Advances in Botanical Research, Volume 33

Advances in

BOTANICAL RESEARCH incorporating Advances in Plant Pathology Editor-in-Chief J.A.CALLOW

School of Biological Sciences, University of Birmingham, UK

Editorial Board J. S. HESLOP-HARRISON M.KREIS R.A. LEIGH E. LORD D. G. MANN P.R.SHEWRY I. C. TOMMERUP

John Innes Centre, Norwich, UK Universite de Paris-Sud, Orsay, France University of Cambridge, Cambridge, UK University of California, Riverside, USA Royal Botanic Garden, Edinburgh, UK IACR-LongAshton Research Station, UK CSIRO, Perth, Australia

CONTRIBUTORS TO VOLUME 33

P. W. CROUS Department of Plant Pathology, University of Stellenbosch, Private Bag XI, Matieland, 7602, South Africa P. S. DYER Microbiology Division, School ofBiological Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK A. R. ENNOS School ofBiological Sciences, University ofManchester, 3.614 Stopford Building, Oxford Road, Manchester M13 9PT, UK M. J. HAWKESFORD IARC-Rothamsted, Biochemistry and Physiology Department, Harpenden, Hertfordshire AL5 2JQ, UK D. KOLLER Plant Biophysics Laboratory, Institute of Life Sciences, The Hebrew University, Jerusalem 91904, Israel W.-M. KRIEL Department of Plant Pathology, University of the Orange Free State, Bloemfontein, 9300, South Africa J. A. LUCAS IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol BS41 9AF, UK T. D. MURRAY Department of Plant Pathology, Washington State University, Pullman, WA 99164-6430, USA W. J. SWART Department of Plant Pathology, University of the Orange Free State, Bloemfontein, 9300, South Africa J. L. WRAY Plant Sciences Laboratory, Sir Harold Macmillan Building, Division of Environmental and Evolutionary Biology, University of St Andrews, St Andrews, Fife KY16 9TH, UK

CONTENTS OF VOLUMES 21-32

Contents of Volume 21 Defence Responses of Plants to Pathogens E. KOMBRINK and I. E. SOMSSICH On the Nature and Genetic Basis for Resistance and Tolerance to Fungal Wilt Diseases of Plants C. H. BECKMAN and E. M. ROBERTS Implication of Population Pressure on Agriculture and Ecosystems A. H. EHRLICH

Plant Virus Infection: Another Point of View G. A. DE ZOETEN The Pathogens and Pests of Chestnuts S. L. ANAGNOSTAKIS Fungal Avirulence Genes and Plant Resistance Genes: Unraveling the Molecular Basis of Gene-for-Gene Interactions P. J. G. M. DE WIT Phytoplasmas: Can Phylogeny Provide the Means to Understand Pathogenicity? B. C. KIRKPATRICK and C. D. SMART Use of Categorical Information and Correspondence Analysis in Plant Disease Epidemiology

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CONTENTS OF VOLUMES 21-32

S. SAVARY, L. V. MADDEN, J. C. ZADOKS and H. W. KLEINGEBBINCK

Contents of Volume 22 Mutualism and Parasitism: Diversity in Function and Structure in the 'Arbuscular' (VA) Mycorrhizal Symbiosis EA. SMITH and S. E. SMITH Calcium Ions as Intracellular Second Messengers in Higher Plants A. A. R. WEBB, M. R. McAINSH, J. E. TAYLOR and I. M. HETHERINGTON The Effects of Ultraviolet-B Radiation on Plants: A Molecular Perspective B.R.JORDAN Rapid, Long-Distance Signal Transmission in Higher Plants M.MALONE Keeping in Touch: Responses of the Whole Plant to Deficits in Water and Nitrogen Supply A. J. S. McDONALD and W. J. DAVIES

Contents of Volume 23 PATHOGEN INDEXING TECHNOLOGIES The Value of Indexing for Disease Control Strategies D. E. STEAD, D. L. EBBELS and A. W. PEMBERTON Detecting Latent Bacterial Infections S. H. DE BOER, D. A. CUPPELS and R. GITAITIS

CONTENTS OF VOLUMES 21-32

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Sensitivity of Indexing Procedures for Viruses and Viroids H.HUTIINGA Detecting Propagules of Plant Pathogenic Fungi S. A MILLER Assessing Plant-Nematode Infestations and Infections K. R. BARKER and E. L. DAVIS

Potential of Pathogen Detection Technology for Management of Diseases in Glasshouse Ornamental Crops I. G. DINESEN and A VAN ZAAYEN

Indexing Seeds for Pathogens J. LANGERAK, R. W. VAN DEN BULK and A A J. M. FRANKEN A Role for Pathogen Indexing Procedures in Potato Certification S. H. DE BOER, S. A SLACK, G. VAN DEN BOVENKAMP and I. MASTENBROEK A Decision Modelling Approach for QuantifYing Risk in Pathogen Indexing C. A LJ3VESQUE and D. M. EAVES Quality Control and Cost Effectiveness of Indexing Procedures C.SUTULAR

Contents of Volume 24 Contributions of Population Genetics to Plant Disease Epidemiology and Management M. G. MILGROOM and W. E. FRY

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CONTENTS OF VOLUMES 21-32

A Molecular View through the Looking Glass: the Pyrenopeziza brassicae-Brassica Interaction A.M. ASHBY The Balance and Interplay Between Asexual and Sexual Reproduction in Fungi M. CHAMBERLAIN and D. S. INGRAM The Role of Leucine-Rich Repeat Proteins in Plant Defences D. A. JONES and J. D. G. JONES Fungal Life-Styles and Ecosystem Dynamics: Biological Aspects of Plant Pathogens, Plant Endophytes and Saprophytes

R. J. RODRIGUEZ and R. S. REDMAN Cellular Interactions between Plants and Biotrophic Fungal Parasites M. C. HEATH and D. SKALAMERA Symbiology of Mouse-Ear Cress (Arabidopsis thaliana) and Oomycetes E. B. HOLUB and J. L. BEYNON Use of Monoclonal Antibodies to Detect, Quantify and Visualize Fungi in Soils F. M. DEWEY, C. R. THORNTON and C. A. GILLIGAN Function of Fungal Haustoria in Epiphytic and Endophytic Infections P. T. N. SPENCER-PHILLIPS Towards an Understanding of the Population Genetics of PlantColonizing Bacteria B. HAUBOLD and P. B. RAINEY

CONTENTS OF VOLUMES 21-32

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Asexual Sporulation in the Oomycetes A. R. HARDHAM and G. J. HYDE Horizontal Gene Transfer in the Rhizosphere: a Curiosity or a Driving Force in Evolution? J. WOSTEMEYER, A. WOSTEMEYER and K. VOIGT The Origins of Phytophthora Species Attacking Legumes in Australia J. A. G. IRWIN, A. R. CRAWFORD and A. DRENTH

Contents of Volume 25 THE PLANT VACUOLE The Biogenesis of Vacuoles: Insights from Microscopy F. MARTY Molecular Aspects of Vacuole Biogenesis D. C. BASSHAM and N. V. RAIKHEL The Vacuole: a Cost-Benefit Analysis J.A.RAVEN The Vacuole and Cell Senescence P.MATILE Protein Bodies: Storage Vacuoles in Seeds G. GALILI and E. M. HERMAN Compartmentation of Secondary Metabolites and Xenobiotics in Plant Vacuoles M. WINK

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CONTENTS OF VOLUMES 21-32

Solute Composition of Vacuoles R.A. LEIGH The Vacuole and Carbohydrate Metabolism C. J. POLLOCK and A. KINGSTON-SMITH Vacuolar Ion Channels of Higher Plants G. J. ALLEN and D. SANDERS The Physiology, Biochemistry and Molecular Biology of the Plant Vacuolar ATPase U. LUTTGE and R. RATAJCZAK The Molecular and Biochemical Basis of Pyrophosphate-Energized Proton Translocation at the Vacuolar Membrane R.-G. ZHEN, E. J. KIM and P. A. REA The Bioenergetics of Vacuolar u+ Pumps J.M.DAVIES Transport of Organic Molecules Across the Tonoplast E. MARTINOIA and R. RATAJCZAK Secondary Inorganic Ion Transport at the Tonoplast E. BLUMWALD and A. GELLI Aquaporins and Water Transport Across the Tonoplast M. J. CHRISPEELS, M. J. DANIELS and A. WEIG

Contents of Volume 26 Developments in the Biological Control of Soil-borne Plant Pathogens J.M. WHIPPS

CONTENTS OF VOLUMES 21-32

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Plant Proteins that Confer Resistance to Pests and Pathogens P.R. SHEWRY and J. A. LUCAS The Net Primary Productivity and Water Use of Forests in the Geological Past D. J. BEERLING Molecular Control of Flower Development in Petunia hybrida L. COLOMBO, A. VAN TUNEN, H. J. M. DONS and G. C. ANGENENT

The Regulation of C4 Photosynthesis R. C. LEEGOOD Heterogeneity in Stomatal Characteristics J. D. B. WEYERS and T. LAWSON

Contents of Volume 27 CLASSIC PAPERS The Structure and Biosynthesis of Legume Seed Storage Proteins: A Biological Solution to the Storage of Nitrogen in Seeds D. BOULTER and R. R. D. CROY Inorganic Carbon Acquisition by Marine Autotrophs J.A. RAVEN

The Cyanotoxins W. W. CARMICHAEL Molecular Aspects of Light-harvesting Processes in Algae T. LARKUM and C. 1. HOWE

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CONTENTS OF VOLUMES 21-32

Plant Transposable Elements R. KUNZE, H. SAEDLER and W.-E. LONNIG

Contents of Volume 28 Protein Gradients and Plant Growth: Role of the Plasma Membrane H+·ATPase M.G. PALMGREN The Plant Invertases: Physiology, Biochemistry and Molecular Biology Z. TYMOWSKA-LALANNE and M. KREIS Dynamic Pleomorphic Vacuole Systems: Are They Endosomes and Transport Compartments in Fungal Hyphae? A. E. ASHFORD Signals in Leaf Development T. P. BRUTNELL and J. A. LANGDALE Genetic and Molecular Analysis of Angiosperm Flower Development V. F. IRISH and E. M. KRAMER Gametes, Fertilization and Early Embryogenesis in Flowering Plants C. DUMAS, F. BERGER, J. E.-FAURE and E. MATTHYS-ROCHON

Contents of Volume 29 The Calcicole-Calcifuge Problem Revisited J.A.LEE

CONTENTS OF VOLUMES 21-32

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Ozone Impacts on Agriculture: an Issue of Global Concern M. R. ASHMORE and F. M. MARSHALL Signal Transduction Networks and the Integration of Responses to Environmental Stimuli G. I. JENKINS Mechanisms ofNa+ Uptake by Plants A. AMTMANN and D. SANDERS The NaCI-induced Inhibition of Shoot Growth: The Case for Disturbed Nutrition with Special Consideration of Calcium Nutrition D. B. LAZOF and N. BERNSTEIN

Contents of Volume 30 Nitrate and Ammonium Nutrition of Plants: Physiological and Molecular Perspectives B. G. FORDE and D. T. CLARKSON Secondary Metabolites in Plant-Insect Interactions: Dynamic Systems of Induced and Adaptive Responses J. A. PICKETT, D. W. M. SMILEY and C. M. WOODCOCK Biosynthesis and Metabolism of Caffeine and Related Purine Alkaloids in Plants H. ASHIHARA and A. CROZIER Arabinogalactan-Proteins in the Multiple Domains of the Plant Cell Surface M.D. SERPE and E. A. NOTHNAGEL

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CONTENTS OF VOLUMES 21-32

Plant Disease Resistance: Progress in Basic Understanding and Practical Application N.T.KEEN

Contents of Volume 31 Trichome Diversity and Development E.WERKER Structure and Function of Secretory Cells A.FAHN Monoterpenoid Biosynthesis in Glandular Trichomes of Labiate Plants D. L. HALLAHAN Current and Potential Exploitation of Plant Glandular Trichome Productivity S. 0. DUKE, C. CANEL, A. M. RIMANDO, M. R. TELLEZ, M. V. DUKE and R.N. PAUL Chemotaxonomy Based on Metabolites from Glandular Trichomes O.SPRING Anacardic Acids in Trichomes of Pelagonium: Biosynthesis, Molecular Biology and Ecological Effects D. J. SCHULTZ, J. I. MEDFORD, D. COX-FOSTER, R. A. GRAZZINI, R. CRAIG and R. 0. MUMMA Specification of Epidermal Cell Morphology B. J. GLOVER and C. MARTIN

Trichome Initiation in Arabidopsis

CONTENTS OF VOLUMES 21-32

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A. R. WALKER and M.D. MARKS

Trichome Differentiation and Morphogenesis in Arabidopsis M. HULSKAMP and V. KIRIK Trichome Plasmodesmata: A Model System for Cell-to-Cell Movement F. WAIGMANN and P. ZAMBRYSKI

Contents of Volume 32 Plant Protein Kinases Plant Protein-Serine/Threonine Kinases: Classification into Subfamilies and Overview of Function D. G. HARDIE Bioinformatics: Using Phylogenetics and Databases to Investigate Plant Protein Phosphorylation E. R. INGHAM, T. P. HOLTSFORD and J. C. WALKER Protein Phosphatases: Structure, Regulation, and Function S.LUAN Histidine Kinases and the Role of Two-Component Systems in Plants G. E. SCHALLER Light and Protein Kinases J. C. WATSON

Calcium-Dependent Protein Kinases and their Relatives E.M.HRABAK Receptor-Like Kinases in Plant Development

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CONTENTS OF VOLUMES 21-32

K. U. TORII and S. E. CLARK

A Receptor Kinase and the Self-Incompatibility Response in Brassica J.M.COCK Plant Mitogen-Activated Protein Kinase Signalling Pathways in the Limelight S. JOUANNIC, A.-S. LEPRINCE, A. HAMAL, A. PICAUD, M. KREIS andY. HENRY Plant Phosphorylation and Dephosphorylation in Environmental Stress Responses in Plants _K. ICHIMURA, T. MIZOGUCHI, R. YOSHIDA, T. YUASA and K. SHINOZAKI

Protein Kinases in the Plant Defence Response G. SESSA and G. B. MARTIN SNFl-Related Protein Kinases (SnRKs) -Regulators at the Heart of the Control of Carbon Metabolism and Partitioning N. G. HALFORD, J.-P. BOULY and M. THOMAS Carbon and Nitrogen Metabolism and Reversible Protein Phosphorylation D. TO ROSER and S. C. HUBER Protein Phosphorylation and Ion Transport: A Case Study in Guard Cells J. LI and S. M. ASS MANN

PREFACE

It is now commonly understood that, in nature, plant organs are invariably

associated, to a greater or lesser extent, with one or more endophytic fungi. Fungal endophytes have a very intimate and co-evolutionary relationship with their hosts, and their presence may profoundly affect the physiological processes of the plant. We have a reasonably good understanding of the relationship between fungal endophytes and root tissues in mycorrhizal associations, but relationships with aerial tissues, notably leaves, are less well researched and understood. The review by Kriel et al. takes a fresh look at foliar endophytes, particularly of gymnosperms, and considers their potential significance in latent disease, and in moderating pathogenesis and other possible mutualistic effects. Organs of terrestrial plants are able to carry out a variety of movements to optimize their utilization of environmental resources. Forces for movement originate from biophysical motors that respond to sensed environmental signals by changing their growth or turgor characteristics. Light signals are particularly significant, and the review by Koller considers a number of aspects of phototropic organ movements, from the perception and transduction of the light stimulus, to the types of motor involved, how they work, and how these movements fit into an overall adaptive strategy. The study of root systems has tended to be dominated by their function in acquisition of water and nutrients, and by comparison the study of their other key role - anchorage - has been neglected. The article by Ennos reviews how botanists have more recently taken inspiration from materials science and engineering piles theory and combined this with novel ways of experimentally testing the mechanical properties of roots, to produce a more interdisciplinary synthesis of the anchorage mechanics of roots. The importance of sulphur in promoting yield, quality and stress resistance parameters in plants has been highlighted by the recent increased problems of S-deficiency in agriculture. These deficiencies are, in part, a consequence of reduced atmospheric emissions from industry and the subsequent decreased deposition on agricultural land. Almost all of the genes responsible for uptake, transport and assimilation of sulphate have now been cloned, and many outstanding questions regarding the control of uptake, and of the pathways and intermediates involved in sulphur assimilation, have now been answered. The article by Hawkesford and Wray

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PREFACE

reviews the present status of the molecular genetics of sulphate assimilation and outlines possibilities for manipulation of these pathways. Of the various fungal diseases of cereals the group of eyespot diseases caused by Tapesia spp. has been relatively neglected, and their economic importance has been uncertain. This has been partly due to difficulties in accurate diagnosis and a relative lack of biological research on the genus in comparison with other cereal disease-causing fungi. The article by Lucas et al. reviews progress made over the last 15 years or so in improving our understanding of aspects of genetics, taxonomy, epidemiology, population biology and control of this fungus. These have radically altered our perceptions of its significance and helped in establishing it as a model for trash-borne cereal pathogens. The Editor would like to thank all the contributors to this volume, for their patience and cooperation in making his task easier.

J. A. Callow

Foliar Endophytes and Their Interactions with Host Plants, with Specific Reference to the Gymnospermae

WILMA-MARIE KRIEL, 1 WIJNAND J. SWART1 and PEDRO W. CROUS 2 1

Department of Plant Pathology, University of the Orange Free State, Bloemfonteim 9300, South Africa 2 Department of Plant Pathology, University of Stellenbosch, Private bag XI, Matieland 7602, South Africa

I. Introduction .... .. ..... .. .. .. .. ..... .. .. .. ... .. .. .. .. ... .. .. .. ... .. .. ....... ..... .... .. ....... ....... .. ... ...... II. Diversity of Endophytic Associations.......................................................... A. Diversity Among Host Species (Interspecific Diversity)................... B. Diversity Within Host Species (Intraspecific Diversity).................... C. Diversity Among Fungal Species.......................................................... III. Ecology of Endophytic Associations............................................................ A. The Host Plant: Gyrnnospermae ........................................ .. .... ............ B. The Endophyte........................................................................................ C. Host-Endophyte Interactions............................................................... IV. Summary.......................................................................................................... Acknowledgements ........................................... ...................................... ....... References.......................................................................................................

1 3 3 5 7 9 9 10 19 25 29 29

This review discusses the nature of endophytic fungal relationships of the Gymnospermae and factors affecting their colonization frequencies within Gymnosperm foliage. The roles offungal foliar endophytes in insect herbivory, biological control, latent pathogenesis and other associations are addressed. Specific mention is made of host and fungal diversity, ecology of endophytic colonization, and the physiology of endophytic associations. Aspects of quiescent infection, latent pathogenesis and absolute endophytism are also discussed.

I.

INTRODUCTION

Fungi live in a mutualistic, antagonistic or neutral symbiosis with a wide variety of both autotrophic and heterotrophic organisms. The properties of Advances in Botanical Research Vol33 incorporating Advances in Plant Pathology ISBN 0- l 2-005933-9

Copyright © 2000 Academic Press All rights of reproduction in any form reserved

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W.-M. KRIEL, W. J. SWART and P. W. CROUS

these relationships are diverse, displaying varying degrees of association and nutritional interdependence (Petrini, 1986). Fungi living on the exterior of their hosts are called epiphytes, as opposed to those living within host tissue which are termed endophytes (DeBary, in Petrini, 1986). Endophytes, in contrast to epiphytes, are contained entirely within the host plant substrate, and may have either a parasitic or symbiotic association with the host (Sinclair and Cerkauskas, 1996). At the most basic level, 'endophyte' simply refers to the location of the organism: 'endo' means within, and 'phyte' means plant, therefore describing all organisms that live inside a plant (Wilson, 1995a). The term has, however, evolved to indicate not only the location of the organism, but the actual type of association the fungi or bacteria have with their host. The nature of the interaction described by the term, endophyte, is that such organisms found inside the plants do not elicit symptoms of disease (Wilson, 1995a). Observations of asymptomatic fungal infections were made in various plant species as early as 1947 (Bose, 1947). Petrini (1986) postulated that all living plants probably host endophytic fungi. The latter term describes all organisms that inhabit plant organs and can colonize internal plant tissues at some time in their life, without any immediate deleterious effect on their host (Petrini, 1991). This would also include endophytic organisms with an epiphytic phase and latent pathogens that may have a symptomless phase in their host. According to Wilson (1995a), 'endophyte' describes the type of infection strategy. Kowalski and Kehr (1992) also introduced another term 'phellophyte', for fungi typically colonizing the dead outer bark tissue of tree stems. Endophytes from smaller tree organs such as leaves, petioles and twigs, were termed 'xylotropic endophytes' by Chapela (1989). Carroll (1988) used the term endophyte to describe fungi that form inconspicuous or asymptomatic infections within the leaves and stems of healthy plants. Many endophytes are closely related to virulent pathogens, but have limited, if any, pathogenic effects on their host plants (Carroll, 1988). According to Dorworth and Callan (1996), the length of the latent endophytic stage is directly related to the extent of evolutionary advance or regression from the pathogenic to the mutualistic state. Endophytic foliar pathogens (endophytic antagonistic symbionts ), such as rust fungi have been studied extensively by plant pathologists (Petrini, 1986). In this review, the definition of endophyte, as circumscribed by Petrini (1991 ), will be used. According to Wilson (1995a) the most important question is not whether an organism is an endophyte or not, but why infection by endophytes does not trigger a defence response by the plant? Other important issues are: Why are they there? What are they doing? How do they affect the host plant? According to Wilson (1993), plants do not consist solely of plant tissues, and should be treated as evolving, integrated symbiotic units of plant and fungal cells, which can affect both ecological and physiological processes. Fungal endophytes thus have a very intimate and probably also a co-evolutionary

FOLIAR ENDOPHYTES

3

relationship with their hosts, and thus, have the potential to influence the evolutionary trajectory of plant defences. Endophytes can, for example, protect host plants from insect herbivory (Clay, 1988; Clark eta/., 1989) and other fungal pathogens (Carroll, 1988). They can, therefore, be used as bioregulators to induce resistance against diseases; as biological control agents against certain pathogens (Bissegger and Sieber, 1994); and also in the biological control of undesirable weeds (Dorworth and Callan, 1996). Endophytes can also be used as bio-indicators, reacting to pollutants such as acid rain, ozone and industrial emissions (Helander et al., 1993b, 1996). The occurrence of foliar endophytes is not confined to the phanerogams, and seems to be quite common in pteridophytes (Dreyfuss and Petrini, 1984). A wide variety of coniferous tree species have yielded foliar fungal endophytes (Carroll et al., 1977; Carroll and Carroll, 1978; Petrini and Muller, 1979; Petrini and Carroll, 1981; Petrini, 1986; Suske and Acker, 1987). The aim of this review is to investigate the endophytic fungal populations associated predominantly with aerial tissues of Gymnospermae so as to obtain a better understanding of the effects they may have on their host including aspects such as latent infection, pathogenesis and possible beneficial associations. The fungal endophytes of roots, especially mycorrhizas, have been extensively reviewed and will not be considered here.

II.

DIVERSITY OF ENDOPHYTIC ASSOCIATIONS

In general, foliar endophytes can be divided into two groups: first, those that are ubiquitous and can be isolated from a wide variety of host species in different ecological and geographical conditions, and secondly, species that show a fair degree of host specificity and follow the same patterns characteristic of obligate antagonistic symbionts (such as the Uredinales) (Petrini, 1986). Endophytes commonly isolated from a given host and, less frequently, from other hosts are generally host specific. In contrast, endophytes that are rarely isolated from a given host species appear to be less host specific and may be isolated from a wide variety of hosts (Petrini et al., 1982). Dreyfuss (in Bills, 1996) speculated that endophytic fungi represent one of the largest reservoirs of fungal species. According to Petrini (1996), 'symptomless endophytes' can basically be assembled in two distinct ecological groups: the clavicipitaceous systemic grass endophytes, which live in a mutualistic symbiosis with their hosts; and the endophytes of trees and shrubs, including non-clavicipitaceous grass endophytes. A.

DIVERSITY AMONG HOST SPECIES (INTERSPECIFIC DIVERSITY)

Todd (1988) suggested that susceptibility to infection by endophytes is heritable, thus being a product of kin selection. According to Petrini and

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Carroll (1981), fungal endophytes displayed a degree of host specificity, at least at family level. This tendency may be more important than geographical location of the host plant as far as determining the overall distribution of endophytes. Host-specificity has been shown to be directly correlated with the existence of a symbiotic association between a fungal endophyte and its host (Petrini and Carroll, 1981). Hata and Futai (1996) found the taxonomic position of host pine species to have a strong effect on the mycobiota. In fact, taxonomy had a stronger effect on the distribution patterns of endophytic species in pines than factors such as sampling date, tree age and the location of the sampling tree (Hata and Futai, 1996). Generally occurring foliar endophytes such as Epicoccum nigrum Link and Aureobasidium pullulans (De Bary) Arnaud, are termed host-neutral endophytes (Boddy and Griffith, 1989) as opposed to an endophyte like Rhabdocline parkeri Sherwood-Pike, Stone and Carroll on Douglas fir (Pseudotsuga menziesii (Mirb.) Franco), which is absolutely host specific and has a close relationship with its only host (Sherwood-Pike et al., 1986). In two species of pine, namely Pinus resinosa Aiton and P. banksiana Lamb., commonly isolated endophytes showed a strong preference for their host (Legault et al., 1989). Planted stands of holly oak (Quercus ilex L.) lack characteristic species-specific endophytes that are found in natural stands (Fisher et al., 1994). Occasional isolation of host specific endophytes from other trees usually occurs only when these trees are in the vicinity of the main host (Kowalski and Kehr, 1996). The latter endophytes are able to colonize morphologically similar hosts growing at the same site. Petrini (1984) found that for ericaceous hosts, endophytes exhibited a moderate degree of host specificity. Both qualitative and quantitative differences in infection frequencies of endophytes have been reported in specific host species. In extensively sampled conifer species, up to 110 (mean value= 60) fungal species could be isolated, with the majority (80-90%) observed infrequently or only once (Carroll and Carroll, 1978). The total rate of infection in P. sylvestris L. was relatively high (80.1% ), whereas other Pinus species showed an infection rate of 20-100% (Carroll et al., 1977; Petrini, 1986), and results of studies on five other pine species varied from 46.0% to 92.3% (Carroll and Carroll, 1978). Hata and Futai (1993) found a more extensive endophytic colonization in Pinus densiflora Siebold and Zucc. than in P. thunbergii Pari. Kowalski (1993) isolated seven fungal species with an infection frequency of more than 5% from symptomless needles of Pinus sylvestris, namely Anthostomella fonnosa Kirschst. (28.0% ), Lophodennium seditiosum Minter, Staley et Millar (20.6% ), Cyclaneusma minus (Butin) Di Cosmo, Peredo et Minter (20.5%), Cenangium ferruginosum Fr.: Fr. (15.7%), L. pinastri (Schrad.ex Hook) Chev. (13.0% ), Sclerophoma pythiophila (Corda) Hahn. (6.4%) and A. pedemontana Ferr. et Sacc. (5.5% ).

FOLIAR ENDOPHYTES B.

5

DIVERSITY WITHIN HOST SPECIES (INTRASPECIFIC DIVERSITY)

Factors inherent to the physiological condition of the host, e.g. host genotype and age of foliage often play a significant role in the distribution of certain foliar endophytes within the host itself (Todd, 1988). Old needles are more heavily colonized by endophytes than young ones (Bernstein and Carroll, 1977; Petrini and Carroll, 1981; Fisher et al., 1986; Sieber-Canavesi and Sieber, 1987; Stone, 1987; Hata and Futai, 1993; Kowalski, 1993). One exception to the tendency of increased frequency of infection with increased needle age is Anthostomella fonnosa. This can be attributed to the low competitive ability of the fungus, and its inability to survive for long periods in needles, or the possibility that nutrients in older needles might become inadequate for its survival (Kowalski, 1993). Infection frequencies of Meria parkeri Sherwood-Pike could be positively correlated with the growth speed of trees. Trimmatostroma salicis Corda was only found in the older needles of conifers, which could be attributed to the fact that wax layers on needles are weathered away during ageing (Millar, 1974). T. salicis grows and sporulates on the needle surface as an epiphyte, and, due to the effect of the host ageing, it is frequently isolated as an endophyte from older needles. In studies conducted with Salicornia perennis Mill., significant differences with regard to colonization by different fungal species were found between old and new tissues (Petrini and Fisher, 1986). Fungi such as Pleospora salicorniae have been reported to colonize most parts of the host plant, but P. bjorlingii was mostly confined to older plant tissues. New tissues were colonized mainly by two species of Stagonospora and to a lesser extent by Diplodina salicorniae (Petrini and Fisher, 1986). Barklund and Kowalski (1996) found that the composition of endophytic species gradually changes, qualitatively and quantitatively, with the increasing age of internodes of Norway spruce (Picea abies). The most dominant species, Tryblidiopsis pinastri (Pers.: Fr.) P. Karsten, was most commonly isolated from young internodes, whereas three other common species, Phialocephala scopifonnis Kowalski and Kehr, Geniculosporium serpens Chesters et Greenhalgh, and Tapesia livido-fusca (Fr.) Rehm were most frequently isolated from old internodes. These fungi, called 'phellophytes' by Kowalski and Kehr (1992), were common in the older, thicker barked parts of the branch, which provide more protection for such fungi living near the surface. In contrast, Tryblidiopsis pinastri, which thrives on apical, thin-barked parts of branches and could regularly be isolated from the inner bark, could therefore be described as a true endophyte. In comparison to other endophytes of Norway spruce, T. pinastri has a special relationship with this host revealing high levels of host specificity (Barklund and Kowalski, 1996). As shown above, many endophytes are specific to the tissues and plant organs that they are able to colonize. Some fungi (e.g. Acremonium spp. and

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Fusarium spp.) are confined almost exclusively to roots, whereas others (e.g. Pestalotia spp. and Colletotrichum spp.) can be isolated only from aerial plant organs (Dreyfuss and Petrini, 1984). According to Fisher and Petrini (1990), different plant tissues and organs can be separated on the basis of their endophytic fungal populations. Fisher and Petrini (1987a) recorded 12 fungal species isolated from leaves and stems of Suaeda fruticosa. Of these fungi, Colletotrichum phyllachoroides (Ellis and Everh.) Arx. was confined to leaves, and two species of Camarosporium were isolated mainly from stems, with a higher incidence in whole stems, compared with isolations from the xylem. This demonstrated the ability of these fungi to penetrate deep into host tissue. Fisher and Petrini (1990) confirmed high colonization frequencies for bark and xylem ofAlnus spp. but, in general, the colonization of experimental segments by more than two fungi is rare. Bissegger and Sieber (1994) found endophytes to be confined to the phellem in coppice shoots of Castanea sativa Mill., with no endophytic assemblages in the pith and xylem, and seldom in the bark tissues between the phellogen and cambium. Three to 16 endophytic thalli and one to six species were isolated per cm 2 of phellem tissue. The density of lenticels has had no influence on the frequency of colonization, but the phellem adjacent to lenticels was more frequently colonized than the lenticels themselves. This could be attributed to the more intense surface sterilization with the disinfectant having penetrated into the lenticels (Bissegger and Sieber, 1994). In studies done by Fisher et al. (1995) on Dryas octopetala L., a higher frequency of endophytic taxa was found in the leaves of the host than the twigs or roots. Endophytic fungi are also associated with non-ectomycorrhizal fine roots of forest trees and shrubs, and occur as dark, septate hyphae throughout the root tissue, except for the innermost phellogen (Ahlich and Sieber, 1996). Sieber-Canavesi and Sieber (1987) observed no succession of endophytic species in needles ofAbies alba Mill., in contrast to Carroll et al. (1977), who suggested succession in the endophytic petiole flora of Sequoia, and demonstrated that the needle petiole was more intensively colonized than the apex of the needle. Fungi associated with the petiole of Sequoia were similar to those commonly found in twigs, although they colonized only the cortex of twigs, and not the vascular bundles (Carroll et al., 1977). Infection frequencies of endophytic fungi were the highest at the needle base of some tree species (Bernstein and Carroll, 1977), but in pine needles it tended to be evenly distributed over the entire needle, with a slight increase in the middle section (Kowalski, 1993). Kowalski (1993) recorded distinct differences in differential species colonization throughout the needle. This tendency varied between first and second year needles and can be attributed to different microclimatic conditions that prevailed in different needle sections. The spread of fungi in needles was not only affected by the nutrient content and microclimate of the needles, but also the interaction between fungi, where some fungi such as Sporormiella, Epicoccum,

FOLIAR ENDOPHYTES

7

Cenangium, Lophodermium, and Coniothyrium were able to limit the growth of other fungi (Kowalski, 1993). Substrate utilization tests showed differences between the various fungi and their origin (Carroll and Petrini, 1983). In studies on Pinus densiflora and P. thunbergii, Hata and Eutai (1993) found a distinct distribution pattern of some of the dominant fungi, especially Phialocephala, at the proximal, and more specifically, the basal areas of P. densiflora needles. The higher colonization frequency of endophytes in the basal part of the midrib of mountain birch (Betula pubescens var. tortuosa (Ledeb.) Nyman) leaves could be explained by more favourable conditions created for spore germination, and higher levels of moisture and leachates (Helander et al., 1993a). Another possibility, speculated by Helander et al. (1993a), concerns mycelia already present in the twigs, which might have grown into the leaf petiole, and eventually the leaf blade. In isolations of endophytic fungi from eastern larch (Larix laricina (Du Roi) K. Koch) leaves, no significant difference in the number of isolates could be detected between leaf segments from the petiole to the tip when all isolates were considered together (Dobranic et al., 1995). If one unidentified fungus was excluded from the analysis (by discounting its specific frequency), all the remaining isolates were isolated significantly more frequently from the petiole segment. Species composition in leaves of coastal redwood trees (Sequoia sempervirens (D. Don ex Lamb)), of progressing age in single branches, revealed a patchy pattern of colonization, without showing any obvious sequence of succession (Espinosa-Garda and Langenheim, 1990). Endophytic populations in leaves and sprouts were very similar, however, showing distinct differences in species richness and distribution of certain fungal species such as Pleuroplaconema sp. and Pestalotiopsis funerea (Desm.) Stey. (EspinosaGarda and Langenheim, 1990). Studies of the endophytic flora of sessile oak (Quercus petraea (Matt.) Lieb.) revealed a colonization rate of 97% in leaves and 84% in twigs. Leaves produced 78 different taxa, whereas the twig segments yielded 45. Of these taxa, 98% belonged to the Ascomycetes or their anamorphs (Halmschlager et al., 1993). Fungal assemblages associated with American beech (Fagus grandiflora Ehrh.) and aspen (Populus tremuloides Michx.) were strongly dominated by Ascomycetes and Coelomycetes (Chapela, 1989). C.

DIVERSITY AMONG FUNGAL SPECIES

The degree of host specificity among endophytes does not permit the use of endophytic distribution as a parameter of taxonomic affinity among various members of the same plant family. However, it could provide some useful taxonomic information if the parasites themselves were abundant and widespread (Carroll and Carroll, 1978). In studies based on substrate

8

W.-M. KRIEL, W. J. SWART and P. W. CROUS

utilization tests and electrophoresis of soluble proteins and pectic enzymes, Sieber-Canavesi et al. (1991) found that three distinct species of Leptostroma, morphologically almost indistinguishable from each other, respectively colonized apparently healthy needles of Picea abies (L.) H. Karst., Abies alba and A. balsamea (L.) Mill. Many fungi from foliage of some Cupressaceae were isolated as anamorphs of known conifer-inhabiting Ascomycetes. The scarcity of Basidiomycetes in the endophytic flora could be more apparent than real, and might be due to the isolation and scoring methods used by researchers. Basidiomycetes tended to fruit infrequently in culture, and were therefore scored as 'sterile' fungi in most instances (Petrini and Carroll, 1981). Some endophyte species which have a large host range can be taxonomically differentiated into groups showing preference for specific hosts. Discula umbrinella (Berk. and Broome) Sutton, a common endophyte in leaves of fagaceous trees in Europe and North America, showed distinct preferences for particular hosts (Toti et al., 1992). Isolates derived from beech trees could only adhere to, penetrate and colonize beech leaves, and not the non-host leaves of oak and chestnut trees in the way isolates from these hosts could (Toti et al., 1992). Hata (in Carroll, 1995) found various host-specific races or cryptic species of endophytes that existed in two Pinus spp., namely P. thunbergii and P. densiflora. Distinct patterns of endophytic colonization were also detected in these needles. Considerable genetic diversity exists within natural populations of endophytic fungal species, as demonstrated by Wilson et al. (1994) for Lophodermium pinastri (Schrad.: Fr.) Chev. in Pinus resinosa. Different genotypes were also found among isolates of L. pinastri from the same tree. Frequently occurring endophytic taxa from Alnus spp. are morphologically identical, despite the different environmental conditions in which their host grow (Fisher and Petrini, 1990). McCutcheon and Carroll (1993) used random amplified polymorphic DNA (RAPDs) to prove the genetic diversity between isolates of Rhabdocline parkeri (anamorph of Meria parkeri) isolated from Douglas fir. The diversity was estimated to be at least three times greater in foliage of mature and juvenile trees in natural stands, compared with foliage from a managed stand or from an isolated tree. This could be attributed to the differences in tree age and accessibility of inoculum (McCutcheon and Carroll, 1993). A combination of cultural and biochemical data was used to determine taxonomic relationships of endophytic isolates of Xylaria species from Euterpe oleracea Mart. (Rodrigues et al., 1993). Because of taxonomic complications associated with Xylaria spp., criteria other than morphology had to be used to determine the taxonomic connections between different species. Isoenzyme analysis showed a high degree of variation within and among the putative species examined, which reflected the morphological variation found in pure cultures and confirmed the genetic diversity of the genus (Rodrigues et al., 1993).

FOLIAR ENDOPHYTES

III.

9

ECOLOGY OF ENDOPHYTIC ASSOCIATIONS A.

THE HOST PLANT: GYMNOSPERMAE

Coniferous foliage varies greatly in physical appearance, ranging from the needle-like foliage, typical of Pinus, Abies and Picea, to tiny, compressed leaves of Cupressus, Thuja and Chamaecyparis, and the rudimentary angiosperm-like leaves found onPodocarpus. Although the aforementioned species usually retain their leaves for more than one year, Larix and Metasequoia are deciduous trees (Millar, 1974). Leaves are usually covered by a chemically complex, thick, waxy cuticle which can be covered with tubules. The cuticle may vary between and within species and consist of paraffin, ester and alcohol-soluble fractions and high carbon components (Schuck, in Millar, 1974). These waxes, which cover the whole leaf including the stomata, form an interlaced mat of tubules, and influence the gaseous exchange of the plant (Jeffree et al., in Millar, 1974). These layers also prevent the direct entry of larger fungal spores (Millar, 1974), which in turn affects infection by endophytes and pathogens. The orientation and surface characteristics of the leaf and inoculum concentration reaching a particular host all effect infection, and ultimately fungal colonization of the host (Fitt et al., 1989). Changes in the ultrastructure of the leaf surface due to environmental factors such as air pollution also have an effect on persistence of canopy moisture, which in turn will directly influence spore germination and growth (Helander et al., 1996). In studies done on larch trees, it was evident that the deciduous nature of these leaves resulted in a shorter period available for leaf colonization, compared with the evergreen softwoods (conifers) (Dobranic et al., 1995). The major representative endophytic taxa were therefore also affected, and endophytes represented in larch leaves might be limited to those adapted for rapid leaf colonization. The time needed to gain access to a particular host, and differences in leaf structure, should therefore be taken into account when studying endophytic populations (Dobranic et al., 1995). 1. Physiology Physiology of a host plant greatly influences its colonization by endophytes. Essential oils in healthy leaves of coastal redwood (Sequoia sempnvirens (D.Don ex Lamb.)) trees were an important factor controlling the activity of certain endophytes (Espinosa-Garda et al., 1993). A Pleuroplaconema sp., occurring in these redwood leaves, was stimulated by low essential oil doses, and inhibited by high doses. Essential oils were important inhibitors of Pestalotiopsis funerea (Desmaz.) Steyaert. However, other factors also involved in the inhibition process of fungi are still unknown (EspinosaGarda et al., 1993).

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W.-M. KRIEL, W. J. SWART and P. W. CROUS

2. Phenology

As discussed previously, needle age plays a significant role in the infection frequencies of endophytes (Todd, 1988). Knowledge of the seasonal development of a host, and its effect on needle age, can therefore be very illuminating in the understanding of endophytic colonization patterns associated with that host. Whitehead et al. (1994) examined the seasonal development of the leaf area in young Pinus radiata D. Don plantations in New Zealand. The trees were 6-7 years old, and elongation of age 0 needles (current year needle flush) began in the spring (October), and continued through summer, becoming fully elongated during autumn (early May), approximately 200 days from the onset of elongation. A smaller growth flush started in summer (January), and needles elongated until the end of the growing season. No significant difference in needle density could be detected with change in canopy height or seasonal variation. Needle density would affect the microclimate and inoculum distribution among needles. Needles of the age 1 group (previous years' needle flush) started to decline during midsummer (end of January), and coincided with the time of maximum elongation of age 0 needles. Needles formed during the spring growth flush contributed the majority of new leaf area during the year, with only a small proportion added by the autumn flush, which occurred predominantly on branches at the top of the canopy. The age of these needles and the climatic factors during needle development, would therefore affect the succession of endophytes in needles. Researchers also believe that needle longevity would increase with stand age (Whitehead et al., 1994), and therefore provide a suitable niche for true endophytes. B.

THE ENDOPHYTE

1. Authenticity of the Endophytic Character

Petrini (1984) examined the dependability of the endophytic character of some of the coprophilous fungi isolated as 'endophytes' from ericaceous hosts. He determined that the surface sterilization techniques used were extensive enough to ensure the genuine endophytic character of even these coprophilous fungi. According to Carroll et al. (1977), the sporadic isolation ofAureobasidium pullulans (deBary) G. Arnaud from conifer needles, could be contributed to contamination from epiphytic fungi. Pugh and Buckley (1971), however, frequently isolated endophytic A. pullulans from surfacesterilized living twigs, buds, leaves and seeds of sycamore (Acer pseudoplatanus L.), and from twigs of horse-chestnut and lime. Most common endophytes are seldom collected in the field, because they rarely sporulate on their hosts or form inconspicuous fruiting bodies (Petrini, 1986). Frequently occurring endophytes from a given host were absent among epiphytes, and likewise, epiphytes were uncommon among

FOLIAR ENDOPHYTES

II

endophytes (Fisher and Petrini, 1987b). The fact that endophytes were absent among epiphytes, could be attributed to the methods of isolation, which tend to favour fast growing saprophytes, or in this case epiphytes. Epiphytes, on the other hand, were excluded from endophytic isolations due to extensive surface sterilization. According to Kowalski and Kehr (1996), endophytes may have the same physiological importance for trunk and branch tissues that mycorrhizas have for the roots. Primary characteristics of mutualistic symbiosis include the lack of cell or tissue destruction, recycling of nutrients or chemicals between the fungus and the host, enhanced longevity and photosynthetic capacity of infected tissues, enhanced survival of the fungus, and a tendency of greater host specificity than is evident in biotrophic infections (Lewis, 1973). Endophytes are contained within the plant, and may be either parasitic or symbiotic. True endophytic colonization or infection is asymptomatic and can be described as a mutualistic symbiosis, which includes a lack of destruction of most cell tissues, nutrient or chemical cycling between host and fungus, enhanced longevity and photosynthetic capacity of infected tissues, and enhanced survival of the fungus. Endophytic infections can, therefore, not be considered as causing disease, because plant disease is an interaction between the host, parasite, vector and the environment over time, which results in the production of disease symptoms and/or signs (Sinclair and Cerkauskas, 1996). The distinction between endophyte and pathogen is not always clear, as some diseases are characterized by a long retardation in the development of progressive disease, due to the growth of the potential pathogen being arrested (Swinburne, 1983). This gives rise to latent infection, where the distinction between bona fide endophytes and latent pathogens become more confused. 2. Sporulation, Dispersal and Infection Endophytes can be transmitted from one generation to the next through host seed or vegetative propagules (Carroll, 1988). In this instance it is referred to as a systemic infection or, as described by Wilson (1996), vertical transmission. Horizontal transmission occurs when infection of leaves or needles takes place by means of spores, and these infection levels are closely correlated with the seasonal distribution of rainfall (Wilson, 1996). Inoculum dispersal of infectious fungi can be divided into three stages: removal from the colonized substrate, transport through air, and deposition on a new host. Rain and/or wind may be involved in all three stages and the two modes are not mutually exclusive (Fitt et at., 1989). Spores from fungi that produce their spores in mucilage, are detached from the host by raindrops and dispersed in splash droplets (Fitt et al., 1989). This includes conidia of some endophytic fungi produced in gloeoid masses, which have been encountered in fall samples collected in coniferous stands (Carroll and Carroll, 1978). When canopies become saturated by rain, fog, dew, or mist,

12

W.-M. KRIEL, W. J. SWART and P. W. CROUS

large drops may form on the leaves and, under canopies, drip-splash may be as important as direct rain-splash (Fitt et al., 1989). Survival stages of endophytes are often present on litter trapped between branches within the tree canopy, from where the spores are subsequently dispersed by wind or rain (Carroll et al., 1977). Rain consisting of large drops is the most effective means of dispersing spores. Drops from the canopy foliage can also be effective because they are often large, but their falling speeds are less than their terminal velocity (Fitt et al., 1989). The mucilage surrounding splashborne spores protects them from desiccation and loss of viability during dry weather. This may confine the dispersal of certain fungal spores to periods of rainfall when conditions are also favourable for infection because of the availability of free water on the host surface. Wind is also an important factor in the dispersal of certain fungal spores, especially hyphomycetes. Some fungal spores are actively removed from the host by turbulent winds, and since most endophytes sporulate on litter trapped between branches in the tree canopy, their dispersal is affected by wind or rain within the canopy (Carroll et al., 1977). Although average wind speeds in the lower part of closed canopies are typically only a fraction of the speed above the canopy, gusts of wind with speeds several times higher than the local mean may occur frequently inside plant canopies (Aylor, 1990). In general, spores produced in the lower part of the canopy are exposed to slower wind speeds and less turbulence. This lower amount of turbulence may prevent the escape of large numbers of spores from a closed canopy (Aylor, 1990). This will affect the distribution of fungal endophytes within the canopies of host plants. Hata et al. (1998) also provided other ways in which endophytic infections may take place. Mycelia of the endophytes Phialocephala and Cenangium ferruginosum may infect current-year needles of Pinus thunbergii and P. densiflora via current-year twigs in early summer and Leptostroma infect the needles with spores via the needle sheath (Hata et al., 1998). 3. Colonization Todd (1988) found that there was a direct correlation between site and the infection frequencies of endophytes. This could be attributed to: (i) a microclimate more conducive to fungal colonization where the foliage was more dense; (ii) the relative position of the needles in the canopy; or (iii) other unknown factors. Theoretically, needle infections can originate from systemic infections in twigs and petioles, through penetration of the cuticle or stomata by mycelium of fungi from epiphytic origin, multiple infections by airborne and/or waterborne spores, or through inoculation by various sucking insects (Bernstein and Carroll, 1977). Bernstein and Carroll (1977) suggested that 1-year-old needles became infected by waterborne spores dispersed by rain. Infection thus increases with needle age and the availability of rainfall during the fruiting stages of endophytic fungal species,

FOLIAR ENDOPHYTES

13

which is in contrast to needle pathogens, where most infections are confined to young needles (Carroll, 1995). Other possibilities are systemic infection as in Guignardia philoprina in Taxus needles (Carroll et al., 1977) and seed transmission. The life cycle of seed borne endophytes is inexorably tied to their grass hosts (Wilson, 1993). Rhabdocline parkeri ( teliomorph of M parkeri) an endophyte of Douglas fir, infects healthy foliage by direct penetration of the host epidermal cell walls, accomplished by very fine penetration hyphae (Stone, 1988). According to Sherwood-Pike et al. (1986), the fungus can persist in living host needles for up to 4 years. These intracellular hyphae occupy the entire lumen of a single epidermal or hypodermal cell (Sherwood-Pike et al., 1986), which eventually leads to the death of the colonized cell (Stone, 1988). Although the hyphae do not elongate, they appear to be metabolically active. At the onset of needle senescence, haustoria are produced from the intercellular hyphae (Stone, 1988), so that rapid colonization and sporulation can occur immediately after abscission (Sherwood-Pike et al., 1986). The microconidia! anamorph is the first state to be produced by R. parkeri, followed by the Meria state, which is rapidly produced in the same conidioma. The function of the microconidia is still unknown, but the macroconidia serve to reinfect the host plant (Sherwood-Pike et al., 1986). Cabral et al. (1993) found characteristic mechanisms of penetration and colonization of individual fungal species in the tissue of ]uncus spp. Infections of Stagnospora innumerosa, a Drechslera sp. and an unidentified endophyte of J. bufonius, were limited to a single host epidermal cell. Phaeosphaeria junicola (Rehm) L. Holm. infected the substomatal cavity of Juncus leaves, followed by limited intercellular colonization of the mesophyll. Infections by Cladosporium cladosporioides (Fresen.) G.A. De Vries and Alternaria altemata (Fr.: Fr) Kiess!. are localized to the substomatal chambers, and only A. altemata will colonize the mesophyll tissue intercellularly. The colonization patterns of these two endophytes are typical of opportunistic saprophytes (Cabral et al., 1993). Ascospores of fungi in the Xylariaceae (mostly endophytes) are irreversibly activated for infection, prior to germination, within minutes of contacting a potential host. These spores are able to recognize different plant species due to their ability to distinguish between structurally similar monolignols (Chapela et al., 1991). This suggests the existence of a hostspecific 'signature' present on different plants, and specific receptors for these molecules, within the fungal spores (Chapela et al., 1991 ). Ascomata of two Norway spruce endophytes, Tryblidiopsis pinastri and Lophodermium piceae only develop several years after initial colonization on dead branches and needles, respectively (Barklund and Kowalski, 1996). In contrast, an Ophiognomonia sp. which is an endophyte of Quercus emoryi Torr., naturally occurs at very high levels, but is only present in the leaves for the last 3-4 months before leaf abscission (Wilson. 1996). According to

14

W.-M. KRIEL, W. J. SWART and P. W. CROUS

Carroll (in Wilson, 1993), the co-occurrence of senescence and endophyte growth could lead to competition between the plant and endophyte for the mobilized nutrients destined for recycling inside the host plant. Persistence of endophytic fungal mycelia originating from latent infections in decomposing tissues will depend on their ability to utilize changing energy and nutrient sources, tolerate changing microclimatic conditions, and to defend their territory against invasions by other primary or secondary colonizers (Boddy and Griffith, 1989). Leaf senescence is the process which precedes tissue death, and during which the photosynthetic activity in leaves stops and leaf constituents are broken down and recycled within the host plant (Wilson, 1993). This process is followed by abscission, colonization and decomposition by saprophytic fungi. Endophytic fungi present in these healthy leaves will be the first to capitalize on the senescing and abscised leaves, and therefore the first species on the decomposing succession ladder (Wilson, 1993). Leaf senescence may trigger the growth and colonization of endophytes, but endophyte growth may also trigger the onset of senescence. Heavy fungal infections of Schizothyrium sp. in needles of Douglas fir, resulted in premature senescence and abscission of needles (Sherwood and Carroll, 1974). In contrast, the infection frequencies of needle endophytes such as R. parkeri, was found to increase continuously with needle age, until colonization resumes at the onset of needle senescence (Stone, 1987). The endophytic phase of branch pruning fungi can give them some advantage in colonizing dying branches (Kowalski and Kehr, 1996). Almost all living branches of eleven deciduous and coniferous European tree species investigated by Kowalski and Kehr (1992) were colonized by some species of highly specific fungal endophytes. Most of the common branch pruning fungi found in general were present in living branches, and therefore have an advantage in colonizing the dying tissue (Kowalski and Kehr, 1992). Primary colonizers of dead or attached twigs derive considerable benefit from their endophytic habit, which allows them to respond rapidly to twig death and establish themselves in the resource before the arrival of secondary colonizers (Boddy and Griffith, 1989). Some branch pruning fungi, however, are not adapted to endophytic life and are frequently found in wood of dead, debarked branches, and are not isolated from living branches. Other fungi are totally adapted to an endophytic lifestyle, but are not able to colonize branches extensively after they die. Thus, it may be speculated that these fungi require more constant moisture conditions in the form of larger branch diameters and stumps in order to become established in the succession of decay fungi (Kowalski and Kehr, 1996). Environmental factors influencing colonization. Changes in the environment can influence plants by altering the interactions between microbial symbionts (such as endophytes), plant pathogens and herbivores (Helander etal., 1996).

FOLIAR ENDOPHYTES

15

Air pollution affects trees directly by damaging needles and leaves and causing a decrease in the assimilative capacity of the canopy (Helander eta!., 1996). Indirect effects occur via the soil, due to acid rain that changes the nutrient content of the soil and causes the accumulation of hazardous ions. Microfungi living inside aerial plant parts can thus be affected and changes in the species composition of these microfungi may have various consequences for other role players in the ecosystem, such as the host plant, plant pathogens and herbivorous insects (Helander et al., 1996). Endophytic fungi live most of their life cycle in an environment protected against sudden weather changes and various environmental factors, including air pollution. Air pollutants, however, modify the microhabitat of the leaf surface, and can affect spore germination and hypha! penetration. In the light of this, several researchers have suggested that endophytes can serve as bio-indicators of air pollution. Sieber (1989) suggested that air pollutants are possible causes of changes in endophytic populations of Picea abies (L.) Karst. and Abies alba Mill. in Switzerland. The design of the experiment did, however, not allow the effects of air pollutants to be quantified. Helander et al. (1994) studied the effects of simulated acid rain on the occurrence of endophytic fungi in needles of Scots pine (Pinus sylvestris) from the subarctic region where environmental pollution is low. The frequency of endophytic colonization was reduced on pines treated with spring water acidified with either sulphuric acid alone, or in combination with nitric acid. Nitric acid alone had no effect on endophytic colonization (Helander et al., 1994). Simulated acid rain was also shown to affect the frequency of endophytic colonization in leaves of mountain birch, with a 25% decrease after an acid rain treatment at pH 3. Species composition, however, was not affected (Helander et al., 1993a). Ozone (0 3 ) also has an effect on endophytic colonization. The most common fungal endophyte isolated from Sitka spruce (Picea sitchensis (Bong.) Carriere) needles, Rhizosphaera kalkhoffi Bubak, was found to be increased by 0 1 exposure (Magan et al., 1995). The same fungus showed a trend to increase under higher sulphur dioxide (S0 2) concentrations, although this was not statistically significant. Laboratory studies by Smith (in Magan et al., 1995), suggested thatR. kalkhoffi is tolerant of elevated S0 2 concentrations and the low availability of water, enabling it to compete more effectively in comparison with other needle phyllosphere or endophytic fungi. The general occurrence of Lophodermium species on Scots pine needles was related to the distance from factory complexes producing copper, nickel, sulphuric acid and fertilizers, and to the chemical composition of living needles (Helander et al., 1996). The adverse effect of air pollution was the clearest in the most abundant species, L. pinastri (Schrad.: Fr.) Chev. The decrease in Lophodermium species can probably be attributed to the toxicity of industrial emissions, such as heavy metals, but impoverished vegetation and its associated changes in the microclimate may have played an additional

16

W.-M. KRIEL, W. J. SWART and P. W. CROUS

indirect role in endophytic fungal colonization. Helander et al. (1996) found that the number of endophytic fungi in pine needles was consistently lowest in high intensity acid rain treatments. In general, however, endophytic fungi are protected from the effects of environmental changes such as air pollution, when compared with epiphytic microorganisms, but if endophytic communities are affected by a long-term exposure to pollutants, the change may be more permanent, with implications for resistance and basic tree health for foresters (Helander et al., 1996). Species composition and canopy characteristics. Differences in composition of the endophytic flora in branches of forest trees can be caused by several factors. The diversity of the plant community may greatly influence the extent of colonization by endophytes, and is illustrated by the occurrence of host-specific fungi on non-hosts growing in mixed stands together with the main host (Kowalski and Kehr, 1996). Species composition in the endophyte population in Abies alba is dependent on the type of management of the forest (Sieber-Canavesi and Sieber, 1987). Clear cuttings and plantations eliminate the transmission of endophytic fungi and clear cutting modifies the plant community as well as the microclimate. Where trees arise spontaneously, needle endophytes are found more frequently than in cases where they have been planted (Sieber-Canavesi and Sieber, 1987). Studies conducted on endophytic fungi present in foliage of different Cupressaceae in Oregon revealed differences in the infection rates of endophytes (Petrini and Carroll, 1981). These studies included samples from Calocedrus decuffens (Torr.) Florin,Juniperus occidentalis Hook., Thuja plicata J. Donn ex D. Don and Chamaecyparis lawsoniana (A. Murr.) Pari. Samples taken from pure stands of any particular host showed higher infection rates than those from mixed stands with an open canopy. This was confirmed by Legault et al. (1989) in studies on Pinus banksiana and P. resinosa, which showed a higher colonization rate in a closed canopy. Helander et al. (1996) found different results in Scots pine needles; pine needles of trees with few other pines in their vicinity had none or only a few endophytic fungi. Two types of endophyte dynamics were reported by Widler and Muller (1984 ): (i) fungi showing an increased frequency of occurrence with leaf age, and (ii) fungi showing a decreased frequency of occurrence with leaf age. Hata et al. (1998) isolated endophytes from needles of P. densiflora and P. thunbergii. The two most frequently isolated fungi were the Leptostroma anamorph of Lophodermium pinastri and Phialocephala sp. Leptostroma showed increased frequencies with needle ageing and Phialocephala decreased frequencies. Possible explanations for the increase in Leptostroma with needle ageing are (i) an increased chance of infection with the time after needle flush, (ii) improved habitat condition with the changing needle physiology with needle ageing, and (iii) an increase in microscopic wounds or changes in the physical conditions of needles which may facilitate fungal

FOLIAR ENDOPHYTES

17

infection. Rata et al. (1998) rated (i) and (ii) as the most probable explanations. Probable factors contributing to a decrease in the detection frequency of Phialocephala with needle ageing are (i) earlier fall of needles colonized by Phialocephala, (ii) aggravation of habitat condition for the endophytes with the changing physiology due to needle ageing (such as an increase in antifungal substances), and (iii) competition with other fungi, such as Leptostroma. Rata et al. (1998) found (iii) to be the most probable, since Leptostroma and Phialocephala showed antagonistic interaction in culture. In any particular tree, general infection rates of endophytes increase with increasing age of foliage and decreasing distance from the tree trunk (Petrini and Carroll, 1981). The height of the needles in the tree canopy showed little correlation with the frequency of infections and latent fungal infections were present in all needles examined older than 3 years (Bernstein and Carroll, 1977). Pinus spp. showed higher colonization rates with increasing foliage age, but were not influenced by twig orientation (Legault et al., 1989). Sherwood and Carroll (1974) found parasitized needles were shed from trees prematurely, because results showed a drop in the infection frequencies in needles from old-growth (7-8 years) of Douglas fir; 4-5 yearold needles were most severely infected. Overall intensity of infection did not, however, increase with age or canopy level (Sherwood and Carroll, 1974). More endophytes could be isolated from the lower branches (up to 1 m) of mountain birch than from branches at 2m height, which is possibly due to the inoculum pressure and more favourable microclimate in the lower parts of the canopy (Helander et al., 1993a). More endophytes were isolated from the bottom of the crown in A. balsamea than from the top, but no correlation could be found between the frequency of infections by endophytes and the geographic directional orientation of needles (Johnson and Whitney, 1989). This could be due to the availability of leachates and water within the crown (McBride and Hayes, 1977). The distribution correlates with the movement of propagules from the top to the bottom of the tree, and the fact that most endophytes are dispersed through waterborne spores (Johnson and Whitney, 1989). Geographic and climatic factors. Geographical and local site factors apparently influence the composition and frequency of host-specific fungal species (Kowalski and Kehr, 1996). Changes in endophytic infection rates may be the result of various environmental changes rather than just direct or indirect effects of air pollution and other factors (Helander et al., 1996). According to Carroll (1995), general exposure and geographic continuity are significant factors when overall endophyte assemblages in a given host are compared over several dispersed sites. Carroll and Carroll (1978) suggested that low infection rates seen at high elevations and dry sites could result from the delayed onset of endophytic infections and not lower incidences of internal needle fungi per se.

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Endophytic infections are influenced by precipitation and elevation. Precipitation in the form of rainfall may be a factor in endophyte dispersal, where moist sites show higher incidences of endophytic infections than dry sites. Petrini et al. (1982) proved that the infection rates of endophytes for a specific host species correlate positively with the relative canopy density and the moisture available at the collection site. Carroll and Carroll (1978) found that a lack of rain and relatively open conifer stands may limit the spread of endophytes. Sites which receive less rain and more snow (usually at higher elevations) will also result in a negative correlation between endophyte incidence and elevation (Carroll and Carroll, 1978). Carroll and Carroll (1978) also found that the infection frequencies of endophytes decreased with increasing elevation on western slopes and increased with increasing elevation on eastern slopes, and explained this by differences in the amount and form of precipitation. Bernstein and Carroll (1977) could not find any correlation between the internal canopy infections of endophytes of Douglas fir (Pseudotsuga menziesii (Mirb.) Franco) foliage, and the elevation and exposure of individual trees sampled. Between-site differences in the frequency of colonization of Castanea sativa Mill. by Amphiporthe castanea (Tul.) Barr and a Phomopsis sp. could probably be attributed to differences in climatic factors and abundance of inoculum (Bissegger and Sieber, 1994). Samples of leaves, twigs and roots of Dryas octopetala taken in the subalpine region, are richer in endophytic species than samples collected in the alpine or Arctic regions (Fisher et al., 1995). According to Widler and Miiller ( 1984) endophytes show seasonal patterns in four categories with regard to their distribution in leaves: (i) fungi that appear once a year for a short period; (ii) fungi with a higher frequency in winter than in summer; (iii) fungi with a higher frequency of occurrence in summer than in winter; and (iv) fungi which do not show any apparent seasonal change. Fungi that show high colonization frequencies can usually be classified into the fourth category (Widler and Miiller, 1984). In general, infection frequencies of endophytes seem to be higher in winter than in spring (Carroll et al., 1977). Hata and Futai (1993) found that the colonization rate of endophytes increases with advance of the season, and even differs from year to year. This tendency possibly reflects changes in needle physiology and changes in biotic and abiotic environmental factors such as other microfungi and climatic elements. Kowalski (1993) found winter to be an inhibiting factor for the infection of endophytes, and therefore fewer endophytes were isolated during spring and summer than in autumn. This could be explained by a lower chance for infection during winter. In contrast, Sieber-Canavesi and Sieber (1987) found a higher infection frequency in Abies alba needles during winter especially from endophytes of the Xylariaceae. This was attributed to the lower physiological activity of trees, resulting in a slower reaction of trees to fungal

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infection, and possibly enhanced penetration due to frost damage to the needle cuticle (Sieber-Canavesi and Sieber, 1987). 4. Substrate Utilization Endophytes may develop distinct substrate utilization patterns. For instance, fungi from needle-bearing conifers show specialization in their utilization capabilities. Fungi occurring only in the petioles have a broad range of substrate utilization capabilities, but those occurring in the needle blades have more restricted abilities (Carroll and Petrini, 1983). Even isolates from the same fungal species may differ in their substrate utilization. Differences in utilization also ensure that several endophytes can coexist within a single needle, without competing with each other. This is called 'biochemical partitioning of resources' (Carroll and Petrini, 1983). Pectin can be utilized by almost all fungi, lignin to a limited extent by needle fungi, but not by petiole fungi. Only petiole fungi are able to break down cellulose, hemicellulose, lipids, pectin, xylan, mannan and galactan, which suggest that they are active decomposers, whereas needle fungi, which are not able to utilize some of these complex substrates, are dependent on their host for simple carbon sources (Carroll et al., 1977; Carroll and Petrini, 1983). Carroll and Petrini (1983) suggested that endophytic fungi with restricted substrate utilization capabilities (like needle blade endophytes) are the most likely to have possible symbiotic relationships with their host plants. Fungi with broader substrate utilization patterns (like petiole endophytes) are more likely to be latent pathogens. Endophytes capable of utilizing only the simple carbon sources in living plant cells, will decline rapidly with the depletion of the food source following the death of the host tissues (Boddy and Griffith, 1989). Endophytes which commonly occur in healthy twig bark but are absent from dead wood, have a limited capacity to utilize complex substrates, in particular lignocellulose. These endophytes depend on their host for simple carbon compounds (Boddy and Griffith, 1989). Substrate utilization and growth experiments have no taxonomic relevance for the distinction of some endophytic species, as was shown for conifer-inhabiting Phyllosticta species. However, a comparison of the electrophoretic banding patterns of different enzymes such as pectinase, polygalacturonase, and amylase, nonetheless, allowed a clear differentiation between five Phyllosticta spp., namely P. multicomiculata Bisset et Palm, P. cryptomeriae Kawamura, P. abietis Bisset et Palm, P. pseudostugae L.E. Petrini and Macrophoma piceae L.E. Petrini (Petrini et al., 1991 ). C.

