for mineral stress tolerance, 290 PUE and, 154 Radiation stress factors, 263–64 Radish, 237 Random amplified polymorphism (RAPD), 236 Reactive oxygen signaling aluminum and, 116 drought response regulatory network and, 72 Reactive oxygen species (ROS), 11, 14, 72 accumulation of, 96 aluminum-dependent root growth inhibition and, 137 antioxidants scavenging, 227–28 damage caused by, 228 detox, 237, 266 gene discovery for, 288–89 heat tolerance and, 227–28, 239t–241t low-temperature tolerance and, 187, 192 production of, 266 signaling, 267 Receptor-like kinases (RLK), 15 Regulatory fingerprint, 66–67, 68t Reproduction, plant development and, 265–66 Restricted fragment length polymorphism (RFLP), 236 Reverse functional genomic techniques, 98 RFLP. See Restricted fragment length polymorphism Rice as chilling-sensitive plant, 185 consumption of, 144

INDEX

drought influencing, 55 gene discovery for, 282 heat tolerance and, 223, 234, 237, 245 meristem activity of, 265 NUE and, 175 reproduction of, 265 shoot storage and, 93–94 universal abiotic stress responses and, 274–76, 275f xylem retrieval in, 92 zinc-finger proteins in, 292 RLK. See Receptor-like kinases RNA miRNA, 205, 246–47, 272–73, 286 nutrition and, 146 siRNA, 205, 246–47 small, 99–100, 246–47, 272 Root growth, 45, 84, 148 growth inhibition, aluminum-dependent aluminum exclusion and, 117 Arabidopsis and, 115, 119–22, 124f, 127f, 128–37, 131f, 135f, CP7–CP9 cell cycle progression and, 134, 135f, CP9 hyperaccumulation and, 120 introduction to, 113–14 resistance mechanisms of, 117–20, 128–29 root tip and, 114, 119, 123, 128, 136 ROS and, 137 simplicity of, 138 tolerance mechanisms for, 120–32, 124f, 127f, 131f, CP7–CP8 toxicity mechanisms of, 114–17 heat tolerance of, 230 influx to, 86, 89 plant development, abiotic stress responses and, 264–65 primary length of, 154 system architecture, phosphate use efficiency and, 153–55 ROP10, 21 ROS. See Reactive oxygen species RPK1, 15 RSW2, 37, 48 Rubisco activation, 225 Rye, 119, 191, 229 S1P. See Sphingosine-1-phosphate SA. See Salicylic acid SAGE. See Serial analysis of gene expression Salicylic acid (SA), 226 Saline-sodic soils, 83 Salinity crop tolerance to components of, 85–86 future research on, 100 genomics, signaling, and, 97–100 halophytic crops and, 85 osmotic tolerance and, 96–97 sodium exclusion and, 86–94, 87f–88f, CP5–CP6 tissue tolerance and, 94–96

INDEX

gene discovery and, 286–87 management-based solutions to, 84–85 microtubules and, 48 osmotic adjustment to, 286 primary, 83 root morphology induced by, 37 secondary, 84 significance of, 83 soil reclamation for, 85 TFs and, 99–100 water and vegetation management for, 84–85 Salt glands, 94 Salt stress. See Salinity Secondary metabolites, 226–28 Secondary salinity, 84 Second messengers, 194–95, 233 Seed pretreatment, 247 Serial analysis of gene expression (SAGE), 187 Shoot storage, 93–94 Short interfering RNA (siRNA), 205, 246–47 sHsp. See Small Hsps Signaling. See also Cell signaling mutants with altered stomatal responses ABA, 11–14, 12f, 71–72, 205–7, 283 Ca2+-dependent, 267–69 cell-specific, 100 drought and, 11–14, 12f, 55–56, 71–72, 283 factors, in heat tolerance, 238f in guard cells, 11–14, 12f low-temperature tolerance and, 187, 192–96 meristem activity and, 265 in metabolic and proteomic responses to abiotic stress, 267–69, 268f osmotic stress and, 42–44, 43f pathways in, 98 perception, 233–34 phosphoprotein, 269 PUE and, 146–48, 149f, CP10 reactive oxygen, 72, 116 ROS, 267 in salinity tolerance, 97–100 signal transduction low-temperature tolerance and, 192–96 pathways, in metabolic and proteomic responses to abiotic stress, 266–67 proteins in, 267 Simple sequence repeat (SSR), 236 siRNA. See Short interfering RNA SLAC1. See SLOW ANION CHANNEL-ASSOCIATED 1 SLOW ANION CHANNEL-ASSOCIATED 1 (SLAC1), 13 Slow-release fertilizer, 176 Small GTPase, 21 Small Hsps (sHsp), 229 Small RNAs, 99–100, 246–47, 272 Small ubiquitin-like modifier (SUMO), 233, 239t–241t Snap bean, 223 SnRK protein kinases, 15 SO2. See Sulfur dioxide