HOST-ENDOPHYTE INTERACTIONS

1. Mutualistic Associations Although the ecological status of many endophytes remain undefined, possible benefits of endophytes to coniferous hosts include antagonism

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towards pathogenic needle parasites and surface saprophytes, delay in needle senescence, and a decrease in needle palatability for grazing insects (Carroll and Carroll, 1978). Resistance to diseases. Phytoalexin production by the host plant in reaction to infection by an endophyte can actually render the host resistant to attack by pathogens (Wilson, 1993). The absence of endophytes in glasshouseraised plants may therefore explain their acute susceptibility to insect and fungal pests and diseases, since these plants are protected against natural airborne inoculum of endophytes (Wilson, 1993). Mutual exclusion of endophytes within leaves where infection by one species may inhibit infection by another is also documented. For example, leaves sprayed with Asteromella sp. or Plectophomella sp., which are recognized endophytic fungi, were able to exclude other endophytic fungal infections (Wilson, 1996). Cryptosporiopsis abietina is a stem endophyte of Picea sitchensis, and shows antagonistic activity against Heterobasidion annosum (Fr.: Fr.) Bref. The fungus also behaves as an aggressive seedling pathogen on Picea abies and can be associated with declining mycorrhizae (Holdenrieder and Sieber, 1992). Bissegger and Sieber (1994) also isolated from European chestnut a fungus with antifungal properties, related to Cryptosporiopsis, namely Pezicula cinnamomea (DC.) Sacc. Pezicula cinnamomea inhibited other pathogens, including Cryphonectria parasitica (Murrill) Barr in dual cultures, possibly rendering it as an effective natural biocontrol agent (Bissegger and Sieber, 1994). Due to the fungitoxic effects ofBalansia cyperi Edgerton, an endophyte of Cyperus rotundus L., this fungus is able to exclude pathogens such as Rhizoctonia so/ani Kiihn, from the leaves of its host (Stovall and Clay, 1991). In vitro bioassays with mycelium and culture filtrates of B. cyperi showed inhibition of test fungi which included Fusarium oxysporum Schlechtend.: Fr. and R. so/ani. Solvent extracts made of leaves from B. cyperi-infected plants, also inhibited the growth of fungi including F. oxysporum, Rhizoctonia oryzae Ryker and Gooch and R. so/ani. These results show the ability of B. cyperi to prevent infection of C. rotundus by other pathogenic fungi (Stovall and Clay, 1991). Secondary metabolites produced by fungal endophytes in tomato roots are highly toxic to Meloidogyne incognita, especially strains of F. oxysporum (Hallmann and Sikora, 1996). These toxins were produced by a nonpathogenic strain of F. oxysporum and were highly effective towards sedentary parasites, less effective towards migratory endoparasites, and nonparasitic nematodes were not influenced at all. Metabolites of this fungus also reduced the growth of pathogens such as Phytophthora cactorum (Lebert and Cohn) J. Schrot., Pythium ultimum Trow and Rhizoctonia so/ani in in vitro studies (Hallmann and Sikora, 1996). Biological control of certain diseases, such as chestnut blight caused by Cryphonectria parasitica on Castanea sativa, can be obtained by spreading

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hypovirulence by means of endophytic thalli from hypovirulent strains of Cryphonectria parasitica (Bissegger and Sieber, 1994). Protection from insect herbivory. Endophytic fungi can affect the interaction between their hosts and insect herbivores. Where a mutualistic association exits between fungi and insects, it will result in increased herbivory of host plants, and a mutualistic association between fungi and plants, in reduced herbivory of the host plant (Clay, 1987). When the endophyte-plant symbiosis is strongly mutualistic and the host benefits through increased defence against herbivores, the host may rely largely or wholly on the endophytes for their resistance (Wilson, 1993). Endophyte infections therefore provide a selective advantage to grazed plants. There are four different mechanisms by which these fungi can influence herbivory: (i) by changing the consistency of host tissues; (ii) by inducing resistance; (iii) by depletion of nutrients; and (iv) by the production of certain toxins (Clay, 1987). Systemically infected grasses display an increased level of resistance to a wide variety of insect and mammalian herbivores as a result of alkaloid production by fungi (Clay, 1987). The most clear-cut mutualistic association is that of Balansia spp., which produce substances capable of reducing the palatability of the grasses to various herbivores (Clay, 1988). There are conifer endophytes that have evolved an ecological strategy that involves the production of compounds that limit the herbivory of conifer needles (Clark et al., 1989). This suggests a mutualistic relationship between the fungus and its host. Infection levels of specific endophytic fungi (with beneficial associations) can be effectively manipulated using polyethylene or polyvinyl chloride (PVC) bags to exclude other organisms. Inoculation of the leaves with specific endophytic fungi can be done by spraying spore suspensions onto the protected leaves (Wilson, 1996). Certain endophyte species inhabiting conifer needles produce compounds that could be linked to the mortality or decreased growth of spruce budworm larvae (Clark et al., 1989). Some species are in the genus Leptostroma, but the most toxic strains are not yet identified and could represent new genera. These coniferous endophytes produce compounds that affect spruce budworm, mortality, or retard larval development (Clark et al., 1989). This can have important ecological consequences, and could result in the disruption of mating because affected budworms reach pupation much later than unaffected worms. Furthermore, larvae will be exposed to adverse environmental and predatory factors for longer periods, and thus suffer a higher mortality. The occurrence of 'escaper trees' in budworm-damaged forests could be attributed to the presence of these endophytes (Clark et al., 1989). Calhoun et al. (1992) refined and identified four toxic metabolites produced by endophytes of balsam fir which are effective against spruce budworm. Phyllosticta sp. produced heptelidic acid, heptelidic acid chlorohydrin and hydroheptelidic acid. A fourth compound, ( + )-rugulosin, an anthraquinone,

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was produced by Hormonema dematioides, and exhibits a wide spectrum of biological activity (Calhoun et al., 1992). The most important endophyte of Douglas fir, Meria parkeri SherwoodPike, produces compounds that are toxic to insects (Todd, 1988). Diamandis (1981, in Gange, 1996) found that the larvae of the pine processionary moth (Thaumetopoea pityocampa) avoided endophyte-infected needles of Pinus brutia. Insect death can also be attributed to starvation in the case Quercus ganyana, where the endophytic fungus kills the galls of a cynipid wasp, and deprives the insects of food (Wilson, 1995b). Endophytic fungi in galls caused by the pine needle gall midge (Thecodiplosis japonensis Uchida and Inouye (Diptera: Cecidomyiidae)) show distinct differences from endophytes isolated from healthy needles (Hata and Futai, 1995). A species of Phialocephala was the most frequent endophyte occurring in the base of needles and galls from Pinus densiflora and an F 2 hybrid pine (a cross between P. thunbergii and P. densiflora ). However, species richness increased in the gall-infested needles. Hata and Futai (1995) suggested that fungi occurring in gall-infested and healthy needles could represent different ecological groups of endophytes. Endophytes from healthy and gall-infested needles can be divided into two groups: position-specific fungi such as Phialocephala sp. and Leptostroma spp., which showed a distinct pattern in their needle distribution; and gall-specific fungi such as Phomopsis sp., Pestalotiopsis sp., and to a lesser degree Alternaria alternata, which preferred galls on infected needles (Hata and Futai, 1995). No mutualistic associations between the gall endophytes and the pine needle gall midge could be detected, and no evidence was found of transmission of endophytic fungi by the gall midges (Hata and Futai, 1995). Growth promotion. Some endophytes promote growth of their host plants. Leptodontium orchidicola Sigler and Currah, a dematiacious hyphomycete isolated from roots of subalpine plants, caused a significant increase in host root length of Salix glauca L. seedlings, but the fungus also invaded the stele, causing extensive cellular lysis (Fernando and Currah, 1996). The effects of four different strains of L. orchidicola were strain- and host-specific, and the symbiotic associations varied from mycorrhizal to parasitic. Phialocephala fortinii Wang and Wilcox has an amensal, parasitic or neutral association with its host and, in combination with Potentilla fruticosa L., results in a significant increase in shoot weight (Fernando and Currah, 1996). Rootendophytic Bacillus strains possess specific physiological and (or) biochemical characteristics that facilitate colonization of internal root tissues with subsequent growth-promoting possibilities for the host plant (Shishido et al., 1995). 2. Detrimental Endophytic Associations Latent pathogenesis. Plant pathologists, rigidly following Koch's postulates, have discarded latent pathogens as 'saprophytes' or 'secondary parasites',

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since no symptoms were detected following inoculation of a vigorous host. Alternatively they have labelled latent pathogens as aggressive pathogens without considering possible predisposing factors (Schoeneweiss, 1975). A parasitic relationship usually starts when the infection hypha of a fungus penetrates the host cuticle and then the outer epidermal cell wall (Verhoef, 1974). In some instances, however, some time may pass between penetration and the start of such a parasitic relationship, which is then referred to as a latent, dormant, or quiescent infection (Verhoef, 1974). The latent period is defined as the time from infection until the expression of macroscopic symptoms, or as a prolonged incubation period (Sinclair and Cerkauskas, 1996). Only fungi colonizing living tissue can potentially be termed latent pathogens (Kowalski and Kehr, 1996). Many pathogens undergo an extensive phase of asymptomatic growth along with colonization and then latent infection before symptoms appear (Sinclair and Cerkauskas, 1996). Latent-infecting fungi as well as endophytes can infect plant tissues and become established after penetration, but infection does not imply the production of visible disease symptoms. According to Sinclair and Cerkauskas (1996), latent infection of plants by pathogenic fungi is considered one of the highest levels of parasitism. 'Bona fide endophytism', on the other hand, refers to a latent infection that never results in visible disease symptoms, and a close mutualistic association with the host plant (Sinclair and Cerkauskas, 1996). Expression of symptoms caused by a latent pathogen can be elicited by changes in the host physiology and environmental stress: (a) Symptom expression elicited by host physiological changes. Simmons (1963, in Verhoef, 1974) discussed four possible explanations for the latent nature of infections in banana fruit: (i) a toxin may be present in unripe, but not ripe fruit; (ii) the nutritional requirements of the fungus are not met by the composition of green, unripe fruits; (iii) the energy requirements of the fungus are only met when the metabolism of the host changes from the unripe to the ripening phase; and (iv) the enzyme potential of the fungus is not strong enough to allow the invasion of the immature fruit, but sufficient to allow the invasion of ripe fruit. Thus, changes in the host physiology of fruits may trigger disease expression. Comparative studies by Espinosa-Garda and Langenheim ( 1991) on the effect of essential oils on three pathogenic and one endophytic fungus demonstrated differences in tolerance to essential oils between pathogens and endophytes. The relatively high tolerance showed by the pathogens, Phomopsis occulta (Sacc.) Traverso, Pestalotiopsis funerea and Seiridium juniperi (Allesch.) Sutton to essential oil phenotypes of redwood, reflect their adaptation to the host defence reactions that involve terpenoids. The coniferous endophyte, Cryptosporiopsis abietina, on the other hand, displayed an overall susceptibility to the redwood essential oils (EspinosaGarda and Langenheim, 1991).

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(b) Symptom expression elicited by environmental stress. A significant number of endophytic fungi in healthy plants become pathogenic when their host plants are weakened. In this instance the host-fungus interaction manifests itself as a disease syndrome (Dorworth and Callan, 1996). These fungi are often referred to as opportunists that may pass from a latent or mutualistic mode to a necrotrophic mode when the host plant is predisposed by several factors such as stress (Schoeneweiss, 1975). This latent phase gives the fungus an advantage over genuine saprophytes in colonizing dying branches (Kowalski and Kehr, 1996). There are indications that the natural pruning of tree branches is a process actively enhanced by certain fungi. Kowalski and Kehr (1996) thus concluded that several of the fungi isolated from living branch bases are likely to be weak parasites. Their presence in the tissue may, however, prevent colonization by more aggressive parasites and thus also their spread into the main stem. This gives rise to the possibility that these endophytes may be involved in the natural pruning of stressed tissues (Boddy and Griffith, 1989). Latent infections by endophytes do not result in the formation of disease symptoms, but may weaken the plant, predisposing it to other stresses or diseases (Sinclair and Cerkauskas, 1996). According to Schoeneweiss (1975), the following factors may act as disease inducing or predisposing factors to change a latent infection by a fungus to a disease syndrome: water stress, which can consist of water deficits and drought, as well as excess water and flooding; temperature stress, consisting of low temperatures and freezing, as well as high temperatures; defoliation stress; transplanting stress; nutrient stress; and various other factors such as reduced light, toxic substances (herbicides and other pesticides) and wounding which reduce host vigour. Any unusual factor can therefore predispose plants with latent infections resulting in disease symptoms. Pathogens can survive during latent infection in a quiescent state by adapting either physiologically or morphologically. For example, Colletotrichum gloeosporioides (Penz.) Penz. and Sacc. in Penz., a pathogen of mango and avocado, survives the latent period as dormant appressoria, but persists in blueberry fruit as germinated appressoria and penetration hyphae. In seedling leaves of Citrus natsudaidai Hayata it persists as versicolate hyphae in the intercellular spaces of the leaf epidermis. In detached mature citrus fruit latent appressoria and latent hyphae occur within and beneath the cuticle and in the intercellular spaces of the epidermis (Sinclair and Cerkauskas, 1996). Most other latent pathogens survive as inactive, latent hyphae or mycelia within host tissue and intercellular spaces (Sinclair and Cerkauskas, 1996). Endophytes and weak parasites of Quercus, namely Pezicula cinnamomea and Colpoma quercinum, may contribute to the death of weakened tissue, but the aggressive Fusicoccum quercus Oud., causal agent of annual canker,

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is hardly ever isolated as an endophyte (Kowalski and Kehr, 1996). Latent colonization of oaks by Hypoxylon atropunctatum (Schwein.: Fr.) Cooke, probably accounts for the rapid increase in disease incidence following drought. The greater natural incidence of disease on black oaks compared with white oaks may be related to differences in drought sensitivity (Bassett and Fenn, 1984). Known pathogens of tropical plants, such as Colletotrichum spp., Fusarium spp. and Lasiodiplodia theobromae (Pat.) Griffon and Maubl., as well as strains of these species, can cause severe damage and losses in forests and plantations (Grey, in Dreyfuss and Petrini, 1984). Their isolation from symptomless plants can be an important aspect generally overlooked in plant pathology and epidemiology (Petrini and Dreyfuss, 1981; Smith et al., 1996a). Simple mutations could also give rise to pathogenic varieties of endophytes by inducing biotrophic characteristics in certain strains (Boddy and Griffith, 1989). The causal agent of chestnut blight, Cryphonectria parasitica, was isolated as an endophyte by Bissegger and Sieber ( 1994) from healthy coppice shoots of European chestnut (Castanea sativa Mill.). The fungus comprised a small component of all the endophyte assemblages and all C. parasitica isolates were of the normal phenotype with a high laccase activity, showing its fitness and potential pathogenicity. Bissegger and Sieber (1994) speculated that the fungus remains latent in the host phellem, until unfavourable conditions such as water stress and wounding of the host lead to the expression of pathogenicity. Three other known pathogens were also isolated from chestnut shoots; Amphiporthe castanea (Tul.) Barr, a weak wound parasite causing dieback and canker on C. sativa; Pezicula cinnamomea (DC.) Sacc., the causal agent of bark cankers on weakened red oak (Quercus rubra L.); and Diplodina castaneae, which causes 'Javart' disease of European chestnut (Bissegger and Sieber, 1994). Smith et al. (1996a) found that the endophytic colonization of healthy cones of different Pinus spp. by the pathogen Sphaeropsis sapinea (Fr.: Fr.) Dyko and Sutton, was positively correlated with the relative susceptibilities of the species to the pathogen. Endophytic colonization can thus reflect the inherent susceptibilities of different host genotypes. Kowalski (1993) isolated the pathogen of autumn needle cast, Cyclaneusma minus twice as frequently from symptomless needles of trees that showed symptoms of second year needle cast, than from trees without such symptoms. Trees showing needle cast symptoms had an overall higher susceptibility to fungal infection already on their first year needles (Kowalski, 1993). It is well known that plant pathogenic fungi express an incubation phase before disease symptoms appear. In the case of Cy. minus, this latent phase extends more than 15 months, which might explain its 'endophytic' nature, and high colonization frequency in pine needles (Kowalski, 1993).

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The most frequently isolated endophytic fungi from sessile oak are Apiognomonia quercina, Aureobasidium apocryptum (Ellis and Everh.) Hermanides-Nijhof and Colpoma quercinum (Pers.) Wallr. Although they

are reported as weak parasites, they are also present in healthy plant tissue without causing apparent disease symptoms (Halmschlager et al., 1993). Apiognomonia quercina, the anamorph of Discula quercina, is the causal agent of leaf galls and leaf spots on oak. Aureobasidium apocryptum is the causal agent of leaf spots on oak trees, and Colpoma quercinum causes infections on twigs and stems of stressed oak trees (Halmschlager et al., 1993). Among the dominant species found on aspen stems and branches, three species with presumed pathogenic abilities were isolated, namely, Cryptosphaeria populina, Cytospora chrysosperma (Pers.: Fr.) Fr. and Hypoxylon mammatum (Wahlenberg) J.H. Miller (Chapela, 1989). Cryptosphaeria populina causes bark disease, whereas C. chrysosperma and H. mammatum are associated with cankers of aspen (Chapela, 1989). Cenangium ferruginosum Fr.: Fr. is documented as a pathogen causing shoot dieback of pines, but it also seems to live as an endophyte in the needles of Pinus sylvestris (Kowalski, 1993). Wood decay of dying trees possibly originates from infections of latent fungi present in healthy, living branches (Chapela and Boddy, 1988b). These fungi are in a state of physiological dormancy, which is only broken under appropriate environmental conditions which include the reduction of water content in the xylem of the tree. The variation in endophytic colonization between annual rings could be attributed to variation in tree susceptibility and inoculum potential (Chapela and Boddy, 1988a). Botryosphaeria dothidea (Moug.) Ces. Et de Not. is the causal agent of die-back, canker and leaf spots of Eucalyptus spp. in South Mrica, but it is also able to colonize the xylem and leaves of trees asymptomatically (Smith et al., 1996b ). Disease symptoms develop rapidly at the onset of environmental stress such as frost, hot winds or drought, which can be seen as the trigger for the pathogenic stage of the pathogen (Smith et al., 1996a). Notwithstanding this evidence, the majority of 'true' endophytes are not associated with disease symptoms (Boddy and Griffith, 1989). Knowledge of the latent phase of any fungus, the length of the latency and the mechanisms that trigger the fungus to induce symptoms and to reproduce is, however, important for the improvement of disease control measures (Sinclair and Cerkauskas, 1996). Indirect enhancement of insect colonization and inhibition of host plant growth. The endophyte, R. parkeri, may slightly inhibit the growth of its host,

Douglas fir at high levels of infection, but has no other deleterious effect on the growth of the host (Todd, 1988). On the other hand, some endophytes can actually have a positive effect on insect colonization. Gange (1996) proved that infection of sycamore (Acer pseudoplatanus L.) leaves by an endophytic fungus, Rhytisma acerinum (Pers.) Fries, positively affected the

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27

number of aphids (Drepanosiphum platanoides (Schr.) and Periphyllus acericola (Walk.)) on leaves, especially during summer. This could possibly be attributed to the higher amount of soluble and total nitrogen, and totalled carbon contents of infected leaves. It is possible that the digestive processes of the fungus alter total carbon or nitrogen contents as compounds are moved into or out of leaves by the host, in this way altering the food quality of these tissues. The presence of endophytes may therefore also determine the seasonal patterns of herbivory by these aphids (Gange, 1996). 3. Utilization and Manipulation of Endophytic Associations Biocontrol of weeds. Until now the only recognized means of controlling weeds killing or constraining growth of newly planted forest trees were to use chemical herbicides or by controlled burning. Both of these methods have attracted huge criticism from environmental groups, and thus other means of control have to be investigated (Dorworth and Callan, 1996). Biocontrol agents can be divided into two groups; first-order 00) biocontrol agents, which can be applied as mycoherbicides for single event weed control or as classical bioagents for continuing weed control, and second-order (11°) biocontrol agents, which are opportunistic weak pathogens (Dorworth and Callan, 1996). First-order (JO) biocontrol can be defined as: 'Direct application of living agents which reduce the individuals of target pest populations either in number or in vigour, or both.' Second-order (UO) biocontrol can be defined as: 'Manipulation of environmental conditions, the targeted hosts, the indigenous microflora or all of these in order to induce the natural pathogenicity or stimulate the virulence of the native microflora, thereby yielding biological control.' Some endophytic fungi show promise as no biocontrol agents of forest weeds, but e biocontrol does not involve endophytes. Historically, no biocontrol resorted under categories of crop rotation, mulching and organic amendments, flooding and other techniques. These methods reduce pathogen populations by eliminating nutritional bases, negatively affecting environmental conditions, or by promoting the development of antagonistic microflora. The same principles can be applied to vegetation management (Dorworth and Callan, 1996). In biocontrol, the balance is tipped towards the pathogen, by strengthening the pathogen or weakening the host. Two benefits in the use of indigenous fungi for biocontrol are operator control, where the operator can limit the reaction by controlling the application of a stress factor quantitatively or qualitatively, and thereby reducing host vigour, and the buffer reaction or natural sink rendered by the natural biosphere. Lack of natural buffering by the biosphere can result in the uncontrolled spread of introduced pathogens as in the case of pine blister rust, chestnut blight and oak wilt (Dorworth and Callan, 1996). Research on biocontrol through the application of endophytes has the goal of promoting internal fungi from resident to necrotrophic status by

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stimulating the fungi themselves or by reducing the physiological vigour of the host plant, or reaching a suitable combination of the two. Endophytes themselves may also predispose their hosts to environmental damage by reducing the damage threshold (Dorworth and Callan, 1996). Manipulation of conditions affecting endophytic fungi in order to utilize their potential as pathogens (as biocontrol agents) should involve manipulation of the host. Two approaches can be considered: the target host plant can be used as a way to translocate agents (chemostimulants) that may stimulate the endophyte into necrotrophic activity, or the target host plant can be subjected to various stress agents, including the application of topical chemicals and physical influences such as heat, cold, drought, etc., which may alter the balance between host and endophyte in favour of the endophyte (Dorworth and Callan, 1996).

Biocontrol of other pathogens. An endophytic Cryptosporiopsis sp. isolated from Vaccinium myrtillus L. produced three different antibiotic-containing substances, which are all inhibitory to Candida albicans (C.P. Robin) Berkhout, a common human pathogen (Fisher et al., 1984). Experiments using crude culture filtrate of the fungus indicated antibiotic activity against Aspergillus niger Tiegh., C. albicans, Staphylococcus aureus Rosenbach and Trichophyton mentagrophytes (Robin) Blanchard. The continuing needs for less toxic but more effective drugs which can be administered orally for the treatment of serious Candida infections, indicate that further investigations in antibiotic activity such as produced by Cryptosporiopsis sp. are required (Fisher et al., 1984). Noble et al. (1991) isolated and identified an echinocandin from an endophytic Cryptosporiopsis sp. derived from twigs of P. sylvestris, and a Pezicula sp. derived from twigs of Fagus sylvatica. This compound proved to have antimicrobial properties against certain yeasts. Fungi which produce such potent antifungal properties give them a competitive advantage over other potential fungal colonizers (Noble et al., 1991).

IV. SUMMARY The term 'endophyte' has evolved not only to describe the location of an organism but also the actual association between the organism and its host plant. True endophytes colonize their host without any symptom expression. They are able to colonize a wide variety of hosts but some endophytic species show strong specificity towards specific host plants. Gymnospermae, which have quite unique types of leaves, harbour their own specialized group of foliar endophyte species. In order to understand the role of foliar endophytes completely, it is important to study the adaptation of endophytes to their specific environment, as well as the environmental factors that contribute to the different colonization patterns encountered in the host plant. True

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endophytes have adapted their infection and colonization strategies in order to infect and colonize their hosts without causing any deleterious effect on the host nor evoking the defence mechanisms of the host plant. In this way, they exist as biotrophs or may have active mutualistic associations with their particular host plant. Beneficial effects on the host plant may vary from growth enhancement, resistance against disease or attack by insects, to detrimental effects such as indirect enhancement of insect colonization and disease symptoms as is the case with latent pathogens. Understanding their adaptation and ecological role in gymnosperms may lead to the utilization of foliar endophytes in the holistic management of mixed and monocultural forest ecosystems. They could for example be used as bio-indicators, indicating the effects of air pollution and acid rain. Foliar endophytes can also play an important role in the initial degradation of plant material and debris. Their utilization as biocontrol agents of weeds or other pathogens, or as protectants against disease and insect infestation, is also documented. This review has elucidated many of the interactions between foliar endophytes and their gymnosperm hosts. It will hopefully serve as a useful source of information on which to base future research.

ACKNOWLEDGEMENTS We wish to thank Jim Callow for presubmission reviews and anonymous reviewers for many helpful suggestions. We also thank George Carroll for useful advice, and Jeff Stone for providing us with resourceful articles. Finally we would like to extend our grateful appreciation to Radilene le Grange and her library personnel for extensive support in obtaining articles and other resources for the writing of this review article.

REFERENCES Ahlich, K. and Sieber, T. N. (1996). The profusion of dark septate endophytic fungi in non-ectomycorrhizal fine roots of forest trees and shrubs. New Phytologist 132, 259-270. Aylor, D. E. (1990). The role of intermittent wind in the dispersal of fungal pathogens. Annual Review of Phytopathology 28, 73-92. Barklund, P. and Kowalski, T. (1996). Endophytic fungi in branches of Norway spruce with particular reference to Tryblidiopsis pinastri. Canadian Journal of Botany 74, 673-678. Bassett, E. N. and Penn, P. (1984). Latent colonisation and pathogenicity of Hypoxylon atropunctatum on oaks. Plant Disease 68, 317-319. Bernstein, M. E. and Carroll, G. C. (1977). Internal fungi in old-growth Douglas fir foliage. Canadian Journal of Botany 55, 644-653. Bills, G. F. (1996). Isolation and analysis of endophytic fungal communities from woody plants. In "Endophytic Fungi in Grasses and Woody Plants.

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Systematics, Ecology, and Evolution" (S.C. Redlin and L. M. Carris, eds), pp. 31-66. APS Press, Minnesota. Bissegger, M. and Sieber, T. N. (1994). Assemblages of endophytic fungi in coppice shoots of Castanea sativa. Mycologia 86, 648-655. Boddy, L. and Griffith, G. S. (1989). Role of endophytes and latent invasion in the development of decay communities in sapwood of angiospermous trees. Sydowia 41, 41-73. Bose, S. R. (1947). Hereditary (seed-borne) symbiosis in Casuarina equisetifolia. Nature, London. 159, 512-514. Cabral, D., Stone, J. K. and Carroll, G. C. (1993). The internal mycobiota ofluncus spp.: microscopic and cultural observations of infection patterns. Mycological Research 91, 367-376. Calhoun, L. A, Findlay, J. A, Miller, J. A and Whitney, N.J. (1992). Metabolites toxic to spruce budworm from balsam fir needle endophytes. Mycological Research 96, 281-286. Carroll, F. E., Muller, E. and Sutton, B. C. (1977). Preliminary studies on the incidence of needle endophytes in some European conifers. Sydowia 29, 87-103. Carroll, G. (1988). Fungal endophytes in stems and leaves: From latent pathogen to mutualistic symbiont. Ecology 69, 2-9. Carroll, G. (1995). Forest endophytes: pattern and process. Canadian Journal of Botany 73(Suppl. 1), S1316-Sl324. Carroll, G. C. and Carroll, F. E. (1978). Studies on the incidence of coniferous needle endophytes in the Pacific Northwest. Canadian Journal of Botany 56, 3034-3043. Carroll, G. and Petrini, 0. (1983). Patterns of substrate utilization by some fungal endophytes from coniferous foliage. Mycologia 15, 53-63. Chapela, I. H. (1989). Fungi in healthy stems and branches of American beech and aspen: a comparative study. New Phytologist 113, 65-75. Chapela, I. H. and Boddy, L. (1988a). Fungal colonization of attached beech branches. I. Early stages of development of fungal communities. New Phytologist 110, 39-45. Chapela, I. H. and Boddy, L. (1988b). Fungal colonization of attached beech branches. II. Spatial and temporal organization of communities arising from latent invaders in bark and functional sapwood, under different moisture regimes. New Phytologist 110, 47-57. Chapela, I. H., Petrini, 0. and Hagmann, L. (1991). Monolignol glucosides as specific recognition messengers in fungus-plant symbioses. Physiological and Molecular Plant Pathology 39, 289-298. Clark, C. L., Miller, J. D. and Whitney, N. J. (1989). Toxicity of conifer needle endophytes to spruce budworm. Mycological Research 93, 508-512. Clay, K. (1987). The effect of fungi on the interaction between host plants and their herbivores. Canadian Journal of Plant Pathology 9, 380-388. Clay, K. (1988). Fungal endophytes of grasses: a defensive mutualism between plants and fungi. Ecology 69, 10-16. Dobranic, J. K., Johnson, J. A and Alikhan, Q. R. (1995). Isolation of endophytic fungi from eastern larch (Larix laricina) leaves from Brunswick, Canada. Canadian Journal of Microbiology 41, 194-198. Dorworth, C. E. and Callan, B. E. (1996). Manipulation of endophytic fungi to promote their utility as vegetation biocontrol agents. In "Endophytic Fungi in Grasses and Woody Plants. Systematics, Ecology, and Evolution" (S. C. Redlin and L. M. Carris, eds), pp. 209-216. APS Press, Minnesota.

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Dreyfuss, M. and Petrini, 0. (1984). Further investigations on the occurrence and distribution of endophytic fungi in tropical plants. Botanica Helvetica 94, 33-40. Espinosa-Garda, F. J. and Langenheim, J. H. (1990). The endophytic fungal community in leaves of a coastal redwood population- diversity and spatial patterns. New Phytologist 116, 89-97. Espinosa-Garda, F. J. and Langenheim, J. H. (1991). Effects of sabinene and Tterpinene from coastal redwood leaves acting singly or in mixtures on the growth of some of their fungus endophytes. Biochemical Systematics and Ecology 19, 643-650. Espinosa-Garda, F. J., Saldivar-Garda, P. and Langenheim, J. H. (1993). Dosedependent effects in vitro of essential oils on the growth of two endophytic fungi in coastal redwood leaves. Biochemical Systematics and Ecology 21, 185-194. Fernando, A. A. and Currah, R. S. (1996). A comparative study of the effects of the root endophytes Leptodontium orchidicola and Phialocephala fortinii (Fungi Imperfecti) on the growth of some subalpine plants in culture. Canadian Journal of Botany 74, 1071-1078. Fisher, P. J. and Petrini, 0. (1987a). Location of fungal endophytes in tissues of Suaeda fruticosa: a preliminary study. Transactions. British Mycological Societv 89, 246-249. Fisher, P. J. and Petrini, 0. (1987b). Tissue specificity by fungi endophytic in Ulex europaeus. Sydowia 40, 46-50. Fisher, P. J. and Petrini, 0. (1990). A comparative study of fungal endophytes in xylem and bark of Alnus species in England and Switzerland. Mycological Research 94, 313-319. Fisher, P. J., Anson, A. E. and Petrini, 0. (1984). Novel antibiotic activity of an endophytic Cryptosporiopsis sp. isolated from Vaccinium myrtillus. Transactions. British Mycological Society 83, 145-187. Fisher, P. J., Anson, A. E. and Petrini, 0. (1986). Fungal endophytes in Ulex europaeus and Ulex gallii. Transactions. British Mycological Society 86, 153-156. Fisher, P. J., Petrini, 0., Petrini, L. E. and Sutton, B. C. (1994). Fungal endophytes from the leaves and twigs of Quercus ilex L. from England, Majorca and Switzerland. New Phytologist 127, 133-137. Fisher, P. J., Graf, F., Petrini, L. E., Sutton, B. C. and Wookey, P. A. (1995). Fungal endophytes of Dryas octopetala from a high arctic polar semidesert and from the Swiss Alps. Mycologia 87, 319-323. Fitt, B. D. L., Mccartney, H. A. and Walklate, P. J. (1989). The role of rain in dispersal of pathogen inoculum.Annual Review of Phytopathology 27, 241-270. Gange, A. C. (1996). Positive effects of endophyte infection on sycamore aphids. Oikos 75, 500-510. Hallmann, J. and Sikora, R. A. (1996). Toxicity of fungal endophyte secondary metabolites to plant parasitic nematodes and soil-borne plant pathogenic fungi. European Journal of Plant Pathology 102, 155-162. Halmschlager, Von E., Butin, H. and Donaubauer, E. (1993). Endophytische pilze in blattern und zweigen von Quercus petraea. European Journal of Forest Pathology 23, 51-63. Hata, K. and Futai, K. (1993). Effect of needle aging on the total colonization rates of endophytic fungi on Pinus thunbergii and Pinus dens if/ora needles. Journal of the Japanese Forestry Society 75, 338-341. Hata, K. and Futai, K. (1995). Endophytic fungi associated with healthy pine needles and needles infested by the pine needle gall midge, Thecodiplosis japonensis. Canadian Journal of Botany 73, 384-390.

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Rata, K. and Futai, K. (1996). Variation in fungal endophyte populations in needles of the genus Pinus. Canadian Journal of Botany 14, 103-114. Rata, K., Futai, K. and Tsuda, M. (1998). Seasonal and needle age-dependent changes of the endophytic mycobiota in Pinus thunbergii and Pinus densiflora needles. Canadian Journal of Botany 16, 245-250. Helander, M. L., Ranta, H. and Neuvonen, S. (1993a). Responses of phyllosphere microfungi to simulated sulphuric and nitric acid deposition. Mycological Research 91, 533-537. Helander, M. L., Neuvonen, S., Sieber, T. and Petrini, 0. (1993b). Simulated acid rain affects birch leaf endophyte populations. Microbial Ecology 26, 227-234. Helander, M. L., Sieber, T. N., Petrini, 0. and Neuvonen, S. (1994). Endophytic fungi in Scots pine needles: spartial variation and consequences of simulated acid rain. Canadian Journal of Botany 72, 1108-1113. Helander, M. J., Neuvonen, S. and Ranta, H. (1996). Ecology of endophytic fungi: effects of anthropogenic environmental changes. In "Endophytic Fungi in Grasses and Woody Plants. Systematics, Ecology, and Evolution" (S. C. Redlin and L. M. Carris, eds), pp. 197-208. APS Press, Minnesota. Holdenrieder, 0. and Sieber, T. N. (1992). Fungal associations of serially washed healthy non-mycorrhizal roots of Picea abies. Mycological Research 96, 151-156. Johnson, J. A and Whitney, N. J. (1989). An investigation of needle endophyte colonization patterns with respect to height and compass direction in a single crown of balsam fir (Abies balsa mea). Canadian Journal ofBotany 61, 723-725. Kowalski, T. (1993). Fungi in living symptomless needles of Pinus sylvestris with respect to some observed disease processes. Journal of Phytopathology 139, 129-145. Kowalski, T. and Kehr, R. D. (1992). Endophytic fungal colonization of branch bases in several forest tree species. Sydowia 44, 137-168. Kowalski, T. and Kehr, R. D. (1996). Fungal endophytes of living branch bases in several European tree species. In "Endophytic Fungi in Grasses and Woody Plants. Systematics, Ecology, and Evolution" (S. C. Redlin and L. M. Carris, eds), pp. 67-86. APS Press, Minnesota. Legault, D., Dessureault, M. and Laflamme, G. (1989). Mycoflore des aiguilles de Pinus banksiana et Pinus resinosa. I. Champignons endophytes. Canadian Journal of Botany 61, 2052-2060. Lewis, D. H. (1973). Concepts in fungal nutrition and the origin of biotrophy. Biological Reviews 48, 261-278. Magan, N., Kirkwood, I. A, Mcleod, A R. and Smith, M. K. (1995). Effect of openair fumigation with sulphur dioxide and ozone on phyllosphere and endophytic fungi of conifer needles. Plant, Cell and Environment 18, 291-302. McBride, R. P. and Hayes, A J. (1977). Phylloplane of European Larch. Transactions. British Mycological Society 69, 39-46. McCutcheon, T. L. and Carroll, G. C. (1993). Genotypic diversity in populations of a fungal endophyte from Douglas fir. Mycologia 85, 180-186. Millar, C. S. (1974). Decomposition of Coniferous leaf litter. In "Biology of Plant Litter Decomposition, Volume I" (C. H. Dickinson and G. J. F. Pugh, eds), pp. 105-128. Academic Press, London. Noble, H. M., Langley, D., Sidebottom, P. J., Lane, S. J. and Fisher, P. J. (1991). An echinocandin from an endophytic Cryptosporiopsis sp. and Pezicula sp. in Pinus sylvestris and Fagus sylvatica. Mycological Research 95, 1439-1440. Petrini, 0. (1984). Endophytic fungi in British Ericaceae: A preliminary study. Transactions. British Mycological Society 83, 510-512.

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Petrini, 0. (1986). Taxonomy of endophytic fungi of aerial plant tissues. In "Microbiology of the Phyllosphere" (N.J. Fokkema and J. van den Heuvel, eds), pp. 175-187. Cambridge University Press, Cambridge. Petrini, 0. (1991). Fungal endophytes of tree leaves. In "Microbial Ecology of Leaves" (J. H. Andrews and S. S. Hirano, eds), pp. 179-197. Springer-Verlag, New York. Petrini, 0. (1996). Ecological and physiological aspects of host specificity in endophytic fungi. In "Endophytic Fungi in Grasses and Woody Plants. Systematics, Ecology, and Evolution" (S.C. Redlin and L. M. Carris, eds), pp. 87-100. APS Press, Minnesota. Petrini, 0. and Carroll, G. (1981 ). Endophytic fungi in foliage of some Cupressaceae in Oregon. Canadian Journal of Botany 59, 629-636. Petrini, 0. and Dreyfuss, M. (1981). Endophytische pilze in epiphytischen Araceae, Bromeliaceae, und Orchidacea. Sydowia 43, 135-148. Petrini, 0. and Fisher, P. J. (1986). Fungal endophytes in Salicornia perennis. Transactions. British Mycological Society 87, 647-651. Petrini, 0. and Muller, E. (1979). Pilzliche Endophyten am Beispiel von Juniperus communis L. Sydowia 32, 224-251. Petrini, 0., Stone, J. and Carroll, F. E. (1982). Endophytic fungi in evergreen shrubs in western Oregon: a preliminary study. Canadian Journal of Botany 60. 789-796. Petrini, L. E., Petrini, 0., Leuchtmann, A and Carroll, G. C. (1991). Conifer inhabiting species of Phyllosticta. Sydowia 43, 148-169. Pugh, G. J. F. and Buckley, N. G. (197l).Aureobasidium pullulans: an endophyte in sycamore and other trees. Transactions. British Mycological Society 57. 227-231. Rodrigues, K. F., Leuchtmann, A and Petrini, 0. (1993). Endophytic species of Xylaria: cultural and isozymic studies. Sydowia 45, 116-138. Schoeneweiss, D. F. (1975). Predisposition, stress and plant disease. Annual Review of Phytopathology 13, 193-211. Sherwood, M. and Carroll, G. (1974). Fungal succession on needles and young twigs of old-growth Douglas fir. Mycologia 66, 499-506. Sherwood-Pike, M., Stone, J. K. and Carroll, G. C. (1986). Rhabdocline parkeri, a ubiquitous foliar endophyte of Douglas-fir. Canadian Journal of Botany 64, 1849-1855. Shishido, M., Loeb, B. M. and Chanway, C. P. (1995). External and internal root colonization of lodgepole pine seedlings by two growth-promoting Bacillus strains originated from different root microsites. Canadian Journal of Microbiology 41, 707-713. Sieber, T. N. (1989). Substratabbauverm6rgen endophytischer Pilze von Weizenk6rnern. Zeitschrift fuer Pflanzenkrankheiten und Pflanzenschutz 96, 627-632. Sieber-Canavesi, F. and Sieber, T. N. (1987). Endophytische pilze in tanne (Abies alba Mill.).- Vergleich zweier standorte im Schweizer Mittelland (Naturwald-Aufforstung). Sydowia 40, 250-273. Sieber-Canavesi, F., Petrini, 0. and Sieber, T. N. (1991). Endophytic Leptostroma species on Pice a abies, Abies alba and Abies balsamea: a cultural, biochemical, and numerical study. Mycologia 83, 89-96. Sinclair, J. B. and Cerkauskas, R. F. (1996). Latent infection vs. endophytic colonisation by fungi. In "Endophytic Fungi in Grasses and Woody Plants. Systematics, Ecology, and Evolution" (S.C. Redlin and L. M. Carris, eds), pp. 3-30. APS Press, Minnesota.

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Smith, H., Wingfield, M. J., Crous, P. W. and Coutinho, T. A. (1996a). Sphaeropsis sapinea and Botryosphaeria dothidea endophytic in Pinus spp. and Eucalyptus spp. in South Africa. South African Journal Botany 62, 86-88. Smith, H., Wingfield, M. J. and Petrini, 0. (1996b). Botryosphaeria dothidea endophytic in Eucalyptus grandis and Eucalyptus nitens in South Africa. Forest Ecology and Management 89, 189-195. Stone, J. K. (1987). Initiation and development of latent infections by Rhabdocline parkeri on Douglas-fir. Canadian Journal of Botany 65, 2614-2621. Stone, J. K. (1988). Fine structure of latent infections by Rhabdocline parkeri on Douglas-fir, with observations on uninfected epidermal cells. Canadian Journal of Botany 66, 45-54. Stovall, M. E. and Clay, K. (1991 ). Fungitoxic effects of Balansia cyperi. Mycologia 83, 288-295. Suske, J. and Acker, G. (1987). Internal hyphae in young, symptomless needles of Picea abies: electron microscopic and cultural investigation. Canadian Journal of Botany 65, 2098-2103. Swinburne, T. R. (1983). Quiescent infections in post-harvest diseases. In "Postharvest Pathology of Fruits and Vegetables" (C. Dennis, ed.), pp. 1-21. Academic Press, London. Todd, D. (1988). The effects of host genotype, growth rate, and needle age on the distribution of a mutualistic, endophytic fungus in Douglas-fir plantations. Canadian Journal of Forest Research 18, 601-605. Toti, L., Viret, 0., Chapela, I. H. and Petrini, 0. (1992). Differential attachment by conidia of the endophyte, Discula umbrinella (Berk. and Br.) Morelet, to host and non-host surfaces. New Phytologist 121, 469-475. Verhoef, K. (1974). Latent infections by fungi. Annual Review of Phytopathology 12, 99-110. Whitehead, D., Kelliher, F. M., Frampton, C. M. and Godfrey, M. J. S. (1994). Seasonal development of leaf area in a young, widely spaced Pinus radiata D.Don stand. Tree Physiology 14, 1019-1038. Widler, B. and Muller, E. (1984). Untersuchungen iiber endophytische pilze von Arctostaphylos uva-ursi (L.) Sprengel (Ericaceae ). Botanica Helvetica 94, 307-337. Wilson, D. (1993). Fungal endophytes: out of sight but should not be out of mind. Oikos 68, 379-384. Wilson, D. (1995a). Endophyte- the evolution of a term, and clarification of its use and definition. Oikos 73, 274-276. Wilson, D. (1995b ). Fungal endophytes which invade insect galls: insect pathogens, benign saprophytes, or fungal inquilines? Oecologia 103, 255-260. Wilson, D. (1996). Manipulation of infection levels of horizontally transmitted fungal endophytes in the field. Mycological Research 100, 827-830. Wilson, R., Wheatcroft, R., Miller, J.D. and Whitney, N.J. (1994). Genetic diversity among natural populations of endophytic Lophodermium pinastri from Pinus resinosa. Mycological Research 98, 740-744.

Plants in Search of Sunlight

DOVKOLLER

Plant Biophysics Laboratory, Institute of Life Sciences, The Hebrew University, Jerusalem 91904, Israel

I. Introduction.................................................................................................... II. The Basics of Plant Movements ................................................................... A. The Concept of Movement in Plants.................................................... B. How are Plant Movements Generated?.............................................. C. The Nature of Plant Motors.................................................................. D. Control of Plant Movements................................................................. E. The Motor for Turgor-mediated Movements..................................... III. Synchronization by Solar Timekeeping....................................................... A. Flowers and Inflorescences .................. .... .. .... .. ............ .... ........ .... ...... ... B. Leaves....................................................................................................... IV. Growth-mediated Phototropic Movements................................................ A. Gravity and Light.................................................................................... B. Direct Control of Phototropic Movements ............ ...... .......... .... .. ....... C. Indirect Control of Phototropic Movement........................................ D. Photoreceptors for Phototropism......................................................... V. Solar-tracking by Heliotropism .................................................................... A. Shoot Apices............................................................................................ B. Leaves....................................................................................................... C. The Nocturnal Phase.............................................................................. D. Perception of the Solar Signal............................................................... E. Remote Phototropic Control by Vectorial Excitation....................... F. Logistics................................................................................................... G. Spectral Dependence of Laminar Phototropism................................ VI. Leaf Movements by Pulvinar Phototropism................................................ A. Perception of Directional Light as a Unilateral Signal...................... B. Logistics ..................................................... ....... ...... ... .. .. .. ..... .. ....... .... .. .... C. Coexistence with Photonastic Pulvinar Responses............................. D. Pulvinar Phototropism in Trifoliate Leguminous Leaves.................. E. Cooperation with Laminar Phototropism............................................ F. Modification by Stress............................................................................ G. Spectral Dependence of Pulvinar Phototropism................................ Advances in Botanical Research Vol 33 incorporating Advances in Plant Pathology ISBN 0- I 2-005933-9

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Copyright C 2000 Academic Pn.::>;\

All right;;; of reproduction in any form reserved

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VII. Adaptive Strategies of Plant Movements in Search of Light.................... A. Adaptations to the Terrestrial Environment....................................... B. Diurnal Movements................................................................................ C. Diaphototropism of Growing Shoots ................................................... D. Diaphototropism of Expanding Leaves ............................................... E. Laminar Diaheliotropism ...................................................................... F. Pulvinar Heliotropism............................................................................ G. Stress-modified Pulvinar Response...................................................... H. Diaheliotropism in Flowers................................................................... VIII. Perspectives..................................................................................................... References.......................................................................................................

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Terrestrial plants perform a variety of movements, by which they optzmtze their utilization of the existing environmental resources. Developing buds, flowers, inflorescences, fruits, leaves, leaflets, or even entire shoots, reorient by means of biophysical motors that sub tend them. The motors operate by performing differential and anisotropic changes in the volume of tissues located in their opposite sides, resulting in increase, or decrease in the radius of curvature of the motor. These changes are either mediated by growth, or by turgor. The lifetime of the motor for growth-mediated movements is limited by the capacity of the tissue to grow. Turgor-mediated movement is characteristic of mature leaves and is accomplished by means of a special motor organ - the pulvinus - situated in strategic junctions within the leaf, principally at the base of the leaf(let) lamina. The pulvinar motor remains operative throughout the active life of the leaf. Light is the major environmental requirement of the shoot. Consequently, most of these movements are driven by specific light signals. Photonastic movements take place in a predetermined direction and are independent of the direction of the light signals. In pulvinated leaves these signals are perceived in the pulvinus. Phototropic movements take place in a direction that is tightly coupled to the direction of light. The direction of light may be perceived either by differential of interception in opposite flanks of the motor itself, or as a vector. Some pulvinated leaves perceive the direction of light in the pulvinus, as a unilateral signal, and exhibit pulvinar phototropism. Other leaves perceive the direction of light in the lamina as a vectorial excitation, and exhibit laminar phototropism. The vectorial signal is transmitted to the subtending pulvinus. Heliotropic movements track the daily solar transit. After sunset, they reverse direction (in total darkness) to face the anticipated direction of the next sunrise. Phototropic movements of pulvinated leaves may be modified by environmental stresses. Plant movements are adaptive strategies for enhancing the photosynthetic performance, water use efficiency and reproductive efficiency.

I.

INTRODUCTION

Light is the sole source of energy to support plant growth. As a consequence of this intimate association, light has assumed an additional role in the existence of plants, as the most prevalent environmental signal by which they control the rate, or timing of different, essential phases of their physiological activities, as well as of their development. The most universal physiological activity controlled by light signals is the movement of stomatal guard cells.

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Seed germination, shoot (stem and leaf) growth, transformation of the shoot apex from vegetative to reproductive activity, different phases of flowering, fruit set and bud dormancy are the most familiar examples of control of developmental processes by light signals (photomorphogenesis ). Different components of the light environment are used by plants as signals to control these processes. For instance, seed germination of many plants is controlled by presence, or absence of light, its spectral composition, its total fluence, or its duration (Frankland and Taylorson, 1983; Mayer and Poljakoff-Mayber, 1989). Morphogenetic control of internode elongation (Smith, 1994) and leaf expansion (Van Volkenburgh et al., 1990) depend on the spectral composition of the light intercepted by these organs. Morphogenesis in the shoot apices (Vince-Prue, 1993), such as induction of flowering and dormancy, may be controlled by the photoperiod sensed by the leaves (critical duration of the uninterrupted dark period; spectral composition of a light interruption during the critical stage of the dark period). The spectral composition of the 'end-of-day' (EOD) light, at the transition from light to darkness may also play a role (Smith, 1994). Leaves of many terrestrial plants perform 'sleep movements', assuming a compactly folded configuration during the night and an unfolded one in daytime, increasing interception of sunlight by the lamina. These leaf movements are controlled by light H dark transitions, and may be characterized as diurnal light-induced movements synchronized by solartime-keeping. They take place in directions that are determined by endogenous factors and are therefore independent of the direction of incident light. Shoots of most plants perform light-guided movements in search of a more adequate and reliable source of photosynthetically active radiation (PAR) for their leaves. To be able to search for light, plants have evolved sensory 'direction-finding' systems by which they can locate the direction of maximal prevalent PAR in their environment, or determine the direction of solar radiation incident on them. These sensory systems provide guidance to biological 'motors' that move the apical bud and its complement of developing leaves, or individual mature leaves, to enhance the photosynthetic performance of the plant. In general, these movements optimize the interception of PAR, but do not necessarily maximize it. The flux of solar radiation intercepted by the leaves of many plants may exceed their capacity to cope with the thermal energy absorbed by their tissues and with the light energy that is absorbed by their chloroplasts. These excesses may cause overheating and photoinhibition of the photosynthetic apparatus, respectively, and result in transient, or even permanent damage to the chloroplasts. In terrestrial plants, excess thermal energy may be dissipated through re-radiation, convection and evaporative cooling. Excess light energy may be dissipated by photobiological mechanisms within the chloroplasts, that become operative under such

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conditions, in the form of the components of the xanthophyll de-epoxidation cycle (Bjorkman and Demmig-Adams, 1994). Many plants are also capable of performing movements that reduce their interception of the incident radiation. Chloroplasts may exhibit movements by which they avoid interception of excessive PAR. Membrane-bound photoreceptors in the cytoplasm sense changes in the fluence rate of light, causing the cytoskeleton to move the chloroplasts so as to present either their face, or their profile to the incoming radiation (Britz, 1979; Haupt and Hader, 1994). Much more prevalent are movements of leaves that are capable of controlling the interception of radiant energy under conditions of stress by varying the orientation of their laminae with respect to the incident radiation, thus evading potential damage to their photosynthetic apparatus by excess PAR. The ability to sense light H dark transitions, differences in photon fluence rate (abbreviated to pFR, to avoid confusion with the Pfr form of phytochrome), and particularly in direction of light, has expanded beyond optimization of photosynthesis. These capabilities have been adopted by some entomophilous plants to synchronize the opening and closing of their flowers/inflorescences with the diurnal activity of their pollinating vectors, or to improve their attractiveness for their pollinating vectors by reorienting their flowers/inflorescences to face the sun.

II.

THE BASICS OF PLANT MOVEMENTS A

THE CONCEPT OF MOVEMENT IN PLANTS

Objects move when their mass is displaced. According to this concept, a plant moves continuously as it grows, because the apices of its shoot and root are displaced in space by growth (elongation) of their subtending tissues. Likewise, leaves move as their petioles elongate, and as their laminae expand in space. These movements facilitate the acquisition of environmental resources that are essential for the existence of the plant. Root growth enhances the acquisition of water and minerals. Shoot elongation improves the competitiveness for light. Leaves expand their laminae and elongate their petioles, thereby improving interception of PAR and uptake of carbon dioxide. However, when plant scientists describe, or study plant movements, they do not include these universal phenomena. The acceptable definition of plant movements is restricted to those that change the spatial orientation, or configuration of the plant in response to specific signals. B.

HOW ARE PLANT MOVEMENTS GENERATED?

Higher plants can move stomatal guard cells, entire leaves, the leaf lamina, its leaflets, or its opposite halves, apical buds and their subtending cluster of

PLANTS IN SEARCH OF SUNLIGHT

39

developing leaves, flowers, or inflorescences, fruits, or entire shoots. Such movements invariably take place by anisotropic changes in cell volume. Anisotropic volume changes are polarized in one (commonly) preferred dimension and thus modify the shape of the cell, or entire tissue. Changes in cell volume may result from irreversible growth, or from reversible changes in hydrostatic (turgor) pressure (potential). The resulting movements are therefore defined as growth-mediated, or turgor-mediated, respectively. An object can be moved only by applying a force. Plants move single, or aggregate organs of their shoot by means of forces generated in motor tissues. Transport of osmotically active solutes, accompanied by water, across the membranes of the cells is an integral and essential part of the motor in turgor-mediated, as well as in growth-mediated movements. Cell growth involves auxin-mediated increases in extensibility of the cell walls, concomitantly with uptake of osmotically active solutes, followed by water (Cosgrove, 1987). Growth may be inhibited by endogenous substances (Bruinsma and Hasegawa, 1990). Any of these processes can be considered a prime target for the signals that drive growth-mediated movements. Darwin and Darwin (1881) noted that as ' ... growth is preceded by ... increased turgescence ... it does not appear to be advisable to separate [them] into two distinct classes'. von Sachs (1887) argued that as growth itself is also mediated by turgor, the only difference between growth- and turgormediated movements lies in the extensibility versus the elasticity of the cell walls of the motor tissue. C.

THE NATURE OF PLANT MOTORS

Motor tissues occupy discrete regions of the plant. As a rule, motors are cylindrical. Movement is the result of differential, anisotropic changes in cell volume in tissues (generally) situated in parallel, opposite flanks of the motor, thus increasing or decreasing its radius of curvature. Motors of growth-mediated movements are not distinguishable from their neighbouring tissues, or delimited from them, except by their capacity for growth. Motors for turgor-mediated leaf movements are generally organized in a specialized, well-defined organ- the pulvinus (Section II.E). Growth-mediated movements take place by differential changes in rates of growth. Cell growth is a direct, irreversible consequence of increased extensibility of the cell wall, resulting in irreversible expansion of cell walls by osmotic uptake of water, balanced by osmotic adjustment of solutes (Cosgrove, 1987). Growth-mediated movement may also be induced in nongrowing parts that retain a potential for growth. For instance, the leaf-sheath base at the grass internode (also known as a 'pulvinus') retains a latent capacity for growth-mediated curvature in response to gravistimulation (Dayanandan et al., 1976). Similarly, leaves of certain insectivorous plants

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retain a potential for rapid, growth-mediated movements that is expressed when their tactile organs are stimulated by contact with prey (Williams and Bennett, 1982). Turgor-mediated movements take place by differential, fully reversible changes in volume (expansion, or contraction) of mature cells as a result of transport of osmotically active solutes, followed by water, from, or into their vacuole. Walls of mature cells can only expand, or contract elastically. Therefore, volume changes in such cells depend on the elastic modulus of their wall and are associated with corresponding changes in their turgor pressure. Expanding/contracting cells may also exhibit deformation that enables large changes in cell volume in response to relatively smaller changes in turgor pressure (Section II.E.1). Growth is irreversible, therefore, maturation of cells comprising the motor tissue puts an end to its capacity for growth-mediated movements. The growth-mediated motor is therefore characterized by inherent obsolescence. However, younger tissues may then take over motor function. In this case, the motor simply moves forward (distally). Growth-mediated curvature may be reversed only by compensatory growth in the opposite (concave) flank, but only as long as the motor tissue retains its capacity for growth. In contrast, the turgor-mediated motor is composed of fully differentiated cells. Its movements do not involve growth and are fully reversible and repeatable throughout maturity. The turgor-mediated motor is stationary and has a 'lifetime warranty'. Turgor-mediated movements are much less costly than growth-mediated ones in resources and metabolic energy. Both types of movements invest metabolic energy in the transport of osmotically active solutes across cell membranes. In addition, turgormediated movements invest metabolic energy in membrane turnover that is a consequence of the extensive changes in volume, whereas growthmediated movements invest metabolic energy, as well as resources in the biosynthesis of new cell walls and cytoplasmic components. Shoot organs and their aggregates are moved passively by their subtending motors. Mature leaves move their lamina by means of opposite volume changes (contraction/ expansion) in opposite sectors of a pulvinus at their base (and in some case in other strategic locations in the leaf as well). Immature parts of the plant move by differential growth of their sub tending motor tissue. Such growth results from acceleration, or inhibition of elongation in one flank of the support, that is usually, but not necessarily accompanied by opposite changes in the opposite flank (Firn, 1994). The resulting change in the radius of curvature of the subtending support changes the spatial orientation of the more distal part( s) of the plant. The leaf lamina may reorient by curvature of its subtending pulvinus. The apical bud, and its cluster of developing leaves (or cotyledons) may reorient by curvature of their subtending young stem (or hypo-/epicotyl); the expanding leaf lamina may reorient by curvature of its petiole; flowers and inflorescences may reorient by curvature of the stalks bearing them.

PLANTS IN SEARCH OF SUNLIGHT

41

However, a developing leaf lamina, or floral organ (petals, sepals, floral bracts, inflorescence bracts) may also move by deformation of their spatial configuration as a result of differential growth in their own opposite tissues. Leaf movements frequently involve torsional rotation (of the petiole, or pulvinus). The structural features of such torsions are far from clear (cf. Snow, 1959). One possibility is that the cells along the lateral flanks of the (cylindrical) organ are structurally constrained to elongate, expand, or contract preferentially along their diagonal axis, from the lower (abaxial) corner of their proximal end to the upper (adaxial) corner of their distal end. In the absence of unilateral stimulus, the diagonal forces in the opposite lateral flanks of the motor tissue are in balance with each other. But when differential volume changes take place in the opposite lateral flanks, these forces are unbalanced, resulting in torsional rotation. D.

CONTROL OF PLANT MOVEMENTS

Plant movements are driven by signals that may be endogenous, originating autonomously within the plant, or exogenous, originating from the environment to which the plant is exposed, such as temperature alternation, mechanical stimulation, but principally gravity, light H dark transitions and directional or unilateral light. Certain plant movements take place in response to changes in atmospheric humidity. Stomatal guard cells are a typical example. Certain grass leaves perform hygroscopic movements, by means of specialized, inflated bulliform cells in their adaxial epidermis, along the vein(s). Lateral shrinkage of these cells when the leaf is waterstressed results in rolling of the lamina (Begg, 1980), or its longitudinal folding. In many plants, dispersal of spores and seeds is accomplished by hygroscopic movements of the organ in which they are contained. Endogenous movements are under unique control by the universal biological oscillator ('clock') and take place in circadian cycles ( -24 h). The biological clock is genetically determined for each individual. An individual chronometer controls the free-running, circadian activity of each organism/ cell. The autonomous nature of these movements is expressed in constant environmental conditions (temperature, humidity, diffuse light or dark). Their direction is invariable, predetermined endogenously by the fixed, opposite location and the bilateral structure of the motor tissue that responds to these signals. Growth-mediated movements of stems and tendrils, known as (circum )nutations, are an exception to this generalization, because they take place by helical displacement of the subapical motor around the organ, resulting in harmonious oscillations. In nature, autonomous movements become synchronized to solar timekeeping by the diurnal 24-h cycle of high H low temperature and (most commonly) light H dark transitions. These transitions repeatedly rephase

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D. KOLLER

the circadian oscillations of leaves (Bunning, 1959, 1973; Satter, 1979; Satter and Morse, 1990) and stomatal guard cells (Gorton, 1990) to diurnal cycles. Circadian and diurnal movements that are growth-mediated are repeated in each cycle, but their life-span is constrained by the irreversible nature of growth. Those that are turgor-mediated may be repeated virtually indefinitely. Diurnal movements retain the constant, predetermined direction of their autonomous origin. These movements are defined as nastic (from the Greek nastos, tight pressed), and are entirely independent of the direction of any external signal that controls them. The signal may also be non-directional. Diurnal movements may also be modified by brief exposures to light, or by changes in irradiance. Skotonastic (from the Greek skoto, dark) movements take place in response to a light ~ dark transition, and the reverse photonastic movements take place in response to the opposite, dark ~ light transition. Leaves assume the skotonastic, folded configuration around nightfall, which led to the term 'sleep', or nyctinastic (from the Greek nyx, nyctos, night) movements. Leaves of the 'sensitive' plant Mimosa pudica exhibit such diurnal movements in response to light H dark transitions, as well as thigmonastic movements, in response to mechanical perturbation. Movements may be guided by unilateral, or directional signals. The direction of these movements is tightly linked to the direction of the signal (stimulus), because specific sensors are assigned to specific targets in their motor tissues. These movements are therefore designated as tropic (from the Greek tropos, turn). Positive and negative tropisms describe curvature towards and away from the source of the signal, respectively. Phototropic movements are guided by differential interception of unilateral light (unilateral excitation), or by interception of directional (more or less collimated) light at an oblique angle (vectorial excitation). The movement is diaphototropic (from the Greek dia-, through) when curvature results in reorientation (of the coleoptile tip, shoot apex and its cluster of young leaves, leaf or leaflet lamina, flower, or inflorescence) to face the light source. Growth-mediated phototropic curvature may be modified by an opposing gravitropic response (Firn, 1990) (Section IV.B.1). Phototropic movements of leaves, flowers, or inflorescences, whose direction changes throughout the day with the changing direction of the sun (solar transit) are heliotropic (Darwin and Darwin, 1881), 'sun-tracking' (Wainwright, 1977), or 'solar-tracking' (Mooney and Ehleringer, 1978). Such movements are diaheliotropic when the moving organ(s) remain normal with respect to the direction of the sun. Leaves and their leaflets may also exhibit plagioheliotropic (from the Greek plagio-, oblique), or paraheliotropic (from the Greek para-, along) movements, depending on the orientation maintained by their lamina with respect to the direction of the sun: oblique, or parallel, respectively. The distinction between 'the action of light in modifying the periodic movements of leaves, and in causing them to

PLANTS IN SEARCH OF SUNLIGHT

43

bend towards its source' is attributed to Julius von Sachs. Darwin and Darwin (1881) wrote: 'heliotropic movements are determined by the direction of light, whilst periodic movements are affected by changes in its intensity and not by its direction. The periodicity of the ... movements often continues for some time in darkness ... whilst heliotropic bending ceases very quickly when the light fails'. E. THE MOTOR FOR TURGOR-MEDIATED MOVEMENTS

Turgor-mediated movements operate by means of a hydraulic motor, powered by bioelecricity. The most universal of these motors is that in stomatal guard-cells. However, of all the plant organs, only the leaf has evolved specialized tissues that are structurally adapted to facilitate its rapid, turgor-mediated reorientation in space. These tissues are organized in a structurally distinct, well-delimited pulvinus. Leaves of many plants belonging to several unrelated taxonomic groups (principally Leguminosae, Oxalidaceae and Malvaceae) exhibit turgor-mediated movements by means of a pulvinus. 1. Structural Features The pulvinus exhibits unique structural features. It differs distinctly in structure from its neighbouring parts on either side, and is clearly delimited from them. It is a (commonly) short cylinder, consisting of a multilayered sheath ('cortex') of thin-walled, anatomically undifferentiated cells, that surrounds a central vascular core and is enclosed by a single layer of epidermal cells. Each of these tissues is uniquely adapted to the function of the pulvinus in leaf movements, contributing to the means for reorienting the leaf(let) lamina, the entire leaf, or its parts (Morse and Satter, 1979; Werker and Koller, 1987; Werker et al., 1991). The pulvinus is strategically located for moving the part of the leaf which it subtends. It forms a flexible joint at the junction between the leaf(let) lamina and its axial support (petiole, rachis, rachilla), but may also be found at the leaf base, or intermediate junctions of pinnate leaves. It acts as a crane, consisting of a hydraulic motor, within a flexible 'pivot' that connects a rigid stationary 'post' (petiole, or rachis) to a movable, rigid 'boom' (midvein of the lamina). The motor operates by simultaneously generating opposite stresses along its opposite sectors, expressed by a corresponding reduction and concomitant increase in turgor pressure. This turgor differential produces sufficient torque to displace the mass of the laminar 'boom' over considerable angles, relative to the stationary rachis (post), by changing the radius of curvature of the pulvinus. Curvature takes place by opposite changes in volume of its cortical tissue in opposite sides of its vascular core. The contracting sector becomes concave, its opposite, expanding sector

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D. KOLLER

becomes convex. These changes in volume of the pulvinar motor tissue, and the resulting changes in its curvature, are fully reversible and repeatable throughout the active life of the leaf. The parenchymatous motor cells of the pulvinar cortex are structurally adapted to undergo reversible, turgormediated changes in volume, by elastically stretching or relaxing their walls, as well as by changing their shape. The low bulk modulus of elasticity of the pulvinar motor tissue enables the cells to undergo extensive changes in volume in response to small changes in mechanical stress (turgor pressure) and is thus conducive to efficient conversion of osmotic work into cell expansion (Mayer et al., 1985; Irving et al., 1997). Similar properties have been described in stomatal guard cells (Raschke, 1975; Sharpe et al., 1987). Contracted pulvinar motor cells in Lupinus and Lavatera frequently exhibit transverse folds in the walls parallel to the pulvinar axis (Werker and Koller, 1987; Werker et al., 1991). Volume changes of pulvinar motor cells are structurally constrained along the pulvinar axis, as a result of the orientation of the cellulose microfibrils in their walls transverse to the pulvinar axis, but also because they are ellipsoid, stacked in parallel along that axis (Mayer et al., 1985). In addition, the epidermal sleeve presents mechanical constraints to radial expansion, whereas transverse folds of the epidermis along the pulvinar axis (Satter et al., 1970; Satter and Moran, 1988) contribute to reduce mechanical resistance to axial expansion/contraction of the subtending motor tissue. The central vascular core (see Fig. lO(A)) is formed by coalescence of the vascular tissues of the veins, and separates into a number of peripheral bundles at the transition to the subtending petiole, or rachis (see Fig. 10(B)). It is flexible, but non-extensible, allowing the pulvinus to change its radius of curvature, without changing its length (Koller and Ritter, 1994). Contraction of pulvinar motor cells of Mimosa pudica (Weintraub, 1951; Campbell and Thomson, 1977) andAlbizzia julibrissin (Satter et al., 1970), as well as of stomatal guard cells of Opuntia (Thomson and De Journett, 1970) has been associated with fragmentation of the large central vacuole(s) into numerous small vacuoles, or vesicles. The multivacuolate state is apparently reversed by fusion during expansion of the cell. Vacuolar fragmentation may conserve the tonoplast membrane to cope with the extensive, rapid and reversible changes in cell volume (Campbell and Garber, 1980). There is no information on how the plasmalemma copes with such changes. The multivacuolate state may be a manifestation of vesicle trafficking of solutes across the tonoplast (MacRobbie, 1999) (Section II.E.7). Pulvinar motor cells (of bean) exhibit features indicative of high metabolic activity. The presence of large, prominent nuclei, abundance of welldeveloped mitochondria, with tightly packed cristae, numerous polysomes and an extensive endoplasmic reticulum (rough and smooth), are all indicative of a high capacity of the motor cells for respiratory and synthetic activity. Numerous prominent plasmodesmata traverse the walls, indicating

PLANTS IN SEARCH OF SUNLIGHT

45

a substantial capacity for symplastic transport (D. Koller and E. Zamski, unpublished observations). Large functional chloroplasts (Koller et a/., 1995) become increasingly abundant towards the periphery of the motor tissue. They exhibit prominent grana stacks but little starch. However, stomata are entirely absent from the pulvinar epidermis. Intercellular spaces are limited in size and are partially filled with fibrous material, rather than air. These features suggest that the major role of pulvinar chloroplasts is to provide photosynthetic electron transport and ATP, not carbon fixation. 2. The Nastic Response The capacity of the pulvinus for fully reversible and repeatable curvatures is virtually unlimited. As a result, circadian and diurnal leaf movements that are turgor-mediated are repeated with remarkable precision in each cycle. In leaves that exhibit nastic movements, the direction of movement is predetermined endogenously. Pulvini of such leaves exhibit a functional bilateral organization of the motor tissue, inherently organized in two opposite sectors that undergo the opposite and reversible volume changes: the extensor expands, while the flexor contracts along the pulvinar axis as the pulvinus curves to unfold the leaf and vice versa. This bilateral organization predetermines the direction of movement (Satter et a/., 1974), which is species specific, but may differ in different leaflets of the same compound leaf, resulting in a great variety of ways in which leaves of different species fold at night (Darwin and Darwin, 1881). In the absence of environmental signals, opposite volume changes take place in the motor cells of the flexor and extensor sectors in circadian rhythmicity, controlled uniquely by the ubiquitous circadian oscillator (the biological clock). Light is the most prevalent exogenous signal that modifies and controls the circadian pulvinar responses. The pulvinus contains the photoreceptors for the diurnal light H dark transitions that repeatedly reset the oscillator to a diurnal 24-h cycle, as well as the transduction chain for responding to them (Section III.B.3). The response is nevertheless restricted to the flexor and extensor, and its direction is therefore independent of the direction from which the light signal is intercepted. Temperature and waterstress may modify these responses.

3. The Phototropic Response All types of turgor-mediated phototropic movements take place by a directional pulvinar curvature in response to a directional light signal. In such phototropic movements, any sector of the motor tissue facing the direction of light (but not necessarily exposed to such light) contracts while its opposite sector expands, resulting in positive phototropic curvature of the pulvinus. However, plants differ in their perception of directional light by their leaves. In some, the direction of pulvinar curvature is dictated by the direction of unilateral light incident on the pulvinus itself (unilateral

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D. KOLLER

excitation, e.g. leguminous leaves). In others, it is determined by the direction from which oblique light is intercepted by the lamina (vectorial excitation, e.g. malvaceous leaves). The difference between these directional sensors will be discussed in detail (Sections V.D.2 and VILA). Leaves of (leguminous) plants that exhibit circadian (autonomous) and diurnal movements, may also exhibit phototropic movements in response to directional (unilateral) light signals (Section VI). This means that the mechanism for detecting and responding to directional light extends to all sectors of the pulvinus, and co-exists with the mechanism for detection and response to non-directional light only in the flexor and extensor sectors (Section VI.C). 4. Operational Aspects Changes in volume of pulvinar motor cells are associated with changes in their water relations. The expanded extensor in the pulvinus of Samanea exhibits a greater osmotic pressure and a more negative water potential !}.if! than the contracted flexor. The contracted extensor and expanded flexor exhibit opposite relationships. The (calculated) turgor pressure is similar in the two sectors when the leaf is unfolded, but is higher in the expanded flexor than in the contracted extensor when the leaf is folded (Gorton, 1987). However, expressed sap was used for measuring osmotic pressure, which therefore represents fluid from the vacuole and apoplast. Measurements in intact motor cells suggest that osmotic pressure remains virtually unchanged during their extensive volume changes. Osmotic pressure (measured microscopically at incipient plasmolysis) of the motor cells remains remarkably stable throughout the volume changes that take place during diaheliotropic movements of the pulvinus of Malva neglecta (Yin, 1938). Similarly, changes in volume of the (abaxial) extensor and (adaxial) flexor of the primary leaf of bean during its circadian movements are associated with changes in [K+], but nevertheless they take place at a relatively stable osmotic pressure of the cell sap. These changes in volume are positively correlated only with [K+]/protein, suggesting that changes inK+ -content are accompanied by concomitant changes in water content per cell (Kiyosawa and Tanaka, 1976). A linear relationship was observed between changes in volume of the guard cells and their K+ -content during stomatal opening (Raschke, 1975). These results support earlier findings that '. .. the concentration of the cell sap remains constant on both ... sides of the joint [pulvinus of Phaseolus] during movement' (Harder et al., 1965, p. 392). Assays of individual motor cells in the terminal pulvinus of the trifoliate leaf of bean in the course of its phototropic response (Section II.E.3), by means of a cell pressure probe, showed that changes in volume throughout pulvinar movement are associated with corresponding changes in turgor pressure, but osmotic pressure remains stable nevertheless (Irving et al., 1997).

PLANTS IN SEARCH OF SUNLIGHT

47

The pulvinus (at least in leguminous plants) appears to be an entirely selfcontained operational unit, equipped with its own circadian clock, and all the components of the apparatus responsible for the volume changes of the motor cells (Satter, 1979). Pulvinar movements take place even after excision of the lamina (Brauner, 1932; Brauner and Brauner, 1947). Excised pulvini maintain autonomous, rhythmical movements under constant environmental conditions, even in darkness. Under such conditions the amplitude of the movements gradually decays, except when the pulvini are supplied with sugar and exposed to red light pulses (Satter and Morse, 1990). Furthermore, although opposite sectors of the pulvinus undergo their (opposite) volume changes with perfect coordination, they do so independently. Excision of either tissue from the pulvinus (Samanea saman) does not prevent the opposite motor tissue in the remaining, intact part of the pulvinus, from continuing rhythmical changes in volume (Palmer and Asprey, 1958). Partial excision of either flexor or extensor of the pulvinus of the primary leaf of bean does not change the period, or the phase of the circadian leaf movements. The excised part of the pulvinus starts to regenerate within 36 h and regenerates completely after 12 days (Millet et al., 1989). It remains to be seen how the export and import of solutes and water are managed in the absence of the opposite sector of the pulvinus. Pulvinar motor cells are also self-contained operational units (Mayer and Hampp, 1995). Protoplasts isolated from pulvinar motor tissue also exhibit circadian oscillations in their volume (Mayer and Fischer, 1994). The pulvinus exhibits precise physiological coordination in the operation of its motor. In the trifoliate leaf of bean, opposite but equivalent volume changes take place simultaneously in opposite sectors of the pulvinus during its curvature: contraction along one sector matches expansion along the opposite sector (Fig. 1). This coupling is supported by the flexibility of the non-extensible vascular core. Export of solutes and water from the contracting sectors and their import into the expanding one take place simultaneously. The volume of water and amounts of osmotically active ions lost from the contracting sector are gained by the opposite, expanding sector. The directions are reversed when the expanded sector contracts and the contracted one expands (Koller and Ritter, 1994; Irving et al., 1997). The same solutes and water are shuttled back and forth, but different cellular processes are involved in expansion and contraction. The transpulvinar transport of ions and water between the contracting sector and its opposite, expanding sector, takes place predominantly through the apoplast (Campbell et al., 1981). This process starts by bioelectric transmembrane transport of solutes between the protoplasts of the motor cells and their apoplast. Solute influx into the vacuole from the water free space (WFS) of the apoplast makes the water potential (!li) more negative in the former and less negative in the latter. The resulting, ingoing gradient (D.!li) leads to uptake of water into the vacuole and expansion of the cell. Solute efflux

48

D. KOLLER

A =- 17• t.., •+ t3•

t20

t80 = + 34•

feo=+58• tiOO: + 73°

---

0 ~

- -LAMINA----Pli..VINULE:-----PETIQ...E - - - • llNinAL)

120,-----------------------------~

-dAb/dAd= 0.913; r = 0.977

8

0

100

80

.c ~60 I

40

20

20

40

60

80

100

120

AAd Fig. 1. Changes in the dimensions of the adaxial and abaxial sectors of the terminal pulvinus of Phaseolus vulgaris (Fabaceae) in the course of its phototropic response to adaxial light. (A) Traces of the adaxial and abaxial crests from time-lapse photographs taken at 20 min intervals. Corresponding angles of laminar elevation are shown in the upper right corner. (B) Linear relationship between opposite changes in adaxial and abaxial length (Md and Mb, respectively, in arbitrary units). Changes in volume of the motor tissue correspond to changes in length. (Reprinted with permission from Koller and Ritter (1994).)

PLANTS IN SEARCH OF SUNLIGHT

49

results in an opposite change in the direction of f1l]f and leads to cell contraction. Most of the water and solutes ( -95%) are sequestered in the vacuole and must therefore be transported across the vacuolar membrane (tonoplast), as well as the cell membrane (plasmalemma). This transport is carried out by means of specialized transmembrane proteins, acting as carriers, or channels (Sections II.E.4 and II.E.7). Signals that control the operation of the pulvinar motor are initially transduced in the motor cells into electrochemical energy, then into osmotic work, and finally into mechanical work. Clearly, mechanisms controlling transmembrane transport are essential components of the transduction path initiated by these signals. A variety of transport mechanisms are involved in the reversible volume changes in pulvinar motor cells and stomatal guard cells. The interaction between them and their precise position in the signal transduction that controls these volume changes are quite complex. The following summary outline is partially based on circumstantial, or indirect evidence and is partially hypothetical. Dr Nava Moran contributed much to these concepts. Bioelectric generation of ion fluxes provides the means for turgormediated volume changes. In general, ions may move across the impermeable cell membranes only by means of specific, transmembrane proteins. Some of these act as carriers and use either metabolic energy (ATP) to move ions against the electrochemical gradient by active transport, or the electrochemical energy gradient of other ions, or organic molecules moving simultaneously, in the same, or the opposite direction (symport and antiport, respectively). Other transmembrane proteins act as channels (varying in their specificity), through which ions move according to their electrochemical gradient, by passive transport. Proton pumps in the cell membranes of pulvinar motor cells and stomatal guard cells are a key component of the mechanism of their reversible volume changes, by their essential contribution to the transport of ions across the membrane (Iglesias and Satter, 1983a, b). Electrogenic H+ -ATPases and H+ -PPases utilize energy released by ATP hydrolysis to pump protons out of the cytosol and into the a poplast, or vacuole, thereby changing the electrical charge of their membranes. Activity of the proton pumps hyperpolarizes the membrane and increases the transmembrane gradient in electric potential (6.pH); their deactivation allows the membrane to become depolarized and decreases 6.pH. Changes in the membrane potential determine the activity of voltage-gated ion channels in the cell membranes. K+ and CI- are the most abundant osmotically active solutes in motor cells. Their transport through these channels makes the greatest contribution to the osmotic changes and consequent movement of water across the pulvinus (Satter and Moran, 1988; Satter and Morse, 1990; Mayer and Hampp, 1995; Irving et al., 1997). Transport of ions across the membrane by means of channels is much more efficient than by means of carriers, because ions move through them

50

D. KOLLER

much more rapidly, by three to four orders of magnitude. Ion channels in the plasma membrane (of motor cells and stomata) are 'gated' and can open or close. Changes in the electric charge of the membrane, normally more negative on the cytoplasmic face, may change the frequency and/or duration of the open state of the channels by several orders of magnitude. Such changes may be brought about by specific conditions of light, specific hormones, or ligands, particularly Ca2 + and inositol1,4,5-trisphosphate (IP 3) (Satter and Moran, 1988). J. I. Schroeder and co-workers have contributed much to define the roles played by mechanisms that control ion transport processes in volume changes of stomatal guard cells. They have identified voltage-gated, hyperpolarization-dependent, inward-rectifying K+ channels (K\n channels) and depolarization-dependent, outward-rectifying K+ channels (K+ out channels) as major pathways for K+ movement during contraction and expansion (reviewed by Thiel and Wolf, 1997; Maathuis et al., 1997). They have shown that contraction involves release of anions (mainly CI- and malate) through 'slow' anion channels, that are activated by increase in concentration of cytoplasmic Ca2 + ([Ca2 +]cy1) and are controlled by their state of phosphorylation. Furthermore, they have shown that [Ca2 +]cyt is controlled by the activity of voltage-dependent Ca2 + channels in the plasma membrane, as well as by mobilization of vacuolar Ca2+ through the ubiquitous 'slow' vacuolar channel. Blatt and Grabov (1997) identify [Ca2 +]cyt, transmembrane b.pH and channel-protein phosphorylation as the signalling pathways that control K+ and anion channel activities during stomatal movement, and suggest that they may integrate stomatal responses to different signals by redundancy. The role of ion channels in signal transduction in guard cells is reviewed by MacRobbie (1997). The Donnan free space (DFS) of walls of pulvinar motor cells, particularly their middle lamella, may serve as a temporary reservoir for cations that are exchanged between the symplast and apoplast. The carboxylic acid residues of the pectin matrix provide an abundance of fixed negative charges and thus a large capacity DFS for exchange of protons and cations (mainly K+) with the symplast. Activation of the proton pumps in the plasmalemma of motor cells acidifies the apoplast, providing the protons to replace these cations, thus releasing them to be taken up into the symplast. Deactivation of the proton pump enables the reversal of this process (Campbell et al., 1981; Starrach et al., 1985; Freudling et at., 1988; Starrach and Mayer, 1989; Mayer, 1990; Irving et al., 1997). The intercellular spaces in the pulvinar motor tissue of bean appear to be at least partially filled with pectins (D. Koller and E. Zamski, unpublished observations). Studies of changes in the elemental composition of pulvinar motor cells of Robinia pseudoacacia during leaflet movement showed that changes in K+ and CI- content take place simultaneously in the apoplast and symplast (Moysset et al., 1991), most probably as a result of transpulvinar transport (Irving et al., 1997).

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Different transport mechanisms operate in expansion and contraction. A great similarity exists between many of these mechanisms in pulvinar motor cells (Lee, 1990) and in stomatal guard cells (Assman, 1993). However, all stomatal guard cells in a single leaf exhibit the same response to a given light signal, whereas in skoto-/photonastic leaves extensor and flexor cells of the same pulvinus exhibit simultaneous, opposite volume changes in response to the same light signals. Extensor cells contract in darkness, probably by similar mechanisms as guard cells, but this does not explain the concomitant expansion of flexor cells, except perhaps as a passive outcome of contraction of the extensor. In phototropic pulvini, solutes and water transported out of the contracting sector in response to its exposure to (blue) light are driven through the water-free space (WFS) to the opposite, shaded sector where their arrival apparently triggers uptake and expansion (Irving et al., 1997). Cell expansion results primarily from influx of K+ and CI-, accompanied by water. The process starts with activation of the electrogenic H+ -ATPase in the plasmalemma, creating a proton-motive force (pmf) and increasing 6pH across the plasma membrane. These changes energize the influx of ions, as follows. The pmf hyperpolarizes the negatively charged membrane, which causes its K+in channels to open and provide a major pathway forK+ influx (Ward et al., 1995), whereas the 6pH provides the electrochemical energy for uptake of cations, enabling influx of K+ through these channels (Moran, 1990). Apoplastic and cytoplasmic pH affects activity of K+in and K+ out channels in the plasma membrane of cultured cells of Arabidopsis (Giro mini et al., 1997). The influx of K+ depolarizes the membrane and also reduces the imbalance in electric charge caused by efflux of protons. However, increase in 6pH also provides the energy for uptake of anions, predominantly CI-, into the cell, which acts to hyperpolarize the membrane. CI- is transported either through CI- channels, or by means of an H+ I CIsymporter. In the absence of CI-, synthesis of malic acid may provide the necessary anions (Bialczyk and Lechowski, 1989). The increased [ionln produces an inward-directed gradient in 6lJ!, which results in influx of water from the apoplast and cell expansion. Cell contraction is controlled by [Ca2 +]cyt and takes place by deactivation of the proton pumps, followed by depolarization of the plasma membrane. Depolarization of the membrane activates selective K+ out channels (Moran et al., 1988, 1990) when its potential becomes more positive than their activation voltage. Efflux of K+ alone through these channels clamps the membrane potential at the K+ equilibrium potential, which prevents further, long-term efflux of K+. However, depolarization also activates CI- channels in the membrane, which enables efflux of CI- down its chemical gradient, thereby providing additional long-term depolarization (Maathuis et al., 1997). Activation of CI- channels, and in particular the S-type (slow) (Ward et al., 1995), also stops influx of K+ by closing K+in channels (Moran, 1990). Thus, Cl- channels play a dual role in contraction, by contributing directly to

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efflux of CI- (and malate) and indirectly to efflux of K+ through activated K\ut channels (Ward et al., 1995). The decreased [ionln produces an outward-directed b.IJ!, efflux of water and contraction. The voltagemediated, electro-osmotic efflux of CI- from its higher intracellular concentration, via anion channels, is a key reaction in contraction, because it is accompanied by efflux of water. The concentration of other ions in the contracting vacuole increases. As vacuolar concentration of these ions exceeds the steady state value, their efflux is enhanced, particularly K+ through opened K\ut channels (Freudling et al., 1980). Guard cell anion channel 1 ( GCAC 1) mediates large, rapid anion efflux and is also an essential element in depolarization of the plasma membrane. Malate, [Ca2 +]cyt and nucleotides control the number and/or probability of opening, the transport capacity, the position of the voltage-sensor, and consequently the voltage threshold of activation of these channels. The voltage range for activity of this channel overlaps that of the K+ out channel (Hedrich and Becker, 1994). Activation and deactivation of channels by depolarization may depend on their phosphorylation (Moran, 1996). Opening of CI- channels and K+ out channels, and closure of K+in channels by depolarization of the plasma membrane may depend on their phosphorylation by a tightly associated protein kinase (PK). The appropriate state of phosphorylation is achieved by activity of the PK, balanced by simultaneous activity of a protein phosphatase (PPase ). These enzymes are activated by an increase in [Ca 2 +]cyt, resulting from import of exogenous Ca2 +, or release from endogenous stores (such as the vacuole). Exogenous Ca 2 +enters via voltagegated Ca2 +channels in the plasma membrane. These channels open transiently upon depolarization of the membrane. The membrane contains a large number of Ca2 +channels, but the majority of these channels are quiescent and are not activated by 'normal' depolarization. These channels are activated by large pre-polarizing pulses, positive to 0 mV, which also induce recovery of the transient activity of the other channels. This 'recruitment' increases with intensity and duration of pre-depolarization. Such modulation might play a role in regulating transport processes that are dependent on [Ca2 +]cyt (Thuleau et al., 1994a, b). 5. Circadian Control of Ion Fluxes Under constant environmental conditions, skoto-/photonastic leaves revert to their autonomous circadian rhythm (Section II.D). They fold at the end of their circadian 'light' phase of the endogenous cycle by expansion of their flexor cells and concomitant contraction of their extensor cells and unfold at the end of their circadian 'dark' period by reversal of these volume changes. Protoplasts isolated from pulvinar motor cells of Phaseolus coccineus exhibit circadian volume oscillations (Mayer and Fischer, 1994). Changes in volume in the extensor and flexor sectors of the pulvinus of Samanea take place with

PLANTS IN SEARCH OF SUNLIGHT

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a circadian periodicity, that is 180° out of phase. The same periodicity is expressed by the behaviour of the K+in channels in protoplasts isolated from these sectors. In uninterrupted darkness, flexor protoplasts have open channels while those from the extensor have closed channels when the leaf is folded (circadian 'dark' period), and this situation is reversed when the leaf is unfolded (circadian 'light' period). These channels are voltage-gated, under control by the activity of the proton pump in their plasma membrane. This suggests that the rhythmic changes in the state of the K+in channels result from the control by the biological clock over the activity of the proton pumps. However, in both cases, closure of these channels is also associated with increases in [IP3] (Kimetal., 1992, 1993). There is no information on the endogenous signals from the biological clock in its two phases, on the cellular receptors for these signals in the flexor and extensor, or on additional transduction steps between them and the rhythmical changes in volume of motor cells. 6. Diurnal Control of Ion Fluxes The circadian volume changes in extensor protoplasts of Phaseolus can be synchronized with diurnal light H dark cycles (Mayer and Fischer, 1994). Circadian unfolding can be advanced by transition from darkness to blue light. Extensor cells expand, and flexor cells contract in blue light. Stomata open in response to blue light, by expansion of their guard-cells. The similar response of pulvinar extensor cells and stomatal guard-cells to blue light suggests a basic similarity in the mechanisms involved in the two systems. Blue light activates the H+ -pump in the guard-cell membranes, thereby driving uptake of ions and water. Calmodulin and calmodulin-dependent myosin light-chain kinase are involved in H+ -pumping by guard-cell pro top lasts from Vicia faba in response to blue light (Shimazaki et al., 1992). Activation of the enzyme phospholipase 2 (PLA2) is probably a component of the transduction of the blue light signal to opening K+in channels and closing K+ out channels, as part of the expansion process. PLA2 activity produces free, polyunsaturated fatty acids (FPFA) and lysolipids, such as lysophosphatidylcholine (LPC), by hydrolysis of phosphatidylcholine (PC). The LPC produced may activate the proton pump in the plasma membrane, whereas the FPFA may open of K+in channels and close K\ut channels. These effects of the products of PLA2 activity may be direct, or by activation of a protein kinase. G-proteins attached to the photoreceptors may become activated by the intercepted light signals, and then activate PLA2 (Lee et al., 1996). The role played by G-protein coupled receptors in signal transduction in plant cells is not clear, but is suggested by the presence in them of Gproteins and of classical downstream signalling elements, such as PLA2 (Millner and Causier, 1996). Skoto-/photonastic leaves fold in response to a light ~ dark transition, and this is facilitated by EOD ('end-of-day' irradiation with red light at the time of transition). Their flexor cells expand,

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and extensor cells contract. A dark ~ light transition results in photonastic unfolding. Extensor cells expand, and flexor cells contract at the end of the nocturnal phase of the endogenous cycle, and this can be advanced by exposure of the pulvinus to blue light. The response of extensor cells to blue light is similar to that of guard cells, but that of flexor cells is opposite. [Ca2 +]cyt plays a major role in contraction of stomatal guard cells and pulvinar (flexor) motor cells by light and other signals, and is probably mediated by [IP3]cyt. Stomatal closure in response to a variety of signals is invariably associated with an increase in [Ca 2+]cyt, acting as a second messenger in the control of ion efflux, by inactivation of the K+ in channel and by activation of the slow anion channel in the plasma membrane. Control of the K+ out channel is independent of [Ca2+]cyt. Membrane depolarization can activate Ca2+channels, as part of signal transduction (McAinsh et al., 1997). [Ca2 +]cyt may also act as a second messenger for a reaction in the leaf movements of Cassia fasciculata that involves calmodulin, or other Ca2+binding enzymes (Roblin et al., 1989). Light-induced expansion of extensor protoplasts in the pulvinus of Phaseolus may require extracellular Ca2+ influx, whereas their contraction in darkness may require IP 3-induced Ca2+ mobilization (Mayer et al., 1997). Influx of Ca2+ into intact cells is controlled by membrane voltage. It is strongly stimulated by depolarization. The number of Ca2+ channels greatly exceeds the requirement for nutrition (Reid et al., 1997). In light- and clock-controlled leaflet movements of Samanea saman, closure of K+in channels is associated with increase in [Ca2 +]cyt, which may result from increase in cytoplasmic, soluble [IP3] (Satter et al., 1988). Light stimulates turnover of inositol phospholipids in the Samanea pulvinus, resulting in increased [IP3]cyt. IP 3 is apparently produced at the plasma membrane by hydrolysis of phosphoinositides, such as phosphatidylinositol 4,5-bisphosphate (PIP), catalysed by phospholipase C (PLC) and possibly activated by a trimeric G protein. Increase in [IP3]cyt may mediate opening of K+ out channels, as well as in closure of K+in channels (Morse et al., 1989a, b; Kim et al., 1992, 1993, 1996). In stomatal guard cells, an increase in [IP3]cyt activates Ca2+ channels in the tonoplast. The resulting elevation of [Ca2 +]cyt reversibly inactivates K+in channels and the H+ATPase, and activates voltage-dependent depolarizing conductance with a permeability to anions in the plasma membrane. The resulting efflux of CIand K+, accompanied by water, leads to contraction (Schroeder and Hagiwara, 1989; Blatt et al., 1990; Gilroy et al., 1990; Hedrich et al., 1990; Kinoshita et al., 1996). IP3 also binds to a specific Ca2+ channel (presumably in the tonoplast), that enables increase of [Ca2 +]cyt by mobilization from intracellular (vacuolar or ER) storage (Muir et al., 1997). IP3 also induces activation of a Ca2 +-dependent PPase that participates in the control of phosphorylation of various proteins, such as ion channels (Cote eta/., 1996; Moran, 1996). On the other hand, exogenous diacylglycerol (DAG) induces stomatal opening. DAG, the other product of phosphoinositide hydrolysis,

PLANTS IN SEARCH OF SUNLIGHT

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may act through protein phosphorylation by protein kinase C (PKC), thereby providing the signals that mediate light-induced H+ -ATPase activation and stomatal opening (Lee and Assman, 1991). (DAGpyrophosphate is a metabolic product of phosphatidic acid during G-protein activation in plant cells (Munnik eta/., 1996).) A calcium-dependent protein kinase present in guard cells phosphorylates the KAT1 K+ channel (Li eta/., 1998). The cytoskeleton, by means of its actin filaments, modulates stomatal opening and the associated activity of K+in channels (Hwang et al., 1997). 7. Role of the Tonoplast Changes in cell volume must start with transport of solutes and water across the tonoplast. Which raises the question: What role does the tonoplast play in the control of these processes? This subject has been recently reviewed by Martinoia (1992). The vacuole is the largest compartment of mature cells, in which are stored most of osmotically active ions, primarily K+ and CI-, as well as mobile Ca2+, available for release for signal transduction. The ion pool in the cytosol remains virtually unchanged. However, it is not clear whether vacuolar transport processes are directly controlled by endogenous and exogenous signals, or as an indirect result of transport processes at the cell membrane (plasmalemma). The signalling chains involved in the regulation of the various ion channels are even less well understood in the tonoplast (MacRobbie, 1999). Considerable information is available on vacuolar transport processes involved in volume changes of stomatal guard cells. and H+ -PPase (by K+) lowers the Activation of vacuolar H+ -ATPase (by vacuolar pH and hyperpolarizes the tonoplast, making the membrane potential more negative on its cytosolic face. The resulting pmf supplies energy for secondary active transport of anions and organic acids into the vacuole, through ion channels. K+ is probably imported into the vacuole by H+ /K+ symport by means of the vacuolar H+ -PPase, but also accumulates via a H+/K+ antiport (Martinoia, 1992; Maeshima eta/., 1996; Davies, 1997). Vacuolar H+ -ATPase activity is greatly enhanced by activation of its closely associated H+-PPase (Fischer-Schliebs eta/., 1997). Studies on the control by light of ion transport across the tonoplast of pulvinar motor cells, or of stomatal guard cells, have not been reported. [Ca2+]cy1 plays a crucial role in the release of ions from the vacuole. Uptake of Ca2+ into the vacuole is predominantly by ATP-dependent transport and its release from the vacuole is mediated by mM [IP3] (Lommel and Felle, 1997). Trans-tonoplast voltage is regulated by the transport of K+ and/or CI-. Increasing vacuolar [Ct-] may activate anion-selective channels, facilitating CI- influx (Davies, 1997). Uptake of CI- and malate into expanding guard cell vacuoles takes place through a tonoplast channel activated by a calcium(and ATP-) dependent protein kinase (CDPK) (Pei eta/., 1996). [Ca2 +]cyt is maintained at 100-200 nM by ATP-dependent Ca2 + pumps, or by Ca2 +/H+ antiport driven by a proton gradient at the plasma membrane or intracellular

en

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D. KOLLER

membranes. Several types of ion channels have been characterized in the tonoplast (reviewed by Allen and Sanders, 1997): VK channels are K+selective, voltage independent vacuolar channels that are activated by increase in [Ca2 +]cyt in the physiological range. Opening of VK channels results in mass efflux of K+ from the vacuole down its electrochemical potential gradient. This shifts the vacuolar membrane potential to less negative values on the cytosolic side, thereby activating voltage-gated ion channels. Among these are the SV (slow vacuolar) channels, that are highly cation-selective (permeability ratio for Ca2+/K+ - 3 : 1) and impermeable to CI-. They play an important role in Ca 2+-induced release of Ca2 + during contraction of guard cells: they are gated by [Ca2+]cyt, mediated via calmodulin and their activation allows efflux of Ca 2 +from the vacuole (Ward and Schroeder, 1994). IP 3 causes release of Ca2 + from isolated vacuoles of red beet root by voltage-dependent activation of Ca2+ channels in the tonoplast (Alexandre et al., 1990). Activation of the tonoplast H+ -ATPase energizes the efflux of K+ from the vacuole, and activates Ca2+ channels in the tonoplast. The FV (fast vacuolar) channels open at low [Ca2 +]cy1 and are active at physiological tonoplast voltages. These channels are also used for efflux of K+ from the vacuole during Ca2 +-independent guard-cell contraction (Maathuis et al., 1997; McAinsh et al., 1997). Activation of each of these vacuolar ion channels takes place at different levels of [Ca2 +]cyt. FV channels are activated by [Ca2 +]cyt up to 100 nM, VK channels are activated as [Ca2 +] increases beyond 100 nM, while the SV channels are activated when [Cacn]cyt exceeds 600 nM (Allen eta/., 1996). Lysophosphatidylcholine and similar phospholipids stimulate proton transport and phosphorylation of tonoplast -specific polypeptides, one of which may be part of the tonoplast ATPase (Martiny-Baran et al., 1992). Ligand-gated Ca2 + channels provide a possible mechanism for linking signal perception to intracellular Ca2 + mobilization. Three types of vacuolar Ca2 + channels that are insensitive to [Ca2 +]cyt have been identified in guard cells: one is gated by hyperpolarization of the tonoplast, another by IP3 and a third by cyclic ADP-ribose (cADPR), a metabolite of NAD+. The latter releases Ca2 + by activating a 'ryanoside receptor'. None of these are likely to mediate amplification of the [Ca2 +]cyt signal, or its long-term duration. However, the first and last of these trigger release of Ca2 + through the SV channel, thus indirectly amplifying this signal (Allen et al., 1996; Kim eta/., 1996; Muir eta/. , 1997). There is accumulating evidence that movement of solutes across the tonoplast may take place by vesicle trafficking. Some of the kinetics (of Clinflux into the vacuole) cannot be accounted for by independent processes of single ion transfer at the plasmalemma and tonoplast. They may be explained in terms of transfer of salt-filled vesicles from the cytoplasm to the vacuole by budding from the endoplasmic reticulum, at a rate related to influx of ions into the cell. Vesicle trafficking may play a role in the massive,

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stimulus-triggered losses of vacuolar solute in pulvinar motor cells and in stomatal guard cells (MacRobbie, 1999). 8. Role of Water Channels Transmembrane transport of water is the direct cause of changes in cell volume. The bulk of water in the cell is in its vacuole, enclosed by the tonoplast. Transport of water during contraction, or expansion of the cell takes place across the tonoplast and plasmalemma. The water permeability of these membranes acts as resistances in series to this transport. Thus, flow of water is controlled by the greater resistance. The lipid bilayer of these membranes is highly impermeable to water and makes up the bulk of their resistance. However, a number of intrinsic proteins have been identified in plant membranes (PIP in the plasmalemma, TIP in the tonoplast) that act as transmembrane channels, specifically for the rapid transport of water. These aquaporins (reviewed by Tyreman et at., 1999) have an estimated diameter of 0.3-0.4 nm. Polar groups lining these channels appear to be similar to those of the bulk solution (water). As a result, the osmotic water permeability, or filtration coefficient (Pr) of the membrane exceeds that of its phospholipid bilayer (Pct), and the transport of water takes place at a lower activation energy (EJ, similar to that of self-diffusion of water, or of its viscous transport. Aquaporins are generally much more abundant in the tonoplast. Pr is higher by two orders of magnitude in the tonoplast (690 ~-tm s- 1) than in the plasmalemma (6.1 ~-tm s- 1). Ea of the tonoplast is about one fifth that in the plasmalemma (2.5 and 13.7, respectively), as measured by osmotic contraction kinetics of microsomal fractions from tobacco suspension cells (purified by free flow electrophoresis). Water transport through the tonoplast is inhibited by mercuric chloride, a specific inhibitor for many aquaporins, suggesting a dominant role for aquaporins. The high Prof the tonoplast suggests a novel osmoregulatory role for water channels in the vacuole in buffering osmotic fluctuations in the cytoplasm, in case of sudden changes in osmotic pressure of the apoplast. Transport of water in pulvinar motor cells and stomatal guard cells is much more extensive than in tobacco suspension cells. Water permeability of their plasmalemma must therefore be quite considerable. Aquaporins (PIPl) were identified in the plasmalemma of Arabidopsis thaliana mesophyll, where they are concentrated in invaginations of the plasmalemma deep into the vacuolar lumen (plasmalemmasomes), suggesting a role in facilitating water transport between the vacuole and apoplast. In mature tissue, both types of aquaporins are expressed in and around vascular tissue and endodermis. The pulvinus consists of mature tissue with a vascular core, separated from the surrounding motor tissue by a (single layer) starch sheath ( endodermal in origin). Aquaporins may be activated by phosphorylation. A major intrinsic protein of the plasma membrane (PM28A) exhibits water-channel activity upon phosphorylation of a serine

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residue (S-247), that is dependent in vivo on an increase in apoplastic b.lf/ and in vitro upon sub-~tM [Ca2+]. There is as yet no information on the regulation of aquaporin activity in pulvinar motor cells, or in stomatal guard cells.

III. SYNCHRONIZATION BY SOLAR TIMEKEEPING A.

FLOWERS AND INFLORESCENCES

The most striking diurnal plant movements are those of flowers and inflorescences. These nastic movements were observed many years ago by Pliny and by Linnaeus (Darwin and Darwin, 1881 ). Flowers ofwaterlilies and species of Oenotheraceae, Cactaceae, Convolvulaceae and Oxalidaceae exhibit diurnal cycles of closing by inward curvature (epinasty) of the perianth leaves at their base and opening by reversing the curvature (hyponasty). Inflorescences of Compositae with ligulate ray florets open and close their floral disc diurnally by similar movements. Such diurnal movements are growth-mediated, taking place only during (part of) development of the flower/inflorescence, and are generally controlled by the diurnal light ~ dark transitions, and/or (less commonly) by the diurnal temperature alternation. The diurnal, growth-mediated movements of flowers and inflorescences are very spectacular. However, flowers or inflorescences of some plants are open only during certain times during the day (dependence on changes in pFR ?). Others are closed in daytime and open at night. Furthermore, these movements may be repeated only a few times, or not at all, by virtue of the ephemeral active life-span of the flowers. The mechanism of these lightdriven diurnal movements, their relationship to light ~ dark transitions, their spectral dependence, or the location of their photoreceptors are unknown. B. LEAVES

Diurnal, growth-mediated leaf movements generally take place only in young leaves that lack discrete pulvini at maturity (von Sachs, 1887; Wetherell, 1990). Diurnal movements of mature leaves are exhibited by many plants (particularly in the Leguminosae, or Oxalidaceae). They may be less spectacular than diurnal opening and closing of flowers and inflorescences, but are none the less very common, much more widespread and prevalent and have also been known for many years (von Sachs, 1875, 1887; Pfeffer, 1875, 1881; Darwin and Darwin, 1881). Movements of pulvinated leaves are turgor-mediated, and repeatable with great precision throughout the active life of the leaf. The nocturnal folded configuration is

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apparently the universal manifestation of the skotonastic response in leaves. However, in Oxalis oregana, adapted to deep shade, the trifoliate leaves are downfolded at night and unfolded during daytime, but also fold down very rapidly in response to an abrupt increase in irradiance (sun-fleck) (Bjorkman and Powles, 1981) (Sections III.B.3 and VII.G). Interaction with the Biological Clock Diurnal movements of pulvinated leaves may continue rhythmically for several cycles under strictly controlled, constant environmental conditions (even in total darkness, when sucrose is provided), but the diurnal cycle is gradually modified to a 'free-running', circadian cycle (Satter and Morse, 1990). Such leaf movements are therefore of circadian origin (attributed by Darwin to 'circumnutation'), controlled by the endogenous biological clock, which is reset repeatedly by the diurnal light H dark transitions (Roennenberg and Foster, 1997). The interaction of the biological clock with the diurnal light H dark transitions is an adaptive strategy in the search for light, by which the presumably meaningless, and energetically wasteful circadian leaf movement are harnessed to coincide the phase of leaf unfolding with the diurnal light period (Section VI.C). The movements reverse each other precisely in response to the opposite transitions, but the interactions between the opposite transitions with the phases of the clock differ. Skotonastic folding can be induced arbitrarily during daytime, by transfer to darkness, with progressively reduced effectiveness, but photonastic unfolding by the reverse transfer to light cannot take place for several hours after normal folding (Burkholder and Pratt, 1936a). Similar phenomena take place in stomata. The rhythm of stomatal opening in Phaseolus vulgaris in continuous darkness is phased primarily by the preceding dark -7 light transition, while that of stomatal closure is phased by the light -7 dark transition (Holmes and Klein, 1986). 1.

2. Perception and Transduction of Solar Timekeeping The location of the mechanism for the perception and transduction of nondirectional light signals in leaves exhibiting growth-mediated diurnal movements is unknown, as are the location of the biological oscillator and the transduction of its signals to circadian movements. In contrast, leaves exhibiting turgor-mediated diurnal movements contain all these elements within their pulvinus (Satter, 1979). To obtain skoto-/photonastic responses it is necessary, as well as sufficient to expose the pulvinus itself to the corresponding light H dark transition at the appropriate time. In a compound leaf, each leaflet responds independently (Koukkari and Hillman, 1968; Watanabe and Sibaoka, 1973; Satter et al., 1981). Localization of the site of perception of the diurnal (non-directional) light within the pulvinus is controversial. The responses of the extensor and flexor to light H dark transitions (as well as to the biological clock) are expressed

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in protoplasts isolated from them (Kim et al., 1992; Mayer et al., 1997). This suggests that the photoreceptors are located within the motor cells. However, the nastic pulvinar response to non-directional light activates simultaneous, opposite volume changes in the extensor and flexor. Therefore, the flexor and extensor must independently be capable of perceiving blue, as well as EOD red light signals and exhibit opposite responses to them (Section III.B.3). On the other hand, excitation of either the flexor, or the extensor must be capable of dictating indirectly the response of its partner. At the same time, in pulvini that also respond phototropically, every pulvinar sector, including its extensor and flexor, must be capable of perceiving these directional light signals and exhibit the same (qualitative) response to blue light (contraction). The distinction between photonastic responses of the extensor and flexor to blue light and their phototropic response to the same light is generally overlooked. A great deal of attention has focused on the control of ion transport processes in pulvinar motor cells by the biological clock and by its interaction with diurnal light ~ dark transitions. Both are apparently controlled by activity of K+in and K\ut channels. Interaction between the biological clock and light ~ dark transitions), pp. ontrols opening and closing of ~n channels in flexor and extensor protoplasts of Samanea. Hyperpolarization of the cell membrane is associated with expansion of the flexor during skotonastic folding, as well as of the extensor during photonastic unfolding. Hyperpolarization takes place by activation of a proton pump in the cell membranes, which enables uptake of K+ by the cells (and their resulting expansion) by opening ~n channels (Lee, 1990). K\n channels in flexor protoplasts are open during the circadian 'dark' phase and close upon exposure to blue light toward the end of this phase, while those in the extensor are closed in the dark and open in light (Kim et al., 1992). EOD phototransformation of phytochrome to its active form, Pr,, enhances skotonastic folding. In response to EOD, influx of K+ into expanding flexor cells takes place through K+ in channels and a concomitant efflux of K+ from contracting extensor cells takes place through K+ out channels (Lowen and Satter, 1989). K+in channels in extensor protoplasts that are open in light, close upon premature transfer to dark, with or without EOD. In contrast, K+ in channels in flexor protoplasts that are closed in light remain closed after premature transfer to darkness, but open in response to EOD. Phototransformation of phytochrome controls the opening of K+ in channels in flexor pro toplasts, thereby enabling their uptake of K+. This suggests that the enhancement of the nyctinastic folding in Samanea leaves by EOD is mediated by the response of K+ in channels in the flexor cells (Kim et al., 1993). Photonastic unfolding of Samanea in blue light appears to be associated with the same effects on membrane polarization in extensor and flexor motor cells, but opposite effects on the state of their K\n channels, expressed by opposite changes in their volume. In darkness, a brief exposure

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to blue (or white, but not red) light transiently hyperpolarizes membranes in protoplasts from both extensor and flexor. Following hyperpolarization by blue light, protoplasts from extensor, but not flexor, are rapidly depolarized by addition of K+, indicating that closed K+;n channels in the extensor had opened, while open ones in the flexor had closed. In contrast, addition of K+ in darkness, or following exposure to red light, results in rapid depolarization of motor cell protoplasts from flexor, not the extensor, indicating that K+;n channels in the flexor remain open in darkness, and are unaffected by EOD. Hyperpolarization by blue light is inhibited by vanadate, suggesting that it results from activation of H+ -ATPase, but the subsequent K+ -mediated depolarization is not similarly inhibited, suggesting that activation of the proton pump is not the sole factor controlling opening of K+;n channels (Kim et al., 1992). Studies on ion transport processes involved in autonomous, or photonastic pulvinar movements have focused on K +. Transport of o- is at least as important. [Ca2 +lcyt may act as a messenger in the photocontrol of ion fluxes in pulvinar motor cells. Increase of [Ca2 +]cyt mimics the effects of EOD on folding in leaves ofAlbizzia lophanta and counteracts its reversion by far-red light. It also mimics the effects of EOD on phase shift of the circadian movement of Robinia leaflets. Phytochrome regulates transmembrane fluxes of Ca 2+ by controlling the activity of Ca2 + channels (Tretyn et al., 1991). Action of an intracellular Ca2 + channel antagonist depends on phototransformation of phytochrome to pFR (Moysset and Simon, 1989; Gomez and Simon, 1995). Ca 2 +-chelators inhibit the nyctinastic folding, as well as the photonastic unfolding responses in the leaf of Cassia fasciculata, whereas a Ca 2+ ionophore increases their rate (only marginally for the unfolding response). However, Ca 2 + channel blockers inhibit the BODmediated nyctinastic folding, but not their blue light-mediated unfolding, suggesting that activation of Ca2+ channels enhances the former, but has no effect on the latter. Ca 2 + may be mobilized in different ways for these opposite movements, possibly from external sources for the phytochrome response, internal sources for the blue light response (Roblin et al., 1989). However, studies using Ca2 + channel antagonists should be regarded with caution, because these substances may also block K+ out channels (Thomine et al., 1994). Dark-adapted extensor protoplasts expand upon transfer to light. Increase in [Ca 2+]cy1 from exogenous sources is required for expansion and takes place via Ca2 + channels in the plasma membrane that open in response to the light. Light-induced expansion in these cells is prevented when uptake of extracellular Ca2+ is inhibited (by verapamil, or LaH), whereas inhibition of intracellular transport (by TMB-8) has no effect. Extensor protoplasts can be induced to expand in the dark, in presence of Ca 2+ ionophores (A 23187), or Ca 2 + agonists (Bay K-8644), or inhibitors of Ca 2 +-ATPase at endomembranes (thapsigargin). These results suggest that dark~ light

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transitions induce opening of Ca2 + channels in the plasma membrane. The resulting increase in [Ca2+]cy1 from extracellular sources acts as a signal in the transduction chain that ends in light-mediated expansion, because it activates H+ -ATPase and opens K+ channels, leading to ion uptake. In contrast, contraction of light-adapted extensor protoplasts upon transfer to darkness occurs in the absence of extracellular Ca2+, but is inhibited by TMB-8, but not by verapamil. The latter is reversed by A-23187, or BAY8644, which by themselves have no effect. Contraction of light-adapted extensor pro top lasts in response to a light ~ dark transition is also inhibited by inhibitors of the phosphoinositide pathway for transmembrane signalling (neomycin, u+). The response to such transition probably takes place by inducing hydrolysis of phosphoinositol (PI). The increase in [IP3]cyt that is generated as a result may mobilize Ca2+ from intracellular stores, leading to closing of K+in channels and activation of outwardly rectifying, voltagedependent Cl- channels (Kim et al., 1992, 1996; Mayer et al., 1997). Increase in soluble [IP3] may activate the proton pump that controls the activity of K\n channels, by stimulating a protein kinase (Cote et al., 1996). The rate of turnover of PI is also controlled by the phases of the circadian clock (Satter et al., 1988). Contraction of stomatal guard cells in the dark is associated with closing ofK\n channels and opening of outgoing CI- channels, both of which may take place as intracellular Ca2+ is mobilized by IP 3 (Schroeder and Hagiwara, 1989; Hedrich et al., 1990). Red light regulates Ca2+ -activated K+ channels in the filamentous alga Mougeotia (Lew et al., 1990). The phytochrome-regulated swelling of protoplasts from etiolated wheat leaves (probably responsible for unrolling the leaf) is Ca2+ -dependent, but independent of K+ -uptake, and involves a G-protein (Bossen et al., 1988, 1990). 3. Pulvinar Photoreceptors for Solar Timekeeping Phytochrome and a blue light-absorbing pigment system (BAP) perceive the different non-directional light signals that interact with the biological clock in the control turgor-mediated, nastic leaf movements. The skotonastic folding response takes place in response to a light ~ dark transition and therefore is not mediated by photoreceptors. However, it is enhanced by EOD, which increases the rate, as well the extent of folding (Fondeville et al., 1966, 1967; Jaffe and Galston, 1967; Satter et al., 1974; Satter, 1979). Enhancement of skotonastic folding by EOD is characteristic of other photomorphogenic responses to phytochrome. It is saturated by brief exposure of the pulvinus to red light at low fluence rates, is reversible by subsequent far-red light (Jaffe and Galston, 1967; Evans and Allaway, 1972). Photonastic unfolding is under control by a BAP.It takes place in response to blue light (.Xmax at 470 and >720 nm) at a relatively high irradiance and requires continuous exposure to maintain the unfolded configuration

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(Burkholder and Pratt, 1936b; Williams and Raghavan, 1966; Fondeville et al., 1967; Satter et al., 1981; Watanabe and Sibaoka, 1983). Phototropic responses of the pulvinus exhibit similar characteristics (Section VI .A). This similarity makes the discrimination between photonastic and phototropic responses of the pulvinus more difficult (Section VI. G). Responsiveness to each of these photosystems changes during each phase of the circadian oscillator (Hillman and Koukkari, 1967; Gomez and Simon, 1995). The identity of the BAP involved in photonastic unfolding is unknown. The action spectrum for photonastic unfolding in leaves ofAlbizzia julibrissin and Vicia faba (in the presence or absence of 660 nm background light) exhibits two major peaks in the blue (440 and 480 nm), falling off sharply beyond 500 nm. Far-red (720 nm) light is also effective, but after a considerable lag phase. On the basis of its action spectrum, photonastic unfolding was attributed to a BAP, as well as to the high irradiance response (HIR) of phytochrome (Evans and Allaway, 1972). Skotonastic folding of Albizzia leaflets is delayed by blue (430-470 nm) and far-red (710 nm) light. Red (660 nm) light and longer wavelength far-red (>730 nm) light are each ineffective by itself, but in combination they delay folding. Green (550 nm) light is also ineffective by itself, but reverses completely the delay in folding caused by 710 nm far-red. These results suggest activity of an additional photoreceptor with Amax 710 nm, plus broad band activity at A > 660 nm (Tanada, 1982, 1984). The action spectrum for photonastic unfolding of Oxalis oregana leaves following a dark ---7 light transition exhibits Amax at 450 and 485 nm, a sharp cutoff at A > 500 nm and no response at A 700-2400 nm. The action spectrum for their rapid downfolding following a sharp increase in irradiance is identical (Bjorkman and Powles, 1981). Clearly, the same photoreceptors must be involved in the opposite responses to light at low and high irradiance. The low irradiance probably excites only the adaxial (flexor) photoreceptors, whereas the higher irradiance is able to traverse the pulvinus sufficiently to excite the abaxial (extensor) ones as well. The transpulvinar differential in osmotic relations, which maintains the unfolded configuration at low irradiance, collapses at the higher irradiance, resulting in downfolding.

IV.

GROWTH-MEDIATED PHOTOTROPIC MOVEMENTS A.

GRAVITY AND LIGHT

The primary stem generally exhibits negative orthogravitropism, growing vertically upward. Branches normally exhibit plagiogravitropism, growing at predetermined angles (;;:: 90°) to the gravity vector, at least for some time. Leaves grow out of the stem at a predetermined angle and in different radial directions, but may eventually exhibit dia- or

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plagiogravitropism, with their lamina at angles ~ 90° to the gravity vector, thus exposing their expanded lamina to overhead light. Although these growth-mediated movements represent an inherent search for light, their direction is not determined by the light vector, but by the gravity vector. Fortuitously, in most habitats the prevailing direction of incoming PAR, integrated throughout the day, happens to be opposite to the direction of the gravity vector. However, the intrinsic gravitropic control over growth of the stem and its leaves is susceptible to modification by phototropic control. Phototropic control manifests itself under limiting availability (low pFR, or short duration) of PAR in the micro-environment. Under such conditions, de-etiolated dicotyledonous plants reorient their apical bud and its complement of young leaves diaphototropically to face the predominant direction of light by growth-mediated, positive phototropic curvature of their subtending growing stem. Individual developing leaves exhibit similar responses, by means of their petiole. B.

DIRECT CONTROL OF PHOTOTROPIC MOVEMENTS

Young, actively growing parts of the de-etiolated stem (hypocotyl; epicotyl) generally exhibit phototropic curvature in response to their direct exposure to unilateral light. The actively growing motor tissue that subtends the apical bud performs the phototropic, as well as gravitropic response. However, phototropic competence may precede gravitropic competence, since the part subtending the phototropically curved stem responds to the gravitropic signal and reverses the preceding phototropic curvature. This separation between the phototropically responsive part of the stem and the gravitropically responsive part that subtends it may be related to the maturation of the starch sheath, the site of graviperception. Negative gravitropic curvature and positive phototropic curvatures of growing stems takes place by differential growth of the opposite flanks, primarily a result of unilateral growth inhibition that may be accompanied by accelerated growth in the opposite flank. The stem integrates phototropic (and gravitropic) signals acting on it from different directions and responds accordingly (Firn, 1990; Gleed et al., 1994). 1.

Seedlings

Direct exposure of the sunflower hypocotyl to unilateral light is necessary and sufficient to cause its phototropic curvature. Shading the apical bud and cotyledons, or their excision, does not affect this response. Curvature is accompanied by redistribution of xanthoxin (a growth inhibitor formed upon de-etiolation), which is significantly higher in the exposed flank (Franssen and Bruinsma, 1981 ). Redistribution of auxin was not observed (Bruinsma et al., 1975; Bruinsma and Hasegawa, 1990).

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The phototropic response of the hypocotyl to direct exposure to unilateral light depends on its de-etiolation. The sunflower hypocotyl, but not its apical bud, or cotyledons, must be de-etiolated first, by overhead light (blue, or white, but not red) (Franssen and Bruinsma, 1981). The phototropic response of the hypocotyl to exposure of the entire seedling (cress, lettuce, mustard, radish) to unilateral blue light, is greater in green than in etiolated seedlings. The cotyledons and apical bud contribute to the phototropic response of the hypocotyl, because shading them inhibits curvature in etiolated seedlings, but only delays and reduces curvature in green seedlings (Hart and MacDonald, 1981). The requirement for de-etiolation for the phototropic response of the hypocotyl has not been studied. Actively growing petioles may also exhibit control of their positive phototropic responses by direct exposure to unilateral light (Section IV.C.1). Phototropic curvature of the stem that is directly exposed to unilateral light has been attributed to excess transpiration in its exposed over its shaded flank (Mcintyre, 1980), but this hypothesis has been challenged (Franssen et al., 1982). 2.

Negative Phototropism and Skototropism Stems of climbing plants (such asHedera, Parthenocissus, Monstera spp.) may exhibit negative phototropism, by which they locate and become appressed to their vertical support. The young shoot of ivy (Hedera helix) curves away from unilateral (blue) light, thus becoming appressed to vertical supports (walls, trees) (Negbi et al., 1982). Seedlings of the tropical vine Monstera gigantea detect and grow in the direction of the trunk of their prospective host tree from a distance exceeding 100 em. This response is attributable to skototropism, not to negative phototropism, because growth is in the direction of the darkest sector of the horizon, rather than away from the brightest sector, and the magnitude of the response increases with the diameter of the target tree. However, when light is too low the shoot may revert temporarily to positive phototropism, in search of light. Some time after the stem has started climbing its host it transforms permanently to positive phototropism, and this coincides with a change in the morphology of its new leaves (Strong and Ray, 1975). In contrast, the prostrate shoot of the fern Selaginella grows along the ground by differential positive phototropism, with higher sensitivity in its 'ventral' underside. Growth of the 'dorsal' side of the shoot is also greater in the dark, irrespective of the direction of gravitropic stimulus. The response does not take place in the absence of the small dorsal leaves (Bilderback, 1984a, b), but their role remains unknown. 3.

The Dual Role of Blue Light Blue light controls elongation of the stem, as well as its phototropic curvature. Direct exposure of the growing part of the stem (internodes,

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hypocotyl) to blue light equally from all sides, or bilaterally, results in suppression of its elongation. Direct unilateral exposure to blue light results in positive phototropic curvature of the stem by differential growth. Unilateral blue light imposes a transverse light-gradient across the stem. This may account for the unilateral inhibition of growth and the resulting differential elongation of opposite flanks and positive phototropic curvature (Iino, 1990). However, growth-mediated positive phototropism in response to unilateral blue light cannot be simply accounted for by the more general suppression of growth by blue light, because these responses are kinetically distinguishable: the former exhibits a 4.5 h lag phase, whereas the latter takes place within 30 s. Rapid suppression of growth of cells that intercept blue light is characterized by reduced extensibility of their wall, with little change in its yield threshold. It is not accompanied by corresponding changes in their turgor pressure (measured by cell pressure probe). Hydraulic conductivity of growing cells is too large to limit their expansion (Cosgrove, 1983, 1988). The considerable time-lag that characterizes the phototropic response to blue light may probably be accounted for by the participation of additional downstream elements to the transduction chain, such as translocation of the components of growth (auxin, ions, water) across the stem, from the exposed flank to its opposite, shaded flank. It has been calculated that the 5- to 6-fold difference in pFR of (unilateral) blue light measured across the hypocotyl should have caused measurable curvature within 30-60 min (Cosgrove, 1985). Support for the concept of separate responses comes from experiments with seedlings of mustard (Sinapis alba) grown under low-pressure sodium lamps, to eliminate growth responses to phytochrome. Bilateral exposure of such seedlings to blue light does not suppress hypocotyl elongation, but their exposure to unilateral blue light results in positive phototropic curvature of the hypocotyl. Growth inhibition is apparently constrained to the cells that actually intercept (blue) light (Rich et al., 1985). These results lead to the conclusion that while the primary direct response of the exposed cells to blue light may be similar, if not identical, in phototropism and growth suppression, they probably differ in downstream parts of the transduction pathway (Spalding and Cosgrove, 1989). However, it appears that different blue light photoreceptors, with different chromophores, control stem elongation and phototropism (Section IV.D). The blue light photoreceptors for stomatal opening may also be different and with a different chromophore. Several transduction elements associated with the suppression of elongation by non-directional blue light and with the positive phototropic response to unilateral blue light have been identified. A pivotal role for Ca2 + is indicated. Growth inhibition of cucumber hypocotyls by exposure to blue (but not green, or red) light is preceded by a large, albeit transient depolarization of the exposed cells. Such depolarization initially involves inactivation of the plasma membrane H+ -ATPase, with subsequent

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activation of Ca2 + channels, and/or Cl- channels, allowing these ions to move down their electrochemical gradient (Spalding and Cosgrove, 1989, 1992). An anion channel in the plasma membrane of Arabidopsis hypocotyls is activated by blue light (Cho and Spalding, 1996) and may be a step in signaltransduction leading to growth inhibition. Such activation probably takes place via a pathway that is indirectly dependent on [Ca2+]cyt, by means of intermediates, such as Ca2+ -dependent kinases and/or phosphatases (Lewis et al., 1997; cf. Reymond et al., 1992). Phototropic curvature (maize coleoptiles and de-etiolated sunflower hypocotyls) is associated with an increase in proton efflux along the shaded flank, prior to onset of curvature (Mulkey et al., 1981). Phototropic curvature of sunflower hypocotyls is associated with a reduction in [Ca2+]cyt and in the activity of calmodulin and of protein kinases in the flank that is exposed to light (Ma and Sun, 1997). Exposure to unilateral blue light generates a directional gradient of protein phosphorylation across the oat coleoptile, reducing it to 32% and 50% of the dark controls, on the exposed and its opposite side, respectively (Salomon et al., 1997). 4. Role of the Cytoskeleton Growth inhibition in response to blue light may be associated with reorganization of the cytoskeleton Differential growth during curvature (sunflower hypocotyls and maize coleoptiles) in response to unilateral (blue) light (or gravity) is accompanied by reorientation of microtubules at the outer epidermal wall: increasingly longitudinal along the concave (growthinhibited) flank, increasingly transverse along the convex (growthstimulated) flank (Nick et al., 1990). Growth is inhibited and microtubules are reoriented from transverse to longitudinal, or oblique, in individual cells of dark-grown gametophytes of the fern Ceratopteris richardii upon their exposure to blue light (Murata et al., 1997). The cytoskeleton, and in particular myosin, may take part in sensory functions and in light signal transduction (Miller et al., 1997; Mermall et al., 1998). Furthermore, organization of the cytoskeleton may change dramatically in blue light in processes that are not directly concerned with growth inhibition, but in ways that might suggest such a role. The helical chloroplasts of the filamentous alga Spirogyra become tightly coiled where they intercept a microbeam of blue light (.\max at 430, 476 and 500 nm, coinciding with those of phototactic migration of chloroplasts in Vaucheria, Selaginella, Lemna, and Funaria ), and their response to centrifugation is modified (Ohiwa, 1977). Microbeam exposure of the filamentous alga Vaucheria sessilis to blue light induces localized reticulation of the longitudinal cortical fibres of the cytoskeleton, by forming cross-linkages, resulting in chloroplast aggregation (Blatt and Briggs, 1980). This response is associated with a light-dependent electric current (Blatt et al., 1981 ). [Ca2+]cyt is a majorfactor controlling actin activity, and may therefore also be involved in reorganization of the cytoskeleton.

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The cytoskeleton may also be involved in control by BAP and phytochrome of the movement of chloroplasts in dark-adapted prothallial cells of the fern Adiantum from the anticlinal walls towards a microbeam spot of red, or blue light (Kagawa and Wada, 1996).

C.