Sodic soils, 83 Sodium exclusion bypass flow and, 90 Ca2+-insensitive influx and, 90 Ca2+-sensitive influx and, 89–90 efflux to soil and, 90–91 genetic determinants associated with, 86, 87f–88f, CP5–CP6 influx to root and, 86, 89 influx to xylem and, 91–92 recirculation in phloem and, 93 retrieval from xylem and, 92–93 salinity tolerance and, 86–94, 87f–88f, CP5–CP6 salt glands and, 94 shoot storage and, 93–94 Soil. See also Salinity acidic, 113 efflux to, 90–91 lime influencing, 113 quality, assessment of, 176 reclamation, 85 saline-sodic, 83 Soil quality indicators (SQI), 176 Solute biosynthesis, 189–90 Somaclonal variation, 236–37 Sorghum, 119, 234, 243 sos mutant pathway, 99 Soybean, 202, 229 Sphingosine-1-phosphate (S1P), 20 Sphingosine kinase (SPHK), 20 Spinach, 224, 229 SQI. See Soil quality indicators SSR. See Simple sequence repeat Starch synthesis genes, 243 Stomatal closure, 97 Stomatal function, drought response of, 7–11, 8f altered, cell signaling mutants with in Arabidopsis, 15–22 farnesyl transferase, 21 genes related to humidity sensing, 21–22 G proteins, 19–21 phosphatases, 17–19 protein kinases, 15–17 of Arabidopsis, 5, 6t, 7, 24–25 ABA biosynthesis in, 9 calcium in, 13–14 cell signaling mutants in, 15–22 genome of, 7 hormones in, 10 ion channels in, 11–13 ROS and NO in, 14 cellular mediators of, 12f signaling events in guard cells and, 11–14, 12f stomatal development and, 23–24 summary of, 24–25 transcriptional regulation in, 22–24 water and, 5, 6t Sucrose, 189

313

314

INDEX

Sugarcane, 223–24, 227, 230 Sugar-nucleotide biosynthesis, 39–40, 39f Sugars, 189–90, 287 Sulfur dioxide (SO2), 264 SUMO. See Small ubiquitin-like modifier Sunflower, 226 Superoxides, 72 Systems biology approaches, 56–63, 58f, 60f–62f, 276, CP1, CP3 field of, 73–74 Tea, 120 TF. See Transcription factor TGG1, 14 Thermotolerance. See Heat tolerance TILLING, 282 Tissue tolerance, to salinity cytoplasmic Na+ tolerance and, 95–96 growth maintenance and, 96 response to damage and, 96 vacuolar storage and, 94–95 Tobacco, 118–19, 168f, 174, 178, 190, 224, CP11 drought and, 178 heat tolerance and, 224, 237, 245 zinc-finger proteins in, 292 Tomato, 194, 224, 229, 234, 291 Top-down transcriptomic meta-analysis workflow, 58f Transcript analysis, in NUE, 174 Transcriptional regulation in low-temperature tolerance CBF-independent regulons and, 201–3 CBF regulon and, 197–201, 200f, CP14 mediation and, 196–97 negative, 152 programs, in transcriptome analysis, 271–72 in stomatal drought response, 22–24 Transcriptional units (TU), 271 Transcription factor (TF) FLC, 265 heat tolerance and, 239t–241t, 241–42 NAC-family of, 203, 242 role of, 271 salinity response and, 99–100 WRKY-family of, 202–3, 287 Transcriptome analysis, of abiotic stress responses association between responses, 272–74 transcriptional regulatory programs and, 271–72 value of, 270 Transcriptomic studies, of drought stress, 57t, 63–66, CP4 Transgenics, lacking a priori evidence for NUE, 175–76, 176t Translational genomics, 246 Transmembrane protein, 125 Transpiration, 5 TU. See Transcriptional units