INDIRECf CONTROL OF PHOTOTROPIC MOVEMENT

1. Expanding Leaves Phototropic curvature of developing leaves may take place by long-distance transmission and is oriented by light signals perceived by foliar organs (vegetative, possibly also floral). Perception of partial shading of the lamina of Sparmannia africana leads to formation of leaf mosaics by shade-evading movements of its leaves. These movements are performed by curvature of the petiole of the developing leaf that moves the lamina out of the shade. The partially shaded lamina apparently produces more auxin in its shaded part, exports this excess to the subtending flank of the petiole and enhances its elongation (Ball, 1923). Leaves of shade-tolerant Hyoscyamus spp. and Urtica spp. exhibit diaphototropism under limiting conditions of PAR, moving their laminae to face the direction of prevailing light, by curvature and/or torsional rotation of the petiole (Section II. C). Similarly, plants growing near walls, cliffs, etc. may exhibit diaphototropism of their developing leaves, and orient them to face the predominant direction of light The stem of climbing plants, such as ivy (Hedera) or Monstera, exhibits negative phototropism and grows parallel and in close proximity to vertical supports, such as walls and trees (Section IV.B.2), but the developing leaves orient their lamina away from the support (unpublished observations). The predominant direction of light is probably detected by the lamina, but the mechanism has not been studied. Young, expanding leaves of Tropaeolum spp. reorient their lamina normal to an oblique light beam by curvature of their subtending petioles toward the light. Exposure of the petiole itself to unilateral light leads to its positive phototropic curvature, by which its (shaded) lamina reorients to face the light. However, exposure of the lamina to directional light also leads to positive phototropic curvature of its (shaded) petiole (Haberlandt, 1905, 1914). Clearly, the petiole exhibits a long-distance phototropic response to directional (oblique) light that is perceived in the lamina, probably as vectorial excitation (Section V.E). This conclusion is supported by results showing that detached leaves of Tropaeolum and Limnanthemum exhibit continuous positive phototropic curvature of the shaded petiole when the lamina is floating on water and continuously exposed to vectorial excitation by oblique light (Jones, 1938). Phototropic curvature of the petiole in response to its direct unilateral exposure to light is growth-mediated and depends on the supply of auxin from the lamina. Curvature is greatly

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reduced in the absence of the lamina and this is reversed by replacing the excised lamina with auxin (Brauner and Vardar, 1950). Lactuca serriola and Silphium spp. are familiarly known as 'compass plants', because the lamina of most of their (cauline) leaves face east-west vertically at maturity (approximately north-south azimuth). In L. serriola, the lamina of newly emerged leaves is vertically appressed to the stem, and its azimuth orientation is phyllotactic. As the leaf expands, it reorients its lamina in the course of several consecutive cycles of solar transit across the sky. Expanding leaves with a north-south phyllotactic azimuth rotate their lamina around its midvein, by torsion of the petiole (Section II. C), and/or of the lamina itself; those on an east-facing azimuth remain vertical, facing east, while those on a west-facing azimuth decline. In maturity, most leaves have their lamina facing either the rising, or the setting sun (Dolk, 1931; Werk and Ehleringer, 1984; Zhang et al., 1991 and unpublished observations). The nature of the light signals that control this diaphototropic, growth-mediated leaf movement has not been studied (cf. Dolk, 1931; Zhang et al., 1991). 2. De-etiolated Seedlings De-etiolated dicotyledonous seedlings exhibit diaphototropic reorientation of their apical bud and cotyledons by positive phototropic curvature of the hypocotyl. Curvature of the hypocotyl takes place in response to its direct, unilateral exposure to light, as well as in indirect, long-distance response to exposure of its foliar organs (cotyledons and/or young leaves) to oblique light. Perception of the directional light signal by these foliar organs has been attributed to differential interception of the directional light by their opposite inclination on opposite sides of the stem. This results in a differential supply of growth-regulating substances to their sub tending flank, leading to differential growth that is expressed in positive phototropic curvature (Shibaoka, 1961). This approach is supported by a number of studies, showing that interception of light by one cotyledon inhibits elongation along the subtending flank of its hypocotyl. Hypocotyls of deetiolated seedlings of Helianthus exposed to vertical light, with one of the cotyledons shaded, curve towards the exposed cotyledon. Diffusates from the hypocotyl on the side with the shaded cotyledon exhibit greater growthpromoting activity. In the absence of one cotyledon, the hypocotyl curves away from the remaining cotyledon, but to a lesser extent in light than in darkness (Lam and Leopold, 1966). Curvature of the hypocotyl away from the shaded cotyledon has also been attributed to reduced transpiration by the shaded cotyledon, resulting in higher water potential in the vascular bundles and higher water content of the sub tending peripheral tissues of the hypocotyl (Mcintyre and Browne, 1996) (Section IV.B.1). De-etiolated seedlings of Cucumis sativus and Helianthus annuus exhibit positive phototropism of the hypocotyl in response to its direct unilateral exposure to blue, but not to red light. The hypocotyl also curves away from

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the shaded cotyledon when the other is vertically exposed to red (or white) light. These results suggested direct control by a BAP in the hypocotyl and independent control by phytochrome in the cotyledons. However, this explanation does not account for the fact that vertical exposure of one cotyledon to blue light causes curvature of the hypocotyl toward the shaded cotyledon (Black and Shuttleworth, 1974; Shuttleworth and Black, 1977). D. PHOTO RECEPTORS FOR PHOTOTROPISM

1. A Choice ofChromophores A great deal of research is focused on the identity of the photoreceptor(s) for phototropism in higher plants. Flavins are leading candidates for the chromophore of the blue light photoreceptor for phototropism. Kl, NaN3 and phenyl acetate specifically inhibit blue light-dependent phototropism, probably by interacting with the excited state of flavins. Simultaneous irradiation with phototropically inert light also inhibits this response, probably by depopulating the first triplet state of flavins (Schmidt et al., 1977). Phototropic responses to blue light have been attributed to cooperation between a flavin enzyme (such as NADH-dependent oxidoreductase) and a b-type cytochrome in the plasma membrane (Asard et al., 1995). Evidence of the central role of flavin nucleotides in stem elongation and phototropism in higher plants is given in the following Section (IV.D.2). Quinones and Zeiger (1994) have provided evidence suggesting a role for xanthophylls in phototropism of corn coleoptiles. This suggestion has been refuted by experiments showing that blue light-dependent phototropism, as well as phosphorylation responses to blue light, are the same in seedlings containing normal levels of carotenoids, and in those that are deficient in carotenoids, either through a genetic lesion, or by chemical blocking of carotene biosynthesis (Palmer et al., 1996). Phytochrome may also play a part in phototropism. Light in the blue spectral region is invariably active in growth-mediated phototropism, but >. > 600 nm are also sometimes active. Vertically growing, young leaves (crozier stage) of the fern Adiantum cuneatum exhibit positive phototropic curvature of their midrib in response to unilateral red light, reversible by far red light, as well as in response to blue light, that is not similarly reversible, suggesting joint control by phytochrome, and a BAP (Wada and Sei, 1994). De-etiolated seedlings of cucumber (Cucumis vulgaris) exhibit positive phototropic curvature in continuous exposure to unilateral blue light and negative curvature in continuous exposure to unilateral far-red light. De-etiolated seedlings of the lh mutant, deficient in phytochrome B, exhibit the positive phototropic response to blue light, but not the negative phototropism mediated by far-red. The magnitude of the negative phototropic response to far-red depends on the irradiance, and is apparently

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mediated by the high-irradiance response (HIR) of phytochrome B (BaHan~ et al., 1992). The negative phototropic response to far-red light may be a

unilateral expression of the enhancement of stem elongation by light with a low pFR ratio between red and far-red (Smith, 1994). Phytochrome may interact with a BAP in phototropism. Phytochromes A and B are both required for the phototropic response of Arabidopsis thaliana seedlings to blue light (Janoudi et al., 1997). Etiolated maize coleoptiles exhibit a (timedependent) second positive curvature in response to unilateral blue light only after exposure to red light, which is reversible by subsequent far-red light (Liu and Iino, 1996). 2. A Choice of Photoreceptor Genes

Suppression of stem elongation and phototropic curvature in response to blue light is mediated by different photoreceptors. Different mutants of Arabidopsis thaliana have been identified, that exhibit suppression either of stem elongation by blue light or positive phototropic curvature of the stem in unilateral blue light, but not the other (Khurana and Poff, 1989; Liscum and Hangarter, 1991). The phototropic response cannot be accounted for by suppression of growth of the cells along the exposed flank, because the mutant exhibiting phototropic response to unilateral blue light, does not exhibit suppression of elongation by bi- or multilateral exposure to the same blue light. Studies with non-phototropic hypocotyl mutants have led to identification of the NPHJ locus, that may encode the apoprotein for a dual- or multi-chromophoric holoprotein photoreceptor capable of absorbing UVA, UV-B, blue and green light, and regulating all phototropic responses. This gene is genetically and biochemically distinct from the HY4 gene that encodes the photoreceptor for blue light-mediated inhibition of hypocotyl elongation. Loci NPH2 and NPH3 appear to act as downstream signal carriers for the phototropism-specific pathway. NPH4 acts in gravitropism as well, and may function directly in the control of differential growth (Liscum and Briggs, 1995, 1996). The protein encoded byArabidopsis NPHJ contains two LOV (light, oxygen, or voltage) domains, so named because proteins with similar domains exist in totally unrelated organisms, some of which exhibit responses to light, oxygen, or voltage. The LOV domains of the NPHJ apoprotein bind flavin momonucleotide (FMN) stoichiometrically. The holoprotein formed, nph1, is a a serine-threonine protein kinase that is autophosphorylated by blue light-induced redox changes. Spectral properties of this chromopeptide are similar to the action spectrum for phototropism, suggesting that the LOV domain binds FMN to function as a blue light sensor and that nph1 probably functions as a dualchromophoric flavoprotein photoreceptor regulating phototropic responses in higher plants. It has therefore been named 'phototropin' (Christie et al., 1998, 1999).

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A soluble protein (cryptochrome 1) has been identified as the photoreceptor that mediates blue light-dependent regulation of plant growth and development, specifically hypocotyl elongation in Arabidopsis. Cryptochrome 1 is also associated with two chromophores. One is flavin adenine dinucleotide (FAD), and the other is probably the pterin methenyltetrahydrofolate (Lin et al., 1995; Malhotra et al., 1995). A flavin may be part of cryptochrome 2. However, the apoproteins of the blue light photoreceptors for growth inhibition show no homology with those for phototropism. A photoreceptor gene in the fern Adiantum capillus-veneris encodes a protein that has been identified as a phytochrome with the features of nph1, the putative photoreceptor for the second positive phototropism (Nozue et al., 1998). The chromoprotein, phy3, exhibits an N-terminal region with sequence homology to the tetrapyrrole-binding region of phytochrome, and a C-terminal region with striking homology to nphl. This extraordinary chromoprotein could act as a dual sensor (Christie et al., 1999) that may mediate the phototropic responses to red, as well as to blue light of leaves of the relatedA. cuneatum (Section IV.D.1). Detailed discussion of the transduction of unilateral light signals for growth-mediated phototropism is outside the scope of this review. Properties and transduction chains of blue light photoreceptors have been reviewed by Horwitz (1994).

V.

SOLAR-TRACKING BY HELIOTROPISM

Heliotropism is a special manifestation of phototropism, and is distinguished by some fundamental features. Phototropism is generally expressed where PAR is limiting and is expressed with respect to the predominant direction of light, integrated over the day. In contrast, heliotropism is expressed under full sunlight and is continuously controlled by on-line information from the solar position. The light requirements (pFR) for the two phenomena differ accordingly. A plant that exhibits a capability for sustained heliotropic movements uses the ever-changing position of the sun to navigate its apical buds, leaves, flowers, or inflorescences, according to the solar transit throughout most of the day ('solar-tracking'). A.

SHOOT APICES

Apical parts of the shoot may exhibit growth-mediated diaheliotropism. The most familiar and conspicuous manifestation of such solar-tracking is exhibited by the domestic sunflower (Helianthus annuus). The (single) apical bud and its cluster of young leaves, and eventually its dish-shaped developing

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Fig. 2. Heliotropism in Crozophora tinctoria (Euphorbiaceae). Post-sunrise (-8:00am; A, C) and pre-sunset (-5:00pm; B, D) configuration of mature (A, B) and young (C, D) plants, photographed from a fixed position; tagged for identification. Note the orientation of the apical buds and the subtending leaves. and the curvature of the stems and petioles.

inflorescence, moves to keep facing the sun with high fidelity during the course of each day. They do so by positive, growth-mediated phototropic curvature of the young, growing part of the subtending stem. Solar-tracking is kept up as long as the stem grows, throughout reproductive development. The developing leaves play a role in the diaheliotropic response of the stem, since their excision results in partial loss of the response (Shibaoka and Yamaki, 1959). Similar diaheliotropic responses have also been observed in the numerous inflorescences of the highly branched Crozophora tinctoria (Fig. 2) and Xanthium strumarium growing in Israel, and wild relatives of H. annuus growing in the southwestern United States (unpublished observations), as well as in flowers of Arctic and alpine plants (Kevan, 1972, 1975; Smith, 1975; Kjellberg et al., 1982; Stanton and Gallen, 1993). B.

LEAVES

The capacity of leaves to continuously reorient their laminae during the day in response to the constantly changing directional signals from the sun (Figs 2, 3 and 4) has been reported in species belonging to diverse taxonomic

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groups (Ehleringer and Forseth, 1980; Rajendrudu and Rama Das, 1981; Koller, 1990; Sailaja and Rama Das, 1996), but there is no information on the mechanism by which heliotropism takes place in most of these species.

1. Developing Leaves Young, developing leaves may exhibit growth-mediated diaheliotropism. Young leaves of sunflower exhibit diaheliotropic movements even before flower initiation (Begg and Torssell, 1974). The laminar orientation of the leaves lags by -12° behind that of the sun, but maximum easterly and westerly orientation of leaves precedes sunrise and sunset, respectively, by

Fig. 3. Heliotropism in leaves of Capparis spinosa (Capparidaceae), branch photographed from a fixed position, tagged for identification, (A) -10:30 am; (B) -4:00 pm. Pulvinus (?) extends over most of the petiole.

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Fig. 4. Heliotropism of leaves in Lupinus pilosus (Fabaceae). Mid-morning ( -9:30 am; A) and mid-afternoon ( -4:00 pm; B) orientation of leaves, photographed from a fixed position, tagged for identification.

several minutes (Shell and Lang, 1976). The amplitude of the diurnal westward reorientation decreases progressively with flower development. Leaves growing facing east and west reorient diaheliotropically by curvature of their petioles and midribs. Those facing north and south do so by axial rotation (torsion) (Lang and Begg, 1979). Expanding leaves of Stachys sylvatica (Snow, 1947), Crozophora tinctoria (Fig. 2) and Xanthium strumarium reorient their laminae diaheliotropically by curvature and/or torsion (Section I. C) of their petiole and/or of the lamina. 2. Pulvinated Leaves Two categories of plants exhibiting heliotropic, turgor-mediated leaf movements are recognized. Plants in one category exhibit laminar phototropism. They perceive the direction of sunlight in their lamina as vectorial excitation and track the position of the sun throughout the day (Section V.D.2). The resulting orientation of the lamina is diaheliotropic, or plagioheliotropic depending on whether its lamina remains normal to the sun, or is capable of tracking the sun at oblique angles. Plants in the other category exhibit pulvinar phototropism. They perceive the direction of sunlight in their pulvinus as unilateral excitation (Section VI.A). Their laminar orientation with respect to the sun changes throughout the day with progress of the solar transit and the resulting changes of interception of light by the pulvinus. It is a passive outcome of their pulvinar phototropism and is neither diaheliotropic, nor plagioheliotropic (Section VI.D.3).

D. KOLLER

76

180,---------------~--------------------~

SUN 160 140 120

w ...J 160

SIMULATED

100 80 60

21

23

01

03

05 07

09

II

13

15

17

19

Tl ME (h)

Fig. 5. Diurnal diaheliotropism in leaves ofLavatera cretica (Malvaceae). Timecourse of laminar reorientation in response to solar transit nature ('SUN' daytime 05:00--19:00) and in response to transit of simulated 'sun' in a vertical arc at an angular velocity of 15° h-1 ('SIMULATE D' daytime 07:00--19:00). Changes in the angle of laminar elevation (LE) during daytime (empty bar) and night (filled bar) when the leaf azimuth faces sunrise (e; .6.) and when the leaf azimuth faces sunset (o; .&). Diagonals= 'solar' elevation. (Reprinted with permission from Koller and Levitan (1989).)

PLANTS IN SEARCH OF SUNLIGHT C.

77

THE NOCTURNAL PHASE

Growth-mediated and turgor-mediated diaheliotropism are associated with nocturnal reorientation. An outstanding feature of the diaheliotropic response of sunflower is its intimate association with nocturnal reorientation. Some time after sunset the apical bud, or the developing inflorescence starts reorienting in the opposite direction. Nocturnal reorientation starts with the apical bud/inflorescence facing sunset and ends with them facing in the anticipated direction of sunrise. It is more rapid (-26oh- 1) than tracking the sun (at -15oh- 1) during the day, and ends several hours before sunrise, but the movement to face sunrise is completed only at sunrise. The direction of nocturnal reorientation appears to be dictated by the preceding solar-tracking. In mature plants, the direction of the nocturnal reorientation is maintained even when the preceding day was overcast, and for 3-4 days after the (potted) plant had been rotated 180° around its axis (Leshem, 1977). Sunflower exhibits diurnal heliotropism. Malvaceous leaves exhibit a remarkably similar nocturnal reorientation. After sunset, leaves of Malva neglecta start to reorient their sunset-facing lamina, to end facing the direction of the anticipated sunrise several hours before it occurs. Plants that are rotated by 180° (or 90°) at sunset persist in their original direction of nocturnal reorientation to face the preceding sunrise (Yin, 1938). Facing directly opposite to the new sunrise, their adaxial face is not exposed to the sun for several hours, during which they are unable to start their daytime heliotropic movement. As a result, the leaves resume normal nocturnal reorientation to face the 'new' sunrise only after the plants had adapted to their new position during several cycles. The direction of nocturnal reorientation appears to be predetermined by that of the preceding sunrise and may therefore be considered as the nocturnal phase of diaheliotropism. These results were confirmed and expanded in time-course studies with the relatedLavatera cretica, under field conditions, as well as during several consecutive cycles of diurnal diaheliotropism under simulated conditions, by means of a 'solar simulator' (Schwartz and Koller, 1986; Koller and Levitan, 1989) (Fig. 5). Analysis of the results identified three phases in nocturnal reorientation of the lamina: (i) pulvinar relaxation from the strained, sunset-facing configuration (duration of this phase depends on the extent of laminar displacement required); (ii) pulvinar equilibrium (time measuring); and (iii) reorientation to face sunrise. Cotyledons acquired the capacity for nocturnal reorientation after the seedlings had performed three to four cycles of diaheliotropic movements under simulated conditions. In these plants the nocturnal reorientation complements the daytime solar-tracking with remarkable precision to exhibit diurnal heliotropism. In navigational terms, malvaceous species guide their leaf laminae, and

78

D. KOLLER

sunflower species guide the orientation of the apical bud complex (subsequently the inflorescence) by the solar transit during the day and by an automatic pilot during the night.

D.

PERCEPTION OF THE SOLAR SIGNAL

1. Growth-mediated Heliotropism The site, or mechanism for perception of directional signals from the sun for growth-mediated, heliotropic movements have not been studied. In Helianthus, the diaheliotropic response of the apical bud and its subtending cluster of young leaves has been ascribed to an inhibitor of stem elongation and of auxin transport that is produced by the young, growing leaves and is translocated more rapidly on the flank exposed to light (Shibaoka, 1961). Alternatively, control of the phototropic curvature of the young stem may be indirect, by differential interception of directional light by leaves on opposite sides of the shoot (Section IV.C.2). There is no information on the site, or mechanism of perception of the directional signals that are responsible for growth-mediated diaheliotropism of expanding leaves. As a working hypothesis, it may be assumed that laminar phototropism may be involved (next section). Vectorial Excitation in Laminar Heliotropism of Pulvinated Leaves Phototropism was discovered and most extensively studied in coleoptiles of grass seedlings and subsequently also in hypocotyls and epicotyls of dicot seedlings, exposed to unilateral light. Light direction is perceived as differential interception by the exposed and its opposite, shaded sectors of the seedling. Diaheliotropic responses of leaves, such as those of Malva spp. and Lavatera spp. (Malvaceae ), cannot be accounted for by differential interception of light. These leaves reorient their laminae virtually normal to the sun throughout every clear day with remarkable accuracy, during most of their lifetime (Vochting, 1888; Yin, 1938; Schwartz and Koller, 1978), in nature (Schwartz and Koller, 1986) and under simulated conditions, obtained by means of a 'solar simulator' (Fig. 5). The normal to the lamina trails the moving oblique beam of 'sunlight' by a minimal threshold angle (Koller and Levitan, 1989). A similar lag characterizes growth-mediated, diaheliotropic leaf movements in sunflower (Section V.A). The leaf must therefore be capable of detecting the azimuth, as well as the elevation angles of the sun continuously. Selective shading of the periphery, or centre of the lamina (which includes the pulvinus) does not interfere with the diaheliotropic response, a result that led to the conclusion that directional light is perceived over the entire lamina, not by the pulvinus (Yin, 1938). Therefore, these leaf movements take place by laminar phototropism. Laminar reorientation takes place by curvature of the subtending pulvinus,

2.

PLANTS IN SEARCH OF SUNLIGHT

79

Fig. 6. Lavatera cretica (Malvaceae). (A) Leaf lamina (palmate venation); (B) diaphototropic laminar orientation in response to a constant vectorial photoexcitation (light beam maintained tip-oriented at + 30° for the older leaf, base oriented at -30° for the younger, opposite leaf). Note the phototropic response of petioles exposed to unilateral light. (Reprinted with permission from Koller et a/. (1985a).)

80

D. KOLLER

that may extend to the petiole (Fig. 6(B)). The directional signal must therefore be transmitted from the lamina, in a transduced form, to the pulvinar site of response. The resulting curvature is positively phototropic and reorients the lamina diaheliotropically to face the light. Perception of directional light by the quasi co-planar lamina of diaheliotropic leaves presented a challenge to the classical concept of phototropism by differential interception of unilateral light. Several studies have been addressed to other mechanisms by which the leaf lamina may perceive directional light signals, without invoking differential interception. Specialized cells in the upper leaf epidermis of certain leaves act as an optical lens (Haberlandt; 1914; Vogelmann et al., 1996) and it has been suggested that they contribute to the perception of oblique light by focusing it on specific receptive areas in the cytoplasm. To test this hypothesis, it was assumed that the refractive index of water was similar to that of these epidermal cells and that covering them with a flat layer of water may eliminate, or weaken their lens effect. One half-lamina of Tropaeolum was covered with water under a thin sheet of mica, and the opposite half left uncovered and dry. When the opposite halves of the lamina were exposed to equivalent, but opposite oblique beams, the lamina reoriented towards the oblique beam incident on its dry half, supporting this hypothesis (Haberlandt, 1914; Smith, 1984). However, previous results by Kniep (1907) showed that the lamina reorients towards the light even when the lens effect was similarly eliminated over its entire surface by means of paraffin oil, suggesting that focusing of directional light by the lens-shaped epidermal cells could not explain the perception of oblique light in the diaphototropic response (Vogelmann et al., 1996). Moreover, lens-shaped cells are not a common feature in the upper epidermis of phototropic leaves. Attempts were also made to account for laminar perception of directional light by invoking non-planar topography of the laminar surface, resulting in local differences in angle of light incidence and a differential pattern of interception of light. This approach was based on similar reasoning to account for indirect phototropic responses of seedlings by invoking differential interception of oblique light by leaves/cotyledons inclined in opposite, or otherwise divergent azimuth angles (Ball, 1923; Lam and Leopold, 1966). It was assumed that when the lamina is nonplanar, the differential interception becomes progressively accentuated as the angle of incident light is more oblique, and changes with its azimuth angle. On this basis, perception of directional light signals in the lamina of Lavatera was attributed to increasingly differential interception of PAR by opposite surfaces on either side of the vein as the azimuth angle of the light beam diverges more from that of the vein. It was assumed that the resulting pattern of assimilate partitioning is therefore differential and these differences are somehow transmitted to different sectors of the pulvinar motor tissue, causing the pulvinus to curve (Fisher and Fisher, 1983). This

81

PLANTS IN SEARCH OF SUNLIGHT

hypothesis was eventually retracted because the vascular connections did not conform to the prediction (Fisher et al., 1987). Nevertheless, C0 2 (Fisher and Wright, 1984) and a certain level of photosynthetic activity (Fisher et al., 1989) may be required for expression of the diaheliotropic response. A series of studies has led to the conclusion that diaheliotropic leaves perceive the direction of light as a vector, rather than by differential interception. The lamina of Lavatera cretica forms an incomplete, nearly circular disc, with the pulvinus at its centre, from which these veins diverge palmately in azimuthal directions that differ from each other by -50° (Fig. 6(A) ). When one half the lamina is shaded and its opposite half is exposed to light, the lamina reorients when the light is oblique to its surface, not when the light is normal to it, a result that is incompatible with the concept of

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Elapsed Time (min)

Fig. 7. Diaphototropism in Lavatera cretica (Malvaceae). Time-course of changes in the laminar elevation (LE) under a constant vectorial excitation. Angle of light incidence, AI, maintained at + 30° ( o, A.) and -30° ( e, 1::..) in the vertical plane of the mid-vein. VE(1), VE(2) =initial and subsequent excitation in the opposite direction (empty and full symbols, respectively). Dotted lines= linear regressions (r > 0.997). Angular velocity (degree h-') ratio VE(2)NE(l): for AI +30°, 55.7/ 31.4 = 1.78; for AI -30°, 42.0/30.1 = 1.40. Note the inertial reorientation following reversal of excitation. (Reprinted with permission from Koller et al. (1989).)

82

D. KOLLER

differential interception of light. These results suggested that directional light, incident on the lamina at an oblique angle, is perceived by the lamina as vectorial excitation (Schwartz and Koller, 1978). To validate this assumption, the lamina was exposed to an oblique light beam that was continuously displaced in an arc along the vertical plane of symmetry of the lamina to maintain a constant angle of incidence with the lamina, as it moved in response to that light. Under such conditions, laminar reorientation proceeds at a constant angular velocity exceeding the 15° h-1 required to track the solar transit (Fig. 7) (Koller et al., 1985a). The perception of vectorial excitation in laminar phototropism is anisotropic. Selective shading of either the central, or the two opposite lateral sectors of the lamina showed that the diaheliotropic response takes place only if the oblique beam is directed along the exposed sector( s) and not at all when oriented transversely to it (Fig. 8). The only obvious structurally directional tissues in the lamina are those associated with the veins. Selective shading of laminar strips along the major veins (of cotyledons, for technical reasons), or of the areas between them, suggested that the directional light is perceived as vectorial excitation in tissues associated with the major veins (Schwartz and Koller, 1978). This conclusion is supported in experiments using leaves from which both lateral sectors of lamina were excised, leaving

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I

N.R.

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.

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--+ --+

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Fig. 8. Diaphototropic reorientation of lamina of Lavatera ere tica (Malvaceae) with selectively shaded sectors, in response to base-oriented (top row) and lateral (bottom row) light beam at 45° to the lamina. Numbers are initial rates (degree h-1). N.R.*, no response (laminar orientation did not deviate by >3°). (Reprinted with permission from Schwartz and Koller (1978).)

PLANTS IN SEARCH OF SUNLIGHT

83

only the mid-sector. Such mutilated leaves responded diaheliotropically to an oblique beam along the plane of symmetry of the midrib. These results led to the hypothesis that the photoreceptors for vectorial excitation are oriented anisotropically in as yet unidentified cell files along the major veins, with their transition moments aligned preferentially parallel to the plane of symmetry of the vein. As a consequence of this anisotropic orientation of the photoreceptors, vectorial excitation is maximal for the photoreceptors associated with the vein located along the oblique beam. Therefore, these photoreceptor arrays enable the lamina to sense the azimuth of the light vector (solar radiation). The diaheliotropic lamina also senses and tracks solar elevation. Accordingly, its response to vectorial excitation at a constant irradiance should also be a function of angle of light incidence. To validate this assumption, the response (steady-state angular velocity of reorientation) of the lamina was measured under continuous vectorial excitation over a range of constant irradiances and constant angles of light incidence. Under such conditions, the response depends on irradiance, as well as on the angle of interception of light by the lamina, confirming the vectorial nature of the excitation. The response increases to a maximum between 40° and 50°. This angular dependence is not consistent with a paradermal localization of the photoreceptors. Analysis of the angular dependence suggests that the photoreceptors might be localized adjacent to the transverse walls of the cell files along the vein, with their transition moments inclined at some preferential angle to these walls, in addition to being preferentially parallel to the plane of symmetry of the vein (Koller et al., 1985b). Yin's results with partial shading suggest that the photoreceptors are located along the entire the vein. Vectorial excitation along the plane of symmetry of the midrib at equivalent, but opposite angles, results in diametrically opposite responses: increase, or decrease in laminar elevation when the beam is directed towards the tip of the vein ('tip oriented'), or its base ('base oriented'), respectively (Fig. 6(B)). When the vectorial excitation is maintained constant, laminar reorientation takes place at constant angular velocities (Fig. 7). This polarity suggests that the array of photoreceptors along each vein is equally capable of perceiving tip-oriented and base-oriented vectorial excitation. According to the model that has evolved from these studies, the opposite responses to opposite vectorial excitations could be accounted for by assuming that the photoreceptors that are immobilized at or in the cell membrane, adjacent to transverse walls of each cell along the file, have an opposite orientation of their transition moments at the proximal and distal poles (Fig. 9). Directional light signals are therefore perceived as differential excitation of the photoreceptors at opposite poles of each cell along the file. This differential may create a potential gradient between the two poles of each cell, as well as across the junction between neighbouring cells, which may be

84

D. KOLLER

Fluence Rate "R"

Fluence Rate II

("L"= SOJ.!mol m·2s-1)

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PLANTS IN SEARCH OF SUNLIGHT

85

expressed as a signal current that is transmitted along the cell file to the pulvinus. The intracellular anisotropic arrangement of the photoreceptors enables the detection of the angle of elevation of the light vector (incident solar radiation), as well as its polarity with respect to the axis of the vein. Laminar diaheliotropism exhibits action dichroism The concept that vectorial excitation depends on anisotropic orientation of immobilized photoreceptors in the lamina is supported by the action dichroism exhibited when plane-polarized light is used for vectorial excitation. Preliminary kinetic analysis showed that the integrated net response (angular velocity of laminar reorientation) of leaves of Lavatera to two simultaneous, diametrically opposite vectorial excitations (transverse to the plane of symmetry of the midvein) is proportional to their pFR ratio. The lamina is capable of discriminating between opposite directional light that differs by as little as 10% in pFR (Fig. 9(A)). This relationship provided the basis for studying the dependence of the responsiveness to vectorial excitation with polarized light on the plane of its polarization. When the lamina is similarly exposed to two diametrically opposite vectorial excitations by polarized light, with identical pFRs, one with its plane of polarization parallel to the plane of symmetry of the midvein (II) and the other transverse to that plane (.l), it invariably reorients towards the (II) beam. When pFR of the more effective (II) beam is reduced, the rate and direction oflaminar reorientation are linearly related to its ratio with respect to the opposite beam (.l). The two beams are balanced at a 11/.l ratio of 0.62 (Fig. 9(B)). The action dichroism could not be accounted for by dichroic optical properties of the lamina (Koller et al., 1990). Similar results were obtained with the plagioheliotropic leaf of Lupinus palaestinus (Koller and Ritter, 1991). The vascular bundles of the major veins coalesce to form the vascular core of the pulvinus, but each maintains its identity and continuity and is

Fig. 9. Lavatera cretica (Malvaceae). Top: schematic responses to differential, bilateral vectorial excitation, transverse to the mid-vein (AI = 30°). Middle: angular velocity of the laminar reorientation, measured over a range of photon fluence rates of one beam, with the opposite beam at a constant 50 rtmol m-2 s-1• (A) Both beams unpolarized; (B) one beam polarized in the vertical plane of the beam (II) and its opposite polarized in the plane transverse to it (l_). Bottom: hypothetical organization of photoreceptors for vectorial photo-excitation. Perception of the light azimuth is by anisotropic arrays of photoreceptors located in a cell file( s) along each of the major veins (Fig. 6). Photoreceptors are immobilized in cytoplasm (dotted lines), adjacent to the transverse walls, with their transition moment predominantly along the plane of symmetry of the vein, at a similar angle to the wall, opposite at the opposite poles. This arrangement is compatible with the perception of the angle of light elevation, as well as with differentiation between tip- and baseoriented light. The thickness of the photoreceptors indicates the level of excitation. (Reprinted with permission from Koller eta/. (1990).)

86

D. KOLLER

associated with a specific sector of the motor tissue (Fig. lO(A, B)). This enables the signal generated by excitation of the photo receptors along a vein to be transmitted selectively to its subtending (target) sector of motor tissue in the pulvinus, where it is transduced into osmotic activity by controlling transmembrane transport of ions and water. Since the direction of the vectorial light signal (base- or tip-oriented) determines the direction of the (presumptive) potential gradient it also determines the direction of the pulvinar response. The signal has opposite consequences for its target sector when the excitation is tip-oriented (expansion), or base-oriented (contraction) (Koller et al., 1985a, b). 3. After-effects of Vectorial Excitation Vectorial excitation, or the response to it, appears to have after-effects. The lamina of Lavatera continues to increase its elevation (inertially) in response to tip-oriented excitation and to decrease it in response to base-oriented excitation for a short time after the excitation has been terminated, suggesting inertial transpulvinar transport of solutes and water, or residual excitation. In both cases, when excitation is terminated by darkness, laminar elevation decreases to a low steady-state. This is apparently an inevitable feature of this transition (Koller and Cohen, 1989). Inertial reorientation of the lamina also takes place when the original vectorial excitation is terminated by reversal of its direction. Subsequent laminar reorientation following reversal of the direction of vectorial excitation is in the new direction, but its rate (angular velocity) is enhanced considerably (by -40% and -70% for base- and tip-oriented excitation, respectively). The enhancement depends on the preceding opposite vectorial excitation (Fig. 7), but is not a result of the preceding diaphototropic movement. By elimination, it may be a result of change( s) at the site of perception (Koller et al., 1989). E.

REMOTE PHOTOTROPIC CONTROL BY VECTORIAL EXCITATION

The response to vectorial excitation can be transmitted beyond the pulvinus. The hypocotyl of seedlings of Lavatera cretica exhibits positive phototropic curvature in response to its direct exposure to unilateral light, resulting in diaphototropic reorientation of its (shaded) apical bud and cotyledons. The cotyledons themselves exhibit laminar phototropism. Exposure of their lamina to vectorial excitation reorients it normal to the light by pulvinar curvature, while the (shaded) hypocotyl remains upright. When the entire seedling is exposed to directional light, positive phototropic curvature of the hypocotyl satisfies the diaphototropic requirement of the cotyledons, without pulvinar intervention. In explants of seedlings (hypocotyl and a single cotyledon), the (shaded) hypocotyl curves

PLANTS IN SEARCH OF SUNLIGHT

87

Fig. 10. Lavatera cretica (Malvaceae ). (A) Photomicrograph of pulvinar crosssection (diameter -1.5 ~m). (Reprinted with permission from Schwartz and Koller (1978).) (B) Photomicrograph of vascular tissues in pulvinus and adjacent portions of lamina and petiole, cleared with lactic acid.

88

D. KOLLER

away from the remaining cotyledon when its lamina is in darkness, or exposed to vertical light. Exposure of the lamina alone to continuous vectorial excitation with blue, but not with red light, results in curvature of the (shaded) hypocotyl (as well as of the shaded petiole) in the direction determined by the direction of vectorial excitation: enhanced curvature away from the cotyledon when the excitation is tip-oriented, and toward the cotyledon when it is base-oriented (Fig. 11; Schwartz and Koller, 1980).

Fig. 11. Remote control of phototropic hypocotyl curvature in explants of Lavatera cretica (Malvaceae) by vectorial excitation of the cotyledonary lamina. Explants consist of a single cotyledon with its lamina exposed and its petiole and hypocotyl in darkness. The hypocotyl (A) curved away from the cotyledon to the same extent whether the lamina was exposed to vertical light (B) or was in darkness (C). This curvature was enhanced when the lamina was exposed to a tip-oriented light beam (D), whereas exposure to a base-oriented beam reversed the curvature (E). This reversal was eliminated when the lamina was free to reorient vertically to the beam (F). (Reprinted with permission from Schwartz and Koller (1980).)

PLANTS IN SEARCH OF SUNLIGHT

89

These results suggest that the signal generated by the vectorial excitation of the lamina may be transmitted to the hypocotyl, causing it to curve by differential growth, possibly by driving fluxes of solutes and water from its concave to its convex flank, as in turgor-mediated pulvinar curvature. This concept is supported by instances where growth-mediated curvature in response to unilateral stimulation by light (Rich et al., 1985), or by gravity (Kohji et al., 1995), results from contraction along the concave flank, coupled with elongation along the convex flank. F.

LOGISTICS

Structural studies of the pulvinar response have been reported in Lavatera (Werker and Koller, 1987) and Lupinus (Werker et al., 1991). The mechanisms for transport of solutes and water between the contracting and expanding sectors of the pulvinus are probably similar to those reported in leguminous pulvini. Osmotic pressure of the cell sap in the contracting and expanding motor tissue of Malva remains unchanged during pulvinar curvature (Yin, 1938), as in the bean pulvinus (Irving et al., 1997). However, the transduction pathway between vectorial excitation at the laminar site of signal perception and the response at the pulvinar motor tissue must include exceptional downstream elements: signal transduction in the receptor cells to element(s) that can be transmitted to the site of response along the intervening tissue, and probably once more into element(s) that elicit the response in the motor cells (solute transport). G.

SPECTRAL DEPENDENCE OF LAMINAR PHOTOTROPISM

Detailed action spectra for laminar phototropism are not available. Continuous vectorial excitation with blue light is required to drive laminar heliotropism (Yin, 1938; Schwartz and Koller, 1978, 1980; Vogelmann and Bjorn, 1983). Exposure of the leaf lamina, or cotyledon of Lavatera to directional red light is equivalent to absence of vectorial excitation (darkness). The lamina reorients normal to its petiole (Koller et al., 1985a; Schwartz and Koller, 1980). The response (angular velocity of laminar reorientation) increases with pFR (Koller et al., 1985b). Leaves of Malva neglecta respond diaphototropically to blue light, even at relatively low irradiances, but the response requires additional PAR in excess of the light compensation point. The rate of response increases progressively with irradiance of total PAR. A role for photosynthesis-mediated translocation of the directional signal from the leaf to the pulvinus has been suggested (Fisher et al., 1989). Normal levels of C0 2 are required to sustain the response to vectorial excitation (Fisher and Wright, 1984), supporting this suggestion.

90

D.KOLLER

VI. A.

LEAF MOVEMENTS BY PULVINAR PHOTOTROPISM PERCEPTION OF DIRECTIONAL LIGHT AS A UNILATERAL SIGNAL

Heliotropic movements of pulvinated leaves of many leguminous plants operate by pulvinar phototropism. Whereas in laminar phototropism directional light is perceived in the lamina as vectorial excitation and the signal is transmitted to the pulvinar site of response, in leguminous leaves that exhibit pulvinar phototropism perception of directional light, as well as the response to it, are localized within the pulvinus. This conclusion is based on effects of selective shading of the pulvinus, or the lamina, from directional light, as well as on the pulvinar responses of debladed leaves to directional light (Wien and Wallace, 1973; Sato and Gotoh, 1983; Sheriff

Fig. 12. Pulvinar phototropism in terminal leaflet of Phaseolus vulgaris (Fabaceae). Lateral view. (A) Low-light adapted pulvinus; (B) response to abaxial light; (C, D) intermediate and final stages of response to adaxial light. (Pulvinar diameter -2 mm.)

PLANTS IN SEARCH OF SUNLIGHT

91

Fig. 13. Pulvinar phototropism in Phaseolus vulgaris (Fabaceae ). Abaxial view of terminal pulvinus before (A) and after (B) lateral exposure to light and of the two lateral pulvini before (D) and after exposure to light from the rear (C) or front (E). (Pulvinar diameter -2 mm.)

and Ludlow, 1985; Donahue and Berg, 1989). Pioneering studies showed that the primary leaf of Phaseolus multijlorus and the pinnate leaf of Robinia pseudoacacia, respond to unilateral exposure of the upper (adaxial), or lower (abaxial) surface of the pulvinus to light by increase, or decrease of laminar elevation, respectively, whereas lateral exposure results in rotation of the lamina toward the light (Brauner, 1932; Brauner and Brauner, 1947). Clearly, the pulvinus itself is phototropic. Similar laminar reorientations were observed in several trifoliate leguminous species upon unilateral exposure of their leaf to adaxial, or lateral light. The resulting movement

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combined changes in azimuth, elevation with axial rotation (Wien and Wallace, 1973; Meyer and Walker, 1981; Reed and Travis, 1984; Sheriff and Ludlow, 1985; Berg and Hsiao, 1986). In the trifoliate leaf of Phaseolus, differential exposure of the pulvinus to light results in a positive phototropic curvature. Pulvinar responses become more complex when they combine curvature with torsional rotation, as in lateral exposure, particularly in the two lateral pulvini that are inherently curved (Section VI.D.2, Figs 12 and 13). These pulvinar responses to unilateral exposure to light result in corresponding passive reorientation of the lamina (Fig. 14). There is thus a clear distinction between perception of light direction in laminar and pulvinar phototropism, as well as between their heliotropic consequences (Section VII.F). These aspects are usually overlooked in studies of heliotropic movements of leguminous leaves and do not support the concept of their dia- or paraheliototropic nature. Leaves exhibiting pulvinar phototropism are heliotropic, because they change their laminar orientation in response to the solar transit. However, they are neither diaheliotropic, nor paraheliotropic. They do not track the solar transit throughout the day, because their lamina is moved passively by pulvinar phototropism (Koller and Ritter, 1994). Exposure of the adaxial, or abaxial sectors to equivalent light treatments may result in phototropic responses of different magnitude (Brauner and Brauner, 1947; Sheriff and Ludlow, 1985; Schwartz et al., 1987; Koller and Ritter, 1994), suggesting that the distribution, or effectiveness of these photoreceptors, or their capacity for interception of light, may differ in different pulvinar sectors. The pulvinus responds phototropically to differential interception of light in its opposite sectors. Selective shading of the adaxial, abaxial, or lateral surfaces of the light-adapted pulvinus (Glycine max) results in laminar reorientation, or rotation towards the opposite side (Donahue and Berg, 1989). Conversely, phototropic curvature of the pulvinus (Phaseolus) by exposure of one of its faces to unilateral light is reversed when its opposite face is simultaneously exposed to similar unilateral light. The pulvinus integrates the phototropic responses of different sectors and its curvature represents their net effect (Koller and Ritter, 1994) as in hypocotyls (Section IV.B.1).

Fig. 14. Consequences of phototropic pulvinar responses for laminar reorientation in Phaseolus vulgaris (Fabaceae ). Front view of right lateral leaflet before (A) and after exposure of pulvinus to light from the rear (B) or front (C). Front view of terminal leaflet before (D) and after exposure of pulvinus to light from the left (E). Front view of both lateral leaflets before (F) and after exposure of pulvini to light from above. Note: to avoid interference with pulvinar exposure, other leaflet( s) and laminar tissue were excised as required.

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Each laminar pulvinus in a compound leaf exhibits independent phototropic responses. However, in the trifoliate leaf of Macroptilium atropurpureum, phototropic excitation can be transmitted between primary and secondary pulvini (Sheriff and Ludlow, 1985). B. LOGISTICS

Information on the mechanism by which continuous unilateral excitation by light causes and maintains the differential activities across the pulvinus is fragmentary. The following sequence of events probably takes place in the course of phototropic curvature of the pulvinus in Phaseolus . Continuous exposure of motor cells to (blue) light of sufficient irradiance affects critical components of their membrane transport, which causes efflux of ions, accompanied by water, from their vacuole into their apoplast WFS. Turgor pressure decreases, but osmotic pressure remains unchanged. The added water in the WFS of the apoplast results in a transpulvinar gradient in hydrostatic pressure, to the opposite sector. The resulting bulk flow of solution changes the ionic environment, as well as the water potential of the apoplast WFS in this sector. These changes in the apoplast affect the membrane transport of the motor cells, enhancing uptake of ions, accompanied by water, and result in their expansion. Turgor pressure increases, but osmotic pressure remains unchanged (Irving et al. ,1997). The hypothesis regarding transport processes into these cells is supported by results showing that protoplasts (isolated from either extensor or flexor) of Phaseolus expand in response to increase in [K+Jaut (Erath et al., 1988). In stomatal guard cells, K+in channels may function as a K+ -sensing valves, that open whenever the K+ gradient is inward, allowing K+ uptake only (Maathuis et al., 1997). The activity of K+ channels depends on voltage of the membrane, which in turn is affected by activity, or inactivity of its H+ATPase. Phototropic responses of the pulvinus of Phaseolus are inhibited by activation of H+ -ATPase, as well as by its inactivation, and by TEA, a K+ channel blocker (Cronlund and Forseth, 1995), suggesting that inactivation of the proton pump in the contracting sector, as well as its continued activity in the opposite, expanding one, take part in establishing the turgor differential for the phototropic response of the pulvinus. Continuous adaxial exposure of the primary leaf pulvinus of Phaseolus to blue light changes the electric potential difference between the motor tissue in the exposed and shaded sectors from -40 to +20 mV, in parallel with the change in laminar elevation. This change in potential difference between the contracting and expanding tissue may be important for the movement of ions between them (Nishizaki, 1986). Contraction of the extensor, or flexor of the pulvinus of Phaseolus when exposed unilaterally to blue light is associated with depolarization of their motor cell membranes, supporting the concept that

PLANTS IN SEARCH OF SUNLIGHT

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the phototropic pulvinar response to blue light results from inhibition of H+ATPase activity, causing loss of the electrogenic component of membrane potential. As a result, H+ IK+ fluxes are suppressed and turgor pressure falls. Even a brief exposure to blue light causes transient depolarization and transient alkalization of the extracellular pH (greater in the extensor), as well as a transient change in laminar elevation, and these changes increase with irradiance (Nishizaki, 1992, 1996). These responses differ from those reported above for Samanea (Kim et al., 1992). However, whereas pulvini of Samanea and Phaseolus are both photonastic, only the latter are phototropic as well. This contradiction may therefore be explained by a phototropic response of the latter. Light induces an irradiance-dependent electric polarity across the leaf lamina of Elodea canadensis; acidification of the abaxial side takes place by means of active proton efflux, and concomitant alkalization of the adaxial side by passive QH- efflux. The electric potential difference results in net cation flux from the abaxial to the adaxial side (Elzenga and Prins, 1989). Exposure of the Phaseolus pulvinus to red light causes membrane hyperpolarization, that is inhibited by DCMU (N-(3,4dichlorophenyl)-N'-dimethylurea), while the ionophore CCCP (carbonylcyanide-m-chlorophenylhydrazone) depolarizes the cells and prevents further light-induced changes. Motor cells depolarize under conditions of anoxia, and do not respond to blue light under such conditions. Exposure of the anoxic pulvinus to red light leads to progressive recovery (hyperpolarization) of the membrane potential and of its capacity to depolarize transiently in response to a unilateral blue light pulse. Under anoxia, unilateral exposure to red light results in negative phototropic curvature, presumably by enhancing hyperpolarization in the exposed side. These results support the conclusion that the primary effect of blue light is depolarization of the hyperpolarized membrane, by inactivating a H+ATPase, the activity of which depends on a supply of ATP from respiration. DCMU completely inhibits the hyperpolarization by red light, but has no effect on the transient depolarization by blue light pulses. The proton ionophore CCCP depolarizes the membrane and progressively reduces the magnitude of the transient depolarization by blue light pulses, suggesting that the source of ATP may also be photophosphorylation, probably by pulvinar chloroplasts (Nishizaki, 1996; cf. Koller et al., 1995). Nishizaki (1996) concludes that unilateral blue light induces a decreasing gradient in depolarization of motor cells across the pulvinus, resulting in greater contraction of the exposed sector than in its opposite (actually, the latter expands). Blue light activates a [Ca 2+]cyt and ATP-dependent anion channel in the plasma membrane of mesophyll cells of pea leaves, allowing efflux of CI-. In mesophyll cells, this efflux depolarizes the membrane and activates a proton pump, H+ efflux, hyperpolarization and uptake of K+, which promotes leaf growth (Elzenga and Van Volkenburgh, 1997a,b). If the CI-

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channel also exists in the plasma membrane of pulvinar motor cells, it may be involved in the phototropic pulvinar response (as well as in the photonastic response of its flexor), by enabling the massive efflux of o- from motor cells upon exposure to blue light. C.

COEXISTENCE WITH PHOTONASTIC PULVINAR RESPONSES

Heliotropic leguminous leaves also exhibit diurnal and circadian movements. Their phototropic and diurnal responses are superimposed. The skoto-/ photonastic response of the pulvinus, as well as its phototropic response, take place by transport of ions, primarily K+ and o-, accompanied by water, out of the contracting sector and into the opposite, expanding one (Satter, 1979; Irving et al., 1997). Similar mechanisms of ion transport are probably involved in both responses. However, photonastic and phototropic responses of the pulvinus differ fundamentally and the relationship between them has not been elucidated satisfactorily. (i) Photonastic responses take place by opposite volume changes in the flexor and extensor to the same light, whereas tropic responses invariably result from contraction of any pulvinar sector that is exposed to light and concomitant expansion of its opposite sector (Fig. 1; Koller and Ritter, 1994). (ii) The pulvinus (of Phaseolus) responds phototropically to unilateral light from any direction (Figs 12 and 13), as well as photonastically to a (non-directional) dark~ light signal, suggesting that photoreceptors for the phototropic response of the pulvinus are located in every sector (Koller and Ritter, 1994). Therefore, the mechanisms of control and response for the phototropic movements exist in every pulvinar sector, and coexist with those of the photonastic movements in the extensor and flexor. However, the two species of photoreceptors apparently act cooperatively in the flexor and antagonistically in the extensor. (iii) The presence of the phototropic response of the pulvinus in all its sectors may account for its photonastic unfolding at sunrise. In the natural environment, the overhead flux of light invariably exceeds the opposite flux, even in the absence of direct sunlight, and the lateral fluxes are very similar to each other. Moreover, at sunrise the flexor is expanded and thus intercepts more light than its opposite, contracted extensor. The pulvinus integrates the light signals, unfolding the leaf. The pulvinus starts to respond heliotropically when any of its sectors intercepts light at higher irradiances, that disrupt this balance. (iv) There is evidence suggesting that different photoreceptor systems control photonastic and phototropic responses of the pulvinus (Koller et al., 1996). (v) The phototropic response of the intact pulvinus is not reflected in the response to light of protoplasts isolated from it. Protoplasts isolated from the abaxial (extensor), or adaxial (flexor) sector of the pulvinus of Phaseolus exhibit opposite responses to increase in irradiance: expansion of the former and contraction of the latter (Yu and Berg, 1994), whereas in

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the intact pulvinus, exposure to light of either of these sectors results in its contraction, with concomitant expansion of the opposite, shaded sector (Koller and Ritter, 1994). However, responses of isolated protoplasts cannot be applied to intact tissues, particularly where the cytoskeleton might be involved (Nick, 1999). D.

PULVINAR PHOTOTROPISM IN TRIFOLIATE LEGUMINOUS LEA YES

1. Structural Modification Heliotropic movements in leguminous leaves are most conspicuous in trifoliate species. Phototropic responses of the pulvinus to directional light may be modified by the pulvinar configuration and by changes taking place in it in the course of its response. The (photonastically) unfolded leaflet lamina is horizontal, but its pulvinus is curved upwards (Wien and Wallace, 1973; Sheriff and Ludlow, 1985). In this configuration (Fig. 12), the lamina may obstruct base-oriented directional light from reaching the concave (adaxial) face of the pulvinus. The pulvinus can start to respond phototropically only after light changes its angle of incidence sufficiently to become accessible to the adaxial surface. The lamina of the light-adapted terminal leaflet of Melilotus alba remains horizontal until the angle of light incidence changes sufficiently to expose the adaxial surface of the upcurved pulvinus (Schwartz et a/., 1987). Pulvinar configuration of the pulvinus changes in the course of its phototropic response. Changes in pulvinar configuration may determine the angle and the extent of interception of directional light by the lamina, and the resulting steady-state of the response by feedback control (Koller and Ritter, 1994). Such changes may also determine the penetration of light into the pulvinus. Studies with an optical microprobe showed that penetration of (blue) light into the adaxial motor tissue in the pulvinus of Glycine max is affected by the angle of light incidence, as well as by the configuration of the pulvinus (Donahue et a/.. 1990). 2. Morphological Constraints Each leaflet in a trifoliate leaf moves independently of the others and has a different azimuth angle (approximately at right angles to each other) and each pulvinus intercepts the same directional light differently. Moreover, the configuration of pulvini of the lateral leaflets also differs from that of the terminal leaflet. They are inherently curved, as well as rotated to the front (away from the leaf base) and this superimposes additional torsional rotation over their positive phototropic response to exposure from all directions (Fig. 13). The resulting laminar reorientations reflect these asymmetries (Fig. 14; Section VI.A). When the entire trifoliate leaf is exposed to directional light from the front, rear, or side, the different

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leaflets exhibit independent, different phototropic responses (Sheriff and Ludlow, 1985). 3. Apparent Solar-tracking Laminar reorientation of trifoliate leaves, as a passive result of pulvinar phototropism, has different consequences for interception of incident light by the lamina of individual leaflets: Adaxial, or abaxial exposure of the terminal pulvinus results in upward, or downward curvature, respectively. The lamina changes its orientation upward or downward, respectively, in both cases becoming less normal to the light (interception decreases). Lateral exposure to light results in positive curvature of the pulvinus (laminar azimuth changes), combined with torsional axial rotation. The change in azimuth reorients the lamina less normal to the light (interception decreases), but its concomitant rotation towards the light reorients it more normal to the light (interception increases). Field-grown trifoliate legumes exhibit leaf movements that appear to track the sun diaheliotropically in the morning and late afternoon, and paraheliotropically, to varying extents, around midday (Wilson and Greenman, 1892-97; Kawashima, 1969; Berg and Hsiao, 1986). It is widely accepted that the primary diaheliotropic movements of such leaves are modified to paraheliotropic when the leaf experiences water stress, or stress by high irradiance, or supra-optimal temperature around midday (Wilson and Greenman, 1892-97; Dubetz, 1969; Begg and Torssell, 1974; Shackel and Hall, 1979; Forseth and Ehleringer, 1980; Travis and Reed, 1983; Ludlow and Bjorkman, 1984). However, with few exceptions, reports of dia- or paraheliotropic movements in leaves (Ehleringer and Forseth, 1980; Rajendrudu and Rama Das, 1981) have not been supported by time-course studies of the relationship between leaflet orientation and the solar transit throughout the day. The leaf of Dolichos lablab has been designated paraheliotropic, but in contrast with all diaheliotropic species, its azimuth remains stationary throughout the changes in the solar azimuth (Sailaja and Rama Das, 1996). The trifoliate leaf of Erythrina herbacea is downfolded at night. After sunrise, the leaflet laminae incline upwards well beyond the horizontal and also rotate axially. This orientation remains virtually unchanged until nightfall, exhibiting no dependence on the changes in the angle of light incidence throughout the day (Herbert, 1984). The leaf exhibits an apparent diaphototropism in the morning and afternoon, and an apparent paraphototropism around midday, despite the fact that its lamina remains stationary. The consequences of the differences in configuration and azimuth of the pulvini for their heliotropic movements were studied in the trifoliate leaf of Phaseolus vulgaris, exposed to a transit of a simulated 'sun' in a vertical arc (12 hat 15° h-1), transverse, or along its major axis. Individual leaflets move independently during different phases of the 'solar' transit and their fidelity of 'solar' tracking is considerably less than in malvaceous leaves

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exhibiting laminar heliotropism. The imperfect fidelity of tracking the sun is reflected in the capacity of the leaf for intercepting solar radiation (Ritter and Koller, 1994b ). E.

COOPERATION WITH LAMINAR PHOTOTROPISM

Leaflet movements of Lupinus palaestinus are controlled by laminar, as well as pulvinar phototropism. The palmately compound lamina of Lupinus spp. exhibits diaheliotropic movement during daytime (Fig. 3), as well as nyctinastic downfolding and photonastic unfolding. The leaf of L. succulentus responds diaphototropically to directional light only if its centre is not shaded, leading to the conclusion that perception of directional light signals is localized in the pulvinus (Vogelmann, 1984). However, the leaf of L. palaestinus exhibits sustained diaheliotropic reorientation when exposed to constant vectorial excitation, even when the pulvinar crown (composed of the pulvini of the individual leaflets) is shaded from the oblique beam. The magnitude of the response (angular velocity of laminar reorientation) is reduced as the diameter of the shaded circle extends to cover a greater laminar segment. Shading the pulvinar crown at the centre of the lamina does not interfere with the sustained diaheliotropic response, but causes the characteristic skotonastic downfolding of the leaflets (Fig. 15(A)). Conversely, the diaheliotropic response takes place when vectorial excitation is confined within such a circle, but is reduced as the diameter of the exposed circle becomes smaller (Fig. 15(B)). The diaphototropic response also takes place when vectorial excitation is confined to a narrow ring at the base of the leaflets, leaving in shade the pulvinar crown, and the rest of the leaflet laminae (Fig. 15(C)) (Koller and Shak, 1990). Individual leaflets exhibit diaphototropic responses when their pulvinus is unilaterally exposed to light from different directions, after excising their neighbours (Werker et al., 1991). Therefore, at least in L. palaestinus, the diaheliotropic response depends on perception of vectorial excitation in the basal part of the lamina of each leaflet, not in the pulvinus (Koller and Shak, 1990). A similar role for the laminar base is also suggested by results with selective shading in leaflets of Phaseolus (Sa to and Gotoh, 1983) and Macroptilium (Sheriff and Ludlow, 1985). In the intact leaf of Lupinus, the leaflet pulvini are arranged vertically, side-by-side in a tight 'crown' over the juncture with the petiole, so that their lateral flanks are shaded by their neighbours. As the entire lamina moves to remain normal to the sun, only the adaxial surfaces of the pulvini, facing the centre of the crown, remain exposed to direct sunlight (Werker et al., 1991). When the entire lamina is exposed to vectorial excitation, but high irradiance light is piped into the centre of the pulvinar crown by means of a shielded optical fibre, the entire lamina responds diaphototropically to the

,.....

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120 180 240 60 Elapsed Time (min}

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Fig. 15. Localization of the perception of vectorial excitation in the leaf lamina of Lupinus palaestinus (Fabaceae ). Time-course of changes in the angle of elevation of the compound lamina (llLE) and of the change in the angle of skotonastic downfolding ('cupping') of its individual leaflets (llNA) under a constant level of vectorial photo-excitation (angle of light incidence +30° or -30°) in the median plane of the lamina. (A) Beam excluded from the circle, centred on the leaflet junction. (B) Beam confined to the circle, centred on the leaflet junction; no downfolding (llNA = 0°), leaflets remained co-planar. Diameter of circle 11 (0), 6 (.A.) or 3 mm (~).(C) Base-oriented beam confined to the circle (e) or ring ( 0 ), centred on the leaflet junction. (Reprinted with permission from Koller and Shak (1990).)

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former, but at the same time the leaflets exhibit 'cupping', by simultaneous and equivalent increase in laminar elevation (Koller and Shak, 1990), probably by a simultaneous phototropic response to increased irradiance. The phototropic response of the pulvinus to high irradiance modifies the diaheliotropic movements of the individual leaflets to plagioheliotropic ones (Section VI.F).

F.

MODIFICATION BY STRESS

Pulvinar phototropism of leguminous leaves may be strongly modified when the leaf experiences a variety of stresses. Suboptimal leaf water potential and supra-optimal leaf temperature are the major stresses that modify heliotropic movements of pulvinated leaves. Steady-state laminar elevation of light-adapted primary leaves of Phaseolus is greater at higher irradiance, and increases progressively with leaf water stress, expressed by increasingly negative leaf water potential (Berg and Hsiao, 1986; Berg and Heuchelin, 1990). Phototropic curvature of excised pulvini of bean increases progressively with temperature (Yu and Berg, 1994). Heliotropic leaf movements of light-adapted leaves of Phaseolus are progressively modified by an increase in ambient air temperature, under constant atmospheric humidity and [C0 2], or mesophyll [C0 2]. Selective exposure of the pulvinus to radiant heat showed that modification of heliotropic leaf movements by supra-optimal air temperature depends uniquely on pulvinar tissue temperature, and is not mediated by effects of temperature on leaf water potential, transpiration, or conductance. Furthermore, modification of such leaf movements by temperature and water stresses take place only in light (Fu and Ehleringer, 1989). It may therefore be assumed that these stresses enhance the processes by which ions are transported from the exposed to the shaded sector of the pulvinus. This enhancement may take place by means of increases in levels of abscisic acid (ABA) in the exposed sector, mediated by stress. ABA inhibits circadian leaf movements of Oxalis regnelii, suggesting effects on membrane permeability (Skrove et al., 1982). ABA may play a similar role in the exposed sector of the pulvinus and in stomatal guard cells. ABA initiates the transduction chain leading to contraction of the guard cells by increasing the level of free Ca 2+ in the cytosol (McAinsh et al., 1997). ABA inhibits the blue light-dependent proton pumping and depolarizes the plasma membrane in pro top lasts from stomatal guard cells of Vicia. This is a first step of contraction in daytime in response to elevated ABA, as an indirect result of inactivation of the plasma membrane H+ -ATPase, and/or inhibition of the blue light signalling pathway (Goh et al., 1996). An increase in ABA promotes stomatal closure in light by increasing the efflux of ions from pulvinar motor cells, possibly by increasing levels of IP 3, acting as a messenger in cascades leading to opening of K+ out and anion channels

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(primarily CI-), and closing K+in channels in the cell membrane of guard cells (Assman, 1993; Cote and Crain, 1994). Mesophyll cell chloroplasts accumulate ABA when pH of their stroma is higher than in the surrounding cytosol. This pH differential results from light-driven proton intake into the grana. The process is reversed as the stroma becomes acidified under stress, releasing ABA into the apoplast, and eventually to the guard cells. Similar processes may take place in motor cell chloroplasts in the exposed sector. The most frequently cited modification is by high irradiance intercepted by the pulvinus, that presumably transforms the (apparent) diaheliotropic orientation of the lamina into an (apparent) paraheliotropic one (Berg and Hsiao, 1986; Berg and Heuchelin, 1990; Fu and Ehleringer, 1991). However, the steady-state laminar elevation is a function of the (adaxial) irradiance intercepted by its pulvinus. It is therefore more likely that the observed change from 'dia-' to 'paraheliotropic' orientation upon exposure to highirradiance is only a manifestation of the phototropic response of the pulvinus, and that the lamina is normal to the light only within some critical, intermediate range of irradiance, but diverges progressively as irradiance increases, or decreases beyond this range (Koller and Ritter, 1994; cf. Sheriff and Ludlow, 1985). Exposure of the palmately compound leaf of Lupinus spp. to supraoptimal irradiance modifies its diaphototropic response to a plagioheliotropic one (Section VI.E). A similar modification takes place in response to water stress (Wainwright, 1977). In the absence of stress, individual leaflets of L. arizonicus move diaheliotropically throughout the day. As water stress develops (expressed as reduced leaf water potential) around midday, movement of the leaflets becomes plagioheliotropic ('cupping') and track the sun at an acute angle (Sections VI.E and VII.G).

G.

SPECTRAL DEPENDENCE OF PULVINAR PHOTOTROPISM

1. Spectral Analysis Pulvinar phototropism is controlled by sustained exposure to relatively high pFR of blue, as well as red and far-red light. Pulvinar phototropism in leguminous leaves is driven by continuous unilateral excitation with blue light (Wilson and Greenman, 1892-97; Brauner and Brauner, 1947; Sato and Gotoh, 1983; Vogelmann and Bjorn, 1983; Sheriff and Ludlow, 1985), with peak activity at 420 nm and a minor one at 470-490 nm (Donahue and Berg, 1989). The action spectrum for the membrane depolarization induced in pulvinar motor cells of Phaseolus by a blue light pulse exhibits a major peak at 460 nm and minor peaks at 380 and 420 nm. No activity was observed at wavelengths <360 and >520 nm (Nishizaki et al., 1997). Sustained exposure of the pulvinus of Phaseolus to blue (450 nm) or far-red (722 nm) light results in proton efflux, increase of osmotically active solutes, a more

PLANTS IN SEARCH OF SUNLIGHT

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negative water potential and expansion of the extensor. Similar changes take place in the flexor in response to continuous exposure to red (671 nm) light. The conclusion that phytochrome is present in both sectors of the pulvinus, but the BAP is only in the extensor (Bialczyk and Lechowski, 1990, 1992), is incompatible with the positive phototropic response exhibited by the pulvinus to adaxial, abaxial and lateral exposure to blue light (Koller et al., 1996). However, the responses to blue light at low and high pFR may be mediated by different photosensory systems. The fluence-response relationships for the phototropic response of light-grown seedlings of Fagopyrum esculentum to blue light exhibit reciprocity at pFRs 0.2 11mol m-2 s- 1 and dependence on total fluence at higher levels (Ellis, 1984). The initial rate (angular velocity) of increase in elevation, as well as the eventual steady-state elevation reached by the terminal leaflet lamina of Phaseolus in response to continuous pulvinar exposure to overhead blue light are linearly related to pFR (50-800 11mol m- 2 s- 1). However, the effectiveness of unfiltered white light equals, or exceeds the effectiveness of blue light at equivalent pFRs. The response to blue light (50 11mol m-2 s- 1) is enhanced progressively by supplementary red light at increasing pFR (200-800 11mol m-2 s- 1). Adding red light to blue light is more effective in accelerating the initial response than adding blue light at equivalent pFR, whereas adding blue light is more effective in increasing the steadystate laminar elevation (Ritter and Koller, 1994a). The pulvinus also responds to overhead exposure to red (500 ~mol m-2 s- 1), or far-red light (880 11mol m-2 s-1 between 700 and 800 nm; E 1760 ~mol m-2 s- 1 > 700 nm), but at substantially lower rates than in blue light (50 11mol m-2 s- 1). The kinetics of the response to red light differ qualitatively from those to far-red light. However, the kinetics of the response to red light, alone or during enhancement of the response to blue light, remain unchanged in the presence of far-red light. In contrast, the response to blue alone, or when enhanced by mixture with red, is partially inhibited by simultaneous exposure to far-red. The kinetics of the inhibition of the response to blue plus red by simultaneous far-red light are similar to those of the positive response to far-red alone. The results suggest that the response to blue is mediated primarily by a BAP, but may involve some absorption by phytochrome (cf Hayami et al., 1986), whereas responses to red or far-red, with and without blue, may be mediated by HIR of phytochromes A and B (Koller et al., 1996; cf. Reed et al., 1993; Parks eta/., 1996). HIR involvement in leaf movements and in flowering of long-day plants has been attributed to interaction between phytochrome and an unidentified 'Heliochrome' (Tanada, 1997). Co-action between phytochrome and BAP is widespread (Mancinelli, 1989; Mohr, 1994). It is exhibited in phototropism of the protonema (Hayami et al., 1986), as well as in directional movement of chloroplasts in dark-adapted cells of the heart-shaped pro thallus of the fern

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Adiantum (Kagawa and Wada, 1996), and in inhibition of hypocotyl elongation of de-etiolated castor bean (Kigel and Schwartz, 1981). Opening of the apical hook in etiolated seedlings of Arabidopsis is stimulated by red, as well as by low-fluence blue light, apparently acting by excitation of phytochrome, because it is inductive, reversible by far-red and exhibits reciprocity. Hook opening is also stimulated by prolonged exposure to farred, apparently acting by means of a HIR, and by high-fluence blue light, apparently acting by means of a BAP. HIR is apparently not involved in the stimulation by red light, presumably because phytochrome B is absent in etiolated tissue (Liscum and Hangarter, 1993). Excitation of phytochrome by red light and of a BAP by blue light controls cell expansion in leaves of Phaseolus. It also controls H+ efflux, hyperpolarization and cation uptake in epidermal strips from pea leaves. In the latter, the effects of near-saturating red and blue light are additive. Enhancement of cell expansion by continuous irradiation exhibits peak activity in blue (460 nm) and red (660 nm) light. However, photosynthesis is not involved, because the enhanced growth takes place whether photosynthesis is active, or inactivated. Activity of the red, but not of the blue light, is reduced in the presence of far-red (730 nm) light (Van Volkenburgh et al., 1990; Staal et al., 1994). Although excitation of either photoreceptor by light enhances leaf growth, their transduction follows

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Fig. 16. Functional differentiation between the activity of blue and red light in the phototropic response of the terminal pulvinus of Phaseolus vulgaris (Fabaceae ). Time-course of changes in the laminar orientation during exposure to blue and red light, separately or together. (A) Changes in the angle of elevation (LE) during abaxial exposure. (B) Changes in the angles of rotation, azimuth and elevation during lateral exposure. Photon fluence rates: B, blue light; R, red light. (Reprinted with permission from Koller et al. (1996).)

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PLANTS IN SEARCH OF SUNLIGHT

different paths. Growth in continuous exposure to blue light is greater and exhibits a significantly shorter lag than in red light. However, the effects of red light on acidification of the leaf surface (normally associated with growth) greatly exceed those of blue light. Inhibition of cation uptake by DCMU in red and blue light suggests a contribution by photosynthesis to growth (Blum et al., 1992). Lights lateral

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Elapsed time (min}

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2. Functional Analysis Functional differences between red and blue light in the control of pulvinar phototropism become apparent when the pulvinar responses to abaxial, or lateral red and blue light, separately and in combination, are compared (Fig. 16(A, B)). Exposure of the pulvinus to abaxial, adaxial, or lateral blue light cause positive phototropic responses in each case. Laminar elevation increases in adaxial exposure and decreases in abaxial exposure, while lateral exposure to blue light causes laminar rotation and azimuth to change toward the light, without affecting laminar elevation. Laminar elevation increases in response to adaxial, abaxial, or lateral exposure to red light. However, laminar rotation, or azimuth are virtually unaffected by lateral red light. The response to adaxial blue light is enhanced in the presence of red light, but the opposite response to abaxial blue light is reversed in the presence of red light. In contrast, lateral red, which by itself has no effect on laminar azimuth and rotation, enhances the azimuth change, but not the rotation caused by lateral blue light. Lateral blue, which by itself has little or no effect on laminar elevation, enhances the increase in elevation caused by lateral red light. This comparative analysis suggests that red light controls the photonastic unfolding of the pulvinus, whereas blue controls its phototropic responses. These responses coexist in the same tissue, but are separate and additive (cf. Kagawa and Wada, 1966). Exposure to red light has the opposite effects on motor cells in the extensor (expansion) and flexor (contraction) sectors of the pulvinus (as expected in photonastic responses of the pulvinus) whereas exposure to blue light has the same effect (contraction) on either (Koller et al., 1996). 3. Role of Pulvinar Chloroplasts Well developed chloroplasts with multilayered grana stacks are present in pulvinar motor cells of bean. These pulvinar chloroplasts contribute to its response to red light. Their pigment composition and activity of the xanthophyll cycle are similar to those of mesophyll chloroplasts. They perform photosynthetic electron transport and quench fluorescence nonphotochemically, showing that they build up a considerable transthylakoid proton gradient in the light. Results of pretreatment of the pulvinus with DCMU on its response to red (500 J.Lmol m-2 s-1), and blue light (50 J.Lmol m-2 s-1), and with the ionophore (uncoupler) CCCP on its response to red light, suggest that the pulvinar response to red, but not to blue light, requires non-cyclic electron transport and the resulting generation of ATP (Koller et al., 1995). Red light acts in stomatal opening by stimulating electrogenic proton pumping at the plasmalemma, using ATP supplied by photophosphorylation in the guard cell chloroplasts, but other products of photosynthesis may also be involved (Serrano et al., 1988). Blue light has different effects than red light on membrane polarization of the motor cells in the exposed (adaxial) sector of the pulvinus in the primary leaves of

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Phaseolus. DCMU inhibits their hyperpolarization by red light, but has no effect on their depolarization by blue light. CCCP depolarizes the membranes and prevents the changes in polarization by red, or blue light. These results support the concept that blue light inhibits activity of a protonATPase, causing loss of the electrogenic component of membrane potential. As a result, H+ /K+ fluxes are suppressed, turgor falls and the exposed sector contracts (Nishizaki, 1990, 1992).

VII. ADAPTIVE STRATEGIES OF PLANT MOVEMENTS IN SEARCH OF LIGHT A.

ADAPTATIONS TO THE TERRESTRIAL ENVIRONMENT

Shoots exhibit orthogravitropic growth of the stem. This is one of the basic adaptive strategies of plants in the terrestrial environment, by which the light-harvesting opportunities of the photosynthetic organs are enhanced. Adaptation of these tropisms was a consequence of the universal generalities that in the terrestrial environment the prevailing light is incident from above and that the higher you are above ground level, the more light you can expect to intercept. Therefore, the signal that controls the direction of these movements is gravity, whose vector is perpendicular to the earth's surface and similar to that of prevailing light. However, in the natural environment the availability of light is diurnal and its direction changes throughout the day. Moreover, in many microenvironments, the spatial availability of PAR is not evenly distributed. Plants have adapted to these constraints by developing capabilities for sensing and using specific components of this environment as signals for moving their photosynthetic organs to optimize their interception of the available light. The evolution of biological vectors for pollination has resulted in expansion of these adaptations to include movement of flowers and inflorescences to optimize their interaction with these vectors. The major light signals that are sensed and used in this way are (i) the diurnal light f-t dark transitions (including changes in irradiance ); (ii) the predominant direction of light; (iii) the changes in direction of incident solar radiation; and (iv) changes in the spectral composition of incident light (mainly by reflection from neighbouring plants). B.

DIURNAL MOVEMENTS

The nastic folding and unfolding of leaves, flowers etc. in diurnal periodicity are the most prevalent movements of plants. The adaptive value of (photonastic) unfolding of leaves during the day for their primary function is self-evident. Unfolding of flowers and inflorescences coincides, more or less, with the period of activity of their pollinators (day or night) and makes the reproductive organs at their centre accessible to them. The adaptive value, if

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any, of the diurnal folding is not as evident. It can be argued that the folded configuration of flowers protects their fragile, but attractive organs against unwelcome visitors. The advantages of the nocturnal (skotonastic) folding of leaves are even more debatable. It has been suggested that nocturnal folding affords protection against freezing, or chilling injuries, by reducing exposure of the lamina to the cold night sky and the resulting radiative heat loss. This explanation does not account for the prevalence of nyctinastic leaf folding among plants of warm climates. Alternative suggestions are equally unsatisfactory (Gorton and Satter, 1983). A different approach to understand the adaptive value of nocturnal folding takes into account that the origin of diurnal leaf movements lies in the circadian rhythmic movements of the leaf. Each living organism exhibits an individual circadian rhythm, controlled by the universal biological oscillator, in which an active phase alternates with an inactive one. The latter is characterized by reduced metabolism, as well as by minimizing the interaction with the environment. When mammals sleep, their senses of sight, smell, hearing and touch are virtually inactivated, and they also curl up to a compact configuration. It can be assumed that plant leaves employ a similar strategy. They minimize their interaction with the environment during the metabolically less active phase of their circadian cycle by folding into a compact configuration, and seal up their internal surface by stomatal closure. Plants use the diurnal light H dark transition to reset their individual circadian rhythm daily to a diurnal 24-h cycle, in which their active phase (leaves unfolded, stomata open) coincides with available light, and their inactive phase (leaves folded, stomata closed) coincides with darkness. The designation of the diurnal folding/unfolding of leaves as 'sleep movements' is therefore justifiable. The phytochrome pigment system has been harnessed to accentuate the termination of the active phase by providing an BOD signal. The BAP system has similarly been harnessed to give notice of sunrise, as in stomatal guard cells (Zeiger eta/., 1981 ). C.

DIAPHOTOTROPISM OF GROWING SHOOTS

The adaptive value of the universal positive phototropic response of etiolated seedlings is not self-evident. This response is superimposed on the inherent negative gravitropism of the seedling stem. Very low pFRs are required and the perception of unilateral light takes place at the extremity of the seedling shoot (coleoptile, or hook). The adaptive value of this response may therefore be ascribed to guiding the tip of the (negatively gravitropic) shoot through the larger pores in the soil to the surface, thereby reducing the mechanical resistance that it encounters. Under limiting PAR, de-etiolated seedlings and vigourously growing, apical parts of the shoot, exhibit diaphototropic reorientation of the apical buds and their associated cluster

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of young leaves passively, by positive phototropic curvature of the subtending stem. The perception of phototropically active light by deetiolated seedlings requires considerably higher pFR and takes place in the subapical, actively growing part of the growing stem. By facing the predominant direction of light the expanding photosynthetic organs maximize their interception of PAR. The phototropic curvature of the stem is eventually reversed by its positive gravitropic response. Therefore, the adaptive value of this response is limited to the expanding leaves that are the most exposed to light. D.

DIAPHOTOTROPISM OF EXPANDING LEAVES

Individual leaves may also reorient in the course of their development, moving their lamina to face the predominant direction of light, by growthmediated curvature and/or axial rotation of their petiole and base. The diaphototropic lamina may move laterally, to form leaf mosaics. These growth-mediated curvatures become permanent when the leaf (petiole) matures, and their adaptive value (maximizing interception of PAR) carries over for the lifetime of the leaf. Laminae of cauline leaves of compass plants face east-west vertically and intercept most of the total daily irradiance in the early morning and late afternoon, at near-saturation PAR. Interception of solar radiation is minimal around midday, resulting in lower leaf temperature and transpirational water loss. Photoinhibition and damage from supraoptimal temperature are minimized. Thus, the specific laminar orientation of the cauline leaves of compass plants (Section IV.C.1) optimizes the interception of solar radiation (including PAR) and has adaptive advantage in terms of carbon gain and water use efficiency (Werk and Ehleringer, 1985, 1986a, b; Smith and Ullberg, 1989; Juriketal., 1990). E.

LAMINAR DIAHELIOTROPISM

Solar-tracking enhances the water use efficiency. It may also provide competitive advantage during early growth, when mutual shading is minimal, and enhance solar heating of the young leaves in the early hours (Bonhomme et al., 1974; Mooney and Ehleringer, 1978; Shackel and Hall, 1979; Forseth and Ehleringer, 1983b). Diaheliotropic leaves are exposed to a virtually constant flux of solar radiation (PAR, as well as thermal) throughout most of the day (Ehleringer and Forseth, 1980; Rajendrudu and Rama Das, 1981). Two distinct adaptive strategies have been distinguished. Malvaceous leaves (Malva, Lavatera) track the sun with high fidelity from 1-2 h after sunrise to 1-2 h before sunset, in the natural environment (Yin, 1938; Schwartz and Koller, 1986), as

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well as under simulated conditions (Koller and Levitan, 1989). Laminar orientation deviates from the normal to the sun by «15°, which reduces intercepted radiation by «3%. In addition to maximizing light-harvesting throughout the day, their diaheliotropic response maintains the input of photons into the chloroplasts at a constant level. Photosynthetic rates are not saturated at pFRs exceeding 2.0 mmol m-2 s-1 (Forseth and Ehleringer, 1983b). Diaheliotropic leaves maximize light-use efficiency throughout most of the day by maintaining a constant photosynthetic rate and efficiency of photosystem II (expressed by their chlorophyll fluorescence ratio, F jFm• and photosynthetic oxygen evolution). Leaves with fixed spatial orientation exhibit midday depression in these activities (Sailaja and Rama Das, 1996).The adaptive value of this response is in maximizing photosynthesis and improving water-use efficiency, while economizing by simplifying the photosynthetic apparatus. Diaheliotropic leaves of Malvastrum rotundifolium exhibit a high photosynthetic capacity, unsaturated at full solar irradiances. As water becomes less available, they control leaf water potential throughout by progressive lowering of their osmotic potential (by 1.86 MPa). They continue to exhibit diaheliotropism virtually until they wilt (at leaf water potential of - -4.0 MPa) albeit at progressively decreasing leaf conductance. They also exhibit a high temperature optimum for photosynthesis, which enables them to tolerate the elevated leaf temperatures resulting from the progressive reduction in evaporative cooling (Mooney and Ehleringer, 1978; Forseth and Ehleringer, 1982, 1983a). Lupinus exhibits a different adaptive strategy when exposed to water stress, by modifying the diaheliotropic movement of its palmately compound leaf to a plagioheliotropic one (Section V.F). Leaves of L. arizonicus exhibit progressive 'cupping' (by -31°), accompanied by reduction in leaf conductance, as leaf water potential decreases (from --1.0 to --1.8 MPa), with virtually no change in osmotic potential. However, the concomitant reduction in intercepted solar radiation act as a modulated response by which PAR (as well as thermal radiation) that is intercepted by each leaflet is equally reduced just below saturation of the (reduced) capacity of the photosynthetic apparatus (Mooney and Ehleringer, 1978; Forseth and Ehleringer, 1983a). F. PULVINAR HELIOTROPISM

Trifoliate leaves of many legumes exhibit heliotropic movements throughout the day. These movements appear diaheliotropic during the morning and late afternoon, and paraheliotropic during the midday hours (Darwin and Darwin, 1881; Wilson and Greenman, 1892-97; Stevenson and Shaw, 1971; Varlet-Grancher and Bonhomme, 1972; Ludlow and Bjorkman, 1984). However, the lamina is oriented passively by pulvinar phototropism,

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according to the unilateral perception of light by the pulvinus. This is expressed in their reduced capacity to track the sun and intercept its radiation throughout the day. The individual leaflets of bean, exposed by means of a solar simulator to a simulated 'solar' transit in a vertical arc, across or along its major axis (tip H base), reorient their laminae in different directions with respect to the 'solar' position, that only occasionally coincide. These movements enhance their interception of 'solar' radiation, but to a considerably lesser degree than in (malvaceous) leaves that exhibit laminar heliotropism (Ritter and Koller, 1994) (Fig. 5). Diaheliotropism may be exhibited in different species of the same genus (e.g. Lupinus: Wainwright, 1977; Vogelmann and Bjorn, 1983; Werker et al., 1991) or in several related genera (e.g. Malva and Lavatera: Yin, 1938; Schwartz and Koller, 1978), or it may be species specific (e.g. Gossypium: Ehleringer and Hammond, 1987). Ehleringer and Forseth (1989) have reviewed the significance of these movements to the productivity of leaf canopies. G. STRESS-MODIFIED PULVINAR RESPONSE

The midday transformation of the (apparent) diaheliotropic response to an (apparent) paraheliotropic one may be ascribed to modification of the pulvinar phototropic responses by stresses resulting from inadequate water relations, supra-optimal temperature and high irradiance around midday (Dubetz, 1969; Kawashima, 1969; Wien and Wallace, 1973; Begg and Torssell, 1974; Sato and Gotoh, 1983; Berg and Hsiao, 1986; Fu and Ehleringer, 1989; Berg and Heuchelin, 1990). Stress-modified laminar movements reduce the angle of incidence of sunlight on the leaf and the flux of solar energy into its lamina, and provide protection against hazards resulting from such stresses to primary photosynthetic reactions. Leaves prevented from stress-mediated reorientation, by restraining them horizontally, become overheated and exhibit lower leaf water potentials (Berg and Hsiao, 1986), as well as progressive symptoms of photoinhibition (Powles and Bjorkman, 1981; Ludlow and Bjorkman, 1984; Sheriff and Ludlow, 1985). However, these responses have also been observed in wellwatered plants (Kawashima, 1969; Forseth and Ehleringer, 1980, 1988; Berg and Hsiao, 1986). Furthermore, they occur several hours in advance of the midday increase in xylem water potential and are eventually reversed even when xylem water potential remains low (Travis and Reed, 1983). In soybean (Glycine max) they are associated with an increase in adaxial stomatal resistance, while the abaxial resistance remains low ( Oosterhuis eta/., 1985). Therefore, they are not necessarily mediated by reduced leaf water potential, transpiration, or conductance and are probably attributable to direct effects of elevated pulvinar temperature (Ludlow and Bjorkman, 1984; Fu and Ehleringer, 1989). The dependence of stress-modified

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movements on pulvinar temperature was the same at 355 and <3 cm3 m-3 ambient carbon dioxide, suggesting that photosynthetic carbon fixation is not involved in such stress-modification (Fu and Ehleringer, 1989). The trifoliate leaves of the shade plant Oxalis oregana fold down (their skotonastic orientation) in response to a sudden increase in irradiance (sunfleck) by the same blue light that causes their photonastic unfolding (Section VI.F). Exposing the leaflets to the full irradiance, by restraining them horizontally, results in photoinhibition. This is the only unequivocal example of an adaptation of a photonastic response for avoidance of high irradiance stress (Bjorkman and Powles, 1981) that does not involve pulvinar phototropism. Stress-related laminar movements and stomatal closure reduce evaporative loss of water from the leaf, by different means and with different consequences. Whereas stomatal closure aggravates the energy balance of the lamina by reducing its evaporative cooling, leaf movements improve its energy balance by reducing the interception of radiant energy, at the cost of reduced photosynthesis (Shackel and Hall, 1979; Forseth and Ehleringer, 1980, 1982). High irradiance probably poses a stress to the leaf only when photosynthetic activity is inhibited, by supra-optimal temperature or by leaf water-stress. Stomatal closure resulting from water stress reduces photosynthesis by its effects on availability of carbon dioxide, as well as by reducing evaporative cooling, that elevates leaf temperature. In dense canopies, the reduced photosynthesis resulting from the stress-related (apparent) paraheliotropic modification is compensated by increased penetration of light to the underlying leaves (Wien and Wallace, 1973; Travis and Reed, 1983). H.

DIAHELIOTROPISM IN FLOWERS

Diaheliotropism of flowers (inflorescences) of plants in Arctic ( > 80° N) and high elevation (3550 feet (1082 m) in the Andean Paramo) regions contributes to their reproductive efficiency by focusing solar radiation, resulting in elevated temperature of, or just above their reproductive organs. In Oritrophium limnophilum (Compositae), air temperature immediately above the flower disc exceeded the ambient, and this was correlated with the number of insect visits. In Papaver radicatum (Papavaraceae) and Dryas integrifolia (Rosaceae) the corolla acts as a spherical/parabolic mirror of a solar furnace and its highly reflective adaxial surface focuses solar radiation on the gynoecium, raising its temperature (in Papaver by as much as lOoC) above the ambient. In Papaver, the gynoecium is exposed to such focused light as long as the flower tracks the sun within ~24°. The focused solar radiation also raises the body temperature of anthophilous insects visiting the flower. Under the ambient conditions

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prevailing in such habitats, the elevated temperature may accelerate metabolism of the reproductive organs of the plant, as well as that of the insect visitors (Kevan, 1972, 1975). These findings were expanded to show that (in Dryas octopetala) solar tracking enhances seed development (Kjellberg et al., 1982). In warmer habitats, diaheliotropism of entomophilous flowers (inflorescences), such as sunflower, increases the interception of solar radiation, as well as its reflection throughout the day. These features may enhance attraction of pollinators and possibly accelerate flower and seed development as well.

VIII.

PERSPECTIVES

Phototropic responses of plants have attracted the attention of many scientists for a considerable time. The resulting studies were multidisciplinary, and have provided a remarkable amount of spin-off. Great progress has been made in research of the classical phototropic response by coleoptiles of grass seedlings and hypocotyls of dicotyledonous seedlings. Such progress is particularly noteworthy in the identification of the photoreceptor responsible for phototropism, its chromophore and the molecular consequences of its excitation by blue-UV (and green) light. This photoreceptor (phototropin) is incapable of suppressing elongation in response to blue light. A different photoreceptor for blue-UV light is responsible for suppression of hypocotyl elongation by reduction in cell wall extensibility, but is incapable of causing phototropic curvature. Phototropic curvature cannot be accounted for by inhibition of elongation (reduced extensibility) of the cells that intercept blue light. An alternative possibility is that activity of phototropin in growth-mediated phototropism depends on the existence of a light gradient across the hypocotyl. Turgor-mediated pulvinar phototropism depends on the existence of a transpulvinar light gradient and takes place by creation of transpulvinar transport of solutes and water. A blue-UV light gradient across the hypocotyl may cause a similar transport (but not necessarily of the same ingredients) resulting in the differential growth of phototropic curvature. Movements of pulvinated leaves have also been the subject of numerous researches. Attention was first focused on the circadian movements, as possible keys with which to unlock the mysteries of the biological clock. When this objective proved elusive, attention shifted to studies of the mechanisms of ion transport by which turgor-mediated leaf movement take place. At first, these studies were still focused on the circadian activity, but were soon expanded to the signal transduction events by which light H dark transitions modify the circadian movements to diurnal movements. These studies benefited much from symbiosis with the extensive and productive activity in the related field of stomatal physiology. Blue light causes

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expansion of stomatal guard cells and leaf unfolding, both of which involve turgor-mediated cell expansion, but only one sector of the pulvinus expands during unfolding, while its opposite contracts. It is still not quite clear whether the flexor and extensor sectors of the pulvinus indeed respond directly, in opposite ways, to their exposure to the phytochrome signal that controls skotonastic folding, and to the BAP signal that controls photonastic unfolding. Alternatively, the flexor and extensor may have direct control of either folding, or unfolding, and their opposite sector participates passively. Studies of mechanisms involved in growth-mediated diurnal movements, particularly of floral organs, appear to be absent. Phototropic responses of the pulvinus contribute an additional complexity, because unilateral exposure of any pulvinar sector, including the extensor and flexor, to blue light results in its contraction. The relationship between the phototropic and photonastic response, as well as the similarity between the BAP photoreceptors, remain to be clarified. Different photoreceptors, with different downstream elements in their transduction chain may be involved, as in hypocotyls. An obvious starting point for such studies is with mutants. This may prove more difficult than in seedlings, because plants with mature leaves are required. For obvious reasons, studies of the ion transport mechanisms associated with volume changes of pulvinar motor cells have lagged behind those of stomatal guard cells. Much remains to be learned about the differences in the transduction sequences and transport mechanisms of the flexor and extensor in their nastic response to phytochrome and blue light. Even less is known about these aspects in the phototropic response. The capacity of perceiving directional light signals as vectorial excitation appears to be limited to a few species with solar-tracking leaves. This view may be deceptively narrow. Vectorial excitation may also operate in perception of the solar signal by foliar organs in growth-mediated solartracking, in solar-tracking by flowers and inflorescences, as well as in diaphototropic responses of developing leaves. Vectorial excitation takes place by means of an unidentified BAP. In view of the involvement of red light and chloroplasts in pulvinar phototropism, these aspects should also be studied in vectorial excitation. The hypothesis explaining the opposite responses to oppositely oriented vectorial excitations awaits experimental evaluation. Solar-tracking and nocturnal reorientation appear to be complementary components of diurnal diaheliotropism. Studies of the nocturnal phase might prove rewarding. The control of the direction of nocturnal reorientation by the preceding solar-tracking experience is intriguing, and amenable to experimentation. Directional light signals perceived by foliar organs appear to be able to exert remote control of phototropism of their support. The nature of the transmitted signal and the mechanism for such remote control are unknown.

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REFERENCES Alexandre, J., Lassalles, J.P. and Kado, R. T. (1990). Opening of Ca 2+ channels in isolated red beet vacuole membrane by inositol 1,4,5-trisphosphate. Nature (London) 343, 567-570. Allen, G., Muir, S. and Sanders, D. (1996). Control of Ca2 + channels at the vacuolar membrane of Plant Cells (Abstract, SEB Lancaster Meeting). Journal of Experimental Botany 47, 65. Allen, G. J. and Sanders, D. (1997). Vacuolar ion channels of higher plants. Advances in Botanical Research 25, 217-252. Asard, H., Horemans, N., Briggs, W. R. and Caubergs, R.J. (1995). Blue light perception by endogenous redox components of the plant plasma membrane. Photochemistry and Photobiology 61,518-522. Assman, S. M. (1993). Signal transduction in guard cells. Annual Review of Cell Biology 9, 345-375. Ball, N. G. (1923). Phototropic movements of leaves. The functions of the lamina and the petiole with regard to the perception oft he stimulus. Proceedings of the Royal Dublin Society, N.S. 17, 281-286. Ballare, C. L., Scope!, A. L., Radosevich, S. R. and Kendrick, R. E. (1992). Phytochrome-mediated phototropism in de-etiolated seedlings. Occurrence and ecological significance. Plant Physiology 100, 170-177. Begg, J. E. (1980). Morphological adaptations of leaves to water stress. In: "Adaptations of Plants to Water and High Temperature Stress" (N.C. Turner and P. J. Kramer, eds), pp. 33-42. Wiley. New York. Begg, J. E. and Torssell, B. W. R. (1974). Diaphotonastic and parahelionastic leaf movements in Stylosanthes humilis HBK (Townsville Stylo). Bulletin of the Royal Society New Zealand 12, 277-283. Berg, V. S. and Heuchelin, S. (1990). Leaf orientation of soybean seedlings. I. Effects of water potential and photosynthetic photon flux density. Crop Science 30, 631-638. Berg, S. V. and Hsiao, T. C. (1986). Solar tracking, Light avoidance influenced by water stress in leaves of kidney bean seedlings in the field. Crop Science 26, 980-986. Bialczyk, J. and Lechowski, Z. (1989). Malic acid synthesis in relation to K+ and CJavailability in Phaseolus coccineus L. pulvini. Biochemie und Physiologie der Pflanzen 184, 79-86. Bialczyk, J. and Lechowski, Z. (1990). Effects of light quality on malic acid synthesis in Phaseolus coccineus pulvini. Plant Physiology and Biochemistry 28, 315-320. Bialczyk, J. and Lechowski, Z. (1992). Influence of different spectral regions of light and Ca2+ channel blockers on Ca2 + and K+ levels in Phaseolus coccineus L. pulvini. Acta Societatis Botanicorum Poloniae 61, 241-252. Bilderback, D. E. (1984a). Phototropism in Selaginella, the differential response to light. American Journal of Botany 71, 1323-1329. Bilderback, D.E. (1984 b). Phototropism in Selaginella, the role of the small dorsal leaves and auxin. American Journal of Botany 71, 1330-1337. Bjorkman, 0. and Demmig-Adams, B. (1994) Regulation of photosynthetic light energy capture, conversion and dissipation in leaves of higher plants. In "Ecophysiology of Photosynthesis" (E-.D. Schultze and M. M. Caldwell, eds), Chapter 2, pp. 17-47. Springer, Berlin. Bjorkman, 0. and Powles, S. B. (1981). Leaf movement in the shade species Oxalis oregana. I. Response to light level and light quality. Carnegie Institute of Washington Yearbook 80, 59-62.

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Black, M. and Shuttleworth, J. E. (1974). The role of the cotyledons in the photocontrol of hypocotyl extension in Cucumis sativus L. Planta 117, 57-66. Blatt, M. and Briggs, W. R. (1980). Blue light induced cortical fiber reticulation concomitant with chloroplast aggregation in the alga Vaucheria sessilis. Planta 147, 355-362. Blatt, M. R. and Grabov, A (1997). Signal redundancy, gates and integration in the control of ion channels for stomatal movement. Journal of Experimental Botany 48, 529-537. Blatt, M., Weisenseel, M. H. and Haupt, W. (1981). A light-dependent current associated with chloroplast aggregation in the alga Vaucheria sessilis. Planta 152, 513-526. Blatt, M. R., Thiel, G. and Trentham, D. R. (1990). Reversible inactivation of K+ channels of Vicia stomatal guard cells following the photolysis of caged inositol1,4,5-trisphosphate. Nature (London) 346, 766-769. Blum, D. E., Elzenga, T. M., Linnemeyer, P. A and Van Volkenburgh, E. (1992). Stimulation of growth and ion uptake in bean leaves by red and blue light. Plant Physiology 100, 1968-1975. Bonhomme, R., Varlet-Grancher, C. and Artis, P. (1974). Utilizationde l'energie solaire par une culture de Vigna sinensis. II. Assimilation nette et acroisement de matiere seche, influence du phototropisme sur Ia photosynthese des premieres feuilles. Annates Agronomiques 25, 49-60. Bossen, M. E., Dassen, H. H. A, Kendrick, R. E. and Vredenberg, W. J. (1988). The role of calcium ions in phytochrome-controlled swelling of etiolated wheat (Triticum aestivum L.) protoplasts. Planta 174,94-100. Bossen, M. E., Kendrick, R. E. and Vredenberg, W. J. (1990). The involvement of a G-protein in phytochrome-regulated, Ca2+ -dependent swelling of etiolated wheat protoplasts. Physiologia Plantarum 80, 55-62. Brauner, L. and Brauner, M. (1947). Untersuchungen uber der Mechanismus der phototropischen Reaktion der Blattfiedern von Robinia pseudacaia. Revue de Ia Faculte des Sciences de l'Universite Istanbul12B, 35-79. Brauner, L. and Vardar, Y. (1950). Uber die Funktion der Lamina bei der phototropischen und geotropischen Reaktion des Tropaeolum-blattes. Revue de Ia Faculte des Sciences de l'Universite Istanbul15B, 269-299. Brauner, M. (1932). Untersuchungen uber die Lichtturgorreaktionen des Primiirblattgelenkes von Phaseolus multijlorus. Planta 18, 288-337. Britz, S. J. (1979). Chloroplast and nuclear migration. In "Physiology of Movements. Encyclopedia of Plant Physiology (NS), Volume 7" (W. Haupt and M. E. Feinleib, eds), pp. 170-206. Springer. Berlin. Bruinsma, J. and Hasegawa, K. (1990). A new theory of phototropism- its regulation by a light-induced gradient of auxin-inhibiting substances (Review). Physiologia Plantarum 79, 700-704. Bruinsma, J., Karssen, C. M., Benschop, M. and Dort, J. B. V. (1975). Hormonal regulation of phototropism in the light-grown sunflower seedling, Helianthus annuus L., Immobility of endogenous indoleacetic acid and inhibition of hypocotyl growth by illuminated cotyledons. Journal of Experimental Botany 26, 411-418. Bunning, E. (1959).Tagesperiodische Bewegungen. In "Encyclopedia of Plant Physiology, Volume XVII/1" (W. Ruhland, ed.), pp. 579-656. Springer. Berlin. Bunning, E. (1973). "The Physiological Clock" 3rd edition. The English Universities Press, London.

PLANTS IN SEARCH OF SUNLIGHT

117

Burkholder, P. K. and Pratt, R. (1936a). Leaf movements of Mimosa pudica in relation to light. American Journal of Botany 23, 46-52. Burkholder, P. K. and Pratt, R. (1936b). Leaf movements of Mimosa pudica in relation to intensity and wavelength of the incident radiation. American Journal of Botany 23, 212-220. Campbell, N. and Thomson, W. W. {1977). Multivacuolate motor-cells in Mimosa pudica L.Annals of Botany 41, 1361-1362. Campbell, N. A. and Garber, R. C. {1980). Vacuolar reorganization in the motor cells ofAlbizzia during leaf movement. Planta 148, 251-255. Campbell, N. A., Satter, R. L. and Garber, R. G. (1981). Apoplastic transport of ions in the motor organ of Samanea. Proceedings of the National Academy of Sciences of the USA 78, 2981-2984. Cho, M. H. and Spalding, E. P. {1996). An anion channel inArabidopsis activated by blue light. Proceedings of the National Academy of Sciences of the USA 93, 8134-8138. Christie, J. M., Reymond, P., Powell, G. K., Bernasconi, P., Raibekas, A. A., Liscum, E. and Briggs, W. R. {1998). Arabidopsis NPH1: a flavoprotein with the properties of a photoreceptor for phototropism. Science 282, 1698-1701. Christie, J. M., Salomon, M., Nozue, K., Wada, M. and Briggs, W. R. (1999). LOY {light, oxygen, or voltage) domains for the blue-light photoreceptor phototropin (nph 1): binding sites for the chromophore flavin ononucleotide. Proceedings of the National Academy of Sciences of the USA 96, 8779-8783. Cosgrove, D. J. (1983). Photocontrol of extension growth. Proceedings of the Royal Society, London 303, 453-465. Cosgrove, D. J. (1985). Kinetic separation of phototropism from blue light inhibition of stem elongation. Photochemistry and Photobiology 42, 745-751. Cosgrove, D. J. (1987). Wall relaxation and the driving forces for cell expansive growth. Plant Physiology 84, 561-564. Cosgrove, D. J. (1988). Mechanism of rapid suppression of cell expansion in cucumber hypocotyls after blue light irradiation. Planta 176, 109-116. Cote, G. G. and Crain, R. C. (1994). Why do plants have phosphoinositides? BioEssay 16, 39-46. Cote, G. G., Yueh, Y. G. and Crain, R. C. (1996). Phosphoinositide turnover and its role in plant signal transduction. In "Subcellular Biochemistry, Volume 26" (B. B. Biswas and R. Biswas, eds), pp. 317-343. Plenum, New York. Cronlund, S. L. and Forseth, I. N. (1995). Heliotropic leaf movement response to H+/ATPase activation, H+/ATPase inhibition, and K+ channel inhibition in vivo.American Journal of Botany 82, 1507-1513. Darwin, C. and Darwin, F. (1881 ). "The Power of Movement in Plants". D. Appleton & Co. (reprinted 1966, De Capo Press), New York Davies, J. M. (1997). Vacuolar energization, pumps, shunts and stress. Journal of Experimental Botany 48, 633-641. Dayanandan, P., Hebard. F. V. and Kaufman, P. B. (1976). Cell elongation in the grass pulvinus in response to geotropic stimulation and auxin application. Planta 131, 245-252. Dolk, H. E. (1931). The movement of the leaves of the compass plant Lactuca scariola. American Journal of Botany 18, 195-204. Donahue, R. and Berg, V. S. (1989). Leaf orientation of soybean (Glycine max) seedlings. II. Receptor sites and light stimuli. Crop Science 30, 638-643. Donahue, R. A., Berg, V. S. and Vogelmann, T. C. (1990). Assessment of the potential of blue light gradient in the soybean pulvini as a leaf orientation signal. Physiologia Plantarum 19, 593-598.

118

D. KOLLER

Dubetz, S. (1969). An unusual photonastism induced by drought in Phaseolus vulgaris. Canadian Journal of Botany 47, 1640-1641. Ehleringer, J. and Forseth, I. (1980). Solar tracking by plants. Science 210, 1094-1098. Ehleringer, J. R. and Forseth, I. (1989). Diurnal leaf movements and productivity in canopies. In "Plant Canopies, Their Growth, Form and Function" (G. Russell, B. Marshall and P. G. Jarvis, eds) SEB Seminar Series, 31 (1988), pp.129-142. Cambridge University Press, Cambridge. Ehleringer, J. and Hammond, S. D. (1987). Solar tracking and photosynthesis in cotton leaves. Agricultural and Forest Meteorology 39, 25-35. Ellis, R. J. (1984). Kinetics and fluence response relationships of phototropism in the dicot Fagopyrum esculentum Moench (Buckwheat). Plant and Cell Physiology 25, 1513-1520. Elzenga, J. T. M. and Prins, H. B. A. (1989). Light induced polar pH changes in leaves of Elodea canadensis. I. Effects of carbon concentration and light intensity. Plant Physiology 91, 62-67. Elzenga, J. T. M. and Van Volkenburgh, E. (1997a). Characterization of a lightcontrolled anion channel in the plasma membrane of mesophyll cells of pea. Plant Physiology 113, 1419-1426. Elzenga, J. T .. M. and Van Volkenburgh, E. (1997b). Kinetics of Ca 2+- and ATPdependent, voltage-controlled anion conductance in the plasma membrane of mesophyll cells of Pisum sativum. Planta 201, 415-423. Elzenga, J. T. M., Staal, M. and Prins, H. B. A. (1997). Calcium-calmodulin signaling is involved in light-induced acidification by epidermal leaf cells of pea, Pisum sativum L. Journal of Experimental Botany 48, 2055-2060. Erath, F., Ruge, W. A., Mayer, W.-E. and Hampp, R. (1988). Isolation of functional extensor and flexor protoplasts from Phaseolus coccineus L. pulvini, potassium induced swelling. Planta 173,447-452. Evans, L. T. and Allaway, W. G. (1972). Action spectrum for the opening ofAlbizzia julibrissin pinnules, and the role of phytochrome in the closing movements of pinnules and stomata of Vida faba.Australian Journal ofBiological Sciences 25, 885-893. Firn, R. D. (1990). Phototropism- The need for a sense of direction. Photochemistry and Photobiology 52, 225-260. Firn, R. D. (1994). Phototropism. In "Photomorphogenesis in Plants" 2nd edition (R. E. Kendrick and R. H. M. Kronenberg, eds), pp. 659-681. Kluwer, Dordrecht. Fischer-Schliebs, E., Mariaux, J.-B. and Liittge, U. (1997). Stimulation of H+ transport activity of vacuolar H+ -ATPase by activity of H+ -PPase in Kalanchoe blossfeldiana. Biologia Plantarum 39, 169-177. Fisher, F. J. F. and Fisher, P.M. (1983). Photosynthetic patterning, a mechanism for sun-tracking. Canadian Journal of Botany 61, 2632-2640. Fisher, F. J. F. and Wright, M. (1984). The dependence of sun-tracking in Lavatera cretica L. upon carbon dioxide availability. New Phytologist 98, 241-248. Fisher, F. J. F., Ebert, D. L. and Hollingdale, J. (1987). The pattern of vascular deployment near the pulvinus of the solar-tracking leaf of Lavatera cretica (Malvaceae ). Canadian Journal of Botany 65, 2109-2117. Fisher, F. J. F., Ebert, D. L. Lister, G. R. and Hollingdale, J. (1989). Light quality and sun tracking in Malva neglecta. Canadian Journal of Botany 67, 515-520. Fondeville, J. C., Borthwick, H. A. and Hendricks, S. B. (1966). Leaflet movements in Mimosa pudica L. indicative of phytochrome action. Planta 69, 357-364.

PLANTS IN SEARCH OF SUNLIGHT

119

Fondeville, J. C., Schneider, M. J., Borthwick, H. A. and Hendricks, S. B. (1967). Photocontrol of Mimosa pudica leaflet movement. Planta 15, 228-238. Forseth, I. and Ehleringer, J. (1980). Solar tracking response to drought in a desert annual. Oecologia 44, 159-163. Forseth, I. and Ehleringer, J. (1982). Ecophysiology of two solar-tracking desert winter annuals. II. Leaf movements, water relations and microclimate. Oecologia 54,41-49. Forseth, I. and Ehleringer, J. (1983a). Ecophysiology of two solar-tracking desert winter annuals. III. Gas exchange espouses to light, C02, and VPD in relation to long-term drought. Oecologia 57, 344-351. Forseth, I. and Ehleringer, J. (1983b ). Ecophysiology of two solar-tracking desert winter annuals. IV. Effects of leaf orientation on calculated daily carbon gain and water use efficiency. Oecologia 58, 10-18. Frankland, B. and Taylorson, R. (1983). Light control of seed germination. In "Photomorphogenesis. Encyclopedia of Plant Physiology (NS), Vol. 16A" (W. Shropshire, Jr and H. Mohr, eds), pp. 428-456. Springer. Berlin. Franssen, J. M. and Bruinsma, J. (1981). Relationships between Xanthoxin, phototropism and elongation growth in the sunflower seedling Helianthus annuus L. Planta 151, 365-370. Franssen, J. M., Digby, J. and Firn, R. D. (1982). An investigation of the importance of transpiration gradients in the control of phototropic curvature in coleoptiles of Avena sativa cultivar Victory. Australian Journal of Plant Physiology 9, 583-586. Freudling, C., Meyer, W.-E. and Gradmann, D. (1980). Electrical membrane properties and circadan rhythm in extensor cells of laminar pulvini of Phaseolus coccineus L. Plant Physiology 65, 966-968. Freudling, C., Starrach, N. Flach, D., Gradmann, D. and Meyer, W.-E. (1988). Cell walls as reservoirs of potassium ions for reversible changes in pulvinar motor cells during rhythmic leaf movements. Planta 175, 193-203. Fu, Q.A. and Ehleringer, J.R. (1989). Heliotropic leaf movements in common beans controlled by air temperature. Plant Physiology 91, 1162-1167. Fu, Q. A. and Ehleringer, J. R. (1991). Modification of paraheliotropic leaf movements in Phaseolus vulgaris by photon flux density. Plant, Cell and Environment 14, 339-344. Gilroy, S., Read, N.D. and Trewavas, A. J. (1990). Elevation of cytoplasmic calcium by caged calcium or caged inositol trisphosphate initiates stomatal closure. Nature (London) 346, 769-771. Giromini, L., Camattari, G., Cerana, R. and Colombo, R. (1997). Effect of apoplastic and cytoplasmic pH on inward and outward rectifying plasma membrane K+ channels of Arabidopsis thaliana cultured cells. Journal of Plant Physiology 151, 682-688. Gleed, D., Firn, R. D. and Digby, J. (1994). How is phototropic stimulus perceived by hypocotyls? Journal of Experimental Botany 45, 409-412. Goh, C.-H., Kinoshita, T., Oku, T. and Shimazaki, K. (1996). Inhibition of blue lightdependent H+ pumping by abscisic acid in Vicia guard cell protoplasts. Plant Physiology 111, 433-440. Gomez, L. A. and Simon, E. (1995). Circadian rhythm of Robinia pseudoacacia leaflet movements, role of calcium and phytochrome. Photochemistry and Photobiology 61, 210-215. Gorton, H. L. (1987). Water relations in pulvini from Samanea saman. I. Intact pulvini. Plant Physiology 83, 945-950.

120

D. KOLLER

Gorton, H. L. (1990). Stomates and pulvini, A comparison of two rhythmic, turgormediated movement systems. In "The Pulvinus, Motor Organ for Leaf Movement" (R. L. Satter, H. L. Gorton and T. C. Vogelmann, eds), pp. 223-237. American Society of Plant Physiologists, Rockville. Gorton, H. L. and Satter, R. (1983). Circadian rhythmicity in leaf pulvini. BioScience 33, 451-457. Haberlandt, G. (1905). "Die Lichtsinneorgane der Laubbliitter". Leipzig. Haberlandt, G. (1914). The motor system. Optical sense organs. In "Physiological Plant Anatomy", 1965 reprint edition (translated from the 4th German edition by M. Drummond), pp. 613-631. Today and Tomorrow's Printers & Publishers, India. Harder, R., Schumacher, W., Firbas, F. and von Denffer, D. (1965). "Strassburger's Textbook of Botany", New English Edition (translated from the 28th German edition). Gustav Fischer, Berlin; Longmans Green, Berlin. Hart, J. W. and MacDonald, I. R. (1981). Phototropic responses of hypocotyls of etiolated and green seedlings. Plant Science Letters 21, 151-158. Haupt, W. and Hader, D-.P. (1994) Photomovement. In "Photomorphogenesis in Plants" 2nd edition (R. E. Kendrick and R. H. M. Kronenberg, eds), pp. 707-732. Kluwer. Dordrecht. Hayami, J., Kadota, A and Wada, M. (1986). Blue-light-induced phototropic response and the intracellular photoreceptive site in Adiantum protonemata. Plant and Cell Physiology 21, 1571-1577. Hedrich, R. and Becker, D. (1994). Green circuits- the potential of plant-specific ion channels. Plant Molecular Biology 26, 1637-1650. Hedrich, R., Bush, H. and Raschke, K. (1990). Ca2+ and nucleotide dependent regulation of voltage dependent anion channels in plasma membrane of guard cells. EMBO Joumal9, 3889-3992. Herbert, T. J. (1984). Axial rotation of Erythrina herbacea leaflets. American Journal of Botany 11, 76-79. Hillman, W. S. and Koukkari, W. L. (1967). Phytochrome effects in nyctinastic leaf movements ofAlbizzia julibrissin and some other legumes. Plant Physiology 42, 1413-1418. Holmes, M.G. and Klein, W. H. (1986). Photocontrol of dark circadian rhythms in stomata of Phaseolus vulgaris L. Plant Physiology 82, 28-33. Horwitz, B. A (1994). Properties and transduction chains of the UV and blue light photoreceptors. In "Photomorphogenesis in Plants" 2nd edition (R. E. Kendrick and R. H. M. Kronenberg, eds), pp. 327-350. Kluwer, Dordrecht. Hwang, J. U., Sub, S., Kim, J. and Lee, Y. (1997). Actin filaments modulate both stomatal opening and inward K+ channel activities in guard cells of Vicia faba L. Plant Physiology 115, 335-342. Iglesias, A and Satter, R.L. (1983a). H+ fluxes in excised Samanea motor tissue. I. Promotion by light. Plant Physiology 72, 564-569. Iglesias, A and Satter, R. L. (1983b ). H+ fluxes in excised Samanea motor tissue. II. Rhythmic properties. Plant Physiology 72, 570-572. Iino, M. (1990). Phototropism, mechanics and ecological implications. Plant, Cell and Environment 13, 633-650. Irving, M. S., Ritter, S., Tomos, A. D. and Koller, D. (1997). Phototropic response of the bean pulvinus: movement of water and ions. Botanica Acta 110, 118-126. Jaffe, M. J. and Galston, A W. (1967). Phytochrome control of rapid nyctinastic movements and membrane permeability in Albizzia julibrissin. Planta 77, 135-141.

PLANTS IN SEARCH OF SUNLIGHT

121

Janoudi, A.-K., Konjevic, R. and Whitelam, G. (1997). Both phytochrome A and phytochrome B are required for the normal expression of phototropism in Arabidopsis thaliana seedlings. Physiologia Plantarum 101, 278-282. Jones, W. N. (1938). Observations on the response of leaves of Limnanthemum and Tropaeolum to light and gravity. Annals of Botany N.S. 2, 819-825. Jurik, T. W., Zhang, H. and Pleasants J. M. (1990). Ecophysiological consequences of non-random leaf orientation in the prairie compass plant, Silphium laciniatum. Oecologia 82, 180-186. Kagawa, T. and Wada, M. (1996). Phytochrome and blue-light-absorbing pigmentmediated directional movement of chlorioplasts in dark-adapted prothallial cells of the fern Adiantum as analyzed by microbeam irradiation. Planta 198, 488-493. Kawashima, R. (1969). Studies on the leaf orientation-adjusting movement in soybean plants. I. The leaf orientation-adjusting movement and light intensity on leaf surface. Proceedings of the Crop Science Society of Japan 38, 718-729. Kevan, P. G. (1972). Heliotropism in some Arctic flowers. Canadian Field Naturalist 86, 41-44. Kevan, P. G. (1975). Sun-tracking solar furnaces in high Arctic flowers, significance for pollination and insects. Science 189, 723-726. Khurana, J.P. and Poff, K. L. (1989). Mutants of Arabidopsis thaliana with altered phototropism. Planta 178, 400-406. Kigel, J. and Schwartz, A. (1990). Co-operative effects of blue and red light in the inhibition of hypocotyl elongation of de-etiolated castor bean. Plant Science Letters 21, 83-88. Kim, H. Y., Cote, G. G. and Crain, R. C. (1992). Effects of light on the membrane potential of protoplasts from Samanea saman pulvini. Plant Physiology 99, 1532-1539. Kim, H. Y., Cote, G. G. and Crain, R. C. (1993). Potassium channels in Samanea saman protoplasts controlled by phytochrome and the biological clock. Science 260, 960-962. Kim, H. Y., Cote, G. G. and Crain, R. C. (1996). Inositol 1,4,5-trisphosphate may mediate closure of K+ channels by light and darkness in Samanea saman motor cells. Planta 198, 279-287. Kinoshita, T., Nishimura, M. and Shimazaki, K. (1996). Cytosolic concentration of Ca 2+ regulates the plasma membrane H+ -ATPase in guard cells of the fava bean. Plant Cell7, 1333-1342. Kiyosawa, K. and Tanaka, H. (1976). Changes in potassium distribution in a Phaseolus pulvinus during circadian movement of the leaf. Plant and Cell Physiology 17, 289-298. Kjellberg, B., Karlsson, S. and Kestensson, I. ( 1982). Effects of heliotropic movements of flowers of Dryas octopetalla on gynoecium temperature and seed develoP.ment. Oecologia 54, 10-13. Kniep, H. (1907). Uber die Lichtperzeption der Laubblatter. Biologische Zentralblatt 27,97-106; 129-142. Kohji, J. Yamamoto, R. and Masuda, Y. (1995). Gravitropic response of Eichhornia crassipes (water hyacinth) I. Process of gravitropic bending in the peduncle. Journal of Plant Research 108, 387-393. Koller, D. (1990). Light-driven leaf movements (Review). Plant, Cell and Environment 13, 615-632. Koller, D. and Cohen, M. (1989). After-effects of vectorial photo-excitation in solartracking leaves of Lavatera cretica L. A kinetic analysis. Journal of Plant Physiology 133, 702-709.

122

D. KOLLER

Koller, D. and Levitan, I. (1989). Diurnal phototropism in leaves of Lavatera cretica L. under conditions of simulated solar-tracking. Journal of Experimental Botany 218, 1059-1064. Koller, D. and Ritter, S. (1991). Diaphototropic responses to polarized light in the solar-tracking leaf of Lupinus palaestinus Boiss. (Fabaceae). Journal of Plant Physiology 138, 322-325. Koller, D. and Ritter, S. (1994). Phototropic responses of the pulvinule and associated laminar reorientation in the trifoliate leaf of bean Phaseolus vulgaris (Fabaceae ). Journal of Plant Physiology 143, 52-63. Koller, D. and Shak, T. (1990). Light-driven movements in the solar-tracking leaf of Lupinus palaestinus Boiss. (Fabaceae ). Photochemistry and Photobiology 52, 187-195. Koller, D., Levitan, I. and Briggs, W. R. (1985a). The vectorial photo-excitation in solar-tracking leaves of Lavatera cretica (Malvaceae). Photochemistry and Photobiology 42, 717-723. Koller, D., Levitan, I. and Briggs, W. R. (1985b). Components of vectorial photoexcitation in solar-tracking leaves of Lavatera cretica (Malvaceae ). Physiologie Vegetale 23, 913-920. Koller, D., Shak, T. and Briggs, W. R. (1989). Enhanced diaphototropic response to vectorial excitation in solar-tracking leaves of Lavatera cretica by an immediately preceding opposite vectorial excitation. Journal of Plant Physiology 135, 601-607. Koller, D., Ritter, S., Briggs, W. R. and Schafer, E. (1990). Action dichroism in perception of vectorial photo-excitation in the solar-tracking leaf of Lavatera cretica. Planta 181, 184-190. Koller, D., Ritter, S. and Bjorkman, 0. (1995). Role of pulvinar chloroplasts in lightdriven leaf movements of the trifoliate leaf of bean (Phaseolus vulgaris L.). Journal of Experimental Botany 46, 1215-1222. Koller, D., Ritter, S. and Fork, D. C. (1996). Light-driven movements ofthe trifoliate leaf of bean (Phaseolus vulgaris L. ). Spectral and functional analysis. Journal of Plant Physiology 149, 384-392. Koukkari, W. L. and Hillman, W. S. (1968). Pulvini as the photoreceptors in the phytochrome effect on nyctinasty in Albizzia julibrisin. Plant Physiology 43, 698-704. Lam, S.-L. and Leopold, A C. (1966). Role of leaves in phototropism. Plant Physiology 41, 847-851. Lang, A P. G. and Begg, J. E. (1979). Movements of Helianthus annuus leaves and heads. Journal ofApplied Ecology 16, 299-305. Lee, Y. (1990). Ion movements that control pulvinar curvature in nyctinastic legumes. In "The Pulvinus, Motor Organ for Leaf Movement" (R. L. Satter, H. L. Gorton and T. C. Vogelmann, eds), pp. 130-141. American Society of Plant Physiologists, Rockville. Lee, Y. and Assman, S. (1991). Diacylglycerols induce both ion pumping in patchclamped guard-cell protoplasts and opening of intact stomata. Proceedings of the National Academy of Sciences of the USA 88, 2127-2131. Lee, Y., Sub, S., Moran, N. and Crain, R. C. (1996), Phospholipid metabolism and light regulation of stomatal opening and leaf movement. In "Regulation of Plant Growth and Development by Light" (W. R. Briggs, R. L. Heath and E. Tobin, eds), pp. 89-97. American Society of Plant Physiologists, Rockville. Leshem, Y. Y. (1977). Sunflower, a misnomer? Nature (London) 270, 102. Lew, R. R., Serlin, B.S., Schauf, C. L. and Stockton, M. E. (1990). Red light regulates calcium-activated potassium channels in Mougeotia plasma membrane. Plant Physiology 92, 822-830.

PLANTS IN SEARCH OF SUNLIGHT

123

Lewis, B. D., Karun-Neumann, G., Davis, G. and Spalding, E. P. (1997). Ca2+activated anion channels and membrane depolarization induced by blue light and cold inArabidopsis seedlings. Plant Physiology 114, 1327-1334. Li, J., Lee, Y.-R. J. and Assman, S. M. (1998). Guard cells possess a calciumdependent protein kinase that phosphorylates the KAT1 potassium channel. Plant Physiology 116, 785-795. Lin, C., Robertson, D. E., Ahmad, M., Raibekas, A A, Jorns, M. S., Dutton, L. and and Cashmore, A R. (1995). Association of a flavin adenine dinucleotide with theArabidopsis blue light photoreceptor CRYl. Science 269, 968-970. Liscum, E. and Briggs, W. R. (1995). Mutations in the NPH1locus of Arabidopsis disrupt the perception of phototropic stimuli. Plant Cell1, 473-485. Liscum, E. and Briggs, W. R. (1996). Mutations of Arabidopsis in potential transduction and response components of the phototropic signaling pathway. Plant Physiology 112, 291-296. Liscum, E. and Hangarter, R. P. (1991). Arabidopsis mutants lacking blue lightdependent inhibition of hypocotyl elongation. Plant Cell 3, 685-694. Liscum, E. and Hangarter, R. P. (1993). Light-stimulated apical hook opening in wild typeArabidopsis thaliana seedlings. Plant Physiology 101,567-572. Liu, Y. J. and Iino, M. (1996). Phytochrome is required for the occurrence of timedependent phototropism in maize coleoptiles. Plant, Cell and Environment 19, 1379-1388. Lommel, C. and Felle, H. H. (1997). Transport of Ca2+ across the tonoplast of intact vacuoles from Chenopodium album L. suspension cells, ATP-dependent import and inositol-1,4,5-trisphosphate-induced release. Planta 201, 477-486. Lowen, C. Z. and Satter, R. L. (1989). Light-promoted changes in apoplastic K+ activity in the Samanea saman pulvinus, monitored with liquid membrane electrodes. Planta 179, 421-427. Ludlow, M. M. and Bjorkman, 0. (1984). Paraheliotropic leaf movement in Siratro as a protective mechanism against drought-induced damage to primary photosynthetic reactions damage by excessive light and heat. Planta 161, 505-518. Ma, L.-G. and Sun, D.-Y. (1997). The involvement of calcium in light signal transduction chain for phototropism in sunflower seedlings. Biologia Plantarum 39, 569-574. Maathuis, F. J. M., Ichida, A.M., Sanders, D. and Schroeder, J. I. (1997). Roles of higher plant K+ channels. Plant Physiology 114, 1141-1149. MacRobbie, E. A C. (1997). Signaling in guard cells and regulation of ion channel activity. Journal of Experimental Botany 48, 515-528. MacRobbie, E.A.C. (1999). Vesicle trafficking: a role in trans-tonoplast ion movements. Journal of Experimental Botany 50, 925-934. Maeshima, M., Nakanishi, Y., Matsuura-Endo, C. and Tanaka, Y. (1996). Proton pumps of the vacuolar membrane in growing plant cells. Journal of Plant Research 109, 119-125. Malhotra, K., Kim, S.-T., Batschauer, A., Dawut, L. and Sankar, A. (1995). Putative blue light photoreceptors from Arabidopsis thaliana and Sinapis alba with a high degree of sequence homology to DNA photolyase contain the two photolyase cofactors but lack DNA repair capacity. Biochemistry 34, 6892-6899. Mancinelli, A L. (1989). Interaction between cryptochrome and phytochrome in higher plant photomorphogenesis. American Journal of Botany 76, 143-154. Martinoia, E. (1992). Transport processes in vacuoles of higher plants (Review). Botanica Acta 105, 232-245.

124

D. KOLLER

Martiny-Baron, G., Manolson, M. F., Poole, R. J., Hecker, D. and Scherer, G. F. E. (1992). Proton transport and phosphorylation of tonoplast polypeptides from zucchini are stimulated by the phospholipid platelet-activating factor. Plant Physiology 99, 1635-1641. Mayer, A M. and Poljakoff-Mayber, A (1989). "Germination of seeds" 4th edition, pp. 56-67. Pergamon, Oxford. Mayer, W.-E. (1990). Walls as potassium reservoirs in Phaseolus pulvini. In "The Pulvinus, Motor Organ for Leaf Movements" (R. L. Satter, H. L Gorton and T. C. Vogelmann, eds), pp. 160-174. American Society of Plant Physiologists, Rockville. Mayer, W.-E. and Fischer, C. (1994). Protoplasts from Phaseolus coccineus L. pulvinar motor cells show circadian volume oscillations. Chronobiology Internationalll, 156-164. Mayer, W.-E. and Hampp, R. (1995). Movement of pulvinated leaves. Progress in Botany 56, 236-162. Mayer, W.-E., Flach, D., Raju, M. V. S., Starrach, N. and Wiech, E. (1985). Mechanics of circadian pulvini movements in Phaseolus coccineus L. Shape and arrangement of motor cells, micellation of and bulk moduli of extensibility. Planta 163, 381-390. Mayer, W.-E., Hohloch, C. and Kalkuhl, A (1997). Extensor protoplasts of the Phaseolus pulvinus, light-induced swelling may require extracellular Ca2 + influx, dark-induced shrinking inositol 1,4,5-trisphosphate-induced Ca 2+ mobilization. Journal of Experimental Botany 48, 219-228. McAinsh, M. R., Brownlee, C. and Hetherington, A M. (1997). Calcium ions as second messengers in guard cell signal transduction. Physiologia Plantarum 100, 16-29. Mcintyre, G. I. (1980). The role of water distribution in plant tropisms. Australian Journal of Plant Physiology 7, 401-413. Mcintyre, G. I. and Browne, K. P. (1996). Effect of darkening the cotyledons on the growth and curvature of the sunflower hypocotyl, evidence of hydraulic signalling. Journal of Experimental Botany 47, 1561-1566. Mermall, V., Post, P. L. and Mooseker, M. S. (1998). Unconventional myosins in cell movement, membrane traffic, and signal transduction. Science 279, 527-533. Meyer, W. S. and Walker, S. (1981). Leaflet orientation in water-stressed soybean. AgronomyJournal73, 1071-1074. Miller, D., Hable, W. and Gottwald, J. (1997). Connections, The hard wiring of the plant cell for perception, signaling, and response. Plant Cell9, 2105-2116. Millet, B., Coillot, L. and Agosti, R. D. (1989). The rhythmic leaf movements after regeneration of the partially excised pulvinus in Phaseolus vulgaris L. Plant and Cell Physiology 30, 643-648. Millner, P. A and Causier, B. E. (1996). G-protein coupled receptors in plant cells (Review). Journal of Experimental Botany 47, 983-992. Mohr, H. (1994) Coaction between pigment systems. In "Photomorphogenesis in Plants, 2nd edition" (R. E. Kendrick and R. H. M. Kronenberg, eds), pp. 353-373. Kluwer. Dordrecht. Mooney, H. A. and Ehleringer, J. (1978). The carbon gain benefits of solar tracking in a desert annual. Plant, Cell and Environment l, 301-311. Moran, N. (1990). The role of ion channels in osmotic volume changes in Samanea motor cells analyzed by patch-clamp methods. In "The Pulvinus, Motor Organ for Leaf Movements" (R. L. Satter, H. L Gorton and T. C. Vogelmann, eds), pp. 142-159. American Society of Plant Physiologists, Rockville.

PLANTS IN SEARCH OF SUNLIGHT

125

Moran, N. (1996). Membrane-delimited phosphorylation enables activation of outward-rectifying K+ channels in motor cell protoplasts of Samanea saman. Plant Physiology 111, 1281-1292. Moran, N., Ehrenstein, G., Iwasa, K., Mischke, C., Bare, C. and Satter, R. L. (1988). Potassium channels in motor cells of Samanea saman. A patch-clamp study. Plant Physiology 88, 643-648. Moran, N., Fox, D. and Satter, R. L. (1990). Interaction of the depolarizationactivated K+ channel of Samanea saman with inorganic ions. A patch-clamp study. Plant Physiology 94, 424-431. Morse, J. M. and Satter, R. L. (1979). Relationships between motor cell ultrastructure and leaf movements in Samanea saman. Physiologia Plantarum 46, 338-346. Morse, J. M., Crain, R. C., Cote, G. G. and Satter, R. L. (1989a). Light-stimulated inositol phospholipid turnover in Samanea saman leaf pulvini, increased levels of diacylglycerol. Plant Physiology 89, 724-727. Morse, J. M., Satter, R. L., Crain, R. C. and Cote, G. G. (1989b ). Signal transduction and phosphatidylinositol turnover in plants. Physiologia Plantarum 16, 118-121. Moysset, L. and Simon, E. (1989). Role of calcium in phytochrome-controlled nyctinastic movements of Albizzia lophanta leaflets. Plant Physiology 90, 1108-1114. Moysset, L., Sole-sugrafies, L. and Simon, E. (1991). Changes in morphometry and elemental composition of Robinia pseudoacacia pulvinar motor cells during leaflet movement. Journal of Experimental Botany 42, 1315-1323. Muir, S. R., Bewell, M.A., Sanders, D. and Allen, G. J. (1997). Ligand-gated Ca 2+ channels and Ca2+ signaling in higher plants. Journal of Experimental Botany 48, 589-597. Mulkey, T. J., Kuzmanoff, K. M. and Evans, M. L. (1981). Correlations between proton-efflux patterns and growth patterna during geotropism and phototropism in maize and sunflower. Planta 152, 239-241. Munnik, T., de Vrije, T., Irvine, R. F. and Musgrave, A. (1996). Identification of diacylglycerol pyrophosphate as a novel metabolic product of phosphatidic acid during G-protein activation in plants. Journal of Biological Chemistry 271, 15708-15715. Murata, T., Kadota, A. and Wada, M. (1997). Effects of blue light on cell elongation and microtubule orientation in dark-grown gametophytes of Ceraopteris richardii. Plant and Cell Physiology 38, 201-209. Negbi, M., Zamski, E. and Ze'evi, 0. (1982). Photo- and thigmomorphogenetic control of the attachment of the ivy (Hedera helix) to its support. Zeitschrift for Pflanzenphysiologie 108, 9-19. Nick, P. (1999). Signals, motors, morphogenesis- the cytoskeleton in plant development. Plant Biology 1, 169-179. Nick, P., Bergfeld, R., Schafer, E. and Schopfer, P. (1990). Unilateral reorientation of microtubules at the outer epidermal wall during photo- and gravitropic curvature of maize coleoptiles and sunflower hypocotyls. Planta 181, 162-168. Nishizaki, Y. (1986). Rhythmic and blue light-induced turgor movements and electric potential in laminar pulvinus of Phaseolus vulgaris L. Plant and Cell Physiology 27, 155-162. Nishizaki, Y. (1990). Effects of anoxia and red light on changes induced by blue light in the membrane potential of pulvinar motor cells and leaf movement in Phaseolus vulgaris L. Plant and Cell Physiology 31, 591-596.