Ultraviolet B radiation (UV-B), 263–64 Unfolded protein response (UPR), 42–44, 43f, 230, 239t–241t Universal abiotic stress responses, 274–76, 275f Upper threshold temperature, 223 UPR. See Unfolded protein response UV-B. See Ultraviolet B radiation Vacuolar storage, 94–95 Vacuolar transporters, constitutive overexpression of, 94–95 Vegetative responses, to abiotic stress, 265 Viability, cell, 225 Violaxanthin, 9 Vitamin C, 227 Water ground-, 167 leaf senescence and, 265 management, for salinity, 84–85 NUE influenced by, 178 in photosynthesis, 5 stomatal function and, 5, 6t WUE and, 174–75, 178, 285 Watermelon, 227 Water replacement hypothesis, 189 Water use efficiency (WUE), 174–75, 178, 285 Wet-bench research, 75 Wheat aluminum resistance in, 118 consumption of, 144 drought and, 236 evolutionary conservation and, 291–92 heat tolerance and, 223, 225–26, 229, 245 hormones in, 270 meristem activity of, 265 proteomic reference maps for, 243 reproduction of, 265 Woody perennials, winter survival of, 187 World food security, 143–44, 178, 185, 263, 281 WRKY-family, of transcription factors, 202–3, 287 WUE. See Water use efficiency Xanthoxin, 9 Xylem influx to, 91–92 retrieval, 92–93 Yield increase in, 281 NUE influencing, 169 PUE influencing, 144–45, 158 Zinc-finger proteins, 292

Figure 3.2 The DREB/CBF circuit anchored with cis-regulatory elements. This view of the CBF portion of cold/drought response is centered on 4 members of DREB A-1 subfamily. Genes are shown as proteins (colored ovals) and promoters (black lines) with cis-regulatory elements (colored small rectangles). Regulators and elements are color matched. The ICE protein (inducer of CBF; blue oval) binds to the ICEr3 and ICEr4 elements (blue rectangles) in ZAT12, NAC72, HOS9, and CBFs 1-3. HOS9 binds to the HOS9r1 element (yellow ovals and rectangles), possibly acting as a repressor by displacing ICE since the elements are adjacent on the promoter. CBFs and DREB2 all bind to the DRE element (green) to induce downstream genes (light green) and the growth regulator ZAT10 (pink). CBF2 suppresses CBF1 and 3, but probably does not bind to the DRE element itself. ZAT10, which is also induced by ROS and other abiotic stresses, has 2 ICE elements, and suppresses the activity of DREbinding genes through an unknown mechanism. NAC72 is both upstream and downstream of CBFs, having both DRE and ICE elements. It binds to NAC72r1 element in CBF2, 3 and HOS9 to suppress expression. DREB2 is activated by drought, CBF4 is activated by increased ABA and interacts with ABRE binding factors (ABF). GA oxidases possess DRE elements and are activated by CBF1 to reduce GA levels, which leads to DELLA mediated dwarfism.

Genes for Plant Abiotic Stress Editors Matthew A. Jenks and Andrew J. Wood © 2010 Blackwell Publishing ISBN: 978-0-813-81502-2

CP–1

Figure 3.3 Assembly of osmosensor, ABA and DREB/CBF regulatory circuits. Individual interactions identified in this chapter were assembled into an excel file, identifying the locus identifier for each gene, and downloading attributes from the Arabidopsis information resource (TAIR), the subcellular localization database for Arabidopsis (SUBA), and input into the network visualization tool cytoscape 2.6.1 (www.cytoscape.org) using the automated hierarchical layout. Shapes represent genes (triangles as hormones), colors are localizations (blue = nucleus; red = endomembrane; green = chloroplast, tan = cytosole, pink = unknown. Edges represent interactions (arrowhead = induces or activates, bar = suppresses).