126

D. KOLLER

Nishizaki, Y. (1992). Effects of inhibitors on the light-induced changes in electric potential in pulvinar motor cells of Phaseolus vulgaris L. Plant and Cell Physiology 33, 1073-1078. Nishizaki, Y. (1996). Effects of blue light on electrical potential and turgor in pulvinar motor cells of Phaseolus. Journal of Plant Research 109, 93-97. Nishizaki, Y., Kubota, M., Yamamiya, K. and Watanabe, M. (1997). Action spectrum of light-pulse induced membrane depolarization in pulvinar motor cells of Phaseolus. Plant and Cell Physiology 35, 526-529. Nozue, K., Kanegae, T., Imaizumi, T., Fukuda, S., Okamoto, H., Yeh, K.-C., Lagarias, J. C. and Wada, M. (1998). A phytochrome from the fernAdianthum with features of the putative photoreceptor nphl. Proceedings of the National Academy of Sciences of the USA 95, 15826-15830. Ohiwa, T. (1977). Response of Spirogyra chloroplast to local illumination. Planta 136, 7-11. Oosterhuis D., Walker S. and Eastham J. (1985). Soybean (Glycine max) leaflet movements as an indicator of crop water-stress. Crop Science 25, 1101-1106. Palmer, J. H. and Asprey, G. F. (1958). Studies in the nyctinastic movement of leaf pinnae of Samanea saman (Jacq.) Merrill. II. The behavior of upper and lower half pulvini. Planta 51, 770--785. Palmer, J. M., Warpeha, K. M. F. and Briggs, W.R. (1996). Evidence that zeaxanthin is not the photoreceptor for phototropism in maize coleoptiles. Plant Physiology 110, 1323-1328. Parks, B. M. Quail, P. H. and Hangarter, R. P. (1996). Phytochrome A regulated redlight induction of phototropic enhancement in Arabidopsis. Plant Physiology 110, 155-162. Pei, Z.-M., Ward, J. M., Harper, J. F. and Schroeder, J .1. (1996). A novel chloride channel in Vicia faba guard cell vacuoles activated by the serine/threonine kinase, CDPK. EMBO JournallS, 6564-6574. Pfeffer, W. (1875). "Die Periodischen Bewegungen der Blattorgane". Engelmann, Leipzig. Pfeffer, W. (1881). "The Physiology of Plants, Volume 3" (translated into English and edited by A J. Ewart). Clarendon, Oxford. Powles, S. B. and Bjorkman, 0. (1981 ). Leaf movement in the shade species Oxalis oregana. II. Role in protection against injury by intense light. Carnegie Institute of Washington Year Book 80, 63-66. Quinones, M. A and Zeiger, E. (1994). A putative role of the xanthophyll, zeaxanthin, in blue light photoperception of corn coleoptiles. Science 264, 558-561. Rajendrudu, G. and Rama Das, V. S. (1981). Solar tracking and light interception by leaves of some dicot species. Current Science 50, 618-620. Raschke, K. (1975). Stomatal action.Annual Review ofPlant Physiology 26, 309-340. Reed, J. W., Nagpal, P., Poole, D. S., Furuya, M. and Chory, J. (1993). Mutations in the gene for red/far-red light receptor phytochrome Balter cell elongation and physiological responses throughout Arabidopsis development. Plant Cell 5, 147-157. Reed, R. and Travis, R. L. (1984). Phototropic leaflet orientation in alfalfa in response to vectorial light. Crop Science 24, 593-597. Reid, R. J., Tester, M. A and Smith, F. A (1997). Voltage control of calcium influx in intact cells. Australian Journal of Plant Physiology 24, 805-810. Reymond, P., Short, T. W. and Briggs, W. R. (1992).Blue light activates a specific protein kinase in higher plants. Plant Physiology 100, 655-671.

PLANTS IN SEARCH OF SUNLIGHT

127

Rich, T. C. G., Whitelam, G. C. and Smith, H. (1985). Phototropism and axis extension in light-grown mustard (Sinapis alba) seedlings. Photochemistry and Photobiology 42, 789-792. Ritter, S. and Koller, D. (1994a). Light-driven movements of the trifoliate leaf of bean (Phaseolus vulgaris L.). Activity of blue light and red light. Journal of Experimental Botany 45, 335-341. Ritter, S. and Koller, D. (1994b). Movements ofthe trifoliate leaf of bean (Phaseolus vulgaris L.) during a simulated day, and their consequences for solar-tracking fidelity and interception of solar radiation. Journal of Plant Physiology 143, 64-71. Roblin, G., Fleurat-Lessard, P. and Bonmort, J. (1989). Effects of compounds affecting calcium channels on phytochrome- and blue pigment-mediated pulvinar movements of Cassia fasciculata. Plant Physiology 90, 697-701. Roennenberg, T. and Foster, R. G. (1997). Twilight times, light and the circadian rhythm. Photochemistry and Photobiology 66, 549-561. Sailaja, M. V. and Rama Das, V. S. (1996). Leaf solar tracking response exhibits diurnal constancy in photosystem II efficiency. Environmental and Experimental Botany 36,431-438. Salomon, M., Zacher!, M. and Rudiger, W. (1997). Phototropism and protein phosphorylation in higher plants. Unilateral blue light irradiation generates a directional gradient of protein phosphorylation across the oat coleoptile. BotanicaActa 110,214-216. Sato, H. and Gotoh, K. (1983). Studies on leaf orientation movements in kidney beans (Phaseolus vulgaris L.).l. The response to light intensity and location of the photoreceptor. Japanese Journal of Crop Science 52, 515-520. Satter, R. L. (1979). Leaf movements and tendril curling. In "Physiology of Movements Encyclopedia of Plant Physiology, Volume 7" (W. Haupt and M. E. Feinleib, eds), pp. 442-484. Springer, Berlin. Satter, R. L. and Moran, N. (1988). Ionic channels in plant cell membranes. Physiologia Plantarum 12, 816-820. Satter, R. L. and Morse, M. J. (1990). Light-modulated circadian rhythmic leaf movements in nyctinastic legumes. In "The Pulvinus, Motor Organ for Leaf Movements" (R. L. Satter, H. L Gorton and T. C. Vogelmann, eds), pp. 10-24. American Society of Plant Physiologists, Rockville. Satter, R. L., Sabnis, D. D. and Galston, A. W. (1970). Phytochrome controlled nyctinasty inAlbizzia julibrissin. I. Anatomy and fine structure of the pulvinule. American Journal of Botany 51, 374-381. Satter, R. L., Geballe, G. T., Applewhite, P. B. and Galston, A. W. (1974). Potassium flux and leaf movement in Samanea saman. I. Rhythmic movement. Journal of General Physiology 64, 413-430. Satter, R. L., Guggino, S. E., Lonergan, T. A. and Galston, A. W. (1981 ). The effects of blue and far red light on rhythmic leaf movements in Samanea andAlbizzia. Plant Physiology 67, 965-968. Satter, R. L., Lee, Y., Morse, M. J., Crain, R. C., Cote, G. G. and Moran, N. (1988). Light- and clock-controlled leaflet movements in Samanea saman. A physiological. biophysical and biochemical analysis. Botanica Acta 101, 205-213. Schmidt, W., Hart, J., Filner, P. and Poff, K. L. (1977). Specific inhibition of phototropism in corn seedlings. Plant Physiology 60, 736-738. Schroeder, J. I. and Hagiwara, S. (1989). Cytosolic calcium regulates ion channels in plasma membrane of Vicia faba guard cells. Nature (London) 338, 427-430. Schwartz, A. and Koller, D. (1978). The phototropic response to vectorial light in leaves of Lavatera cretica L. Plant Physiology 61, 924-928.

128

D. KOLLER

Schwartz, A. and Koller, D. (1980). Role of the cotyledons in the phototropic response of Lavatera cretica seedlings. Plant Physiology 66, 82-87. Schwartz, A. and Koller, D. (1986). Diurnal phototropism in the solar-racking leaves of Lavatera cretica. Plant Physiology 80, 778-781. Schwartz, A., Gilboa, S. and Koller, D. (1987). Photonastic control of leaflet orientation in Melilotus indicus (Fabaceae ). Plant Physiology 84, 318-323. Serrano, E. E., Zeiger, E. and Hagiwara, S. (1988). Red light stimulates an electrogenic proton pump on Vicia guard cells. Proceedings of the National Academy of Sciences of the USA 85, 436-440. Shackel, K. A. and Hall, A. E. (1979). Reversible leaflet movements in relation to drought adaptation of cowpeas, Vigna unguiculata (L.) Walp. Australian Journal of Plant Physiology 6, 265-276. Sharpe, P. J. H., Wu, H. and Spence, R. D. (1987). Stomatal mechanics. In "Stomatal Function" (E. Zeiger, G. D. Farquhar and I. R. Cowan, eds), pp. 91-114. Stanford University Press, Stanford. Shell, G. S. G. and Lang, A. R. G. (1976). Movements of sunflower leaves over a 24-h period. Agricultural Meteorology 16, 161-170. Sheriff, D. W. and Ludlow, M. M. (1985). Diaheliotropic responses of leaves of Macroptilium atropurpureum cv. Siratro.Australian Journal of Plant Physiology 12, 151-171. Shibaoka, H. (1961). Studies on the mechanism of growth-inhibiting effect of light. Plant and Cell Physiology 2, 175-197. Shibaoka, H. and Yamaki, T. (1959) Studies on the growth movement of sunflower plant. Scientific Papers, College of General Education, Tokyo 9, 105-126. Shimazaki, K., Kinoshita, T. and Nishimura, N. (1992). Involvement of calmodulin and calmodulin-dependent myosin light-chain kinase in blue light-dependent H+ pumping by guard cell protoplasts from Vicia faba. Plant Physiology 99, 1416-1421. Shuttleworth, J. E. and Black, M. (1977). The role ofthe cotyledons in phototropism of de-etiolated seedlings. Planta 136, 51-55. Skrove, D., Rinnar, T. and Johnsson, A. (1982). Effects of abscissic acid on the circadian leaf movements of Oxalis regnelli. Physiologia Plantarum 55, 221-225. Smith, A. P. (1975). Insect pollination and heliotropism in Oritrophium limnophilum (Sch. Bip.) Cuatr. (Compositae) of the Andean paramos. Biotropica 1, 284-286. Smith, H. (1984). Plants that track the sun. Nature (London) 308, 774. Smith, H. (1994). Sensing the light environment, the functions of the phytochrome family. In "Photomorphogenesis in Plants", 2nd edition (R. E. Kendrick and R. H. M. Kronenberg, eds), pp. 377-416. Kluwer, Dordrecht. Smith, M. and Ullberg, D. (1989). Effect of leaf angle and orientation on photosynthesis and water relations in Silphium terebinthinaceum. American Journal of Botany 16, 1714-1719. Snow, R. (1959). Some tests of theories of plant torsions. Proceedings of the Royal Society, London 151B, 26-38. Snow, R. A. (1947). A test for Sachs's theory of the plagiotropism of laminae. New Phytologist 46, 258-261. Spalding, E. P. and Cosgrove, D. J. (1989). Large plasma membrane depolarization precedes rapid blue light-induced growth inhibition in cucumber. Planta 178, 407-410. Spalding, E. P. and Cosgrove, D. J. (1992). Mechanism of blue light-induced plasmamembrane depolarization in etiolated cucumber seedlings. Planta 188, 199-205.

PLANTS IN SEARCH OF SUNLIGHT

129

Staal, M., Elzenga, J. T. M., van Elk, A. G., Prins, H. B. A. and Van Volkenburgh, E. (1994). Red and blue light-stimulated proton efflux by epidermal leaf cells of the Argenteum mutant of Pisum sativum. Journal of Experimental Botany 45, 1213-1218. Stanton, M. L. and Galen, C. (1993). Blue light controls solar tracking by flowers of an alpine plant. Plant, Cell and Environment 16, 983-989. Starrach, N. and Mayer, W.-E. (1989). Changes in the apoplastic pH and K+ concentration in the Phaseolus pulvinus in situ in relation to rhythmic leaf movements. Journal of Experimental Botany 40, 856-873. Starrach, N., Flach, D. and Mayer, W.-E. (1985). Activity of fixed negative charges of isolated extensor cell walls of the laminar pulvinus of the primary leaves of Phaseolus. Journal of Plant Physiology 120, 441-445. Stevenson, K. R. and Shaw, R. H. (1971). Effects of leaf orientation on leaf resistance to water vapor diffusion in soybean (Glycine max L. Merr.) leaves. Agronomy Journal63, 327-329. Strong, D. R. and Ray, T. S. (1975). Host tree location behavior of a tropical vine (Monstera gigantea) by skototropism. Science 190, 804-807. Tanada, T. (1982). Effect of far-red and green irradiation on the nyctinastic closure ofAlbizzia leaflets. Plant Physiology 70, 901-904. Tanada, T. (1984). Interactions of green or red light with blue light on the dark closure ofAlbizzia pinnules. Physiologia Plantarum 61, 35-37. Tanada, T. (1997). The photoreceptors in the high irradiance response of plants. Physiologia Plantarum 101, 451-454. Thiel, G. and Wolf, A. H. (1997). Operation of K+ -channels in stomatal movement. Trends in Plant Science 2, 339-345. Thomine, S., Zimmermann, S., Vanduijn, B., Barbierbrygoo, H. and Guern, J. (1994 ). Calcium channel antagonists induce indirect inhibition of the outward rectifying potassium channel in tobacco protoplasts. FEBS Lettters 340, 1-2. Thomson, W. W. and Journett, R. D. (1970). Studies on the ultrastructure of guard cells of Opuntia. American Journal of Botany 51, 309-316. Thuleau, P., Moreau, M. Schroeder, J. I. and Ranjeva, R. (1994a). Recruitment of plasma membrane voltage-dependent Calcium permeable channels in carrot cells. EMBO Journa/13, 5843-5847. Thuleau, P., Ward, M. J., Ranjeva, R. and Schroeder, J. I. (1994b). Voltagedependent calcium-permeable channels in the plasma membrane of a higher plant cell. EMBO Journa/13, 2970-2975. Travis, R. L. and Reed, R. (1983). The solar tracking pattern in a closed alfalfa canopy. Crop Science 23, 664-668. Tretyn, A., Kendrick, R. E. and Wagner, G. (1991). The role(s) of calcium ions in phytochrome action. Photochemistry and Photobiology 54, 1135-1155. Tyreman, S.D., Bohnert, S. J., Maurel, C., Steudle, E. and Smith, J. A. C. (1999). Plant aquaporins: their molecular biology, biophysics and significance for plant water relations. Journal of Experimental Botany 50, 1055-1071. Van Volkenburgh, E., Cleland, R. E. and Watanabe, M. (1990). Light-stimulated cell expansion in bean (Phaseolus vulgaris L.) leaves. II. Quantity and quality of light required. Planta 182, 77-80. Varlet-Grancher, C. and Bonhomme, R. (1977). Utilization de l'energie solaire par unc culture de Vigna sinensis. I. Etude thcorique de !'influence de l'hcliotropisme des feuilles sur !'interception des rayonenments. Annates Agronomiques 23,407-417. Vince-Prue, D. (1994). The duration of light and photoperiodic responses. In "Photomorphogenesis in Plants", 2nd edition (R. E. Kendrick and R. H. M. Kronenberg, eds), pp. 447-490. Kluwer, Dordrecht.

130

D. KOLLER

Vochting, H. (1888). Uber der Lichtstellung der Laubbliitter. Botanische Zeitung 46, 501-560. Vogelmann, T. C. (1984). Site of light perception and motor cells in a sun-tracking lupine (Lupinus succulentus). Physiologia Plantarum 62, 335-340. Vogelmann, T. C. and Bjorn, L. 0. (1983). Response to directional light by leaves of a sun-tracking lupine (Lupinus succulentus). Physiologia Plantarum 59, 533-538. Vogelmann, T. C., Bornman, J. F. and Yates, D. J. (1996). Focusing of light by leaf epidermal cells. Physiologia Plantarum 98, 43-56. von Sachs, J. (1875). Uber das Bewegungsorgan und die periodischen Bewegungen der Blatter von Phaseolus und Oxalis. Botanische Zeitung 15, 793-802, 809-815. von Sachs, J. (1887). "Lectures on the Physiology of Plants" (translated by H.M. Ward from "Vorlesungen der Pflanzenphysioiogie", Wiirzburg). Clarendon, Oxford. Wada, M. and Sei, H. (1994). Phytochrome-mediated phototropism in Adiantum cuneatum young leaves. Journal of Plant Research 107, 181-186. Wainwright, C. M. (1977). Suntracking and related leaf movements in a desert lupin (Lupinus arizonicus).American Journal of Botany 64, 1032-1041. Ward, J. M., Pei, Z.-M. and Schroeder, J. I. (1995). Roles of ion channels in initiation of signal transduction in higher plants (Review). Plant Cell?, 833-844. Ward, J. M. and Schroeder, J. I. (1994). Calcium-activated K+ channels and calciuminduced calcium release by slow vacuolar ion channels in guard cell vacuoles implicated in the control of stomatal closure. Plant Cell 6, 669-683. Watanabe, S. and Sibaoka, T. (1973). Site of photo-reception to opening response in Mimosa leaflets. Plant and Cell Physiology 14, 1221-1224. Watanabe, S. and Sibaoka, T. (1983). Light- and auxin-induced leaflet opening in detached pinnae of Mimosa pudica. Plant and Cell Physiology 24, 641-647. Weintraub, M. (1951). Leaf movement in Mimosa pudica. New Phytologist 50, 357-382. Werk, K. S. and Ehleringer, J. (1984) Non-random leaf orientation in Lactuca serriola L. Plant, Cell and Environment 1, 81-87. Werk, K. S. and Ehleringer, J. (1985). Photosynthetic chracteristics of Lactuca serriola L. Plant, Cell and Environment 8, 345-350. Werk, K. S. and Ehleringer, J. (1986a). Effects of nonrandom leaf orientation on reproduction in Lactuca serriola L. Evolution 40, 1334-1337. Werk, K. S. and Ehleringer, J. (1986b). Field water relations of a compass plant, Lactuca serriola L. Plant, Cell and Environment 9, 681-683. Werker, E. and Koller, D. (1987). Structural specialization of the site of response to vectorial photo-excitation in the solar-tracking leaf of Lavatera cretica. American Journal of Botany 14, 1339-1349. Werker, E., Shak, T. and Koller, D. (1991). Photobiological and structural studies of light-driven movements in the solar-tracking leaf of Lupin us palaestinus Boiss. (Fabaceae ). Botanica Acta 104, 144-156. Wetherell, D. F. (1990). Leaf movements in plants without pulvini. In "The Pulvinus, Motor Organ For Leaf Movement" (R. L. Satter, H. L. Gorton and T. C. Vogelmann, eds), pp. 72-78. American Society of Plant Physiologists, Rockville. Wien, H. C. and Wallace, D. H. (1973). Light induced leaflet orientation in Phaseolus vulgaris L. Crop Science 13, 721-724. Williams, C. W. and Raghavan, V. (1966). Effects of light and growth substances on the diurnal movements of the leaflets of Mimosa pudica. Journal of Experimental Botany 17, 742-749.

PLANTS IN SEARCH OF SUNLIGHT

131

Williams, S. E. and Bennett, A B. (1982). Leaf closure in the venus flytrap, An acid growth response. Science 218, 1120-1121. Wilson, W. P. and Greenman, J. M. (1892-97). Preliminary observations on he movement of the leaves of Melilotus alba (L.) and other plants. Contributions from the Penn State University Botanical Laboratories 1, 66-73. Yin, H. C. (1938). Diaphototropic movement of the leaves of Malva neglecta. American Journal of Botany 25, 1-6. Yu, F. and Berg, V. S. (1994). Control of paraheliotropism in two Phaseolus species. Plant Physiology 106, 1567-1573. Zeiger, E., Field, C. and Mooney, H. A (1981). Stomatal opening at dawn, possible role of the blue light response in Nature. In "Plants and the Daylight Spectrum" (H. Smith, ed.), pp. 391-407. Academic Press, New York. Zhang, H., Pleasants, J. M. and Jurik, T. W. (1991). Development of leaf orientation in the prairie compass plant, Silphium laciniatum L. Bulletin of the Torrey Botanical Club 118, 33-42.

The Mechanics of Root Anchorage

A. R. ENNOS

School of Biological Sciences, University of Manchester, 3.614 Stopford Building, Oxford Road, Manchester M13 9PT, UK

I. II.

III.

IV.

V. VI.

Introduction: Early Research and Misconceptions.................................... Root Systems which Resist Uprooting ........................................................ A. Theory of Single Root Extraction......................................................... B. Practical Test of Theory......................................................................... C. Implications of the Practical Test......................................................... D. Methods of Minimizing Anchorage Costs........................................... E. Anchorage Systems of Mature Plants .................................................. Root Systems which Resist Overturning..................................................... A. Theory of Anchorage ............................................................................. B. Experimental Methods for Studying Anchorage................................ C. Mechanics of Anchorage Systems......................................................... Uses of Models of Anchorage...................................................................... A. Understanding Root System Morphology........................................... B. Understanding the Thigmomorphogenic Responses of Roots......... C. Improving the Stability of Crop Plants................................................. Areas for Future Research............................................................................ A. The Effect of Soil Properties................................................................. B. Numerical Models of Anchorage.......................................................... Conclusions..................................................................................................... References.......................................................................................................

134 135 135 137 137 138 140 141 141 142 143 147 147 148 150 151 151 152 153 154

The past 20 years has seen the re-emergence of the study of root anchorage. The research has combined theory from composite materials and foundations technology with experimental observations and mechanical testing. This has shown that the mechanical role of roots is restricted to the basal root system, but that it nevertheless greatly influences both the size and shape of root systems. Procumbent and climbing plants, which must resist being uprooted vertically, are most efficiently anchored by a fibrous root system. In contrast, self-supporting plants require rigid elements in their anchorage systems to prevent them toppling. Small herbaceous dicots tend to possess tap root systems, Larger Advances in Botanical Research Vol 33 incorporating Advances in Plant Pathology ISBN 0-1 HJ05933-9

Copyright © 2000 Academic Press All rights of reproduction in any form reserved

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herbaceous dicots and trees possess plate systems, and monocots possess coronal orprop root systems. The mechanics ofanchorage ofeach system has been determined in a range of species, methods used including direct observations of the motions of roots systems, and measurements of the torque required and of surface strains around the root system as plants were pulled over. Serial destruction of elements of the anchorage system allowed the relative importance ofthe various components ofanchorage to be estimated. The new understanding ofanchorage has thrown light on various aspects ofroot biology, such as their distribution of strength, lack ofprestress, and their sensitivity to mechanical stimulation. It has also helped point the way towards the development of more stable cereal crops and forestry trees. It is hoped that future work on the effect of soil properties and on the modelling of anchorage could help explain other observations: why, for instance, trees develop different root systems in different soil types; and why trees replace tap root systems with plate systems as they grow.

I.

INTRODUCTION: EARLY RESEARCH AND MISCONCEPTIONS

Despite being acknowledged as one of the two primary functions of roots - the other being of course acquisition of water and nutrients - root anchorage was until recently more or less completely ignored as a subject of pure research. Indeed, the only well-known experiments in the area were those of Pfeffer (1893). Though interested mostly in root growth, he examined not only the axial force the radicles of beans and maize could exert as they grew into small blocks of plaster-of-Paris but also the force required to pull them out. This study was later repeated using peas (Pisum sativum) growing into prepared soil cores (Stolzy and Barley, 1968). It was found that friction of the hairless tip of the radicle with the soil could resist pullout forces of around 104 Pa, while areas covered by root hairs could resist forces around five times that figure. Unfortunately, the results of this study, as of Pfeffer's, rather than shedding light on the design of root systems, have tended merely to perpetuate several myths about anchorage. First, the mere fact that they investigated the pulling resistance of the roots tended to reinforce the misconception that roots are only ever loaded in tension. The fact that they examined very short radicles, that were coated with extensive root hairs, reinforced the view that root hairs are always important elements of anchorage. Finally, the finding that even a single root could withstand large forces seemed to support the impression that adequate anchorage could be achieved by root systems merely as a by-product of their absorption function. With a root area of over 200m2 (Dittmer, 1937), for instance, a rye plant should be able to resist upward forces of around 2 tonnes, and so would have no trouble anchoring itself. Therefore it was assumed that the design of root systems would be influenced only by their absorption function, and that there was little point in studying anchorage.

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Meanwhile agronomists and foresters continued to observe anchorage failure, as their cereals lodged and their trees suffered windthrow. Constrained by the traditional approach to anchorage they tended to measure the pullout resistance of whole plants (Nass and Zuber, 1971; Rogers et al., 1976; Donovan et al., 1982) or of individual roots (Neenan and Spencer-Smith, 1975). Most studies gave only weak correlations with lodging resistance (Arihari and Crosbie, 1982; Kevern and Hallauer, 1983; Melchinger et al., 1986; Beck et al., 1987). In any case, such correlative methods are clearly rather inefficient ways of working out how anchorage could be improved. The last 20 years has seen the emergence of a new science of plant anchorage which has combined theoretical ideas based on composite materials and engineering piles theory, with direct observation and mechanical testing. As we shall see, the new approach has overturned many of the misconceptions of the past. It has shown how the need for anchorage has influenced the overall shape of root systems and has been extremely successful in understanding how roots anchor plants. Agronomists, plant breeders and foresters now have a much better framework to allow them to increase the stability of their crops. The challenge for the future is to try to understand why plants of different sizes, like small herbs and trees, have such different anchorage systems, and how soil type influences anchorage morphology and mechanics. On the physiological and developmental side, almost nothing seems to be known about how root system morphology (and hence anchorage) is controlled.

II.

ROOT SYSTEMS WHICH RESIST UPROOTING A

THEORY OF SINGLE ROOT EXTRACTION

The simplest anchorage systems to study are those, like the single radicles of seedlings or the root systems of prostrate or climbing plants, which must resist only axial uprooting forces, such as those caused by grazing herbivores or weeding gardeners. In fact, even the simplest possible case of a single root being pulled out of a uniform soil (Fig. 1) is far from easy to analyse (Ennos, 1989). The process is analogous to the extraction of a fibre from the matrix of a composite material, and depends critically on the mechanical properties of the materials. Unstrengthened roots in tension behave more-or-less elastically up to strains of around 5%; they have a Young's modulus of around 107 Pa, and stretch by around 10% of their original length before breaking at a stress of around 106 Pa. Agricultural soils, in contrast, have an unusual plastic behaviour in shear. They have a very high initial stiffness (Ennos, 1989), but fail at very low strains and hence stresses (in the region of 103-105 Pa for damp clay loams). But the soil does not 'break' like the root; it

136

A R. ENNOS

continues to resist shear long after failure because of the friction and cohesion between the soil particles. These unusual properties mean that when roots are pulled from the soil the sequence of events is quite complex. Because the roots are less stiff than the soil, when they are pulled up, the regions closest to the surface will tend to stretch and shear past the surrounding soil. Shear stresses will therefore be concentrated around the top of a root, and the soil (or the bond between the root and the soil) will fail first in this region. However, the root will not simply pull out without any force. The friction within the soil or between the root and the soil will

t

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Fig. 1. Predicted (A) and actual (B) shape of the force-displacement curve when an unstrengthened root is pulled out of the soil. Short roots (solid lines) pull out, whereas long roots (broken lines) break. The curves in Bare for leek roots 34 mm (solid line) and 41 mm (broken line) long. The actual curves follow predictions closely, and roots resist forces of only up to 0.3 N. (Redrawn from Ennos (1990).)

MECHANICS OF ROOT ANCHORAGE

137

continue to resist uprooting and tension will be transferred from the root to the soil. The greater the upward force applied to the root the greater the area of soil which will fail and the further down in the soil will the root be stretched. The force required will therefore rise, rapidly at first (Fig.1), but more slowly as uprooting proceeds (Ennos, 1990). The system will eventually fail in one of two ways (Ennos, 1990). If the root is short and strong and the soil or the bond between the root and soil is weak, the root will be pulled out of the ground. In contrast if the root is long and narrow and the soil strong, the root will break at the top before the soil around lower regions of the root is disturbed. B.

PRACTICAL TEST OF THEORY

A series of simple uprooting tests has been carried out to test this theory on what are essentially the simplest possible roots: the unstrengthened cylindrical radicles of leeks (Allium porrum) (Ennos, 1990). The roots showed the predicted pattern of extraction force with upward displacement of the root (Fig.l); the force increased rapidly initially, before reaching a peak. The force then fell, either catastrophically as the root broke, or gradually as it was pulled out. The roots also showed the predicted relationship between length and mode of failure; short roots broke whereas long ones pulled out. These tests, therefore, confirmed the model of uprooting but they also showed something very important for anchorage. In practice, unstrengthened roots more than a few millimetres long will break before they are pulled out of even wet soil and at forces of around 0.1-0.3 N. C.

IMPLICATIONS OF THE PRACTICAL TEST

The results of these tests have important implications for the design of anchorage systems. A plant cannot improve its anchorage simply by increasing its root length or strengthening the bond between the root and the soil; the root must also be strengthened, especially towards its base, and the material needed to do this will represent a real cost to the plant. In fact, all other roots which have been studied show strengthening, usually by lignification of the stele (Ennos, 1989, 1991a) and, in dicots, by additional secondary thickening. The cost of strengthening the root system against uprooting is not, in fact, particularly high because roots only have to withstand forces up to the tensile strength of the stem. No part of the root system need therefore be very strong. However, plants with improved anchorage and reduced anchorage costs will be chosen by natural selection, so anchorage considerations must have influenced the morphology of root

138

A. R. ENNOS

systems, contrary to the received wisdom. In fact the design of the basal part of the root systems of prostrate plants and climbers do show a number of adaptations which reduce the cost of anchorage. D.

1.

METHODS OF MINIMIZING ANCHORAGE COSTS

Strengthening Only Basal Areas

Because upward forces are transmitted from the root rapidly into the soil, only the regions of the root system near the surface need be strengthened. The root system of a rye plant needs to mobilize only a tiny fraction of the 2 tonnes of force its 200 m2 area should be capable of resisting. Therefore only a tiny part of the root system proximal to the surface need be strengthened. The great majority of the roots can never be stressed by pulling on the top of the plant, since the stem or upper roots will have failed long before. So whatever forces are placed on a plant from above, its water and nutrient uptake is very unlikely to be affected. The distal roots can be specialized for absorption functions alone. Mechanical tests show that the vast majority of roots are indeed strengthened progressively towards their base, i.e. proximal to the surface (Fig. 2) (Ennos and Fitter, 1992), and it is possible to separate basal 'anchorage roots' with strengthened steles from distal unstrengthened 'absorption roots' which may be easily pulled off the fibrous part of the root system. 2.

Using Basal Root Hairs

Another way of reducing anchorage costs is to increase the anchoring capabilities of the uppermost root system so that forces are transmitted less far into the soil. One way of doing this is to coat the top of a root with root hairs. A good example of the use of root hairs in anchorage is seen in the radicles of sunflowers (Ennos, 1989). The strong, basal 25 mm of the radicle is covered with a dense covering of unusually strong root hairs which are glued firmly to the soil. The root hairs improve anchorage both by increasing the effective area of the root and by improving its contact with the soil. They allow the region to withstand upward forces of over 0.6 N even in very weak soils (Ennos, 1989). These basal root hairs clearly have an important role in the establishment of seedlings. First, they prevent uprooting of the young seedlings by external forces, even when little root is in the soil. Perhaps even more important for a plant developing from a seed which is lying on the surface, is that the root hairs can produce the anchorage required to drive the tip of the developing radicle further into the soil. However, it is far less likely that root hairs have a major role in the anchorage of mature plants. Root hairs are produced near the tip of

139

MECHANICS OF ROOT ANCHORAGE

elongating roots, well away from the areas which are likely to be stressed when external forces disturb the plant. The major mechanical role of root hairs is therefore almost certainly in root growth. They anchor the root just like the chaetae of worms, allowing the root tip to force its way through the soil. 3. Root Branching and Adventitious Root Production A final way of reducing anchorage costs is to produce a fibrous root system with many roots radiating away from the base of the plant rather than relying on a single 'tap' root. This is because many narrow roots have a larger surface area than a single root of equivalent cross-sectional area, and hence will transfer tension more rapidly into the soil. Consequently, they need not be strengthened so far from their base and will use less strengthening material. Theoretical analysis (Ennos, 1993a) has shown, however, that there is an optimal number of roots. If there are too many roots the soil will tend to fail in shear and tension around the whole system and a 'root ball' will be removed from the soil at a very low force.

1.5

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150

Distance from root base (mm)

Fig. 2. The distribution of strengths down the five seminal roots of a spring wheat plant at age seven days. The roots are all progressively strengthened towards their base.

140

A. R.ENNOS

E.

ANCHORAGE SYSTEMS OF MATURE PLANTS

Most climbing and procumbent plants certainly have fibrous root systems with large numbers of roots (Ennos and Fitter, 1992), formed either by rapid branching of the main axis, or production of adventitious roots. The roots are also strengthened towards their base and, despite making up only a tiny fraction of the root length, these strengthened areas constitute a large fraction of root mass. The anchorage roots of prostrate and climbing annuals studied by Ennos and Fitter accounted for 1-5% of the dry mass of the annual herbs studied (Fig. 3), comparable to the 5-12% invested in unstrengthened absorption roots. Taller climbing plants tended to have a lower percentage dry mass in their anchorage roots, probably because of the way in which their stems scale (Ennos, 1993a). Because their stems do not thicken isometrically as they extend their anchorage systems need not be much stronger than those of younger, shorter plants. Unfortunately, no detailed experimental studies have been carried out on the anchorage of the mature root systems of climbers or procumbent plants, so there has been no testing of the theoretical models of anchorage. The

Procumbent or climbing

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Fig. 3. Percentages of total dry mass invested in anchorage in annuals of differing form grown in a glasshouse. Hatched area = tap root; open area = fibrous root. Error bars show standard deviation of anchorage investment. Free-standing plants have the most anchorage material, much of it invested in a rigid tap root.

MECHANICS OF ROOT ANCHORAGE

141

most complex system investigated has been the seminal root system of wheat (Ennos, 1991a). In this plant the five roots combine to resist almost as much force as the sum of the individual roots, and there is no sign of a root ball forming. Studies in this area would be important in determining the factors which help grasses resist being uprooted by sheep, cattle, and other herbivores. The pullout resistance of grasses and a few other prostrate herbs has been examined by Bailey (1998) but only to investigate the scaling of the anchorage force, and to determine if grasses have grazing ecotypes with stronger anchorage. No evidence was found of differences between grasses grown in grazed and ungrazed swards.

III.

ROOT SYSTEMS WHICH RESIST OVERTURNING A.

THEORY OF ANCHORAGE

It was shown in the last section that the optimal root systems to resist uprooting by vertical forces are those composed of a large number of fibrous roots. However, many plants only rarely have to resist such forces. It is hard to imagine a tree being pulled up by a herbivore, for instance, and though gardeners do pull up weeds, they are an evolutionary novelty! Tall, selfsupporting plants are more likely to be pulled or pushed sideways by a herbivore or blown sideways by the wind. These forces will be transmitted down to the root system by the stem and will cause it to rotate in the soil. Failure to prevent this will result in crop plants leaning over permanently, a process called 'lodging', or in trees being blown right over, a process called 'windthrow'. The anchorage systems of self-supporting plants must therefore be adapted to transmit rotational torques into the soil rather than simple upward forces. The fibrous root systems which are so good at resisting uprooting will be useless at preventing lodging or windthrow because each root would simply bend at its base. To provide reasonable resistance to rotation a more expensive system is required containing at least one rigid element at the base of the stem to act as a lever (Ennos and Fitter, 1992). The self-supporting annual crucifers examined by Ennos and Fitter (1992) used one of two strategies to support themselves: some used shoot elements, such as a rosette of leaves around the base of the plant or multiple stems; the rest used a rigid tap root. Both types had a significantly higher investment in strengthened anchorage roots than procumbent plants: 3-7% of dry mass in rosette and multistemmed plants, and 8-12% in tap-rooted plants, compared with 1-5% in procumbent plants (Fig. 3). Examination of root systems (Coutts, 1983; Ennos, 1991b) has identified two other types of anchorage systems in self-supporting plants: the plate systems of many trees, and the coronal or prop root systems of many cereals.

142

A. R.ENNOS

Though some thought has been put into the theory of the interactions of all these root systems with soil (Coutts, 1983; Ennos, 1993a), the mechanics of anchorage of self-supporting plants has been investigated using a largely experimental approach. This contrasts with the extremely theoretical framework used to investigate uprooting, and is mainly due to the extreme complexity of most mature anchorage systems and hence the difficulty in modelling them. B.

EXPERIMENTAL METHODS FOR STUDYING ANCHORAGE

Almost all the experimental work on anchorage against overturning has followed the methods pioneered by Coutts (1983, 1986) in his study of the anchorage of unstable Sitka spruce (Picea abies) trees growing on periodically waterlogged peat in upland Britain. Coutts outlined a range of techniques to identify various 'components of anchorage' and quantify their relative importance. The first stages in determining the components of anchorage involve observation of the morphology of the root system, and determination of how the anchorage system fails when the plant is pushed over. This is best achieved by cutting vertical trenches into the soil alongside the stem or trunk to expose a 'cross-section' of the root system (Coutts, 1983). This has two advantages. It allows direct observation of the roots in situ and allows the movements of the roots and surrounding soil to be observed. Coating the sides of the trench with spray paint shows up the cracks and accentuates the movements. Sounds of breaking can also be recorded, and give further clues about how failure is proceeding. Of course, trenching experiments only involve about half the root system, so other tests are needed on intact plants. One such step is to measure the torque required to pull plants over. Measurement of the torque throughout the process allows further information about the mechanics to be gleaned, as well as the total anchorage strength of the plant. To do this, trees can be pulled over with a winch mounted with a force transducer (Coutts, 1986; Crook and Ennos, 1996a), and smaller plants can be pulled over by hand with a force gauge (Kushibiki, 1979; Koinuma eta/., 1990; Terashima eta/., 1992, 1995; Ennos eta/., 1993a, b), or pushed over with a custom-built lodging apparatus (Fouere eta/., 1995) or by an attachment of a universal testing machine (Ennos, 1991b; Crook and Ennos, 1993). The state of tissues around the surface of the roots and stem base can also be examined during uprooting by attaching strain gauges (Crook and Ennos, 1996a). Once possible components of anchorage have been identified, their relative importance can be determined by carrying out consecutive pulling tests, between which individual anchorage components are destroyed. Typical experimental procedures can involve cutting through roots or

MECHANICS OF ROOT ANCHORAGE

143

trenching within the soil (Coutts, 1986). Investigations can also study the force required for real roots or groups of roots to be pulled out of the soil in the correct orientation (Anderson et al., 1989; Ennos, 1991b). The force required to push or pull mechanical models of root system components, such as rods or cones, into and out of the soil can also be measured (Crook and Ennos, 1993). Finally, mechanical tests on roots or soil can also be carried out in the laboratory to help to build up an understanding of the anchorage system. C.

MECHANICS OF ANCHORAGE SYSTEMS

To date, detailed investigations of anchorage mechanics have been completed on only a few species of trees and herbaceous plants. However, cursory examination of a much wider range of plants can help us to work out their likely anchorage mechanics. Most root systems can be classified as 'plate', 'tap' or 'coronal' systems, though there are also many plants whose systems show intermediate behaviour. Already it is possible to see trends both with the size of plants and their taxonomic status.

1. Plate Systems Most mature trees seem to rely on a plate root system such as that shown in Fig. 4(A). (Coutts, 1983, 1986; Crook and Ennos, 1996a; Crooket al., 1997). The main elements of the structural root system are the large lateral roots which radiate more or less horizontally out from the trunk before tapering and branching, ramifying into the narrower absorption roots which grow out many metres from the trunk. The other important elements in these root systems are the vertical sinker roots which emerge from the laterals usually a short distance from the trunk and taper and branch downwards into the subsoil, and in some cases the remaining tap root. In these systems, the resistance of the soil to downward movement of the laterals will be very high because of their large area and the large compressive strength of soil. Consequently failure mostly occurs on the windward side, the plant being levered up about a leeward hinge (Fig. 4(A) ). Exactly what happens depends on the soil conditions. In wet, weak soil a large plate may be levered out of the ground together with the intact sinker roots, whereas in stronger soil the plate may be smaller and windward roots may be broken and sinker roots sheared off. It is possible to identify three main components of anchorage: the resistance of the leeward hinge to bending; the resistance of the windward roots, especially the sinkers, to uprooting; and the weight of the root-soil plate. The majority of the anchorage is usually due to the uprooting resistance of the windward roots. For instance this was responsible for 75% of the anchorage strength of deep-rooted larch (Crook and Ennos, 1996a),

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A. R. ENNOS

compared with only around 25% due to the leeward hinge. The figure is lower for Coutts's (1986) spruce, and it is clear that their poor anchorage performance and hence lack of stability is due to their lack of sinker roots in the waterlogged soils in which they are grown. The soil can easily break below the root plate, and the windward laterals are inefficiently positioned to resist upward forces. The buttressed trees of the tropical rainforest seem to have a similar anchorage system, though they tend to retain a large tap root (Crook et al., 1997). The sinker roots of these trees also tend to emerge further from the trunk, and the laterals are strengthened, allowing them to resist the large bending forces they have to withstand by developing tall buttresses (Mattheck, 1991). Tap Root Systems Anchorage systems dominated by a single central tap root are characteristic of small dicots, such as many Brassicaceae (Ennos and Fitter, 1992) and a few pioneer trees from tropical rainforests (Crook et al., 1997). Recent experiments carried out in our laboratory (A.M. Goodman, M. J. Crook and A. R. Ennos, unpublished observations) have shown that in oil-seed rape (Brassica napus ), anchorage is provided almost entirely by the vertical root which acts rather like a foundation pile. As the plant is pushed over the tap root is bent and rotates about a point some way below the soil surface, the top moving to leeward and the bottom to windward (Fig. 4(C)). These movements are resisted by two components of anchorage: the compressive resistance of the soil to lateral motion; and the bending resistance of the tap root itself. As in the plate systems, the exact mode of failure depends on the soil properties. Soft, wet soil fails readily in compression and the plant rotates deep underground pushing the soil sideways and eventually leaning over permanently. In stronger, drier soil, in contrast, the tap rot or even stem are more likely to fail. In our tests on Brassica napus we found that in wet loam around 50% of anchorage was due to the resistance of soil to lateral compression, and 50% to the bending resistance of the root.

2.

Fig. 4. Failure modes due to horizontal forces in three types of root systems. In widely spreading root systems with sinker roots such as those possessed by trees and some herbaceous dicots (A) the system rotates up around a leeward hinge. Uprooting is resisted by three components: the stiffness of the hinge; the resistance to pullout of the windward sinker roots; and the weight of the root/soil plate. In the narrower systems seen in cereals (B) and other monocots rotation occurs about a windward hinge. Lodging is resisted by two components: the resistance of the soil beneath the root/soil cone to compression; and the buckling resistance of the coronal roots. Overturning of simple tap root systems (C) is resisted by the resistance of the soil on either side to compression and the bending resistance of the tap root itself. Movement is centred directly beneath the stem.

MECHANICS OF ROOT ANCHORAGE

Sinker root

c Lateral root

Centre of movement Tap root

...

145

146

A. R.ENNOS

3. Coronal and Prop Root Systems Monocots are incapable of producing tap root systems because their roots cannot undergo secondary thickening. Instead, many monocots, including economically important cereals, such as wheat (Triticum aestivum) and maize (Zea mays), anchor themselves using whorls of thick lignified adventitious roots which emerge from the stem and grow down obliquely into the soil like an inverted crown (Ennos, 1991b; Ennos et al., 1993b; Crook and Ennos, 1993). In these rather narrow anchorage systems the resistance of the windward roots to being pulled out of the soil is usually higher than the resistance of the roots beneath the windward side to being compressed. As a result, when plants are pushed over the system rotates about a windward hinge (Fig. 4(B)) and a cone of roots and attached soil is levered into the ground. As in most plants, the force required is lower in wetter, weaker soil but not by as much as might be expected, because the cone will be larger in weak, wet soil and smaller in dry soil. There are two components of anchorage (Crook and Ennos, 1993): the resistance of the soil to compression; and the buckling resistance of the windward roots. Intermediate Systems Many plants have root systems and exhibit behaviour which are intermediate between the extremes described above, or whose behaviour is slightly different from what might be expected. The tap root system of the rainforest pioneer tree Mallotus wrayi looks and behaves like a scaled-up version of oil seed rape (Crook et al., 1997) but also possesses lateral roots whose resistance to lateral motion makes up 20% of the anchorage strength. In contrast, the buttressed rainforest trees of the genera Aglaia and Nephilium, which basically have a plate system, also possess a well-developed tap root (Crook et al., 1997). This has a major role in anchorage, acting not only like a very large sinker, but also like the tap root of Mallotus, supplying 40% of the anchorage of these trees. The root system of sunflower (Helianthus annuus) is superficially very different from trees; it has a large tap root which seems to dominate the root system (Ennos et al., 1993a), and there are no obvious sinker roots, though the laterals point down obliquely into the soil. Despite this, the anchorage system tends to behave very like the plate systems of trees. When pulled over, the plant rotates about a leeward hinge. In wet soil a ball of roots and soil is pulled out of the ground, though as with trees in drier soil the laterals and tap root are more likely to break. As in trees, anchorage is dominated by the windward roots which have to be pulled from the soil, while the tap root has a somewhat smaller role. Similarly, the adventitious root system of Himalayan balsam (Impatiens glandulifera) behaves like a plate system even though it looks superficially more like the coronal root systems of wheat and maize (Ennos et al., 1993a). It seems that the key in both these cases is that

4.

MECHANICS OF ROOT ANCHORAGE

147

the relatively wide spread of the lateral or adventitious roots effectively prevents lateral or downward motion of the root system. The only way the system can fail is by being levered upwards. Individual species can also show different anchorage behaviour depending on their genetic make-up, on the conditions in which they have been grown and on the conditions in which they are tested. A good example of a system which can show different behaviour depending on its exact morphology is wheat. Most winter wheat plants show the typical behaviour shown in Fig. 4(B). However, in spring wheat plants, which have a lower number of roots, the roots can move independently through the soil rather than in a block (Ennos, 1991 b). It has also been observed (Crook, 1994) that in some winter wheat plants with particularly widely spreading coronals, the soil resists downward forces so much that windward roots are pulled from the soil instead, just as in trees.

IV. A.

USES OF MODELS OF ANCHORAGE UNDERSTANDING ROOT SYSTEM MORPHOLOGY

Perhaps the most important result of the detailed mechanical analyses of anchorage that have been carried out is that we now have a much better idea of the functional considerations which influence root system form. Most crucially, the studies have shown that it is anchorage considerations rather than absorption considerations which effectively constrain the overall shape of root systems. It is becoming clear that root system form depends on the type of anchorage required by a plant, on its size, and on its taxonomic position. However, an understanding of anchorage can also help us to understand finer aspects of root system morphology and anatomy. For example, the rigid lignified endodermis of the adventitious roots of wheat (Crook and Ennos, 1993), maize (Haberlandt, 1914; Ennos eta!., 1993b) and Himalayan balsam (Ennos et al., 1993a) are clearly adaptations that increase the rigidity of roots which are incapable of secondary thickening. The elliptical cross-sections of lateral tree roots will clearly increase their flexural rigidity in the vertical plane. This will improve anchorage both by increasing the hinge component of anchorage and also by increasing the size of root plate they can support (Coutts, 1983). In their investigation of the mechanical properties of the wood of the lateral roots of trees, both Stokes and Mattheck (1996) and Niklas (1999) have found that material strength peaked around 1m from the trunk, just the area where leeward roots are maximally stressed during overturning tests (Crook and Ennos, 1996a). Again, this will clearly maximize anchorage strength.

148

A. R.ENNOS

Another aspect of root mechanics which has recently come under investigation is their degree of prestress. Unlike the trunks of trees in which the outer tissue is held in tensile prestresses of up to 10 MPa (Archer, 1986) and prestrains of up to w-3 (Bonser and Ennos, 1998), the values for the structural roots are much lower. Gartner (1997) in her study of two species of angiosperm trees and two conifers, showed that root prestrains were only some 40% of stem prestrains. These results are explicable bearing in mind that her (fairly distal) roots would only be loaded in tension, and hence the lower pretension would reduce their chances of failure. In contrast with Gartner's roots, the tops of root buttresses, which are more likely to be loaded in compression often have high tensile prestrains (Chouquet et al., 1995), and the leeward roots of maritime pine (Pinus pinaster) can develop compressive prestrains which may help counteract wind forces (Stokes et al., 1998). B.

UNDERSTANDING THE THIGMOMORPHOGENIC RESPONSES OF ROOTS

It is becoming clear that roots are just as sensitive to mechanical stimulation

as shoots. Indeed, herbaceous plants which have been shaken (Gartner, 1994; Goodman and Ennos, 1996) or been allowed to sway freely in the wind (Crook and Ennos, 1996b; Goodman and Ennos, 1997) generally show increased root to shoot ratios compared with unstressed plants. This is due to increased secondary thickening and hence increased biomass of the structural roots. In sunflowers, there was also a change in orientation of the main laterals, which showed a greater spread from the vertical (Goodman and Ennos, 1996). Both changes are clear adaptive responses which should improve the anchorage strength, and indeed in most cases thigmomorphogenesis has been shown to increase the anchorage strength of herbs. Unidirectional bending of the stems of maize and sunflowers (Goodman and Ennos, 1998) caused growth responses which suggested some local control of growth. Increased secondary thickening and the orientation changes in sunflowers occurred only in the plane of stimulation. This slightly increased anchorage strength in this plane. Even more interesting and explicable in terms of anchorage, was that in both species leeward roots, which would be more highly stressed in bending than the windward roots, showed stronger changes in morphology and mechanics (Goodman and Ennos, 1999). Again these changes should increase anchorage strength in the direction of bending. There has been even more interest in the effects of unidirectional stimulation in trees. Two sorts of investigations have been carried out: experiments in which young trees were subjected to direct bending (Stokes et a/., 1997) or put in a wind tunnel (Stokes et al., 1995); and investigations of

MECHANICS OF ROOT ANCHORAGE

149

field-grown trees which had been subjected to prevailing winds (Nicholl and Ray, 1996; Stokes et al., 1998) or were leaning (Niklas, 1999). The studies found increased growth, especially secondary growth, of the roots parallel to the plane of stimulation, a pattern which would increase anchorage strength. Secondary thickening tended to be higher on the leeward side on Sitka spruce trees in a prevailing wind (Nicholl and Ray, 1996) and buttresses were formed since roots grew more on top than below. This pattern of growth would greatly increase the strength of the leeward hinge in these trees. In the maritime pine, in contrast, the roots grew more below than on top, which may be related to the fact that these trees have a tap root system (Stokes et al., 1998). However, the distribution of wood strength around each root does not appear to be optimal from an anchorage point of view but rather reflects taxonomic differences in the way wood is laid down over the year. A study of the angiosperm Acer saccharum found that the regions with slower growth and hence narrower growth rings were weaker and less stiff, probably because less of the denser late wood was laid down (Niklas, 1999). In contrast, Stokes and Mattheck (1996) found that in conifers, the narrower growth rings were stiffer and stronger because they had laid down less of the lighter early wood. In trees, the role of mechanical stress or strain in the local control of secondary growth has been championed by Mattheck (1991, 1993). Using finite element analysis, he was able to mimic the growth of buttresses using the simple growth rule that given a plate anchorage system, and occasional swaying of the tree in the wind, growth would be highest in areas of high strain. He thus suggested that given a primary root system of the right morphology, the complete buttress system would develop automatically (Mattheck, 1993; Ennos, 1993b). Tests we carried out attaching strain gauges around the bases of buttressed rainforest trees showed that when trees were pulled sideways, strain was distributed around the buttress just as Mattheck had predicted (Crook et al., 1997). The buttresses could be growing automatically, therefore, in the manner he suggested. Unfortunately, two pieces of evidence weigh against the simple theory that root growth is totally controlled by mechanical stresses. First, tests carried out on non-buttressed trees showed even higher strains at the join of the lateral roots and trunk when the trunks were pulled laterally than in buttressed trees (Crook eta!., 1997). Second, buttress formation is well advanced in buttress-forming trees even when they are very young and so unlikely to have had appreciable mechanical stimulation (M. J. Crook, unpublished observations). Therefore secondary root growth, though influenced by mechanical stimulation, must also be strongly controlled by genetic factors. Clearly the development of the root systems of trees must be subjected to more studies like that of Coutts and Lewis (1983) before we can hope to understand how anchorage development is controlled.

150

A. R.ENNOS

C.

IMPROVING THE STABILITY OF CROP PLANTS

Perhaps more importantly the recent research has enabled applied researchers to investigate with a much greater chance of success how crop plants and trees can be better stabilized. The research has emphasized how tests of anchorage must be made more realistic. Only pulling or pushing tests will mimic with any reliability the processes of lodging and windthrow. Less realistic tests such as pulling plants vertically from the soil can be abandoned. Similarly, tests in which individual roots are pulled from the soil, though relatively easy to perform (Neenan and Spencer-Smith, 1975) are fairly meaningless; anchorage failure may not involve root pullout, and even if it does roots are more likely to pull out in blocks together. When sensible tests are carried out in the light of knowledge about mechanisms of anchorage failure, progress can be made far more rapidly. In wheat, for instance, it has been shown (Crook and Ennos, 1995) that adding excess nitrogen decreases stability because this reduces the strength of the coronal roots. Similarly, rolling the soil in autumn could increase stability because it increases soil strength and hence the anchorage strength of the wheat (Crook, 1994). In contrast, adding growth regulators makes wheat more stable only because it reduces the height of the stem (Crook and Ennos, 1995). A study of root system development (Crook et al., 1994) also showed why lodging is confined to the period of grain filling; plant weight peaks at this point, long after anchorage strength has reached a plateau, so at this point it is least stable. Similar progress has been made in understanding why different cultivars have different lodging resistance. Lodging-resistant cultivars tend to be shorter, and to have stronger anchorage systems because they have a larger number of stronger, more widely spreading coronal roots (Crook and Ennos, 1994). Specific recommendations can, therefore, be made to plant breeders to develop plants with just these characteristics, though it must be remembered that the effectiveness of increasing root spread is probably limited; if roots spread too widely the mode of failure will change. It is likely that with further research, specific recommendations to breeders and improvements could also be made in the stability of the other major cereals: maize and rice. Excellent progress has been achieved in showing how the stability of plantation trees like Sitka spruce could be improved. It was clear from the original anchorage experiments that increasing the depth of rooting is probably the best way to improve anchorage (Coutts, 1986). Recent research has taken up the challenge and investigated the effect on anchorage of improving the drainage of the waterlogged peat in which they grow (Ray and Nicholl, 1998). The results showed that draining land could increase anchorage strength by around 20%, largely because the trees had thicker root plates, which were harder to bend and lifted up a greater mass of soil.

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This was in spite of a significant reduction in the root/shoot ratio which would enable trees to partition more energy into productive wood. There is also potential for breeding more waterlogging-resistant trees. Finally, data about anchorage strength can be incorporated into complex computer models oflodging and windthrow, to help predict the likelihood of failure (Baker, 1995). This approach has been used successfully on wheat to identify root, shoot and soil characters which may be controlled to reduce lodging risk (Baker et al., 1999).

V. AREAS FOR FUTURE RESEARCH Although great progress has been made over the last 20 years there are still wide areas of ignorance about root anchorage, and important questions that remain to be answered. How stable are plants? How does anchorage change as plants grow? How does soil type affect root growth and anchorage? How do single roots and groups of roots interact with soil? How can numerical models of anchorage be devised? How does root anchorage scale? Why are taproot systems more common in small plants, whereas plate systems are commoner for large plants? The main problem is that far too few studies have been carried out to build up a broad picture of the area. More data from both uprooting and overturning experiments are needed, on a wider range of plants and over the entire span of their development. These data should also be combined with data about the wind forces exerted on them. Unfortunately the field of plant aerodynamics is in an even earlier stage of development than is anchorage mechanics (Ennos, 1999). However, the studies of anchorage have also tended to follow a similar course, so the questions posed above can only be answered if several new areas of research are developed and their results combined. A.

THE EFFECT OF SOIL PROPERTIES

Virtually all the work so far carried out on anchorage has been on plants growing in loam soils at field capacity and with shear strengths of 104-105 Pa. The effect of soil type on the root morphology of plants and their resulting anchorage mechanics is therefore still largely unknown. In this respect it will be important to compare plants growing in three very different types of soil: pure unstructured clay, in which the properties are dominated by cohesion; pure sands, in which the properties are dominated by friction; and structured agricultural soils like the loams already studied. The effect of the water status on anchorage mechanics has also been largely ignored. It is clear that in loams the anchorage strength in wetter.

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weaker soil is lower than in drier stronger soil (Ennos, 1990; Crook, 1994). However, the effect is smaller than might be expected because in wetter soil failure tends to occur further from the stem base. The effect might be expected to be rather higher in clays, because its cohesion is greatly affected by water status, but lower in sand whose friction is altered far less. Controlled studies investigating the relationship between anchorage of plants and the water status of the soil are therefore needed in all types of soil. Such work may best be carried out using plants grown in soil cores lying on sand tables (Crook, 1994). The effect of controlling soil density on root system development and consequently anchorage strength has been examined only once, despite the large literature on the effect of soil density on primary root growth. Goodman and Ennos (1999) showed that plants growing in soil with bulk densities of 1.0 x 103 kg m-3 and 1.4 x 103 kg m-3 had only marginally different anchorage systems. Because the denser soil had over double the shear strength, plants growing in it were much more resistant to lodging. B. NUMERICAL MODELS OF ANCHORAGE

Because of the lack of a theoretical framework for the experimental work carried out on the root systems of self-supporting plants so far, there are no reliable numerical models of anchorage systems. Before sensible models of anchorage can be developed it will be essential to understand the interactions of single roots and groups of roots with the soil. The model could then be built up by adding the elements together. Some understanding already exists about the resistance to movement of structures like piles and foundations within engineering soils like clays and sands, though usually at rather larger scales than roots (see for instance Whitaker, 1970). A study by Stokes et al. (1996) has also investigated the pullout resistance of model root systems embedded in wet sand. It was found that the most efficient type of anchorage system was one with an increased number of horizontal roots located deep in the soil. The results are instructive, though no effort was made to work out the costs of the different systems and pure sand is a very unusual medium for root growth. Much more of this sort of work needs to be performed but in agricultural soils and also examining resistance to downward and lateral movements. A major problem in making repeatable and meaningful tests on agricultural soils is that their behaviour is very complex and depends on a wide range of factors. The behaviour of such soils is best represented by the critical state theory (see, for instance Smith and Mullins, 1999), which is notoriously hard to quantify. Perhaps the most relevant research in this area is that on the movement of farm implements, such as narrow tines through the soil (Spoor, 1973). The soil behaviour depends on a multitude of factors

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such as depth, implement orientation, the water status of the soil and the overburden pressure, and failure modes range from compressive smear to brittle fracture. Much research needs to be carried out on movement of single and multiple rods in agricultural and clay soils. Two investigations of this sort have already shown that the results of these sorts of tests could help to explain the change from tap root systems in small plants to plate systems in large ones. It had been assumed, based on research carried out on piles (Broms, 1964), that the overturning resistance of tap roots was proportional to their diameter and to the square of their length. A study of the scaling of anchorage in the tap-rooted tropical tree Mallotus wrayi, growing in wet clay (Crook and Ennos, 1997) confirmed this relationship. Big trees had longer and thicker tap roots and had stronger anchorage. However, the tap roots did not grow isometrically with trunk diameter but became relatively thinner and shorter as the tree grew. Consequently anchorage became relatively weaker than stem strength as the trees grew taller, so that larger ones pulled over rather than snapped. The question to be answered is why did the roots not grow isometrically? Perhaps growth became more difficult deeper in the soil or as the roots expanded sideways. Clearly work on the resistance of soil to root expansion and the ability of woody roots to exert radial pressure (such as that carried out for newly growing roots by Misra et al. (1986)) is required to test whether this was the case. The second study, investigating the resistance of model tap roots of oilseed rape to rotation through a loam soil was even more instructive. The results showed that rotational resistance was less affected by rod diameter than it would be in clay and more by the depth it was buried (A.M. Goodman, unpublished observations). These results suggest that, at least in agricultural soils, tap roots will lose their effectiveness as anchorage devices as they get thicker. This may be a major reason why in temperate regions trees tend to lose their tap root and develop plate systems as they grow even more readily than in the tropics.

VI.

CONCLUSIONS

Inevitably at such an early stage in the emergence of a new science, there is still much that is unclear about the anchorage mechanics of plants, particularly in relation to root development and the mechanics of agricultural soils. The dynamic nature of anchorage failure has also hardly been considered. Nevertheless, the foundations have been laid for future research which could greatly extend the number of species studied, explain the variability of root system form and root system development, and help promote the stability of plantation and amenity trees and crop plants.

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REFERENCES Anderson, C. J., Coutts, M. P., Ritchie, R. M. and Campbell, D. J. (1989). Root extraction forces measurements for Sitka spruce. Forestry 62, 127-137. Archer, R. (1986). "Growth Stresses and Strains in Trees". Springer-Verlag, New York. Arihara, J. and Crosbie, T. M. (1982). Relationships among seedling and mature root system traits of maize. Crop Science 22, 1197-1202. Bailey, P. H. J. (1998). "Grazing Pressure and Anchorage in Herbaceous Plants". PhD Thesis, University of York. Baker, C. J. (1995). The development of a theoretical model for the windthrow of plants. Journal of Theoretical Biology 175, 355-372. Baker, C. J., Scott, K., Berry, P., Griffin, J., Sylvester-Bradley, R. and Clare, R. (1999) A method for the assessment of the risk of wheat lodging. Journal of Theoretical Biology 194, 587-603. Beck, D. L., Darrah, L. L. and Zuber, M. S. (1987). An improved technique for measuring resistance to root pulling in maize. Crop Science 27, 356-358. Bonser, R. H. C. and Ennos, A. R. (1998). Measurement of prestrain in trees: implications for the determination of safety factors. Functional Ecology 12, 971-974. Broms, B. B. (1964 ). Lateral resistance of piles in cohesive soils. Journal of the Soil Mechanics and Foundations Division: Proceedings of the American Society of Civil Engineers SM2 90, 27-63. Chouquet, I., Fournier, M. and Thibaut, B. (1995). Visual assessment of wood quality in standing trees in Eperua falcata Aublet (Caesalpinaceae): growth stresses in buttresses in relation with their mechanical function. I.A. WA. Journal16, 17-24. Coutts, M.P. (1983). Root architecture and tree stability. Plant and Soil11, 171-188. Coutts, M. P. (1986). Components of tree stability in Sitka spruce on peaty gley soil. Forestry 59, 173-197. Coutts, M.P. and Lewis, G. J. (1983). When is the structural root system determined in Sitka spruce? Plant and Soi/11, 155-160. Crook, M. J. (1994). "Mechanics of Lodging in Wheat". PhD Thesis, University of Manchester. Crook, M. J. and Ennos, A. R. (1993). The mechanics of root lodging in winter wheat Triticum aestivum L. Journal of Experimental Botany 44, 1219-1224. Crook, M. J. and Ennos, A. R. (1994). Stem and root characteristics associated with lodging resistance in four winter wheat varieties. Journal of Agricultural Science 123, 167-174. Crook, M. J. and Ennos, A. R. (1995). The effects of nitrogen and growth regulators on stem and root characteristics associated with lodging resistance in winter wheat. Journal of Experimental Botany 46, 931-938. Crook, M. J. and Ennos, A. R. (1996a). The anchorage mechanics of mature larch Larix europea x L. japonica. Journal of Experimental Botany. 47, 1509-1517. Crook, M. J. and Ennos, A. R. (1996b). Mechanical differences between freestanding and supported wheat plants Triticum aestivum L.Annals of Botany 77, 197-202. Crook, M. J. and Ennos, A. R. (1997). Scaling of anchorage in the tap-rooted tree Mallotus wrayi. In "Plant Biomechanics: Conference Proceedings". University of Reading, Reading. Crook, M. J., Ennos, A. R. and Sellars, E. K. (1994). Structural development of the shoot and root systems of two winter wheat cultivars. Journal of Experimental Botany 45, 857-863.

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Crook, M. J., Ennos, A. R. and Banks, J. R. (1997). The function of buttress roots: a comparative study of the anchorage systems of buttressed (Aglaia and Nephileum ramboutan species) and nonbuttressed (Mallotus wrayi) tropical trees. Journal of Experimental Botany 48, 1703-1716. Dittmer, H. J. (1937). A quantitative study of the roots and root hairs of a winter rye plant (Secale cereale ). American Journal of Botany 24, 417-420. Donovan, L. S., Jui, P., Kloek, M. and Nicholls, C. F. (1982). An improved method of measuring root strength in corn (Zea mays L.). Canadian Journal of Plant Science 62, 223-227. Ennos, A. R. (1989). The mechanics of anchorage in seedlings of sunflowers Helianthus annuus. New Phytologist 113, 185-192. Ennos, A. R. (1990). The anchorage of leek seedlings: the effect of root length and soil strength. Annals of Botany 65, 409-416. Ennos, A. R. {1991a). The mechanics of anchorage in wheat Triticum aestivum L. I. The anchorage of wheat seedlings. Journal of Experimental Botany 42, 1601-1606. Ennos, A. R. (1991b ). The mechanics of anchorage in wheat Triticum aestivum L. II. Anchorage of mature wheat against lodging. Journal of Experimental Botany 42, 1607-1613. Ennos, A. R. (1993a). The scaling of root anchorage. Journal of Theoretical Biology 161, 61-75. Ennos, A. R. (1993b ). The function and formation of buttresses. Trends in Ecology and Evolution 8, 350-351. Ennos, A. R. (1999). The aerodynamics and hydrodynamics of plants. Journal of Experimental Biology 202, 3281-3284. Ennos, A. R. and Fitter, A. H. (1992). Comparative functional morphology of the anchorage systems of annual dicots. Functional Ecology 6, 71-78. Ennos, A. R., Crook, M. J. and Grimshaw, C. (1993a). A comparative study of the anchorage systems of Himalayan balsam Impatiens glandulifera and mature sunflower. Helianthus annuus. Journal of Experimental Botany 44, 133-146. Ennos, A. R., Crook, M. J. and Grimshaw, C. (1993b). The anchorage mechanics of maize Zea mays. Journal of Experimental Botany 44, 147-153. Fouere, A., Pellerin, S. and Duparque, A. (1995). A portable electronic device for evaluating root lodging resistance in maize.Agronomy Journal87, 1020-1024. Gartner, B. L. (1994). Root biomechanics and whole plant allocation patterns: responses of tomato to simulated wind. Journal of Experimental Botany 45, 1647-1654. Gartner, B. L. (1997). Longitudinal growth strains in stems v roots of four woody species. In "Plant Biomechanics: Conference Proceedings". University of Reading, UK. Goodman, A.M. and Ennos, A. R. (1996). A comparative study of the response of roots and shoots of sunflower and maize to mechanical stimulation. Journal of Experimental Botany. 47, 1499-1507. Goodman, A.M. and Ennos, A. R. (1997). The responses of field grown sunflower and maize to mechanical support. Annals of Botany 79, 703-711. Goodman, A. M. and Ennos, A. R. (1998). Responses of the root systems of sunflower and maize to unidirectional stem flexure. Annals of Botany 82, 347-358. Goodman, A.M. and Ennos, A. R. (1999). The effects of soil bulk density on the morphology and anchorage mechanics of the root systems of sunflower and maize. Annals of Botany 83, 293-302. Haberlandt, G. (1914). "Plant Physiological Anatomy". MacMillan, London.