CP–2

Figure 3.4 Systems level view of drought/cold stress network. Genes described in this chapter were identified in the predicted interactome of Arabidopsis (Geisler-Lee et al. 2007), and all interaction between, and were captured with first neighbors using cytoscape. This predicted and neighbor’s network was then merged with known interactions from Figure 3.3 to produce a single network of 323 genes and 1,638 interactions. Genes are identified by shape (octagon = known transcription factor or DNA binding; hexagon = protein, metabolite, or hormone binding; parallogram = structural or transporter; triangle = nucleotide binding; circle = everything else or unknown) and color (blue = nucleus, light blue = mitochondria; green = chloroplast; tan = cytosol; red = endomembrane; pink = unknown). The whole network is shown (A) without gene labels, and two smaller portions show interactions near the CBF/DREB regulatory circuit (B) and the osmosensing ATHK1, ARR phosphor-relay genes and PRR genes of the circadian clock (C).

CP–3

Table 3.1 stresses.

CP–4

Correlation of drought stress transcriptome with other

CP–5

Na+

Na+

Na+

OsHKT2;1

Na+ H+

Na+

Na+

SOS1

Na+

SOS2

H+

NHX1

H +

AVP1

2 Pi

SOS3

Vacuole

PPi

H+

PPi 2 Pi

[Ca2+]cyt

ATP

ADP + Pi

AVP1 ?

V-ATPase

H+

Figure 4.1 The ion transporters, channels, and pumps, which have been characterized as being involved in Na+ exclusion from the shoot. The various proteins are localized on the putative membrane that they are associated with, and all proteins have been localized in a single cell to simply show their putative function in the movement of Na+. Responsible for influx of Na+ into cells are cyclic-nucleotide gated channels (CNGC), glutamate receptors (GLR), nonselective cation channels (NSCC), and HKT transporters (AtHKT1;1, OsHKT1;4, OsHKT1;5 and OsHKT2;1). Responsible for efflux of Na+ from cells is the Na+/H+ antiporter (SOS1), which interacts with the serine/threonine protein kinase (SOS2), which interacts with the calcium binding protein (SOS3), which is activated by [Ca2+]cyt. Vacuolar storage of Na+ is mediated by a vacuolar Na+/H+ antiporter (NHX1) and the proton gradient is provided by the vacuolar H+-pyrophosphatase (AVP1) and the vacuolar H+-ATPase (V-ATPase). AVP1 has also been associated with acidifying the apoplastic space.

NSCCs

GLRs

CNGCs

Cytoplasm

Apoplast

OsHKT1;4 OsHKT1;5 AtHKT1;1

MX

XP

SOS1 ?

AtHKT1;1

OsHKT1;4

OsHKT2;1

NSCCs SOS1 ? GLRs

AtHKT1;1

OsHKT1;5

CNGCs

SOS1 ?

EN

Soil

PR

XP

MX

CO EP

Figure 4.2 Whole plant localization of several Na+ transporters or channels involved in Na+ transport in the shoot (top) or root (bottom). Depicted are proteins putatively involved in Na+ influx from soil to the epidermis: OsHKT2;1, nonselective cation channels (NSCC), glutamate receptors (GLR), and cyclic-nucleotide gated channels (CNGC). Transporters responsible for Na+ retrieval from the metaxylem to the xylem parenchyma in the root include HKT transporters (AtHKT1;1 and OsHKT1;5) and a Na+/H+ antiporter (SOS1). Transporters responsible for Na+ retrieval from the metaxylem to the xylem parenchyma of the shoot include the HKT genes (AtHKT1;1 and OsHKT1;4) and a Na+/H+ antiporter (SOS1). Cell types depicted include epidermis (EP), cortex (CO), endodermis (EN), pericycle (PR), xylem parenchyma (XP), and metaxylem (MX). Also depicted is the apoplastic influx pathway for Na+ (green arrow).

CP–6

CP–7

Figure 5.1 Expression analysis of ALS3 in Arabidopsis. For this analysis, transgenic Arabidopsis T2 plants expressing an ALS3:GUS fusion under the control of the ALS3 promoter were analyzed for patterns of ALS3 expression, as evidenced by manifestation of GUS-dependent blue color. (A) 5d old transgenic seedlings grown in the absence of Al were examined for accumulation of ALS3:GUS in their roots. GUS activity was found primarily in the phloem and epidermis beginning at the region of root elongation. (B–E) Close-up of ALS3:GUS accumulation in the vasculature and epidermis of transgenic Arabidopsis roots, including a cross section of the mature region of a root (D). (F–G) Examination of ALS3:GUS activity in transgenic Arabidopsis roots exposed to either no (F) or 100 mM (G) AlCl3 (pH 4.2) for 24 hours in nutrient solution. (H) Patterns of ALS3:GUS activity in leaves of transgenic Arabidopsis show ALS3 expression in the phloem and hydathodes of the leaf. (I) Close-up view showing ALS3:GUS activity in the epithem of the hydathode, but not the water pore. (J) ALS3:GUS activity was also found in flowers including the floral vasculature, the junction between the filament and anther, and the stylar transmitting tract.