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Kevern, T. C, and Hallauer, A R. (1983). Relation of vertical root-pull resistance and flowering in maize. Crop Science 23, 357-363. Koinuma, K., Inoue, Y. and Kato, A (1990). Evaluation of lodging resistance of maize (Zea mays L.) by the measurement of the horizontal pull resistance. Bulletin of the Natural Grassland Research Institute 43, 23-29. Kushibiki, H. (1979). A simple method of testing lodging resistance of maize. Bulletin of the Hokai Prefecture Agricultural Experimental Station 42, 21-27. Mattheck, C. (1991). "Trees: the Mechanical Design". Springer-Verlag, New York. Mattheck, C. (1993). "Design in der Natur. Der Baum als Lehrmeister". RombachVerlag, Freiburg. Melchinger, A E., Geiger, H. H. and Schmidt, G. A (1986). Vertical root-pull resistance and its relationship to root lodging and forage traits in early maturing European inbred lines and Fl hybrids of maize. Maydica 31, 335-348. Misra, R. K., Dexter, A R. and Alston, A M. (1986). Maximum axial and radial growth pressures of plant roots. Plant and Soil95, 315-326. Nass, H. G. and Zuber, M.S. (1971). Correlation of corn (Zea mays L.) roots early in development to mature root development. Crop Science 11, 655-658. Neenan, M. and Spencer-Smith, J. L. (1975). An analysis of the problem of lodging with particular reference to wheat and barley. Journal of Agricultural Science 85, 495-507. Nicholl, B. C. and Ray, D. (1996). Adaptive growth of tree root systems in response to wind action and site conditions. Tree Physiology 16, 891-898 Niklas, K. J., (1999). Variations of the mechanical properties of Acer saccharum roots. Journal of Experimental Botany 331, 193-200. Pfeffer, W. (1893). Druck und Arbeitsleistung durch Waschende Pflanzen. Abhandlungen der Koniglich Sachsischen Gesellschaft der Wissenschaften 33, 235-474. Ray, D and Nicholl, B. C. (1998). The effect of soil water-table depth on root-plate development and stability of Sitka spruce. Forestry 71, 169-182. Rogers, R. R., Russell, W. A and Owens, J. C. (1976). Evaluation of a vertical-pull technique in population improvement of maize for corn rootworm tolerance. Crop Science 16, 591-594. Smith, K. A and Mullins, C. E. (1999). "Soil Analysis: Physical Methods" 2nd edition. Marcel Dekker, New York. Spoor, G. (1973). Fundamental aspects of cultivation. In MAFF Technical Bulletin 29 "Soil Physical Conditions and Crop Production". HMSO, London. Stokes A and Mattheck C. (1996). Variation of wood strength in tree roots. Journal of Experimental Botany 41, 693-699. Stokes, A., Fitter, A H. and Coutts, M.P. (1995). Responses of young trees to wind and shading: effects on root architecture. Journal of Experimental Botany 46, 1139-1146. Stokes, A Ball, J., Fitter, A H., Brain, P. and Coutts, M.P. (1996). An experimental investigation of the resistance of model root systems to uprooting. Annals of Botany 78, 415-421. Stokes, A, Nicholl, B. C., Coutts, M.P. and Fitter, A H. (1997). Responses of young Sitka spruce clones to mechanical perturbation and nutrition: effects on biomass allocation, root development, and resistance to bending. Canadian Journal of Forest Research 21, 1049-1057. Stokes, A., Bethier, S., Sacriste, S. and Martin, F. (1998). Variations in maturations strains and root shape in root systems of Maritime pine (Pinus pinaster Ait.) Trees- Structure and Function 12, 334-339.

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Stolzy, L. H. and Barley, K. P. (1968). Mechanical resistance encountered by roots entering compact soils. Soil Science lOS, 297. Terashima, K., Akita, S. and Sakai, N. (1992). Ecophysiological characteristics related with lodging tolerance of rice in direct sowing cultivation. 1: Comparison of the root lodging tolerance among cultivars by the measurement of pushing resistance. Japanese Journal of Crop Science 61, 380-387. Terashima, K., Akita, S. and Sakai, N. (1995). Ecophysiological characteristics related with lodging tolerance of rice in direct sowing cultivation. III: Relationship between the characteristics of root distribution in the soil and lodging tolerance. Japanese Journal of Crop Science 64, 243-250. Whitaker, T. (1970). "The Design of Piled Foundations". Pergamon Press, Oxford.

Molecular Genetics of Sulphate Assimilation

MALCOLM J. HAWKESFORD 1 and JOHN L. WRAY 2 1

IACR-Rothamsted, Biochemistry and Physiology Department, Harpenden, Hertfordshire ALS 2JQ, UK 2 Plant Sciences Laboratory, Sir Harold Mitchell Building, Division of Environmental and Evolutionary Biology, School of Biology, University of St Andrews, St Andrews, Fife KY16 9TH, UK

I. II.

Introduction.................................................................................................... Applied Relevance of Studies on Sulphur Metabolism............................. A Sulphur Inputs and Agriculture............................................................ B. Sulphur and Crop Quality and Yield................................................... C. Engineering Sulphur Sinks.................................................................... III. Uptake and Translocation............................................................................. A A Family of Transporters....................................................................... B. Sites of Expression.................................................................................. C. Subcellular Transport............................................................................. D. Regulation of Expression....................................................................... E. Long Distance Transport....................................................................... IV. The Pathway of Reductive Sulphate Assimilation..................................... A Activation of Sulphate............................................................................ B. Reduction of Sulphate ........ ............ ...... .... .. ............. ...... .... .. .................. C. Synthesis of Cysteine.............................................................................. V. Regulation and Interaction with the Environment.................................... A. Control of Flux........................................................................................ B. Allosteric Regulation and Protein-Protein Interaction as a Regulatory Device- Serine Acetyltransferase and 0-Acetylserine ( thiol) Lyase ............................................................................................ C. Sulphur Supply........................................................................................ D. Co-ordination with Nitrogen Metabolism........................................... E. Environmental Stresses, Glutathione and Abscisic Acid................... F. Sensing the Environment and Signal Transduction to the Genes.... G. Studies with Transgenic Plants.............................................................. VI. Conclusions and Future Prospects............................................................... Acknowledgements ............ ......................... ......................... ....... ............... .... References....................................................................................................... Advances in Botanical Research Vol 33 incorporating Advances in Plant Pathology ISBN 0-12-1105933-9

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Copyright © 2000 Academic Pr-::s~ All right:, of reproduction in any form reserved

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The importance of sulphur in promoting yield, quality and stress resistance parameters in plants has been highlighted by the recent increased problems of S-deftciency in agriculture. These deficiencies are in part a consequence of reduced atmospheric emissions from industry and the subsequent decreased deposition on agricultural/and. Contributions from several laboratories, worldwide, have resulted in the cloning of almost all of the genes responsible for uptake, transport and assimilation of sulphate. This has led directly to the resolution of many outstanding questions regarding the control of uptake and of the pathways and intennediates involved in assimilation. Furthennore, the ability to manipulate these pathways is now possible and will allow the engineering of crops with improved sulphur acquisition and utilization traits. This paper reviews the present status of the molecular genetics of sulphate assimilation in plants.

I.

INTRODUCTION

There have been several excellent recent reviews on sulphur metabolism and on the insights molecular biological approaches are providing (Leustek, 1996; Hell, 1997; Bick and Leustek, 1998; Ravanel et al., 1998; Leustek and Saito, 1999). This review focuses on recent insights research on uptake and reductive assimilation to cysteine. In addition practical applications of the molecular approaches are highlighted. Predating the molecular cloning explosion was the review by Schmidt and Jager (1992) entitled 'Open questions about sulfur metabolism in plants'. A huge research effort has followed so that, although there are many answers to old questions, many more fresh questions have evolved. For example, it is not clear as to why there are such large gene families encoding the transporters and other components of the assimilatory pathway. These gene families encode differentially expressed isoforms and little is known of their individual roles in the context of whole plant sulphur metabolism. We are at an exciting juncture with the availability of a vast array of cloned genes and an ability to manipulate expression of sulphur metabolism in ways not previously possible. This review sets out to summarize the current research position and to identify some of the important areas to which molecular approaches will contribute and which are of physiological importance and agronomic relevance for the future.

II.

APPLIED RELEVANCE OF STUDIES ON SULPHUR METABOLISM A.

SULPHUR INPUTS AND AGRICULTURE

Sulphur is frequently referred to as the fourth major nutrient for crop nutrition (Syers et a/., 1987), however until recently research into sulphur nutrition lagged behind the other major nutrients. The increased awareness

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of the importance of sulphur from the agronomic viewpoint has come about because of the developing trend for deficiency problems in agriculture for many areas of the world, particularly in the developed countries. Substantial inputs of S into agricultural land, particularly near to industrialized regions, have for many years, resulted from atmospheric deposition arising from airborne pollution from anthropogenic sources. In addition, prior to the use of high analysis super phosphate fertilizers (with little or no S content), sulphur was also applied in the form of single superphosphate (10-12% S) or as ammonium sulphate (24% S). As a consequence, crop requirements for S were fulfilled and little planned management of S-fertilization was required. However, in recent years substantial progress has been made to decrease pollution levels such that emissions have decreased from 3.2 million tonnes of S in 1970 to around 1.0 million tonnes in 1996 (Department of the Environment, Transport and the Regions, 1998). Aerial deposition varies greatly depending on geographic location, but deposition 1 rates at Woburn, Bedfordshire, UK have decreased from 72 kg ha- in 1970 1 to 15 kg ha- in 1992 (McGrath et al., 1996). These low deposition rates are much less than many crop requirements for S, which is typically 15-20 kg ha- 1 for wheat and around 50 kg ha- 1 for oil-seed rape (McGrath et al., 1996). Even lower targets, and the continuing trend for decreased emissions, will effectively remove this source of S for agriculture and crop losses, both in terms of yield and quality, are likely to result. The clear message for agriculturists is to incorporate S-fertilizer application into management practices. However much is still to be learned about optimum regimes of quantity, timing and type of S-fertilizer. Of additional benefit to the farmer are accurate monitors of S-nutritional status and the early recognition of S-deficiency symptoms. The recent advances in the cloning and analysis of the genes involved inS-uptake and assimilation will facilitate the application of biotechnology to these real agronomic problems. Plant biotechnology is likely to contribute to both enhancing S-utilization efficiency, particularly in terms of preferential S-allocation to sink tissues and the development of diagnostic procedures for the recognition of deficiency, utilizing either chemical analysis or via the use of the so-called 'smart plant' technology. B.

SULPHUR AND CROP QUALITY AND YIELD

Sulphur assimilation and the effects of sulphur nutrition on yield and quality of wheat have recently been reviewed (Zhao et al., 1999a). Improved yield responses to S-fertilizer additions have been shown for wheat (see Zhao et al., 1999a and references therein) and for oil-seed rape (McGrath and Zhao, 1996). The impact of S-deficiency on quality aspects has been recognized, particularly in terms of the effect on the bread-making quality

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of wheat. Poor S-nutrition leads to significant changes in the protein composition of grain, favouring the synthesis of the low-S proteins, such as w-gliadins and high molecular weight subunits of glutenin. In contrast the Srich proteins, such as a- and 1-gliadins and low-molecular-weight subunits of glutenin are decreased in abundance (Wrigley et al., 1984; Fullington et al., 1987; Castle and Randall, 1987; Byers et al., 1987). The roles of cysteine residues and the formation of disulphide bonds in glutenin functionality have been established (Shewry and Tatham, 1997). The direct link between $-nutrition in the field and the influence on breadmaking quality of flour has been demonstrated recently (Zhao et al., 1999b ). Previously the trend to lower S-content of British grain in relation to decreased S-inputs was described (Zhao et al., 1995). It is clear from these studies that an adequate S-input and assimilation in the grain tissues during grain filling is an essential element of optimal crop quality. Studies on sink partitioning and the delivery of S to grain and seed tissues indicate that the delivery of sulphate for in situ reduction and assimilation is important, together with delivery of reduced S as glutathione or as S-methylmethionine (Bourgis et a!., 1999). C.

ENGINEERING SULPHUR SINKS

An approach to improve the nutritional quality of both human foodstuffs and animal feeds is to engineer the protein composition of grains and sink tissues. These approaches would envision over-expression of endogenous proteins or the transgenic expression of foreign genes encoding S-rich proteins in sink tissues. The expression of many of the endogenous $containing sink proteins is controlled by $-availability, as discussed above in relation to gluten biosynthesis. A motif in the promoter region responsible for sensing sulphur-nutritional status has been identified for the ~­ conglycinin gene from soybean (Hirai et al., 1995) and the availability of such promoters will allow the manipulation of sink strength and over-expression of specific proteins in sink tissues. The sink strength may be further manipulated by expression of foreign or even artificial $-containing proteins in tissues. Genes for the 2S albumins of brazil nut and for the sunflower seed have been successfully expressed in Vicia narbonensis (Saalbach et al., 1994; Pickardt et a!., 1995) and lupin (Molvig et al., 1997), respectively. The sunflower albumin protein accounted for 5% of extractable protein and a 94% increase in methionine content of the seed, however totalS content was not significantly altered. Clearly, to increase the overall S-content of seed tissues, either $-delivery to the seed, or the ability to assimilate S in seed tissue, needs to be enhanced in parallel with the modification of sink strength.

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UPTAKE AND TRANSLOCATION

Uptake of sulphate into plant roots and cells has been thoroughly described at the physiological level (see for example Leggett and Epstein, 1956; Lee, 1982; Clarkson et al., 1983, 1993). Transport into roots has been shown to be a multicomponent process, interpreted to be due to multiple carriers with differing affinities for sulphate. Transport across the plasma membrane is thought to be a proton/sulphate cotransport process, an idea supported by the observation of proton gradient-dependent uptake of sulphate into plasma membrane vesicles isolated from roots of Brassica napus (Hawkesford et al., 1993). A fundamental characteristic of sulphate uptake is the enhancement of sulphate uptake capacity observed during sulphurlimiting conditions and a reduction followingS re-supply (see for example, Lee, 1982; Clarkson et al., 1983, Smith et al., 1995a, 1997). The mechanism underlying this response and whether it is at the level of functional allosteric control or at the level of gene expression, has been a long standing question, which has been partly answered following the cloning of transporter genes. A.

A FAMILY OF TRANSPORTERS

The first cloning of a verifiable eDNA for a plant sulphate transporter was achieved by functional complementation of a yeast mutant, isolated for its resistance to selenate and chromate, toxic analogues of sulphate (Breton and Surdin-Kerjan, 1977; Smith et al., 1995b). A yeast eDNA library was expressed in the mutant and eDNA was isolated which was able to restore the ability of the mutant to grow on sulphate as the sole S-source, and also restored sensitivity to selenate and chromate. The predicted amino acid sequence derived from this eDNA showed high homology to that of the only known sequence of a eukaryotic sulphate transporter at that time, the cys14 gene product from Neurospora crassa (Ketter et al., 1991). In addition, sequence similarity between a soybean nodule-expressed protein, a putative human tumour suppressor and the Neurospora sulphate transporter had been documented (Sandal and Marcker, 1994). A yeast deletion mutant, YSD1, was created by excision of 1096 bp of the coding region of the SULJ transporter gene (Smithet al., 1995b), and used to screen plant eDNA libraries of sulphur-starved root of the tropical legume, Stylosanthes hamata (Smith et al., 1995a) and of barley (Smith et al., 1997). Three cDNAs from Stylosanthes (shstl, shst2 and shst3) and one from barley (hvst1) were isolated which functionally complemented the yeast mutant. All of the cDNAs encoded polypeptides which showed homology to one another and to the proposed sulphate transporter family (Sandal and Marcker, 1994). Although the deduced proteins, SHSTl and SHST2 and HVSTl all showed a high degree of similarity to each other, SHST3 was less closely

164

M. J. HAWKESFORD andJ. L. WRAY

related. Expression in yeast also enabled functional characterization of these individually expressed sulphate transporters and all facilitated sulphate transport with apparent Km of around 10 ~-tM with the exception of the SHST3 transporter which had a Km of 100 ~-tM. Analysis of the mRNA abundance for these transporters by Northern blot analysis indicated that expression of SHST1 and SHST2 was specific to the root and that SHST3 showed both root and shoot expression. More recently a number of additional plant sulphate transporters have been cloned (see Tables I and II), most notably in Arabidopsis thaliana (Takahashi et al., 1996, 1997, 1999a, b), but also in the resurrection plant, Sporobolus stapfiana (Ng et al., 1996) and in wheat (Hawkesford and Prosser, 2000). The wheat genes were isolated from the diploid wheat Triticum tauschii and were designated ttstl and ttst2. Additional ESTs (expressed sequence tags) have been identified as fragments of sulphate transporters from maize, rice, potato and Brassica. In Arabidopsis a total of seven sulphate transporter genes have been identified, initially as EST fragments and subsequently as full-length cDNAs and genomic clones. The alignment of the deduced amino acid sequences of these seven Arabidopsis clones is presented in Fig. 1. The sequence similarity that is evident demonstrates that these proteins belong to the same family. The overall percentage of identity, however, is only around 30% and there is considerable divergence present. The evident divergence between the Arabidopsis isoforms is the most likely reason for the small numbers of isoforms of sulphate transporters identified for other species, as discovery is mostly dependent on exploitation of homology. There are no major blocks of identity and even where there are four or five consecutive identical residues in the Arabidopsis family, this is usually lost when comparisons are made with other plant sequences and certainly lost in an overall comparison of all known sulphate transporters from all sources. In spite of some diversity, the whole sulphate transporter family, including members from yeast, filamentous fungi and mammalian sources, share sufficient overall sequence similarity to be classified as a single transporter group. Furthermore, this is a distinct and unique group showing little if any similarity with other transporter groups (Saier et al., 1999). There is considerable variation even within the amino acid sequence motif, which defines members of this family (PROSITE motif database accession number PS01130 (Bairoch and Bucher, 1994)), with up to six variations even at a single residue. Despite the sequence variability, algorithms used to predict secondary structure, and particularly transmembrane helices, suggest similar structures for all sequences. Typically 10 or 12 transmembrane helices are predicted, with no long extramembrane loops, except for long N- and Cterminal regions predicted to have a cytoplasmic location (see Hawkesford and Smith, 1997). Sequence comparison of all the known sulphate transporter sequences places the plant transporters somewhat distinct from

TABLE I Reductive sulphate assimilation genes identified in Arabidopsis thaliana Pathway step

Gene symbol(s)"

Accession number (EMBL)

Protein accession or identification number

Chromosomal Iocationh

Notes/protein locationc

Referenced

Sulphate transport'

AtSTJ-1 (ASTZOJ)

AC002983'

022277

IV

MSGTINI

AB018695

BAA33932

Gnoj eta/. (1997), unpublished Takahashi and Saito (1998), unpublished

AtST2-l (AST68)

AB003590' AB003591

004722

NF

MKERDS'

Takahashi et al. (1997)

AtST2-2 (AST56, ATD14)

AB012047' D85416

064434 P92946

NF

MGIELQf

Takahashi et al. (1996)

AtST3-1 (ASTJ2, ATSTJ,ATD631)

AB012048'

064435

III

MGTEDYf

Takahashi et al. (1998), unpublished

AtST3-2 (AST77)

089631 AB004060

023250 004289

IV

MSSKRN

Yamaguchi et al. (1997) Takahashi et al. (1999a)

AtST3-3 (AST91)

AB023423'

BAA75015 049307

I

MEVHKVf

Takahashi et al. (1999a)

AtST4-1 (AST82)

AB008782

022123

NF

MSYASli

Takahashi et al. (1999b)

APSJ

U05218

Q42519

NF

Plastidic

Leustek et al. (1994)

ATP sulphurylas&

TABLE I Pathway step

APS reductase

Continued

Gene symbol(s)•

Accession number (EMBL)

Protein accession or identification number

Chromosomal locationb

Notes/protein locationc

Referenced

METJ-1 APS2

X79210 006276

Q43870 Q43870

NF

Plastidic? Plastidic?

APS2

059737'

Q43870

ASA1

040715

Q43870

Klonus et al. (1995) Murillo and Leustek (1995) Leustek (1996), unpublished Logan et al. (1996)

APS3

006275

Q42520

APS3

059738'

Q96530

APS4

AJ012586

CAB42640

APS4

AF110407'

AF110407

PRH-19

053864

P92979

APR1 APR1

043412 AF016282'

Q39248 048886

Plastidic? IV

Plastidic?

Murillo and Leustek (1995) Leustek (1996), unpublished

v

Plastidic?

Hatzfeld and Saito (1998), unpublished Lee et al. (1998), unpublished

IV

Plastidic?

Gutierrez-Marcos et al. (1996) Setya et al. (1996) Chen and Leustek (1998)

Plastidic?

PRH-26

U53865

P92980

APR3 APR3

U56922 AF016283'

0 38948 004583

PRH-43

U53866

P92981

APR2 APR2

U5692l AF016284.

0 38947 048887

APSR

AF023167

022554

PRH-73

U96045

004215

NF

Plastidic?

Gutierrez-Marcos eta/. (1997), unpublished

Sulphite reductase

sir sir

Z49217 Yl0157'

042590 0 23650

NF

Plastidic?

Bruhl eta/. (1996) Bork et al. (1998)

Serine acetyltransferase

SAT-A

X80938

042532

III

Plastidic?

SAT/ -6 SAT-I

X82888 U22964

0 42532 042532

SAT/

L78443'

039218

Hell and Bogdanova (1995) Bogdan ova eta/. ( 1995) Roberts and Wray (1996) Ruffet eta/. (1996), unpublished

sat5 SATJ sat5

L34076 L42212 Z34888'

043297 042588 043297

IV

Plastidic? Plastidic?

I

Plastidic? Plastidic?

Plastidic? Plastidic?

I

Cytosolic?

Gutierrez-Marcos et at. (1996) Setya et al. (1996) Chen and Leustek (1998) Gutie rrez-Marcos et al. (1996) Setya et al. ( 1996) Chen and Leustek (1998) Min et al. (1997), unpublished

Ruffet et al. (1995) Murillo et at. (1995) Ruffet eta!. ( 1995)

TABLE I Pathway step

0-Acetylserine(thiol) lyase

Continued

Gene symbol(s)"

Accession number (EMBL)

Protein accession or identification number

Chromosomal Jocationb

Notes/protein Jocationc

Referenced

SAT-52

U30298

Q42538

v

Mitochondrial?

Howarth et at. (1997)

SAT-106

AF112303

Q9ZPJ4

II

Cytosolic?

SAT2

L78444'

L78444

Howarth et at. (1998), unpublished Ruffet et at. (1999), unpublished

AtOAS.S-8 CytACsl

X80376 X81679

P47998 Q43726

IV

Cytosolic? Cytosolic?

cys3A

X84097

Q42570

Cytosolic?

Hell et at. (1994) Hesse and Altmann (1995) Barroso et at. (1995)

AtOAS.7-4 AtCS-B

X80377 X81698

P47999 Q42568

II

Plastidic? Plastidic

Hell et at. (1994) Hesse et at. (1999)

AtCS-C

X81973

Q43725

NF

Mitochondrial

Hesse et at. (1999)

cs26 AtcysCl

AB003041 AB024282

022682 BAA78560

NF

Organellar?

AtcysD2

AB024283

BAA78561

AtcysDJ

AB024284

BAA78562

Nakamura et at. (1997) Yamaguchi et at. (1999), unpublished Yamaguchi et at. (1999), unpublished Yamaguchi et al. (1999), unpublished

APS kinaseh

AKNJ

X75782 005238 059759'

043295 043295 043295

II

Plastidic? Plastidic? Plastidic

Arz et al. (1994) Jain and Leustek (1994) Lee and Leustek (1998)

AKN2

AF043351

049196

IV

Plastidic?

Schiffmann and Schwenn (1998)

aGene symbol is shown where one has been assigned. bLikely chromosomal location was identified by us from an analysis of Genbank data including that available from the Arabidopsis Genome Initiative, NF: chromosomal location not found. 'Likely subcellular location determined by the author,'?' denotes from sequence analysis. "Citations with author names and year but referred to as unpublished, are EMBL etc. database entries ''Seven different sulphate transporters are defined and named according to Takahashi, Watanabe, Smith, Blake-Kalff, Hawkesford and Saito, unpublished. Alternative gene names appearing in the literature or database entries are given in parentheses. 1The six N-terminal amino acids which define the sequence. "ATP sulphurylase, APS reductase, serine acetyltransferase and 0-acetylserine ( thiol) lyase entries are clustered into what are considered to be probable replicate database entries. "APS kinase is included here but, as discussed in the text, is not considered as an intermediate step in the pathway. · denotes genomic clone as opposed to eDNA clone.

TABLE II Reductive sulphate assimilation gene types identified in higher plants other than Arabidopsis thaliana Pathway step

Species

Gene symbola

Sulphate transport

Brassica napus Brassica napus Brassica juncea Glycine max

bnstl bnst2 bjlast nod70

Gene accession number

AJ223495' D13505

Protein accession number

09ZPOO 002920, S34800

Fragment size or protein locationb

Referencec

338 aa 330aa 385 aa

Unpublishedd Unpublishedd Heiss et al. (1999) Kouchi and Rata (1993) Blewitt et al. (1999), unpublished Blewitt et al. (1999), unpublished Smith et al. (1997) Vidmar et al. (1999) Vidmar et al. (1999) Cushman (1999), unpublished Minobe and Sasaki (1997), unpublished Ng et at. (1996) Unpublished' Smith et al. (1995b) Smith et al. (1995b) Smith et al. (1995b) Hawkesford and Prosser (2000) Hawkesford and Prosser (2000)

Gossypium hirsutum

AI730085t

737bp

Gossypium hirsutum

AI730832t

620bp

Hordeum vulgare Hordeum vulgare Hordeum vulgare Mesembryanthemum crystallinum Oryza sativa

hvstl

Sporobolus stapfianus Solanum tuberosum Stylosanthes hamata Stylosanthes hamata Stylosanthes hamata Triticum tauschii

ssulptrp stst shstl shst2 shst3 ttstl

X96761

004001

X82255 X82256 X82454 AJ238244'

P53391 P53392 P53393 CAB42985

Triticum tauschii

ttst2

AJ238245'

CAB42986

X96431 U52867 AF075270' AI823009t

043482 040008 040008 614 bp

D250oot

105 aa 335 aa

ATP sulphurylase

Zea mays

zmstl

AF016306t

048889

233 aa

Bolchi eta/. ( 1999)

Brassica juncea Brassica juncea Brassica napus

A TPS3 ATPS6 LSC680

AJ223498 AJ223499 U68218

0 9ZNZ9 09ZNZ8 096349

Organellar? Organellar? Plastidic?

Brassica oleracea Glycine max

AsBo

U69694 Al437658t

096541 NA

Plastidic?

AB015204

09ZWMO

Plastidic?

Solanum tuberosum Solatium Luberosum Zea mays

Stmet3-J Stmet3-2 ZmASI

X75041 X79053 AF016305

043170 043183 048888

Cytosolic? Plastidic? Plastidic?

Heiss el a/. (1999) Heiss eta/. (1999) Buchanan-Wollasto n and Ainsworth (1997) Hatzfeld eta/. (1997) Shoemaker eta/. (1999), unpublished Nakase-Alvarez et at. (1998), unpublished Klonus eta/. (I 994) Klonus eta/. (1994) Bolchi eta/. (1999)

APS reductase

Brassica juncea Brassica juncea Catharamhus roseus

APSR2 APSR8

AJ001207 AJ001208 U63784

09ZP23 09ZP22 039619

Organellar? OrganeiJar? Plastjdic?

Heiss et al. (1999) Heiss el al. (1999) Prior eta/. ( 1999)

Sulphite reductase

Nicotiana tabacum

gNtSiRI

AB0107L7'

0 82802

Plastidic?

Nicoriana tabacum

NtSiRI

083583

0 82802

Plastidic?

Pnmus armeniaca

AF071890

08 1362

Zea mays

0 50679

023813

Plastidic?

Yonekura-Sakakibara et at. (1998) Yonekura-Sakakibara et a/. ( 1998) Mbeguie et al. ( 1998). unpublished ldeguchi et al. ( 1995)

049535 0 85624' AB006530'

039533 0 39533 039533

Cytosolic? Cytosolic? Cytosolic?

Oryza sativa

Serine acetyltransferase

Citrullus lanatus Citrullus lanalus Citrullus lanatus

Sat Sat Sat

Saito et al. ( 1995) Saito eta/. (1997) Noji eta/. (I 997), unpublished

TABLE II Pathway step

0-Acetylserine( thiol) lyase

Continued

Gene accession number

Protein accession number

Gossypium hirsutum

AI725434t

NA

Spinacia oleracea

D88529

P93544

Y10845 Y10846 Y10847 Al352787t

023733 023734 023735 NA

X64874 AJ006024t

P31300 065747

Citrullus lanatus

D28777 AA660077t

Q43317 NA

Citrullus lanatus

Al563124t

NA

Glycine max

AI461188t

NA

Gossypium hirsutum

Al728743t

NA

Gossypium hirsutum

Al725847t

NA

Species

Brassica juncea Brassica juncea Brassica juncea Brassica napus

Gene symbol"

oas-t/4 oas-t/5 oas-t/6

Capsicum annuum Cicer arietinum Citrullus vulgaris

cysA

Fragment size or protein locationb

Referencec

Blewitt et al. (1999), unpublished Saito and Takagi (1996), unpublished Cytosolic? Mitochondrial Cytosolic?

Schafer et al. (1998) Schafer et al. (1998) Schafer et al. (1998) Fristensky et al. (1996) unpublished Plastidic Romer et al. (1992) Dopico et al. (1998), unpublished Cytosolic? Noji et al. (1994) Ok et al. (1997), unpublished Shin (1999), unpublished Shoemaker et al. (1999), unpublished Blewitt et al. (1999), unpublished Mitochondrial? Blewitt eta/. (1999), unpublished

Gossypium hirsutum

Al7257451

NA

Medicago trunculcua

AA6602571

NA

Medicago trunculata

AA660438t

NA

Mesembryanthemum crystal/inurn Mesembryanthemum crystallin urn Mesembryanthemum crystallinum Oryza sativa Oryza sativa

AA832541 1

NA

AI8226411

NA

Al8227081

NA

AD417

0109571 021271 1

NA NA

Oryza sativa Oryza sativa Oryza sativa Oryza sativa Solanum tuberosum

res] rcs2 rcs3 rcs4 StCS-A

AF073695 AF073696 AF073697 AF073698 AF044172

AA023907 AAD23908 AAD23909 AAD23910 081154

Cytosolic? Organellar? Cytosolic? Cytosolic? Cytosolic?

Solanum tuberosum

StCS-B

AF044173

081155

Plastidic?

Spinacia oleracea Spinacia oleracea Spinacia oleracea Spinacia oleracea Spinacia oleracea Triticum aestivum

CysK

X66860 L05184 037963 010476 014722 013153

P32260 Q33137 Q43153 Q00834 P32260 P38076

Plastidic Plastidic? Mitochondrial Cytosolic? Plastidic Plastidic?

CysC CysA CysB cysl

Blewitt et al. (1999), unpublished Covitz et al. (1997), unpublished Covitz et a[. (1997), unpublished Cushman (1997), unpublished Cushman (1997), unpublished Cushman (1997), unpublished Uchimiya et al. (1992) Uchimiya (1993), unpublished Nakamura et al. (1999) Nakamura et al. ( 1999) Nakamura et at. (1999) Nakamura et al. (1999) Hesse and Hofgen (1998) Hesse and Hofgen (1998) Rolland et al. (1993) Hell et al. (1993) Saito et al. (1994b) Saito et al. (1992) Saito et al. (1993) Youssefian et al. (1993)

TABLE II Continued Pathway step

Species

Gene symbol"

Zea mays

APS kinase'

Catharanthus roseus

CRakn

Gene accession number X85803

Protein accession number P80608

Fragment size or protein locationb Plastidic?

AF044285

049204

Plastidic?

Reference< Brander et al. (1995) Schiffmann and Schwenn (1998)

"Gene symbol is shown where one has been assigned. bLikely subcellular location determined by the author, ?: denotes from sequence analysis. Where only incomplete sequences are known, lengths are given in base pairs (bp) for untranslatable sequence, or as amino acid residues (aa) when the sequence can be translated.
MOLECULAR GENETICS OF SULPHATE ASSIMILATION

175

the fungal, yeast and mammalian transporters, and also clearly shows that the plant group divides into at least four distinct subtypes (see Fig. 2). It is possible to make some broad generalizations about some of these groups and suggest that the sub-types represent functional specialization. Clear grouping of all of the root -expressed transporters is apparent and similarly for the leafspecific transporters. A third group comprises proteins, which are expressed in shoot and root tissues and are most likely to be involved in transport in vascular tissues. Of the remaining non-grouped sequences, only AST82 has been putatively located as a chloroplast transporter (Takahashi et al., 1999a ). B.

SITES OF EXPRESSION

Within a higher plant there is clearly a need to translocate sulphate from the root to the shoot parts of a plant. From uptake to delivery to the various sinks within a plant, sulphate crosses multiple membranes. Transport steps involving both loading and unloading of cells is catalysed by membrane transporters, which may be distinct entities or the same transporters operating in different modes (Fig. 3). The characteristics of transport at the site of uptake in the root cortex, where extracellular sulphate concentrations are likely to be very low and the pH relatively acidic, may differ markedly from extracellular spaces elsewhere within the plant, particularly in the vascular tissues in the root and in the shoot. In the only kinetic study of isolated transporters, the root -expressed SHSTl-type sulphate transporter of Stylosanthes had a high affinity for sulphate and optimum activity at acidic pH, whilst the root and shoot SHST3-type had a lower affinity and a less acidic pH dependency when expressed in the yeast mutant, YSDl (Smith et al., 1995a). To date few data have been published on tissue and subcellular patterns of expression of the sulphate transporters, except in Arabidopsis (Takahashi et al., 1997, 1999a, b and personal communication). The seven Arabidopsis sequences fall into the four subgroups discussed above and shown in Fig. 2. ASTlOl is clearly a member of the root group and studies utilizing green fluorescent protein (GFP) fused to the transporters, indicate expression in the root cap, the root hairs, the epidermis and in the cortex (K. Saito, personal communication). In situ analysis of the expression pattern of the AST68 gene in Arabidopsis demonstrated expression predominantly in the root cap and particularly in the central cylinder of the root, and also in vascular tissue of aerial parts. AST68 falls into a subgroup of transporters separated from the characterized root high affinity transporters (see Fig. 1). The Sporobolus eDNA was isolated from a library constructed from RNA isolated from leaf tissues (Ng et al., 1996) and both AST12 and AST77 have been shown to have leaf-specific expression (Takahashi et al., 1999b ). Further analysis of expression patterns of sulphate transporters is clearly required.

176

M. J. HAWKESFORD and J. L. WRA Y

20 ST2-2 ST2-1 ST3-2 ST3-1 ST3-3 ST1-1 ST4-1

40

60

- -- - -------- - - ------------ MGIELQNHQ S HHEEASPAEE ~ -MSRWL

33 60 18 28 12 31 41

DRSKWL ------------------------------------------ MSSKRASQYHQ - - - - --- - ------------------------ 1-lGTEDYTFPQGAEELHRRHHT E ---------------------- ------------- - - - - - - -- ---- MEVHK ----------------------------- MSGTINPP DGGGSGARN PVVRQ --- - - ------ -------- MSYASLSVK DLTSLVSRSGTGSSSS LK P PGQTRP

MKER D SESFESL S H Q VL P N TS NSTHMIQMA~1ANSG SS AAAQAGQDQ

80

1 00

120

ST2-2

92 119 77 86 70 89 99

s·r2 -1 ST3-2 ST3-1 ST3-3 ST1-1 ST4-1 1 60

1 40

ST2-2 ST2-1 ST3-2 ST3-1 ST3-3 ST1-1 ST4-1

·1· ::

l

:~··l~.i.

: l>.S' l>.S l>.S ,

~-

AS ·

lj lj

AS ~

:i

200

180

PL PL PL PL PL.

;.

.. ~

r;

220

lZ·~ ·

;

'

i

A tlj. A

~ ~~

: · ·:.!'. .~ ::

152 179 137 146 130 149 159

240

ST2-2 ST2-1 ST3-2 ST3-1 ST3-3 ST1-1 ST4-1

212 239 197 206 190 209 217

ST2-2 ST2-1 ST3-2 ST3-1 ST3-3 ST1-1 ST4-1

272 299 256 265 249 269 275

ST2-2 ST2-1 ST3-2 ST3-1 ST3-3 ST1-1 ST4-1

332 359 316 325 309 329 330

Fig. 1. Alignment of the seven sulphate transporter protein sequences of Arabidopsis thaliana. Sequences were aligned using PILEUP in the Wisconsin GCG package (version 10). The resulting MSF file was viewed with GeneDoc (Nicholas et al., 1997). Accession numbers for the indicated sequences are in Table I. Residues in

177

MOLECULAR GENETICS OF SULPHATE ASSIMILATION

ST2-2 ST2-1 ST3-2 ST3-1 ST3-3 ST1-1 ST4-1

392 419 376 385 369 389 390

ST2-2 ST2-1 ST3-2 ST3-1 ST3-3 ST1-1 ST4-1

452 479 436 445 429 449 450

ST2-2 ST2-1 ST3-2 ST3-1 ST3-3 ST1-1 ST4-1

500

54 0

SI SI

EA ET

s

!Y

s TT

SAN

A 600

ST2-2 ST2-1 ST3-2 ST3-1 ST3-3 ST1-1 ST4-l

572 599 552 561 545 565 567

ST2-2 ST2-1 ST3-2 ST3-1 ST3-3 ST1-1 ST4-l

ST2-2 ST2-1 ST3-2 ST3-1 ST3-3 STl-1 ST4-1

512 539 496 505 489 509 510

631 657 611 621 605 624 626 680

700

nAi~ F(~Ll
-PDSPVPEFNNV ---------------------~·~~~·vm~II~KTEPASKNEPWNNV ----------------------

------KGPS LSNV ---------------------A~ns•rE!~ I,OCJT-·---------------------------------

h,,~ .•~vi~II~>JTEDKHLSFTRRYGGSNNNSSSSNALLKEPLLSVEK

658 677 646 658 631 649 685

black boxes are where all seven sequences have an identical or conserved substitution; dark grey: 6 out of 7; light grey: 4 or 5 out of 7 conserved residues, respectively.

178

M. J. HAWKESFORD and J. L. WRAY

hvst1 ttst1 ttst2

Root group

z. mays shst1 shst2 Atst1-1 (ast101)

~'B. nspus(bot1) B. napus (bst2)

S. tuberosum(stst) r--

Atst3-3 (ast91)

J

I

-

Sporobo/us

Leaf group

'----

Atst3-2 (at4060)

J I

Atst3-1 (atd631)

I

rice EST

I

Atst4-1 (ab8782)

G. max(nod70)

I I

Atst2-2 (atd14)

B.juncea shst3

Root and shoot group

Atst2-1 (at3591)

0.1

Fig. 2. Phylogenetic tree for the known members of the plant sulphate transporter family. An alignment was constructed from the derived amino acid sequences using PILEUP in the Wisconsin GCG package (version 10). The resulting MSF file was analysed using Clustal X (Thompson et al., 1997) to produce a bootstrapped tree, which was then displayed as an unrooted tree using Tree View version 1.52 for Windows NT (Page, 1996). Accession numbers are given in Tables I and II. Clusters of sequences are grouped according to probable major sites of expression. The scale bar indicates the branch length representing a rate of substitution of 0.1 per residue.

MOLECULAR GENETICS OF SULPHATE ASSIMILATION

179

In addition to transporters involved in uptake, in some tissues there is an obligate requirement for efflux of sulphate from cells, as shown on Fig. 3. A voltage-dependent channel, activated by sulphate and inactivated by nucleotides would ensure sulphate unloading when sulphate is in excess and cell energy levels are low (Frachisse et al., 1999). Such a channel would have a role in sulphate homeostasis for any cell, but may also operate to unload sulphate in, for example, the xylem parenchyma cells of the root vascular tissues. C.

SUBCELLULAR TRANSPORT

In contrast to the recent progress in the elucidation of the molecular mechanisms of sulphate transport across the plasma membrane, relatively little is known about the intracellular transport processes. The probable movements of sulphate, together with an indication of the type of transporter involved for either influx or efflux from the cell or individual organelle, is summarized in Fig. 3. One role of these transport systems is to

Plasma membrane b.'P= 100 mV sulphate <100 11M, low pH

Uptake H+symport

+

H+ antiport, channel or ATP-driven

Vacuole

Cytoplasmic sulphate =1 mM pH?

Stored sulphate = 1-50 mM

Tonoplast b.'P +1Q-30 mV pH 5

=

H+symport or channel

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Fig. 3. Transmembrane movements of sulphate, driving forces and potential mechanisms. All values of compartment sulphate concentrations, pH and membrane potentials are approximate. Arrows indicate transmembrane fluxes of sulphate and do not necessarily imply the existence of a specific transporter, although possible transporter type is indicated based on the likely local transmembrane driving forces.

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M. J. HAWKESFORD and J. L. WRAY

contribute to cytoplasmic sulphate homeostasis. However, of fundamental importance to whole plant S-assimilation is the effective delivery of sulphate to the plastid, which is necessary for subsequent reductive assimilation. The vacuole is the major intracellular store of excess sulphate, and although concentrations are usually only a few tens of millimolar (see Clarkson eta/., 1993), in certain species tolerant of high sulphur-environments, concentrations can rise to hundreds of millimolar (see Ernst, 1990). Isolated tonoplast membranes are often considered to be relatively impermeable to sulphate (e.g. Churchill and Sze, 1984), although transport across the tonoplast has been demonstrated (Kaiser et al., 1989; Dietz eta/., 1992). Accumulation in the vacuole, which acts as a storage sink for excess sulphate, is likely to be against a membrane potential and against a proton gradient. The most likely mechanism for transport would be an electroneutral proton/ sulphate exchange mechanism. Subsequent efflux might be down a concentration gradient, aided by the membrane potential, a process catalysed by an anion channel. The proteins for these transport processes, and genes which encode them, have not been identified and this is an important area still to be explored at the molecular level. Transport into the chloroplast has been suggested to be via a sulphate/ phosphate counter exchange (Hampp and Ziegler, 1977) and it has been speculated that sulphate transport could be accomplished by the phosphate translocator, although the general conclusion is for a distinct transporter (Mourioux and Douce, 1979; Clarkson eta/., 1993). The gene responsible for phosphate transport has been cloned (Fliigge et a/., 1989) and it is a quite distinct transporter-type compared to the sulphate transporters discussed here. One of the plasma membrane-type sulphate transporters, AST82 from Arabidopsis, has been localized to chloroplasts by GFP (jellyfish green fluorescent protein) fusion studies, indicating that this transporter also may be a candidate for plastid sulphate transport (Takahashi eta/., 1999a). It is possible that transporters in organelles may be quite unrelated to the plasma membrane H+ transporter family shown in Fig. 1 and discussed above. A further type of sulphate transport system which could operate in plants, would be an ABC-type (ATP-binding-cassette) transporter, as is found in prokaryotic systems, including the cyanobacteria, Synechococcus. There are numerous examples of this type of transporter operating in plants, however there is as yet no report of such a transporter for sulphate. There are database entries for ABC-transporter sequences of unassigned function, some of which which fall into sulphate-type sub-groups (T. G. E. Davies, personal communication). D.

REGULATION OF EXPRESSION

The underlying mechanisms controlling the activity of the transporter in response to S-nutrition have been suggested to have transcriptional,

MOLECULAR GENETICS OF SULPHATE ASSIMILATION

181

translational or allosteric components. A rapid response of transporter activity to nutritional circumstances is often seen (Lee, 1982; Clarkson et al., 1983) with the increase in sulphate uptake capacity appearing over a few days after the removal of sulphur supply. This is generally credited as being

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Fig. 4. Down-regulation of the sulphate transporter expression in barley by sulphate. The effect of sulphate re-supply on the transporter activity (uptake of radiolabelled sulphate into roots measured over a 10 min period), the mRNA abundance in root tissues and on sulphate transporter protein abundance (western blot) in the plasma membrane fraction of the roots is shown at intervals over a 24 h period. The plants were grown in hydroponic culture on a complete nutrient solution (Smith et al., 1997) for 10 days and then starved of sulphate for a further 4 days prior to re-addition of 0.25 roM sulphate. The transporter protein was detected using a rabbit polyclonal antibody raised against a 150-amino acid carboxyl-terminal fragment of the HVST1 expressed in Escherichia coli, as a his-tag fusion protein. The uptake measurements (expressed per gram fresh weight of root) are redrawn from Smith et al. (1997), and samples for RNA extraction and membrane isolation were taken in parallel. Both the Northern blot autoradiogram and the western blot (silver-enhanced immunogold labelled) were scanned and quantified using Molecular Analyst for the Macintosh, version 2.1 (BioRad Laboratories, Heme! Hempstead, UK).

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M. J. HAWKESFORD andJ. L. WRAY

due to the removal of repressive signals acting either allosterically or at the level of gene expression. The use of inhibitors of protein synthesis indicated that protein turnover was rapid, in the range of a few hours (Rennenberg et al., 1989; Clarkson eta!., 1992). Availability of the genes for the transporter in several species has enabled a more direct demonstration of the regulation of transporter mRNA abundance in response to S-nutritional status of the plant (Smith et al., 1995a, 1997; Takahashi et al., 1997; Lappartient et al., 1999; Vidmar et al., 1999). The abundance of mRNA approximately correlated with measured sulphate uptake capacities of roots in all of these studies, supporting the model of regulation by control of gene expression, but not ruling out an allosteric component. An example of such an approach shows the kinetics of the repression of the sulphate transporter mRNA, the transporter protein in the plasma membrane and the transporter activity by re-supply of sulphate to previously S-starved hydroponically grown barley roots (Fig. 4). The mRNA abundance decreased very rapidly, indicating a rapid response of gene expression to sulphur availability, which resulted in deceased transcription. The levels of protein and transporter activity decreased soon after the observed decrease in mRNA abundance, indicative of a rapid turnover of the protein in the membrane. No evidence for a mechanism of control involving transfer of the transporter protein to internal membranes was seen (data not shown). The decreased gene expression combined with the rapid protein turnover strongly suggests that these are sufficient mechanisms for the control of sulphate uptake in these circumstances. E.

LONG DISTANCE TRANSPORT

The major form of transported sulphur is sulphate, and the existence of multiple sulphate transporters expressed throughout the plant (discussed above) reflects this. There is clear evidence for re-translocation of sulphate accumulated during vegetative growth for utilization during seed production in soybean, barley wheat and oil-seed (Adiputra and Anderson, 1992, 1995; Sunarpi and Anderson, 1995, 1996a, b, 1997a, b; Blake-Kalff et al., 1998; Fitzgerald et al., 1999a, b). The phases of storage or re-mobilization will depend on availability and sink demand. Clearly, quite different net fluxes in and out of specific organelles, cells and tissues will occur. This will require co-ordinated expression of transporter and assimilatory capacity throughout development, and studies to analyse expression of the genes and the regulatory pathways involved should be undertaken. A general conclusion that can be drawn is that when there is excess sulphur supply, this is stored in vegetative tissues and this can be re-mobilized to supply S-demands for sink tissues such as seed. Plants without stored sulphate reserves will mobilize protein-S (Gilbert et al., 1997).

MOLECULAR GENETICS OF SULPHATE ASSIMILATION

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Reduced sulphur in the form of amino acids or derivatives, either glutathione (Rennenberg et al., 1979) or S-methylmethionine (Bourgis et al., 1999), is transported in the phloem, in addition to sulphate. The question of the form in which S is delivered to sink tissues is an important consideration when engineering S-metabolism for improved nutritional quality of crops (Section II. C). If sulphate were the major form in which sulphate is delivered to the sink tissues, then an adequate reductive capacity would need to exist in the sink tissues. The presence of the enzymes for sulphate reduction in developing sink tissues (for example, Romer et al., 1992), for sulphate transporter expression in wheat ears (our unpublished data) and for pools for sulphate in Brassica napus seeds (Zhao et al., 1993), indicate that sulphate delivery to sink tissues has a role in the S-budget during seed filling. In addition there is clear evidence for a substantial role of reduced sulphur as a major form of S transported to sink tissues (Bourgis et al., 1999). It is quite likely that the relative proportion of S, as sulphate, glutathione, Smethylmethionine or any other S-compound may vary from species to species and may be dependent on the nutritional status of the plant.

IV.

THE PATHWAY OF REDUCTIVE SULPHATE ASSIMILATION

A diagrammatic representation of the currently generally accepted pathway for reductive sulphate assimilation in plants is presented in Fig. 5. In the following section, the molecular evidence to support this pathway is presented. The pathway is unique to plants and, as presented, resolves the long-standing controversies concerning the intermediates in the pathway. In addition a comprehensive catalogue of all the genes isolated to date which represent components of this pathway are presented in Tables I and II. A.

ACTIVATION OF SULPHATE

Subsequent reduction of internal sulphate requires its prior activation to adenosine-5'-phosphosulphate (APS) by the enzyme ATP sulphurylase. A number of eDNA and genomic species encoding this well-characterized enzyme have been cloned from a wide variety of plants (Tables I and II and references therein). In Arabidopsis a small gene family of at least four members has been described (Leustek et al., 1994; Klonus et al., 1995; Murillo and Leustek, 1995). Sequence analysis (plastidic transit peptide), and chloroplast import studies with the recombinant protein in the case of APSl (Leustek et al., 1994) suggest that they encode proteins destined for the plastid. This is consistent with earlier studies showing that isolated plastids are able to perform the light-dependent reduction of sulphate to cysteine (for example Schmidt, 1968). However, other studies, both

184

M. J. HAWKESFORD andJ. L. WRAY

biochemical (spinach, Renosto et al. (1993)) and molecular (potato, Klonus et al. (1994); Brassica juncea, Heiss et al. (1999)), suggest the existence of additional cytosolic (Renosto et al., 1993; Klonus et al., 1994) and perhaps mitochondrial (Heiss et al., 1999) isoforms in these species. Such extrachloroplastic species have not (yet?) been identified inArabidopsis. The Sulphate

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Fig. 5. The reductive assimilation pathway for sulphate in higher plants showing key enzymes, intermediates and possible control loops. Fluxes of metabolites are shown with solid arrows and regulatory loops are dashed. The APS kinase branch is most likely utilized for the production of sulpholipids, flavonol sulphates, glucosinolates, etc., via the action of various sulphotransferases. Abbreviations: ST, sulphate transporter; ATPS, ATP sulphurylase; APSK, APS kinase; APSR, APS reductase; SiR, sulphite reductase; OASTL, 0-acetylserine thiollyase; SAT, serine acetyltransferase, OAS, 0-acetylserine; GSH, glutathione.

MOLECULAR GENETICS OF SULPHATE ASSIMILATION

185

significance of APS production within the cytosol is unclear since present evidence suggests that APS kinase and 'APS reductase', the two enzymes able to utilize APS, are located in the plastid (see below). The presence in plants of APS kinase, able to further activate APS to 3'phosphoadenosine-5'-phosphosulphate (PAPS), led to the notion that activation of sulphate within the reductive sulphate assimilation pathway might be a two-step process (discussed in Schmidt and Jager (1992)). Two members of a small gene family encoding this plastid-localized enzyme (Lee and Leustek, 1998) have been described fromArabidopsis (Arz et al., 1994; Schiffmann and Schwenn, 1998) and one from Catharanthus roseus (Schiffmann and Schwenn, 1998). However, the identification of 'APS reductase', initially fromArabidopsis (Gutierrez-Marcos et al., 1996; Setya et al., 1996) but subsequently from other species (Heiss et al., 1999; Prior eta!., 1999) (discussed below), supports other ideas that PAPS is not a direct intermediate in reductive sulphate assimilation but may rather act as a substrate for sulphotransferases in the biosynthesis of, for example, glucosinolates (Glendening and Poulton, 1990), sulpholipids (KleppingerSparace and Mudd, 1990) and flavonol sulphates (Varin et al., 1992). B.

REDUCTION OF SULPHATE

As suggested above, the subsequent fate of APS has been the subject of controversy (discussed in Schmidt and Jager, 1992; Wrayet al., 1998). Earlier studies led to the idea of a 'bound-intermediate' pathway in which the sulpha group of APSis first transferred to a carrier molecule (glutathione in vivo?) by APS sulphotransferase to form a bound sulphite intermediate which is then further reduced by thiosulphonate reductase to bound sulphide. In contrast, the ability of plants to form PAPS from APS via APS kinase Jed to the idea that the pathway of reductive sulphate assimilation in plants might be the same as the 'free-intermediate' pathway present in enterobacteria such as Escherichia coli. In this organism PAPS, formed by the two-step activation of sulphate by ATP sulphurylase and APS kinase, is converted to sulphite by a thioredoxin-dependent PAPS reductase (Schwenn and Schriek, 1987). Sulphite is then reduced to sulphide by sulphite reductase. A report of a thioredoxin-dependent PAPS reductase in spinach provided some support for this idea (Schwenn, 1986). To confirm the operation of the 'free intermediate' pathway attempts were made to clone an Arabidopsis PAPS reductase eDNA by functional complementation of an E. coli cysH mutant, defective in PAPS reductase activity, to prototrophy with an Arabidopsis eDNA library in the expression vector >..YES (Gutierrez-Marcos et al., 1996). Three, complementing but different, eDNA types (represented by PAPS reductase homologue (Prh)-19, Prh-26 and Prh-43) were identified but they did not encode microbial-type

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M. J. HAWKESFORD and J. L. WRAY

PAPS reductases, rather they encoded novel proteins (PRH proteins). Whereas the central region of the deduced PRH proteins has substantial homology to microbial PAPS reductases, each PRH protein carries an Nterminal extension with characteristics expected of a plastidic transit peptide and a C-terminal extension that has substantial homology with thioredoxin and the thioredoxin-like domain of a number of members of the thioredoxin superfamily. The homology includes the motif CxxC that carries the redoxactive half-cysteine residues present at the active site of thioredoxins but, of particular subsequent interest, the amino acid residues between these redoxactive cysteines are more typical of glutaredoxin (CPFC) than thioredoxin (CGPC) (Holmgren, 1989). Assay of cell-free extracts of the complemented cysH mutant and of the recombinant PRH19 protein, using a conventional PAPS reductase assay (Schwenn and Schriek, 1987) with either [35 S)APS or P5S)PAPS as substrate, showed that APS was preferred to PAPS as an in vivo substrate for the formation of acid-volatile sulphite, suggesting that APS is the in vivo substrate for the PRH proteins (Gutierrez-Marcos eta/., 1996). This ability to use APS as substrate was supported by the demonstration that the PRH cDNAs were able also to complement an E. coli cysC mutant, defective in APS kinase activity, to prototrophy (Gutierrez-Marcos et al., 1996). Four lines of evidence suggest that the activity of the PRH proteins is equivalent to the previously described APS sulphotransferase activity. First, both activity assays depend on the conversion of (35 S]APS to acid volatile 35 [ S]sulphite in the presence of enzyme, APS and thiol (as dithiothreitol or glutathione). Second, the presence of a transit peptide suggests that, like APS sulphotransferase, APS reductase is plastid-localized. Third, the extractable activity of APS sulphotransferase (Brunold et al., 1987) and transcript abundance of each of the PRH proteins (Gutierrez-Marcos et al., 1996), is elevated in sulphate deprivation. Fourth, the value of M, of about 43 kDa for the mature PRH proteins is similar to the subunit size reported for partially purified APS sulphotransferase (for example from Porphyra yezoensis) (Kanno et al., 1996). The presence of the thioredoxin-like domain in the PRH proteins shows APS sulphotransferase activity in an entirely new light. Gutierrez-Marcos et a/. (1996) suggested that, rather than acting as an APS sulphotransferase, each PRH protein might act as an 'APS reductase' in a manner analogous to that described for the Escherichia coli thioredoxin-dependent PAPS reductase but involving the internal thioredoxin-like domain rather than exogenous thioredoxin (Berendt et al., 1995). Proteins encoded by the same (Setya et al., 1996) or homologous (see Table II) cDNAs have been described, without qualification, as APS reductases (but see discussion in Leustek and Saito, 1999). Although the precise reaction mechanism of these proteins has not yet been established, recent evidence shows that the thioredoxin-like domain is able to function as a glutaredoxin, receiving electrons from reduced glutathione (Bick et al., 1998).

MOLECULAR GENETICS OF SULPHATE ASSIMILATION

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These data provide further support for the suggestion that APS can be utilized directly without activation to PAPS, as an intermediary substrate in reductive sulphate assimilation (reviewed in Schmidt and Jager, 1992). The possibility that a pathway involving PAPS as a direct intermediate also exists cannot be completely excluded, but none of the cDNAs isolated by functional complementation of an E. coli PAPS reductase mutant encode a 'conventional' PAPS reductase (Gutierrez-Marcos et al., 1996; Setya et al., 1996). The data are consistent with the idea (Wray et al., 1998) that, rather than proceeding via either the 'bound-intermediate' or 'free-intermediate' pathways outlined above, APS is reduced to sulphite by 'APS reductase', with the sulphite being reduced to sulphide by sulphite reductase (Fig. 5). Sulphite reductase is well-characterized in plants and appears to be exclusively plastid-localized, unlike other pathway enzymes, and in Arabidopsis appears to be encoded by a single gene (Bruhl et al., 1996; Bork et al., 1998) (Table II). There is no role in this scheme of reductive sulphate assimilation (Fig. 5) for either thiosulphonate reductase, described only in one report from Chiarella (Schmidt, 1973) and perhaps due to sulphite reductase (Siegel, 1975), or for the well-characterized APS kinase. In this scheme APS stands at the branch point of two divergent pathways, but it is presently unclear what determines the relative rates at which APS is converted to PAPS (APS kinase Km for APS is ::;1 ~tM), the substrate for subsequent sulphotransferase reactions, or is used within the reductive sulphate assimilation pathway (APS reductase Km for APS -8 ~tM). The presence in plants of a 3'(2'),5'-diphosphonucleoside 3'(2')-phosphohydrolase(3'-phosphonucleotidase) that is able, in vitro, to dephosphorylate PAPS to APS in a relatively specific manner (Goldschmidt et al., 1975; Tsang and Schiff, 1976) must also be considered, especially since rice (Peng and Verma, 1995) and Arabidopsis (Quintero et al., 1996; Gil-Mascarell et al., 1999) li4L2-like genes encoding this enzyme are able to complement functionally yeast met22 and E. coli cysQ mutations, suggesting a role in plant sulphate assimilation. A role for this enzyme in modulating PAPS pool size has been postulated (Goldschmidt et al., 1975; Murguia et al., 1995) but this would imply a plastidic location for the enzyme and our analysis of the li4L2-like RHL and SALJ gene sequences, from rice and Arabidopsis, respectively, does not support this. The significance of the presence of multiple plastidic isoforms of ATP sulphurylase, APS kinase and APS reductase (but apparently not sulphite reductase) is unclear but perhaps may be related in part to differential regulation of particular isoforms by the multitude of environmental signals that appear to affect the expression of these pathway steps (see Section V). It may also be that, within the plastid stroma, associations between one isoform of ATP sulphurylase and APS kinase and between a different isoform of ATP sulphurylase and APS reductase, might play an important role in

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M. J. HAWKESFORD and J. L. WRAY

regulating the partitioning of APS between sulphotransferase reactions on the one hand and the synthesis of cysteine and related metabolites via the reductive sulphate assimilation pathway on the other (Wray et al., 1998). Introduction of the HAL2-like SALI-encoded enzyme into plastids, via a transgenic approach, in an attempt to perturb relative pool sizes of PAPS and APS might throw some interesting light on partitioning of APS between the two competing pathways. C.

SYNTHESIS OF CYSTEINE

Incorporation of sulphide into organic combination as cysteine is catalysed by 0-acetylserine (thiol) lyase (OASTL) with the other substrate, 0acetylserine (OAS), being provided from acetyl-CoA and serine by the enzyme serine acetyltransferase (SAT). Recent evidence suggests that OASTL and SAT may associate together in a complex (Bogdanova and Hell, 1997; Droux et al., 1998) (Section V.B). Biochemical studies have identified isoforms of both OASTL and SAT that are located to organellar, mitochondrial and cytoplasmic sites (Smith and Thompson, 1969; Brunold and Suter, 1982; Lunn et al., 1990; Droux et al., 1992; Rolland et al., 1992; Ruffet et al., 1994, 1995; Kuske et al., 1996). In pea leaves 76% of SAT activity was located in the mitochondrion, 14% in the cytosol and 10% in the chloroplast (Ruffet eta/., 1995). In the case of OASTL, studies with spinach leaves showed that 15% of the total activity was located in the mitochondrion while the remainder was distributed between the chloroplast (42%) and the cytosol ( 44%) (Lunn et al., 1990). Sequence analysis ofArabidopsis cDNAs, isolated by functional complementation of equivalent E. coli mutants, suggests the presence of species encoding putative cytoplasmic, plastidic and mitochondrial isoforms of SAT (Ruffet et al., 1995; Roberts and Wray, 1996; Howarth et al., 1997) (Table I) and of OASTL (Hell et al., 1994; Hesse and Altmann, 1995; Hesse et al., 1999) (Table I). Molecular cloning of SAT and OASTL genes from a wide range of other plant species suggests an apparently similar distribution of isoforms (Table II). Recent studies have attempted to determine directly the subcellular location of the individual members of the Arabidopsis SAT protein family (Noji et al., 1998), rather than relying on sequence analysis alone. These workers examined the transient expression of fusion proteins of GFP with the N-terminal region from either Sat-52 (Howarth et al., 1997), SATI (Murillo et al., 1995) or SAT-A (Hell and Bogdanova, 1995); the primary authors had determined from sequence inspection that these genes were likely to encode either mitochondrial, cytosolic or plastidic isoforms, respectively (Table I), but the GFP fusion data of Noji et al. (1998) suggested cytosolic, plastidic or mitochondrial locations, respectively, for these isoforms. Clearly, further studies of this type are required to unequivocally determine subcellular locations of particular isoforms.