Figure 5.2 Localization analysis for ALS1 in various organs of Arabidopsis. Transgenic Arabidopsis T2 plants expressing an ALS1:GUS fusion under the control of the ALS1 promoter were analyzed for patterns of ALS1 expression. (A) GUS activity found to occur primarily in the root tip and vasculature of 5-day old transgenic seedlings. (B) Cross section of the mature region of an ALS1:GUS root shows that GUS activity is found in the ground tissue of the stele. (C) Analysis of a mature ALS1:GUS leaf shows that GUS activity is strictly localized to the vasculature. (D) Close-up of an ALS1:GUS leaf, which shows GUS activity in both the vasculature and a hydathode. (E) Pattern of GFP fluorescence in transgenic roots expressing ProACT2:GFP or ProACT2:ALS1:GFP. Arrows indicate areas where the vacuolar membrane wraps around the cell’s nucleus, with this analysis indicating that ALS1 is localized to the plasma membrane of the root.

CP–8

CP–9

Figure 5.4 Al-dependent root growth inhibition results from inhibition of cell cycle progression and loss of the quiescent center. (A) Treatment with inhibitory levels of AlCl3 causes a disproportionate increase in the number of cells trapped in the G2 phase of the cell cycle. A cell cycle progression marker representing a truncated version of the CDS of CyclinB1;1, including a predicted mitotic destruction box, fused to the GUS reporter gene was introgressed into both als3-1 and alt1-1. Col-0 wt, als3-1 and alt1-1 lines carrying this CyclinB1;1::GUS reporter were grown in the absence or presence of increasing concentrations of AlCl3 in a soaked gel environment (pH 4.2), following which seedlings were stained for GUS activity. Growth in the absence of AlCl3 resulted in minimal levels of GUS staining, indicating that few cells were trapped in the G2 phase under these conditions. Addition of AlCl3 resulted in a profound increase in GUS staining in roots of Col-0 wt and als3-1, showing that a consequence of Al treatment is an increase in the number of cells trapped in the G2 phase of the cell cycle. Addition of highly inhibitory levels of AlCl3 resulted in complete loss of GUS activity in als3-1 roots, suggesting that cells in the root tip of als3-1 were completely differentiated and not undergoing cell division. This is supported by development of lateral roots immediately behind the primary root tip of Al-treated als3-1. In contrast to Col-0 wt and als3-1, alt1-1 roots had only basal levels of GUS activity even with high AlCl3, indicating that alt1-1 mutant roots do not arrest the cell cycle in response to Al, which likely results from reduced AtATR activity in the alt1-1 mutant. (B) Evans’ blue staining indicates that even though root growth is severely arrested, als3-1 roots are viable following Al treatment. Col-0 wt, als3-1, and alt1-1 seedlings grown in the absence or presence of AlCl3 were stained with the vital stain Evans blue. Since Evans blue staining was minimal in all samples tested, as evidenced by lack of blue color in treated roots, it is likely that Al-dependent root growth inhibition does not result from tissue death but rather arises from terminal differentiation of the root tip. (C) Al treatment results in loss of the quiescent center (QC) in Arabidopsis roots. A GUS-based marker for the root quiescent center, QC46, was introgressed into als3-1 and alt1-1. Col-0 wt, als3-1, and alt1-1 carrying the QC46 GUS promoter trap were grown either in the absence or presence of 1.50 mM AlCl3 in a soaked gel environment (pH 4.2), after which seedlings were stained for GUS activity to assess the status of the QC. In the absence of Al, all samples had normal staining at the position of the QC in the root tip, suggesting that all samples maintained a viable QC. In contrast, treatment with levels of Al that cause severe root growth inhibition resulted in the loss of GUS activity in both Col-0 wt and als3-1 roots. This suggests that Al forces the QC in both of these lines to fully differentiate, with terminal differentiation due to the loss of stem cells in the QC likely resulting in root growth inhibition following Al treatment. Alt1-1 roots had normal GUS activity at the position of the QC even after treatment with Al, which indicates that AtATR activity is required to trigger the differentiation of the QC in the presence of Al.

CP–10

Figure 6.1 Pi signaling pathways in Arabidopsis thaliana. In plants, molecular, metabolic, and morphological changes observed in response to external Pi availability seem to be determinate by local and systemic Pi signaling pathways. Under a high Pi regimen, Pi uptake occurs through low-affinity Pi transporters; under this condition, PHO2, a Pi homeostasis modulator, is induced and could negatively regulate the expression of high-affinity Pi transporters. A similar role has also been suggested for SPX proteins. In contrast, when plants grow under Pi starvation, several signaling pathways are triggered, all of them focused to enhance Pi uptake and optimize internal Pi use. To date, it is unknown how low external Pi availability is perceived and how this signal is transmitted. The PHR1 signaling pathway is the route best described for the activation of Pi-responsive genes. PHR1, a transcriptional factor sumoylated by SIZ1, positively regulates a large sub-set to Pi responsive genes, including the Mir399 gene family, which in turn can systemically modulate PHO2 expression. Other transcriptional factors (bHLH32, WRKY75, ZAT6) recently reported modulated subsets of Pi-responsive genes or control, together with hormones (auxins, cytokinins, and ethylene) and sugar root growth (ZAT6). The expression of the auxin receptor TIR1 is induced by Pi deprivation and plays a role in the increased lateral root density observed under this condition.

CP–11

Threonine

Lysine

Valine

Leucine

Serine

Fumarate

Malate

Tyrosine

Oxalosuccinate

Isocitrate

Microbial activity, NO3 NH4

Alpha-Ketoglutarate Succinate

Shikimate

Ornithine

Citrulline

Arginine

Anthranilate

Proline

Glutamate

Glutamine

Histidine

16 Fatty acids

Alanine

DAHP

9 Amines

Tryptophan

Chorismate

Phenylalanine

5 Amines

Prephenate Erythrose 4phosphate

Citrate

Pyruvate

Oxaloacetate

Acetyl-CoA

Pyruvate

Phosphoenolpyruvate

6-phosphoglycerate

Dihydroxyacetone-P

Glycealdehyde-3-P

Fructose 1,6-bisphosphate

Fructose phosphate

Glucose

Figure 7.1 Metabolite changes related to NUE in GDH transgenic roots. Metabolites in blue boxes were not detected. Metabolites in red boxes were used as internal standards and therefore detected. Metabolites in blue boxes were increased 2–3 fold. Metabolites in black boxes were detected and not changed. Metabolites in green boxes were decreased.

Methionine

Cystathionine

Homoserine

Isoleucine

Aspartate

Glycine

Cysteine

O-Acetylserine

Aspartate 4-semialdehyde

0

1–2

2–3

3–4

4–5

5–10

10–15

Asparagine

Low

High

Fold Increase

3.5

(H) Shannon Index

3 2.5 control 2 urea 1.5 MU 1 0.5 0 15 days

42 days

90

cm

70

50

control MU

30

urea

10

7

14

21

28

35

42

days

Figure 7.3 Microbial populations are altered by N fertilization. Panel A. Shannon indexes (H) for characterizing soil bacterial diversity in soil treated with different N sources show differences that develop over time. Panel B. The alterations contribute to plant growth.

CP–12

Figure 8.1 An outline of processes leading to cold acclimation and development of freezing tolerance (see text for details).

CP–13

Figure 8.2 Control of the CBF regulon in Arabidopsis (see text for details).

CP–14

Figure 9.2 Acquired thermotolerance in Arabidopsis thaliana. Seedlings were heated to 45 °C for 2 hours directly (left) or after pretreatment at 38 °C, 1.5 hour + 22 °C, 2 hours (right), before moving back to 22 °C. Only the acclimated seedlings on the right survived after 5 days. Photo courtesy of Dr. Elizabeth Vierling, University of Arizona, Tucson, Arizona, USA.

Figure 9.3 Symptoms of high-temperature stress can vary. Left: Leaf scalding in maize. Photograph courtesy of Robert L. Croissant, deceased (from the collection of Dr. Howard F. Schwartz, Colorado State University, Bugwood Network Image). Right: Leaf rolling in potato. Photograph courtesy of Dr. Howard F. Schwartz, Colorado State University, Bugwood Network Image.

CP–15