MOLECULAR GENETICS OF SULPHATE ASSIMILATION

189

The databases currently contain around 27 entries of full-length eDNA clones for OASTL from plants, together with a number of EST fragments (Tables I and II). A phylogenetic analysis of the deduced full-length amino acid sequences is presented in Fig. 6. Of the 27 sequences analysed 10 are from Arabidopsis and, although some are either very closely related or are deduced from identical eDNA clones that probably contain DNA sequencing errors, the majority are widely dispersed around the tree. The sequences of monocotyledonous species cluster together, as is also found for the sulphate transporter sequences (Fig. 2). The majority of the sequences possessing longer N-terminal regions, indicative of organellar targeting sequences, also cluster together. Although all of these isoforms are predicted to be organellar in location, no clear grouping of isoforms deduced to be specifically plastidic or mitochondrial is apparent, and three isoforms without this N-terminal region also align within this cluster. As for SAT, most of the evidence for the subcellular localization of the OASTL gene products relies on the analysis of the presequence using computer programs such as P-SORT, but in a number of cases other approaches have been adopted. Thus analysis of the spinach CysC (D37963) (Saito et at., 1994b) and CysB (D14722) (Saito et at., 1993) sequences suggested mitochondrial and plastidic locations for their protein products, respectively; fusion of the nucleotide sequence encoding the N-terminal extension to the gus reporter gene, and analysis of the subcellular distribution of GUS activity in transgenic tobacco transformed with this construct, confirmed this localization (Takahashi and Saito, 1996). The protein product of the Arabidopsis AtCS-C (X81973) (Hesse et at., 1999) gene has been shown to be located to the mitochondrion using in vitro import studies and epitope tagging but curiously it does not align in the same group as other mitochondrial isoforms (see Fig. 6). In the same study, evidence for the localization of the protein product of theArabidopsis AtCys-B (X81698) gene to plastids was presented (Hesse et at., 1999). TheArabidopsis isoform encoded by the AtcysCl (AB024282) gene (Table I), aligns most closely to the spinach mitochondrial isoform (see Fig. 6) suggesting that this is also a mitochondrial isoform, a prediction supported by analysis using P-SORT. As a first step towards understanding the role of the different isoforms of OASTL, Gator et al. (1997) used in situ RNA hybridization to examine the tissue-specific expression ofAtcys-3A, the gene encoding a putative cytosolic isoform of OASTL in Arabidopsis (Table I). A similar study has been performed with two members of the SAT gene family from Arabidopsis (C. Gator, unpublished). In both studies particularly high expression of these genes in the trichomes was observed, supporting a specialized role for cysteine biosynthesis in the function of these organs. Why subcellular compartments other than the plastid have the capacity for cysteine synthesis from sulphide, serine and acetyl-CoA and what the relative contributions these different compartments play in cysteine

190

M. J. HAWKESFORD and J. L. WRAY

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MOLECULAR GENETICS OF SULPHATE ASSIMILATION

191

biosynthesis under the different environmental conditions experienced by a plant during its life cycle, is unknown. It has been suggested (Lunn et al., 1990) that cysteine must be synthesized in each subcellular compartment to compensate for an inability to transport cysteine across intracellular membranes. Some isoforms of 0 ASTL may have substrate specificities which favour reactions other than cysteine synthesis, for example, the synthesis of 13-cyanoalanine as part of a cyanide detoxification mechanism (Maruyama et al., 1998; Warrilow and Hawkesford, 1998)

V.

REGULATION AND INTERACTION WITH THE ENVIRONMENT A.

CONTROL OF FLUX

Regulation of the reductive sulphate assimilation pathway is complex and any attempt to understand control of flux of S through the pathway must be in the context of whole plantS nutrition. It is necessary to account for, not only internal and external signals impinging on individual pathway steps, but also the significance of the spatial arrangement of these steps within the different plant parts and within the cell. Whereas earlier attempts to interpret experimental data concentrated on the identification of ratelimiting steps within metabolic pathways, metabolic control theory predicts that all steps within a pathway contribute to the control of flux through the pathway (Kaeser and Burns, 1973; Fell, 1997). As yet no attempts have been made to assign control strength coefficients to pathway steps, but the availability of genes encoding perhaps all of the pathway steps, at least in Arabidopsis, suggests that the type of transgenic approach used to study the contribution of individual steps to the control of other pathways in plants will soon be carried out. However, a number of recent transgenic studies have taken advantage of the availability of eDNA species encoding steps of the pathway to obtain information on the operation of the pathway that would be difficult to obtain by other means (Youseffian et al., 1993; Saito et al.,

Fig. 6. Phylogenetic tree for the known members of the plant 0-acetylserine (thiol) lyase protein family. An alignment was constructed from the derived amino acid sequences using PILEUP in the Wisconsin GCG package (version 10). The resulting MSF file was analysed using Clustal X to produce a bootstrapped tree, which was then displayed as an unrooted tree using TreeView version 1.52 for Windows NT. Accession numbers are given in Tables I and II. Clusters of sequences within boxes have long N-terminal regions in their predicted proteins, which may be involved in organelle targeting. A closely related group of sequences from monocotyledonous species is also marked. The scale bar indicates the branch length representing a rate of substitution of 0.1 per residue. Putative subcellular locations determined by author; '?' denotes that this is from sequence analysis alone.

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M. J. HAWKESFORD andJ. L. WRAY

1994a; Hatzfeld et al., 1998; Pilon-Smits et al., 1999). It is anticipated that the application of modern approaches to pathway analysis (Schuster et al., 1999) will inform attempts at the metabolic engineering of the reductive sulphate assimilation pathway. Many signals, such as sulphate and nitrate availability and a wide range of environmental stresses, have been implicated, either directly or indirectly, in the regulation of flux through the pathway. These signals act either at the level of gene expression (measured as changes in steady-state level of specific mRNA), or by allosteric regulation of enzyme activity (mainly at the level of SAT/OASTL), as is considered in the next section. B.

ALLOSTERIC REGULATION AND PROTEIN-PROTEIN INTERACTION AS A REGULATORY DEVICE- SERINE ACETYLTRANSFERASE AND 0-ACETYLSERINE (THIOL) LYASE

The incorporation of sulphide into organic combination as cysteine requires the participation of two enzymes: serine acetyltransferase (SAT), which synthesizes the sulphide acceptor, 0-acetylserine, from acetyl-CoA and serine and 0-acetylserine (thiol) lyase (OASTL), which catalyses the transfer of the sulphide onto 0-acetylserine (OAS), thus synthesizing cysteine. A number of lines of evidence suggest that these two steps are closely regulated by protein-protein interaction that is probably modified by allosteric mechanisms. The activity of OASTL is typically many fold higher than that of SAT in cytosol, mitochondrion and plastid (as much as 345-fold) (Lunnetal., 1990; Drouxetal., 1992; Rolland eta/., 1993; Ruffetetal., 1994). Enzyme purification studies suggest that, in at least some plants such as spinach (Droux et al., 1992; Ruffet et al., 1994) and Allium tuberosum (Nakamura and Tamura, 1990), the two proteins associate together in vitro in a complex perhaps analogous to the 'cysteine synthase' complex first identified in E. coli (Kredich and Tomkins, 1966). Since in vitro interactions between these two enzymes are not necessarily significant, Bogdanova and Hell (1997) used the yeast two-hybrid system to search for an in vivo interaction between SAT and 0 ASTL. They found that the cytosolic isoform of OASTL (product of theAtOAS.5-8 gene) (Hell et al., 1994) was able to interact in vivo not only with a putatively cytosolic isoform of SAT (product of theArabidopsis sat5 gene) (Ruffet et al., 1995) but also with a putatively plastidic isoform of SAT (product of theArabidopsis SAT-A gene) (Hell and Bogdanova, 1995) that is approximately 70% identical to the sat5 isoform with respect to its primary amino acid sequence. Analysis, using a series of truncated proteins, identified a hexapeptide motif repeat region towards the C-terminus that is conserved between all SAT proteins. The motif is involved both in interaction with OASTL and in catalysis, as previously suggested by Roberts and Wray (1996) from an analysis of the deduced amino acid sequence ofthe SAT1 protein.

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Further studies by Droux et al. (1998), which examined the interaction between a recombinant SAT protein (product of the Arabidopsis sat5 gene) (Ruffet et al., 1995) and a recombinant OASTL protein (product of the spinach cysK gene) (Rolland et al., 1993, 1996), provided what appears to be an explanation for the high ratio of OASTL to SAT found in nature. In the absence of 0 ASTL the free recombinant SAT was found to aggregate and become unstable. In its presence homotetrameric SAT associated with two molecules of homodimeric OASTL to form a bienzyme complex- the 'cysteine synthase' complex. With respect to SAT, interaction with OASTL caused a transition from a typical Michaelis-Menten model to a model displaying positive kinetic cooperativity with respect to serine and acetyiCoA, whereas interaction with SAT led to a very substantial decrease in the activity of OASTL and the release of SAT into the bulk solution. OASTL appears to behave within the complex as a structural or regulatory subunit of SAT and the excess of free 0 ASTL serves as an 'auxiliary' enzyme to achieve maximal capacity for cysteine synthesis from serine, acetyl-CoA and sulphide. No evidence to support a role for the complex in the substrate channelling of SAT, as suggested earlier (Droux et al., 1992; Saito et al., 1995), was found. The observation (Droux et al., 1998) that OAS promotes dissociation of the complex and that this can be prevented by sulphide, suggests that the SAT-OASTL interaction is a device that contributes to the regulation of cysteine synthesis. Interaction of SAT and 0 ASTL to form the 'cysteine synthase' complex, and thus allow maximal rates of cysteine synthesis, may be a sensor of the relative rates at which, on the one hand. sulphide is produced from sulphate via reductive assimilation and, on the other, OAS is produced by N/C assimilation. If SAT only contributes to cysteine synthesis within the complex, as the above results suggest, then the in vivo significance of studies examining feedback inhibition of recombinant SAT isoforms fromArabidopsis, presumably free from OASTL, by cysteine, which showed that only the putative cytosolic isoform was feedback inhibited, is unclear (Noji et al., 1998). C.

SULPHUR SUPPLY

Sulphate deprivation inArabidopsis leads to the up-regulation of expression of particular members of the gene families encoding the sulphate transporters (AST68 and AST56 (Takahashi et al., 1997) but not ATSTJ (Yamaguchi et al., 1999) ), ATP sulphurylase (APS3 (Yamaguchi et al., 1999) andASAl (Logan et al., 1996) but notAPSl (Takahashi et al., 1997)), APS reductase (PRH-19, PRH-26, PRH-43 and APRJ (Gutierrez-Marcos et al., 1996; Takahashi et al., 1997; Yamaguchi et al., 1999)), SAT (SATJ (Takahashi et al., 1997) and SATI-6 (Bogdanova et al., 1995) but not SAT-52 (Howarth et al., 1997; Takahashi et al., 1997) or SAT-A (equivalent to

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SATJ-6) in the study of Takahashi et al. (1997)), and OASTL (AtCys3A (Barrosa et al., 1995), AtOAS.5-8 (Hell et al., 1994), AtOAS. 7-4 (Hell et al., 1994) and cytACSJ as well asAtCS-B andAtCS-C (Hesse et al., 1999) (but notAtCS-B andAtCS-C in the study of Yamaguchi et al. (1999) ). Expression of the sir gene did not change when plants were deprived of sulphate (Takahashi et al., 1997; Yamaguchi et al., 1999). Data from plants other than Arabidopsis suggest a similar pattern (for example the Stylosanthes and barley sulphate transporters (Smith eta/., 1995a, 1997), isogenes of Zea mays sulphate transporter and ATP sulphurylase (Bolchi et al., 1999) and rice OASTL (Nakamura et al., 1999)). Where tested, changes in mRNA abundance were reversed by re-supply of sulphate (see also Fig. 4). Not all members of all gene families have been examined for their response to sulphate supply and, in some cases, there is a discrepancy between individual reports that is perhaps related to differences in the manner in which the experiment was performed. Despite this, it is clear that differential expression of isogenes does occur, and this suggests some type of metabolic difference between the different protein members of the responsive families. But what these differences might be is presently unclear. One needs to distinguish, on the one hand, between responses of isoforms located in different cellular compartments, where differences in response probably reflect the role the different compartments play in overall cellular sulphate assimilation and, on the other, situations where there are apparently more than one isoform in a particular subcellular location, when differential patterns of expression of different isoforms may perhaps contribute to flexibility of response within that subcellular compartment. At present there does not seem to be any evidence of a co-regulated response to sulphate supply by that full set of genes encoding plastidic, mitochondrial or cytosolic isoforms. Interestingly, expression of APS kinase, unlike that of APS reductase, is not up-regulated on sulphate deprivation (Lee and Leustek, 1998; Yamaguchi et al., 1999). Although only theArabidopsis AKNJ gene has been examined, it does suggest that under conditions of sulphur deprivation APS might be channelled preferentially towards cysteine synthesis and away from secondary metabolism (Yamaguchi eta/., 1999). Associated changes occur outwith the reductive sulphate assimilation pathway. At the gross biochemical level S deficiency leads to a decrease in protein, amino S (membrane) sulpholipids and chlorophyll (Clarkson et al., 1983)- changes that can be reversed by resupply of sulphate. Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) (a 'reserve' protein?) appears to be particularly susceptible to degradation when sulphate limitation is imposed (Gilbert et al., 1997). Restriction of sulphur supply also leads to up-regulation of synthesis of the sulphur-poor 0-subunit of the soybean storage protein 0-conglycinin and down-regulation of the more sulphur-rich glycinin (Hirai et al., 1995). We have suggested (GutierrezMarcos et al., 1996) that plants show a coordinated response to S limitation

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by modulation of expression of a network of genes analogous to the Sresponsive stimulon proposed for E. coli (Kertesz et al., 1993). S-responsive genes within the plant stimulon lie both within and outside the reductive sulphate assimilation pathway; many of those lying outside might be expected to be involved in the synthesis of enzymes that scavenge S and deliver it to the up-regulated reductive sulphate assimilation pathway in an attempt to maintain protein synthesis and growth - at least in the short term. The nature of the signal(s) that is sensed under conditions of sulphate limitation and which is transduced to the members of this stimulon is unclear. However, a number of studies suggest a central role in regulation for OAS, formally a product of C and N assimilation. Thus one consequence of sulphur limitation is that sulphide may become limiting for cysteine biosynthesis and, if there are no compensatory changes in either N or C metabolism, OAS will accumulate. Feeding OAS increases flux through the pathway (Neuenschwander et al., 1991) and more recent studies show that OAS feeding results in increased mRNA abundance of, at least, the sulphate transporter (Smith et al., 1997), whereas the activities of ATP sulphurylase and of APS reductase (APS sulphotransferase) (Neuenschwander et al., 1991) are also up-regulated on OAS feeding. Re-supply of sulphate to Sstarved plants leads to rapid down-regulation of expression of, at least, the sulphate transporters and to an increase in levels of cysteine and glutathione, but which of these, if either, is the signal that down-regulates gene expression under these conditions is not entirely clear. A report from Zea mays (Bole hi et al., 1999) showed that L-cysteine, but not D-cysteine, was able to down-regulate both a sulphate transporter gene and an ATP sulphurylase gene (as measured by a decrease in steady-state mRNA abundance) when supplied to sulphur-deficient seedlings, under conditions where glutathione synthesis was blocked with buthionine sulphoximine, a specific inhibitor of:glutamyl-cysteine synthetase. In contrast, the negative effect of cysteine on the sulphate transporter AST68 and the ATP sulphurylase APS1 mRNA abundance in roots of Arabidopsis was blocked by buthionine sulphoximine (Lappartient eta!., 1999). These latter data would suggest that glutathione rather than cysteine is the regulatory signal and data with split root experiments support the idea of this being an ideal candidate for long distance signalling of S-nutritional status. D.

CO-ORDINATION WITH NITROGEN METABOLISM

The role of nitrogen nutrition in the regulation of expression of pathway genes has been examined by a number of workers. When N is limiting, and the synthesis of OASis expected to be low, up-regulation of the transporters (Reuveny et al., 1980; Clarkson et al., 1989, 1999) and of other pathway steps such as ATP sulphurylase (APS3 (Yamaguchi et al., 1999)), APS reductase

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(PRH-19 (Yamaguchi eta/., 1999)) and OASTL (AtCys3A (Barroso et al., 1995) and rcsl(Nakamura et al., 1999)) is much reduced in response to S limitation. When N status is high or mimicked by feeding with OAS (Neuenschwander et al., 1991; Smith et al., 1997; Clarkson eta/., 1999; Kim et al., 1999) expression is enhanced, even under plentiful S supply and in the presence of increased cysteine and glutathione pools. With respect to the nitrate assimilation pathway, under conditions of nitrate availability, but sulphate limitation, the amides asparagine and glutamine, derived not from net protein degradation but by de novo synthesis (Amancio et al., 1997), accumulate (Karmoker et al., 1991) and the levels of nitrate reductase decline (Friedrich and Schrader, 1978), changes which might be anticipated to lead to a decrease in nitrate assimilation. Presumably these patterns exist to co-ordinate the rates of S and N assimilation and to deliver reduced S and N in the optimum ratio of around 1 to 15 for protein synthesis. Studies on the regulation of reductive sulphate assimilation pathway enzymes in the wide range of mutants defective in nitrate assimilation (Hoff et al., 1994) and amino acid metabolism (Lea and Forde, 1994) might lead to further insights into the interaction between S and N/C assimilation. Attempts to isolate Arabidopsis and barley mutants defective in reductive sulphate assimilation, by selection for resistance to the toxic sulphate analogue selenate, were unsuccessful (A F. Gilkes and J. L. Wray, unpublished). The existence of multigene families encoding the pathway steps was not known at the time the work was carried out. If OASis involved in regulation as a signal metabolite, then intracellular levels would be expected to fluctuate in response to S-nutrition, and such data have recently been presented (Kim et al., 1999). Measurements of cellular OAS in both Arabidopsis siliques and soybean cotyledons were inversely correlated with sulphate supply. Furthermore, OAS content was further enhanced at high nitrate supply, except in the presence of the highest levels of supplied sulphate when levels of OAS were always depleted. In this same study, supplying exogenous OAS to soybean cotyledons resulted in the accumulation of the ~-subunit mRNA and protein of ~-conglycinin and reduced accumulation of glycinin, mirroring the responses seen under Sdeficiency (Hirai eta/., 1995). These data are consistent with OAS being a signal metabolite which regulates the expression of a wide range of genes associated with S-metabolism and links this control to the availability of N and C. In this context we note that pathway flux (Kopriva et al., 1999) and expression of a number of pathway genes is higher in the light than the dark (for example Hell et al., 1997; Kopriva et al., 1999). Sucrose feeding in the dark strongly up-regulates APS reductase in roots (Kopriva eta/., 1999). We speculate that at least part of this effect may be related to increased OAS synthesis from sucrose, especially since OAS feeding in the dark has previously been shown to lead to the up-regulation of APS sulphotransferase (APS reductase) activity (Neuenschwander eta/., 1991).

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ENVIRONMENTAL STRESSES, GLUTATHIONE AND ABSCISIC ACID

A number of environmental stresses such as chilling (Kocsy et al., 1996), heat shock (Nieto-Sotelo and Ho, 1986), pathogen attack (Edwards et al., 1991; May et al., 1996), active oxygen species accumulation (Smith et al., 1985; May and Leaver, 1993), air pollution (Sen Gupta et al., 1991; Guri 1983; Madamanchi and Alscher, 1991 ), drought (Dhindsa, 1991) and salt (Barroso et al., 1999) lead to an increase in the level of the tripeptide, glutathione (1glutamyl-cysteinyl-glycine). Increased levels of glutathione protect against the adverse environment (reviewed in May et al., 1998). Glutathione is often the major pool of non-protein reduced sulphur present in plants and is a precursor of the phytochelatins (PCs), polypeptides based on the structure (1-glutamyl-cysteine)n-glycine (Zenk, 1996) and of the low-molecularweight, cysteine-rich metallothioneins (Robinson et al., 1993), both of which are believed to be involved in vivo in metal homeostasis. Exposure of plants to heavy metals such as cadmium usually leads to at least a small decrease in glutathione levels (Ruegsegger et al., 1990; Schafer et al., 1998; Heiss et al., 1999) but to a relatively large increase in the level of the PCs. In all these environmental situations it might be expected that the requirement for enhanced glutathione synthesis would place increased demand on the provision of cysteine, the level of which is normally limiting for glutathione synthesis (Strohm et al., 1995; Noctor et al., 1996). Examination of the effects of environmental stresses on the reductive sulphate assimilation pathway has concentrated mainly on responses to heavy metals and, more recently, to salt stress. Whether some or all of the changes seen in these cases occur also in response to the other types of environmental stress outlined above is presently unknown, but might be predicted. The increased requirement for cysteine to fuel glutathione and phytochelatin synthesis after cadmium treatment is manifested by an increase in flux of sulphur from [35s]sulphate into higher-molecular-weight compounds (largely as PCs) (Nussbaum et al., 1988) and by an increase in the total extractable activity (Ruegsegger et al., 1990; Lee and Leustek, 1999) and transcript abundance of particular isoforms (Schafer et al., 1998; Heiss et al., 1999; Lee and Leustek, 1999) of sulphate transporter, ATP sulphurylase, APS reductase and a putative cytosolic OASTL isoform in the roots and sometimes leaves, of a number of plants. Where examined the activity increases were reversible on removal of cadmium (Nussbaum et al., 1988). There is some evidence which suggests the nature of the signal that mediates the regulation of the pathway enzymes, and concomitant flux through the pathway on cadmium treatment. When cadmium treatment was carried out together with buthionine sulphoxime, a specific inhibitor of 1glutamyl-cysteine synthetase (1-ECS), the first enzyme of glutathione synthesis, no glutathione or PCs were detected and cysteine levels (21-fold) and the activities of ATP sulphurylase and APS reductase were elevated

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compared with cadmium treatment alone (Riiegseggeret a/., 1990). Coupled with the observations that glutathione levels generally fall on cadmium treatment and that the Arabidopsis cad2-1 mutant (C. Cobbett quoted in May et a/., 1998), defective in the 1-ECS gene and thus in glutathione synthesis, shows a two-fold increase in cysteine, these results support the idea on cadmium treatment at least, that glutathione is a negative regulator of expression of the responsive genes and of flux through the reductive sulphate assimilation pathway. Recent studies inArabidopsis show that glutathione levels also increase on salt stress (Barroso et al., 1999) and are correlated with an increase in expression of a gene (Atcys-3A) encoding a cytosolic isoform of OASTL. Since other pathway steps have not been examined under similar salt stress conditions one cannot conclude from this study that the increased glutathione levels are a consequence of this increased expression of the Atcys-3A gene alone. Of particular significance in this work was the demonstration that exogenous application of abscisic acid (ABA) mimicked the salt-induced response. Additionally, in the Arabidopsis mutants aba-1 (defective in the epoxidation reaction converting zeaxanthin to antheraxanthin within the ABA biosynthetic pathway (Rock and Zeevaart, 1991)), abi1-1 and abi2-1 (both defective in their responsiveness to ABA (Koorneef et al., 1984)), the response was abolished or severely reduced (Barroso et al., 1999). Thus for the first time ABA, a plant growth substance intimately associated with plant adaptation to a wide range of environmental stresses (Giraudat et a/., 1994), is implicated in the regulation of the reductive sulphate assimilation pathway. Perhaps ABA is also involved, via the reductive sulphate assimilation pathway, in mediating other environmental stresses that lead to increased glutathione levels? F.

SENSING THE ENVIRONMENT AND SIGNAL TRANSDUCTION TO THE GENES

1. Sensing Sulphur Status and Signal Transduction in Non-photosynthetic Organisms Considerable progress has been made in elucidating the details of the regulation of expression of genes for sulphate uptake and assimilation in prokaryotes (reviewed in Kredich, 1992, 1993) and in lower eukaryotes such as yeasts (reviewed in Thomas and Surdin-Kerjan, 1997) and filamentous fungi (reviewed in Marzluf, 1997). It is possible that some of the lessons learnt from these organisms may be relevant to higher plants. but one must not be seduced into the idea that any one of these systems represents a paradigm for the higher plant situation. Regulation of expression of the cysteine (cys) biosynthetic genes in E. coli and Salmonella typhimurium is probably the simplest operationally. The metabolite signal that links sulphur status to gene expression appears to be

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N-acetylserine, formed non-enzymatically from 0-acetylserine by 0- toNacetyl migration. Repression of the cys regulon by growth on a readily usable source of sulphur such as cysteine or sulphide is mediated largely by feedback inhibition of serine acetyltransferase by cysteine, which results in a decrease in the synthesis of 0-acetylserine. Transcription of the cys genes is regulated by the trans-acting protein CysB, the positively-acting product of the single regulator gene, cysB. In vitro studies show that N-acetylserine is around one order of magnitude more effective than 0-acetylserine in promoting the binding of the CysB protein to cys promoters and stimulating the formation of transcription initiation complexes. Sulphide and thiosulphate appear to act as anti-inducers and are able to reverse the effects of acetylserine on binding of the CysB protein to cys promoters, and on transcription initiation. These effects are specific for cys promoters and can be overcome by increasing the concentration of acetylserine. Kredich (1992) suggests that although anti-inducers may seem redundant in view of the efficient inhibition of serine acetyltransferase by cysteine, they may be useful in the fine-tuning of the regulatory mechanisms during rapid changes in the availability of environmental sulphur. At least seven different CysB protein binding sites have been identified among the promoters of various cys genes and their role in the regulation of cys gene expression explored (reviewed in Kredich, 1992). In yeasts, expression of the MET genes that encode the pathway components is repressed when high concentrations of methionine, or Sadenosylmethionine (AdoMet), are added to the growth medium. In mutants unable to synthesize AdoMet the presence of methionine does not lead to repression of enzyme biosynthesis. This implies that, in wild-type cells in the presence of added methionine, the increased intracellular AdoMet, rather than methionine, is sensed and acts as the signal for repression of the MET genes (Thomas et al., 1988). At least seven regulatory genes, whose products act in trans to regulate some or most of the MET genes at the transcriptional level, have been identified. Met4p, the major transcriptional activator of the MET genes, and Met28p are bZIP proteins whereas Cbflp (centromere-binding-factor 1) is a bHLH protein. Maximal MET gene expression requires a complex, Cbflp-Met4p-Met28p, of at least these three, which function together as a transcriptional activator. Under repressive conditions, in the presence of increased levels of intracellular Ado Met concentrations, the activation function of the complex is inhibited by interaction, via Met4p, with a further regulatory protein, Met30p. Two other proteins, Met31p and Met32p, have also been implicated in regulation (Blaiseau et al., 1997). A further protein, Gcn4p, that is involved in crosspathway regulation of amino acid biosynthesis mediates expression of a number of the MET genes on amino acid starvation, independently of the sulphur-specific activation mediated by Met4p (Hinnebush, 1992). cis-acting regulatory elements that are the DNA binding sites for the tripartite

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activator complex, and for Gcn4p, have been identified. Transcriptional activation of the MET genes requires at least the sequence TCACGTG, whereas Gcn4 binding requires the sequence ATGA(C/G)TCAT (reviewed in Thomas and Surdin-Kerjan, 1997). In Neurospora crassa our understanding of the regulatory processes involved in sulphur metabolism are derived from a study of the use of secondary sulphur sources, such as choline-0-sulphate and aromatic sulphates, that occurs when primary sulphur sources such as sulphate are not available for growth. Under these conditions expression of reaction steps associated with the catabolism of relatively poor sulphur sources, such as the aromatic sulphate transport system, aryl sulphatase and of sulphate transporters, is switched on. Genetic evidence suggests that cysteine, or a closely related metabolite, rather than methionine represents the true repressing metabolite of the encoding genes under conditions of sulphur sufficiency (sulphur catabolite repression) (Jacobson and Metzenberg, 1977). At least three distinct genes encoding sulphur trans-acting regulators, cys-3, scon1 and scon2 (sulphur controller) have been identified (Fu et al., 1989; Paietta, 1990). CYS3 is a positive-acting member of the bZIP protein family that exhibits significant sequence similarity to both the yeast Met4p and Met28p proteins at the bZIP region. Transcription of cys3 is itself highly regulated by sulphur catabolite repression; expression of cys3 is switched on under sulphur limitation conditions and the CYS3 protein is able to upregulate autogenously its own expression in a positive manner (Coulter and Marzluf, 1998). Both scon-1 and scon-2 act in the negative control of cys-3 because mutations in either leads to constitutive expression of the CYS3 protein and of aryl sulphatase, sulphate transporter and other related proteins. A complex feedback loop exists in which cys3, together with scon-1, also controls expression of scon-2. The SCON1 protein, rather than the CYS3 protein itself, is suggested to be the factor that senses the sulphur repressing metabolite; under conditions of high levels of sulphur it may act to prevent cys3 function by converting the SCON2 protein into a form that binds directly to the CYS3 protein. This interaction prevents further cys3 expression by inhibiting its positive autogenous control. The SCON2 protein has high sequence similarity to the yeast protein Met30p outlined above and they may be functionally equivalent. cis-acting-regulatory elements with the consensus sequence ATGPuPyPuPyCAT have been identified in the promoters of the co-regulated genes and the CYS3 protein is believed to bind to these primarily as a single species rather than as a member of a multicomponent complex as observed in yeast (reviewed in Marzluf 1997). Sensing Sulphur Status and Signal Transduction in Plants In contrast to the detailed information on how the sulphur status is sensed and this signal transduced to the genes in non-photosynthetic organisms, little or nothing is known about these processes in plants. Although many

2.

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environmental parameters appear to affect expression of the reductive sulphate assimilation pathway it is tempting to speculate that signalling to the genes within the S-responsive stimulon is initiated by the perception of a limited number of candidate signal compounds. Only a very few signal transduction pathways may be involved and these probably exhibit cross-talk with each other and with those pathways involved in other aspects of environmental signalling. Molecular evidence of the type outlined above, which firmly identifies signal metabolites in higher plants, is lacking and our ideas are based largely on correlative changes between metabolite levels on the one hand and enzyme/protein levels, and/or steady state levels of mRNA abundance, on the other. Despite this reservation, and without considering the distribution of these metabolites within the cell, candidate signals that need to be accommodated in the context of reductive sulphate assimilation include OAS (Sections V C and V D) and glutathione (or cysteine? (Bolchi et al., 1999)) (Sections V.C and V.E). OAS appears to play a positive role in the regulation of the responsive genes whilst glutathione (or cysteine?) plays a negative role. The product of the Arabidopsis SAT-52 gene, described as mitochondrial by sequence inspection (Howarth et al., 1997) but cytosolic by transient expression of its GFP fusion protein (Noji et al., 1998) is inhibited by the pathway end-product, cysteine. Noji et al. (1998) have speculated that a feedback control loop of the type identified in E. coli might be in operation in the cytosol (if that is where this protein is located). When cysteine is available, SAT is inhibited and OAS levels and flux to cysteine are low. When cysteine levels are low, as on sulphate deprivation, then inhibition of SAT is relieved and the 0 AS level, and flux to cysteine, rises. The same authors speculate that the elevated OAS level might also act to upregulate expression of the pathway genes. These metabolites, glutathione and OAS, may act directly by modulating the binding of presently unidentified trans-acting protein factors to cis-acting-regulatory elements within the promoters of co-regulated genes. Alternatively they may first be sensed by some type of receptor protein, which then transduces the signal, via other protein intermediates, to these trans-acting protein factors. Data from Chlamydomonas reinhardtii discussed below suggest that this latter possibility might be more likely. A number of genomic clones encoding pathway steps have been published (Tables I and II), and a number of other genomic sequences are available via database inspection of information derived from the Arabidopsis Genome Initiative (see Table I for the chromosomal location of several Arabidopsis genes encoding pathway steps), but no cis-acting regulatory elements that might be specifically involved in sulphur-regulation have yet been identified using a functional analysis approach. However, inspection of the approximately 1 kb region upstream of the transcription initiation site of the Citrullus vulgaris Sat gene (Saito et al., 1997), identified two probable ciselement-like structures which are identical or very similar to the SEF4

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recognition sequence found in the promoter of the soybean f3-conglycinin gene (Allen et al., 1989). SEF4 is a trans-acting protein factor that binds to this sequence and is believed to mediate up-regulation of the sulphur-poor f3-subunit of this seed storage protein by sulphate deficiency and its downregulation by methionine (Lessard et al., 1991; Fujiwara and Beachey, 1994; Hirai et al., 1995; Fujiwara et al., 1997). Analysis of the 2 kb region upstream of the transcription start site of the Arabidopsis Prh-26 gene, which encodes one of the four members of the sulphur-regulated APS reductase gene family (Gutierrez-Marcos et al., 1996) (Table I), also identifies a number of cis-element-like structures that have been implicated in transcriptional regulation of other sulphur-related systems (J. F. Gutierrez-Marcos, personal communication). These include (a) GCN4-like motifs which have been implicated in the transcriptional regulation of prolamin synthesis by amino acid availability (Muller and Knudsen, 1993) and which have homology with promoter elements found in certain Saccharomyces cerevisiae MET genes (discussed above), (b) a motif related to the recognition site for the product of the positive-acting cys3 gene implicated in sulphur-regulation in Neurospora crassa (discussed above), and (c) a motif related to the SEF4 recognition sequence. Genomic clones for sulphite reductase have been described from Arabidopsis (Bork et al., 1998) and from Nicotiana tabacum (YonekuraSakakibara et al., 1998). Analysis of the 5' flanking sequence of gNtSiRl revealed the presence of a sequence identical to that required for the binding of the Cbfl protein in yeast, as well as a sequence similar to the yeast MET4 protein binding site. Both sequences were also present (YonekuraSakakibara et al., 1998) in the promoter region of theArabidopsis sir gene as well as that ofArabidopsis ATP sulphurylase genesAPS2 andAPS3 (Table 1). However, whereas expression of ATP sulphurylase genes is up-regulated on sulphate deprivation this does not appear to be the case for sulphite reductase (Takahashi et al., 1997; Yamaguchi et al., 1999). As a means to approach the dissection of the mechanisms involved in sensing the sulphur-status and transducing this signal to the $-responsive genes, a search has been made in Chlamydomonas reinhardtii for mutants that are altered in expression of the aryl sulphatase (ars) gene in response to sulphur deprivation (Davies et al., 1994). Production of this periplasmic enzyme under these conditions allows Chlamydomonas, and a number of other organisms, to use aromatic sulphates as a source of sulphur if they are present in the immediate environment. A very simple screen was used, in which aryl sulphatase activity could be easily detected in Chlamydomonas cells growing on minus-$ agar medium supplemented with thiocyanate, which involved spraying colonies with the chromogenic aryl sulphatase substrate 5-bromo-4-chloro-3-indolyl sulphate and monitoring for the appearance of a blue ring round the colonies. Such a screen led to the identification of three classes of mutant, designated sacl, sac2 and sac3

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(sulphur acclimation) (Davies et al., 1994), that had altered expression of ars, sulphate transporter and ATP sulphurylase genes (Yildiz et al., 1996), on sulphate deprivation. sacl mutants were unable to up-regulate ars and transporter gene expression, and down-regulate photosynthesis, on sulphate deprivation and died much more rapidly than wild-type cells under these conditions (Davies et al., 1996). The sacl gene encodes a protein that has homology to ion transporters and may be involved in sensing the sulphur status (Davies and Grossman, 1998). The sac2 mutant exhibited low ars and transporter gene expression under conditions of sulphate deprivation; the sac2 gene has not been cloned but its mutant phenotype suggests that it is involved either directly or indirectly in the transcriptional regulation of ars genes and possibly other genes induced during sulphate deprivation (Davies et al., 1994). sac3 mutants express the ars gene even in nutrient-replete conditions suggesting that the wild-type SAC3 protein exerts negative control of ars gene expression. In contrast, since the sac3 mutant is unable to induce fully the expression of sulphate transport during sulphur deprivation, it appears that the SAC3 protein exerts positive control of transporter gene expression (Davies et al., 1994). Molecular cloning demonstrates that the SAC3 protein is related to the catalytic domain of the yeast Snfl family of serine/threonine kinases and that it falls within the SnRK2 subfamily of vascular plants (Davies et al., 1999). Members of the SnRK2 subfamily contain kinase domains related to those of Snfl from yeast and AMP-activated kinase from mammals, but the Cterminal domains are unrelated and are usually characterized by stretches of acidic amino acid residues. These data implicate a phosphorylation-driven cascade in signal transduction, although the functions of these kinases are poorly understood (Hardie, 1999). Genetic analysis suggests that sacl and sac2 operate in the same linear pathway and that sacl is epistatic to (acts before) sac2. No clear epistatic relationship exists between sac3 and the other sac genes and Davies et al. (1999) suggest that sac3 may operate in a distinct signalling pathway. Attempts to demonstrate production of an aryl sulphatase enzyme inArabidopsis under sulphate deprivation, and thus use a similar approach in higher plants, proved unsuccessful (J. L. Wray, unpublished) but clearly a search for equivalent higher plant genes is required. Another approach to dissecting the pathway that transduces sulphur status might be that recently described by Ishitani et al. (1997) for the analysis of pathways involved in transduction of osmotic and cold stress. In this study transgenicArabidopsis plant expressing the firefly luciferase (luc) gene, driven by the promoter of the stress-responsive gene RD29A, were constructed. Seed from plants homozygous for the transgene were mutagenized and M 2 seedlings were screened by luminescence imaging to identify individual plants defective in osmotic stress regulation of luc gene

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expression. At least some of the mutant plants identified might be expected to be defective in sensing the osmotic stress or transducing this signal to the stress-responsive genes. Positional cloning could then be used to identify the relevant gene. For such a system to be useful in the context of the reductive sulphate assimilation pathway it would first be necessary to demonstrate sulphur-dependent regulation of luc gene expression from a chimaeric gene construct driven by the promoter of a sulphur-responsive gene, such as a sulphate transporter or APS reductase.

3. ABA Responsive Gene Expression The expression of many genes is altered in plants in response to osmotic stress elicited by water deficit or exposure to salinity (reviewed in Leung and Giraudat, 1998; Campalans et al., 1999) and this may occur via ADAdependent or ADA-independent signal transduction pathways that probably exhibit cross-talk (Ishitani et al., 1997). In the case of the Atcys-3A gene encoding a putative cytosolic isoform of OASTL (Barroso et al., 1999) it would appear that signalling occurs via an ADA-dependent pathway that involves protein phosphatases C, encoded by the abil (Meyer et al., 1994) and abi2 (Leung et al., 1994) loci. Whether other components, such as Ca2+ and the Ca2+-mobilizing second messenger, cyclic adenosine 5'-diphosphate ribose (cADPR), identified elsewhere (reviewed in Campalans et al., 1999) as putative members of the signal transduction pathway leading from the asyet unidentified ABA receptor to the promoter of ABA-responsive genes is also involved here, remains to be determined. Whatever the pathway, it will also be necessary to determine how the sulphur-related ADA-signalling pathway cross-talks with, and integrates into, other ABA-dependent and ADA-independent signalling pathways that transduce other environmental stresses. Functional dissection of the promoters of AHA-responsive genes have identified several cis-acting regulatory elements involved in ABA-induced gene expression. Some of these sequence elements, such as occur in the rice Rabl6 LEA (Onoetal., 1996) and the wheat Em LEA (Guiltinanetal., 1990) genes, share a G-box ACGT core motif that has been designated as an ABA response element (ABRE). Other studies support the idea that the smallest promoter units, ABA responsive complexes (ABRCs ), consist of at least two essential cis-elements. ABRCs may consist of two G-boxes, as in the wheat Em promoter (Vasil et al., 1995), or an ABRE and an unrelated cis element such as Sph (Kao et al., 1996), present in the promoter of the maize C1 gene, a regulator of seed anthocyanin synthesis, or coupling elements (CE) found in the barley HVA22 (Shen and Ho, 1995) andHVAJ (Shenetal., 1996) gene promoters. ABA-regulation of other stress-inducible genes does not involve ABRElike motifs. For example the promoter of the AHA-inducible Arabidopsis gene rd22 contains a region essential for induction that includes several

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conserved motifs for DNA-binding proteins such as MYC and MYB (Iwasaki et al., 1995). Homologues to MYC and MYB have been identified inArabidopsis and are induced by drought and high salinity (Abe et al., 1997). ABRE-related and MYB/MYC-related sequence motifs are present in many other ABA-inducible genes and may also be identified in theAtcys3A gene, and perhaps other genes encoding steps within the reductive sulphate assimilation pathway, but whether they are truly involved in ABA-signalling must be confirmed by functional analysis.

G.

STUDIES WITH TRANSGENIC PLANTS

1. ATP Sulphurylase A number of recent studies have taken advantage of the availability of eDNA species to obtain information on the operation of the pathway using transgene technology. An additional motivation for such approaches is to examine the possibility of metabolic engineering of the reductive sulphate assimilation pathway. ATP sulphurylase, the first enzyme of the pathway after sulphate has entered the plant, has been suggested to be rate-limiting for sulphate assimilation: the enzyme has a high substrate/product ratio, a Km for sulphate and ATP in the micromolar range and is subject to strong inhibition by its product adenosine phosphosulphate (Ki = 0.04 f.LM) (Schwenn, 1994; Leustek, 1996). To examine whether modification of ATP sulphurylase activity might perturb the pathway, Hatzfeld et al. (1998) constitutively overexpressed the Arabidopsis ASAJ eDNA encoding a putative chloroplastic isoform of ATP sulphurylase (Logan et al., 1996) in Bright Yellow 2 tobacco suspension culture cells. Despite the fact that ATP sulphurylase activity and protein in the transformed cells were eight-fold greater than that of control cells, there was no effect on cell growth or sulphate uptake, under non-limiting S. Sensitivity to selenate, a sulphur analogue that is metabolized via the reductive sulphate assimilation pathway to the toxic analogue of cysteine, selenocysteine, was identical in transgenic and control cells, suggesting no increase in flux through the pathway in the transgenic cells. These results imply that, under the conditions tested, A TP sulphurylase activity is either non-limiting for assimilation or that the enzyme activity is under tight control, or both. Of further interest was the observation that, under conditions of sulphur limitation, both tobacco and Arabidopsis ATP sulphurylase protein levels rose. Since it is unlikely that the constitutive 35S CaMV promoter driving expression of theArabidopsis gene is susceptible to sulphur limitation it would appear that some type of posttranscriptional effect is in operation. In other studies, selenium metabolism was examined in transgenic Indian mustard (Brassica juncea) seedlings expressing an approximately 2-2.5-fold increase in the activity of a plastidic ATP sulphurylase, encoded by theAPS I

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gene (Leustek et al., 1994), under the control of the CaMV 35S promoter (Pilon-Smits et al., 1999). Transgenic plants showed increased selenate reduction since they contained mostly organic selenium (selenamethionine?) whereas wild-type plants contained mainly selenate, but selenate levels in transgenic plants were still around twice that of wild-type plants. Although the fact that transgenic plants grew better than wild-type plants in the presence of toxic levels of selenate seems somewhat paradoxical, these studies do show that ATP sulphurylase not only mediates selenate reduction in plants, but that it is also rate-limiting for selenate uptake and assimilation. 2. 0-Acetylserine (Thiol) Lyase Attempts to modulate cysteine biosynthesis by overexpression of OASTL have also been attempted. Tobacco plants transformed with the wheat cysl eDNA, encoding a putative plastidic isoform of OASTL, driven by the CaMV 35S promoter had about 3-5-fold higher OASTL activity than wildtype tobacco plants (Youssefian et al., 1993) and were more resistant to the toxic effects of H 2S gas than wild-type plants. A more detailed analysis was performed in studies in which tobacco was transformed with the spinach CysA eDNA, encoding a putative cytosolic OASTL (Saito et al., 1992), fused to the sequence for a chloroplast-targeting peptide of pea Rubisco and driven by the CaMV 35S promoter (Saito et al., 1994a). However, although these transgenic plants had about 2-3-fold OASTL of wild-type plants and OASTL activity of isolated chloroplasts was several-fold higher than wildtype plants, demonstrating that the spinach OASTL had been targeted correctly to the tobacco chloroplasts, cysteine and glutathione levels of the transgenic plants were the same as wild-type. Although this may suggest that under normal conditions and without sulphur stress, endogenous levels of OASTL are sufficient to maintain maximal rates of cysteine biosynthesis one cannot discount the possibility that there was increased conversion of cysteine into other sulphur compounds, such as protein. Of particular interest was the observation that addition of OAS to isolated chloroplasts resulted in increased cysteine production and this was around an order of magnitude higher in the transgenic plants. These results suggest that, in chloroplasts, supply of OAS is a limiting factor for S flux to cysteine, supporting conclusions reached from earlier studies (Neuenschwander et al., 1991; Brunold, 1993), and that an increase in SAT activity may be essential for any attempt to enhance the flux of S through the reductive sulphate assimilation pathway. 3. Sulphur Metabolism and Engineering Salt Tolerance Interesting implications for plant sulphur metabolism have come from a search for yeast genes whose overexpression is able to confer tolerance to salt. A number of these halotolerant (HAL) genes have been identified but

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the HAL2 gene (Glaser et al., 1993), which is able to complement the E. coli cysQ and the Saccharomyces cerevisiae met22 mutations, is of particular significance here. HAL2 encodes a salt-sensitive 3'(2'),5'-bisphosphate nucleotidase that is able to dephosphorylate 3' -phosphoadenosine-5'phosphate (PAP), generated during PAPS-dependent sulpha group transfer by sulphotransferases. Inhibition of the 3'(2'),5'-bisphosphate nucleotidase by salt leads to a decreased tolerance to salt due to the trapping of adenine nucleotides as PAP (Murguia et al., 1996) and by the inhibition of sulphotransferases (Varin and Ibrahim, 1992) and of RNA processing enzymes (Dichtl et al., 1997) by this metabolite. Overexpression of the 3'(2'),5' -bisphosphate nucleotidase in yeast reduces the salt-induced accumulation of PAP and allows growth at higher salt levels (Murguia et al .. 1996). Overexpression of the HAL2 gene in tomato was also shown to increase the in vitro salt tolerance of transgenic progeny (Arrillaga et a!., 1998). HAL2-like genes have more recently been identified in rice (RHL gene, Peng and Verma, 1995) andArabidopsis (AtSALJ gene, Quintero et al., 1996; AtAHL and AtSAL2, Gil-Mascarell et al., 1999); they all complement the yeast ha/2 mutant and encode protein products that are able to hydrolyse PAP (as well as PAPS). The fact that the PAP phosphatase activity of the protein encoded by AtAHL alone is sensitive to inhibition by salt (GilMascarell et al., 1999) weakens somewhat the idea that these enzymes are cellular targets for salt sensitivity in plants, as they appear to be in yeasts. However, it could still be argued that theAtAHL gene product might play a major part in PAP degradation at certain developmental stages and under particular combinations of environmental stress, and thus under these conditions might act as a major cellular target for salt sensitivity.

VI.

CONCLUSIONS AND FUTURE PROSPECTS

The importance of S-nutrition has recently been acknowledged principally because of the increased incidence of S-deficiency in agriculture. Substantial progress has been made on cloning structural genes for the transporter and the enzymes involved in sulphate uptake and assimilation. Surprisingly many of the components are encoded by small multigene families and much remains to be learnt about the roles of these individual genes and gene products. In some cases there is apparent subcellular compartmentation of the assimilatory enzymes, with fragments of pathways occurring in parallel in separate organelles and again the reasons remain to be elucidated. The availability of transgenic plants with modified expressions of individual components may help in elucidating the roles of these components. Transgenic manipulation of sulphur metabolism will enable the development of plants better able to manage S-reserves with resulting

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improved nutrient use efficiency, with increased stress resistance and with better nutritional quality. Studies on the sensing and signalling pathways and their involvement in the control of gene expression are still in their infancy. These remain veritable 'black boxes' and will undoubtably be the focus of research efforts in the coming years.

ACKNOWLEDGEMENTS Work in the laboratory of M.J.H. is sponsored by grants from the BBSRC, Home-Grown Cereals Authority and by Framework IV of the EU. The IACR receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the UK.

REFERENCES Abe, H., Yamaguchi-Shinozaki, K., Urao, T., Iwasaki, T., Hosokawa, D. and Shinozaki, K. (1997). Role of Arabidsopsis MYC and MYB homologs in drought and abscisic acid regulated gene expression. Plant Cell9, 1859-1868. Adiputra, I. G. K. and Anderson, J. W. (1992). Distribution and redistribution of sulfur taken up from nutrient solution during vegetative growth in barley. Physiologia Plantarum 85, 453-460. Adiputra, I. G. K. and Anderson, J. W. (1995). Effect of sulphur nutrition on redistribution of sulphur in vegetative barley. Physiologia Plantarum 95, 643-650. Allen, R. J., Bernier, F., Lessard, P. A. and Beachey, R.N. (1989). Nuclear factors interact with a soybean f3-conglycinin enhancer. Plant Cell1, 623-631. Amancio, S., Clarkson, D. T., Diogo, E., Lewis, M. and Santos, M. (1997). Assimilation of nitrate and ammonium by sulphur deficient Zea mays cells. Plant Physiology and Biochemistry 35, 41-48. Arrillaga, 1., Gil-Mascarell, R., Gisbert, C., Sales, E., Montesinos, C., Serrano, R. and Moreno, V. (1998). Expresssion of the yeast HAL2 gene in tomato increases the in vitro salt tolerance of transgenic progenies. Plant Science 136, 219-226. Arz, H. E., Gisselmann, G., Schiffmann, S. and Schwenn, J.D. (1994). A eDNA for adenylyl sulfate (APS) kinase from Arabidopsis thaliana. Biochimica et Biophysica Acta 1218, 447-452. Bairoch, A. and Bucher, P. (1994). PROSITE- recent developments. Nucleic Acids Research 22, 3583-3589. Barroso, C., Vega, J. M. and Gotor, C. (1995). A new member of the cytosolic 0acetylserine(thiol)lyase gene family inArabidopsis thaliana. FEBS Letters 363, 1-5. Barroso, C., Romero, L. C., Cejudo, F. J., Vega, J. M. and Gotor, C. (1999). Saltspecific regulation of the cytosolic 0-acetylserine(thiol)lyase gene from Arabidopsis thaliana is dependent on abscisic acid. Plant Molecular Biology 40, 729-736.

MOLECULAR GENETICS OF SULPHATE ASSIMILATION

209

Berendt, U., Haverkamp, T., Prior, A. and Schwenn, J. D. (1995). Reaction mechanism of thioredoxin: 3'-phosphoadenylylsulfate reductase investigated by site-directed mutagenesis. European Journal of Biochemistry 233, 347-356. Bick, J. A. and Leustek, T. (1998). Plant sulfur metabolism- the reduction of sulfate to sulfite. Current Opinion in Plant Biology 1, 240-244. Bick, J. A., Aslund, F., Chen, Y. C. and Leustek, T. (1998). Glutaredoxin function for the carboxyl terminal domain of the plant type 5'-adenylylysulfate reductase. Proceedings of the National Academy of Sciences of the USA 95, 8404-8409. Blaiseau, P. L., Isnard,A. D., Surdin-Kerjan, Y. and Thomas, D. (1997). Met31p and Met32p, two related zinc finger proteins, are involved in transcriptional regulation of yeast sulfur amino acid metabolism. Molecular and Cellular Biology 17, 3640-3648. Blake-Kalff, M. M.A., Harrison, K. R., Hawkesford, M. J ., Zhao, F. J. and McGrath, S. P. (1998). Distribution of sulfur within oilseed rape leaves in response to sulfur deficiency during vegetative growth. Plant Physiology 118, 1337-1344. Bogdanova, N. and Hell, R. (1997). Cysteine synthesis in plants: protein-protein interactions of serine acetyltransferase from Arabidopsis thaliana. Plant Journal11,251-262. Bogdanova, N., Bork, C. and Hell, R. (1995). Cysteine biosynthesis in plants: isolation and functional identification of a eDNA encoding a serine acetyltransferase fromArabidopsis thaliana. FEBS Letters 358,43-47. Bolchi, A., Petrucco, S., Tenca, P. L., Foroni, C. and Ottonello, S. (1999). Coordinate modulation of maize permease and ATP sulfurylase mRNAs in response to variations in sulfur nutritional status: stereospecific down-regulation by Lcysteine. Plant Molecular Biology 39,527-537. Bork, C., Schwenn, J. D. and Hell, R. (1998). Isolation and characterization of a gene for assimilatory sulfite reductase from Arabidopsis thaliana. Gene 212, 147-153. Bourgis, F., Roje, S., Nuccio, M. L., Fisher, D. B., Tarczynski, M. C., Li, C. J., Herschbach, C., Rennenberg, H., Pimenta, M. J., Shen, T. L., Gage, D. A. and Hanson, A. D. (1999). S-Methylmethionine plays a major role in phloem sulfur transport and is synthesized by a novel type of methyl transferase. Plant Cellll, 1485-1497. Brander, K. A., Owttrim, G. W. and Brunold, C. (1995). Isolation of a eDNA (EMBL X85803) encoding a putative chloroplastic isoform of cysteine synthase from maize. Plant Physiology 108, 1748. Breton, A. and Surdin-Kerjan, Y. (1977). Sulfate uptake in Saccharomyces cerevisiae: biochemical and genetic study. Journal of Bacteriology 132, 224-232. Bruhl, A., Haverkamp, T., Gisselmann, G. and Schwenn, J. D. (1996). A eDNA clone from Arabidopsis thaliana encoding plastidic ferredoxin: sulfite reductase. Biochimica et BiophysicaActa 1295, 119-124. Brunold, C. (1993). Regulatory interactions between sulfate and nitrate assimilation. In "Sulfur Nutrition and Assimilation in Higher Plants" (L. J. De Kok, L Stulen, H. Rennenberg, C. Brunold and W. E. Rauser, eds), pp. 61-76. SPB Publishing, The Hague. Brunold, C. and Suter, M. (1982). Intracellular localization of serine acetyltransferase in spinach leaves. Planta 155,321-327. Brunold, C., Suter, M. and Lavanchy, P. (1987). Effect of high and low sulfate concentrations on adenosine 5'-phosphosulfate sulfotransferase activity from Lemna minor. Physiologia Plantarum 70, 168-174. Buchanan-Woolaston, V. and Ainsworth, C. (1997). Leaf senescence in Brassica napus: cloning of senescence-related genes by subtractive hybridisation. Plant Molecular Biology 33, 821-834.

210

M. J. HAWKESFORD and J. L. WRA Y

Byers, M., McGrath, S. P. and Webster, R. (1987). A survey of the sulfur content of wheat grown in Britain. Journal of the Science of Food and Agriculture 38, 151-160. Campalans, A., Messeguer, R., Goday, A. and Pages, M. (1999). Plant responses to drought, from ABA signal transduction events to the action of the induced proteins. Plant Physiology and Biochemistry 37, 327-340. Castle, S. L. and Randall, P. J. (1987). Effects of sulfur deficiency on the synthesis and accumulation of proteins in the developing wheat seed. Australian Journal of Plant Physiology 14,503-516. Chen, Y. C. and Leustek, T. (1998). Three genomic clones fromArabidopsis thaliana encoding 5'-adenylylsulfate reductase (Accession Nos. AF016282, AF016283 and AF016284) (PGR98-030). Plant Physiology 116, 869. Churchill, K. A. and Sze, H. (1984). Anion-sensitive, H+ -pumping ATPase of oat roots- direct effects of CI-, N0 3-, and a disulfonic stilbene. Plant Physiology 76,490-497. Clarkson, D. T., Smith, F. W. and Vandenberg, P. J. (1983). Regulation of sulfate transport in a tropical legume, Macroptilium atropurpureum cv Sirato. Journal of Experimental Botany 34, 1463-1483. Clarkson, D. T., Saker, L. R. and Purves, J. V. (1989). Depression of nitrate and ammonium transport in barley plants with diminished sulphate status. Evidence for co-regulation of nitrogen and sulphate intake. Journal of Experimental Botany 40, 953-963. Clarkson, D. T., Hawkesford, M. J., Davidian, J. C. and Grignon, C. (1992). Contrasting responses of sulfate and phosphate-transport in barley (Hordeum vulgare L.) roots to protein-modifying reagents and inhibition of proteinsynthesis. Planta 187, 306-314. Clarkson, D. T., Hawkesford, M. J. and Davidian, J.-C. (1993). Membrane and long distance transport of sulphate. In "Sulfur Nutrition and Assimilation in Higher Plants" (L. J. De Kok, I. Stu!en, H. Rennenberg, C. Brunold and W. E. Rauser, eds), pp. 3-19. SPB Academic Publishing, The Hague. Clarkson, D. T., Diogo, E. and Amancio, S. (1999). Uptake and assimilation of sulphate by sulphur deficient Zea mays cells: The role of 0-acetyl-L-serine in the interaction between nitrogen and sulphur assimilatory pathways. Plant Physiology and Biochemistry 37, 283-290. Coulter, K. R. and Marzluf, G. A. (1998). Functional analysis of different regions of the positive-acting CYS3 regulatory protein of Neurospora crassa. Current Genetics 33, 395-405. Davies, J.P. and Grossman, A. R. (1998) Survival during macronutrient limitation. In "The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas" (J.-D. Rochaix, M. Goldschmidt-Clermont and S. Merchant, eds), pp. 613-635, Kluwer, Dordrecht. Davies, J. P., Yildiz, F. and Grossman, A. R. (1994). Mutants of Chlamydomonas with aberrant responses to sulfur deprivation. Plant Cell 6, 53-63. Davies, J.P., Yildiz, F. H. and Grossman, A. (1996). Sacl, a putative regulator that is critical for survival of Chlamydomonas reinhardtii during sulfur deprivation. EMBO Journal15, 2150-2159. Davies, J. P., Yildiz, F. H. and Grossman, A. R. (1999). Sac3, an Snfl-like serine/ threonine kinase that positively and negatively regulates the responses of Chlamydomonas to sulfur limitation. Plant Cellll, 1179-1190. Department of the Environment, Transport and the Regions (1998). Digest of Environmental Statistics No. 20. HMSO, London.

MOLECULAR GENETICS OF SULPHATE ASSIMILATION

211

Dhindsa, R. S. (1991). Drought stress, enzymes of glutathione metabolism, oxidation injury and protein synthesis in Tortula ruralis. Plant Physiology 95, 648-651. Dichtl, B., Stevens, A and Tollervey, D. (1997). Lithium toxicity in yeast is due to the inhibition of RNA processing enzymes. EMBO Journa/16, 7184-7195. Dietz, K. J., Brune, A and Pfanz, H. (1992). Trans-tonoplast transport of the sulfurcontaining-compounds sulfate, methionine, cysteine and glutathione. PhytonAnnales Rei Botanicae 32, 37-40. Droux, M., Martin, J., Sajus, P. and Douce, R. (1992). Purification and characterization of 0-acetylserine (thiol) lyase from spinach chloroplasts. Archives of Biochemistry and Biophysics 295, 379-390. Droux, M., Ruffet, M.-L., Douce, R. and Job, D. (1998). Interactions between serine acetyltransferase and 0-acetylserine (thiol) lyase in higher plants. Structural and kinetic properties of the free and bound enzymes. European Journal of Biochemistry 255, 235-245. Edwards, R., Blount, J. W. and Dixon, R. A (1991). Glutathione and elicitation of the phytoalexin response in legume cell cultures. Planta 184, 403-409. Ernst, W. H. 0. (1990). Ecological aspects of sulfur metabolism. In "Sulfur Nutrition and Sulfur Assimilation in Higher Plants" (H. Rennenberg, C. H. Brunold, L. J. De Kok and I. Stulen, eds), pp. 131-144. SPB Academic Publishing, The Hague. Fell, D. (1997). "Understanding the Control of Metabolism". Portland Press, London. Fitzgerald, M. A, Ugalde, T. D. and Anderson, J. W. (1999a). Sulphur nutrition changes the sources of S in vegetative tissues of wheat during generative growth. Journal of Experimental Botany 50, 499-508. Fitzgerald, M. A, Ugalde, T. D. and Anderson, J. W. (1999b). S nutrition affects the pools of S available to developing grains of wheat. Journal of Experimental Botany 50, 1587-1592. Fliigge, U. 1., Fischer, K., Gross, A, Sebald, W., Lottspeich, F. and Eckerskorn, C. (1989). The triose phosphate-3-phosphoglycerate phosphate translocator from spinach-chloroplasts- nucleotide-sequence of a full-length eDNA clone and import of the in vitro synthesized precursor protein into chloroplasts. EMBO JournalS, 39-46. Frachisse, J. M., Thomine, S., Colcombet, J., Guern, J. and Barbier-Brygoo, H. (1999). Sulfate is both a substrate and an activator of the voltage-dependent anion channel ofArabidopsis hypocotyl cells. Plant Physiology 121, 253-261. Friedrich, J. W. and Schrader, L. E. (1978). Sulfur deprivation and nitrogen metabolism in maize seedlings. Plant Physiology 61, 900-906. Fu, Y. H., Paietta, J. V., Mannix, D. G. and Marzluf, G. A (1989). cys-3, the positiveacting sulfur regulatory gene of Neurospora crassa, encodes a protein with a putative leucine zipper DNA-binding element. Molecular and Cellular Biology 9, 1120-1127. Fujiwara, T. and Beachey, R.N. (1994). Tissue-specific and temporal regulation of a ~-conglycinin gene: role ofthe RY repeat and other cis-acting elements. Plant Molecular Biology 24,261-272. Fujiwara, T., Matsui, A, Hirai, M. Y., Furuhashi, A, Awazuhara, M., Honda, C., Kim, H., Noguchi, K., Shibagaki, N., Yasumori, M., Hayashi, H., Naito, S. and Chino, M. (1997). Genetic and physiological approaches toward understanding the mechanisms underlying the sulfur-regulated expression of ~-conglycinin genes. Soil Science and Plant Nutrition 43, 965-969. Fullington, J. G., Miskelly, D. M., Wrigley, C. W. and Kasarda, D. D. (1987). Qualityrelated endosperm proteins in sulfur-deficient and normal wheat grain. Journal of Cereal Science 5, 233-245.

212

M. 1. HAWKESFORD and J. L. WRAY

Gilbert, S.M., Clarkson, D. T., Cambridge, M., Lambers, H. and Hawkesford, M. J. (1997). SO/- deprivation has an early effect on the content of ribulose-1,5bisphosphate carboxylase/oxygenase and photosynthesis in young leaves of wheat. Plant Physiology 115, 1231-1239. Gil-Mascarell, R., Lopez-Coronado, J. M., Belles, J. M., Serrano, R. and Rodriguez, P.L. (1999). TheArabidopsis HAL2-like gene family includes a novel sodiumsensitive phosphatase. Plant Journal11, 373-383. Giraudat, J., Parcy, F., Bertauche, N., Gosti, F., Leung, J., Morris, P.-C., BouvierDurand, M. and Vartanian, N. (1994). Current advances in abscisic acid action and signalling. Plant Molecular Biology 26, 1557-1577. Glaser, H.-U., Thomas, D., Gaxiola, R., Montrichard, F., Surdin,-Kerjan, Y. and Serrano, R. (1993). Salt tolerance and methionine biosynthesis in Saccharomyces cerevisiae involve a putative phosphatase gene. EMBO Journal 12, 3105-3110. Glendening, T. M. and Poulton, J. E. {1990). Partial purification and characterization of a 3'-phosphoadenosine 5'-phosphosulfate: desulfoglucosinolate sulfotransferase from cress (Lepidium sativum). Plant Physiology 94, 811-818. Goldschmidt, E. E., Tsang, M. L. and Schiff, J. A. {1975). Studies of sulphur utilization by algae, 13. Adenosine 5'-phosphosulfate (APS) as an intermediate in the conversion of adenosine 3'5' -phosphosulfate (PAPS) to acid-volatile radioactivity. Plant Science Letters 4, 293-299. Gotor, C., Cejudo, F. J., Barroso, C. and Vega, J. M. (1997). Tissue-specific expression of ATCYS-3A, a gene encoding the cytosolic isoforrn of 0acetylserine (thiol) lyase inArabidopsis. Plant Journalll, 347-352. Guiltinan, M. J., Marcotte, W. R. and Quatrano, R. (1990). A plant leucine zipper protein that recognizes an abscisic acid response element. Science 250, 267-271. Guri, A. (1983). Variation in glutathione and ascorbic acid content among selected cultivars of Phaseolus vulgaris prior to and after exposure to ozone. Canadian Journal of Plant Science 63, 733-737. Gutierrez-Marcos, J. F., Roberts, M. A., Campbell, E. I. and Wray, J. L. {1996). Three members of a novel small gene-family fromArabidopsis thaliana able to complement functionally an Escherichia coli mutant defective in PAPS reductase activity encode proteins with a thioredoxin-Iike domain and 'APS reductase' activity. Proceedings of the National Academy of Sciences of the USA 99, 13377-13382. Hampp, R. and Ziegler, I. ( 1977). Sulfate and sulfite translocation via the phosphate translocator of the inner envelop membrane of chloroplasts. Planta 137, 309-312. Hardie, D. G. (1999). Plant protein serine/threonine kinases.Annual Review of Plant Physiology and Plant Molecular Biology 50, 97-131. Hatzfeld, Y., Logan H. M., Cathala, N. and Davidian J.-C. {1997). Cloning of a gene encoding an ATP-sulfurylase from Brassica oleracea (Accession number U69694) PGR97-005. Plant Physiology 113, 305-307. Hatzfeld, Y., Cathala, N., Grignon, C. and Davidian, J.-C. (1998). Effect of ATP sulfurylase overexpression in Bright Yellow 2 tobacco cells. Regulation of ATP sulfurylase and sulphate transport activities. Plant Physiology 116, 1307-1313. Hawkesford, M. J. and Prosser, I. P. (2000). The plant sulfate transporter family. In "Sulfur Nutrition and Sulfur Assimilation in Higher Plants: Molecular,

MOLECULAR GENETICS OF SULPHATE ASSIMILATION

213

Biochemical and Physiological Aspects" (C. Brunold, H. Rennenberg, L. J. De Kok, I. Stulen and J .-C. Davidian, eds}, pp. 259-260. Paul Haupt Verlag, Bern. Hawkesford, M. J. and Smith, F. W. (1997). Molecular biology of higher plant sulphate transporters. In "Sulphur Metabolism in Higher Plants" (W. J. Cram, L. J. De Kok, I. Stulen, C. Brunold and H. Rennenberg, eds), pp. 13-25. Backhuys, Leiden. Hawkesford, M. J., Davidian, J.-C. and Grignon, C. (1993). Sulfate proton cotransport in plasma membrane vesicles isolated from roots of Brassica napus L. Increased transport in membranes isolated from sulfur-starved plants. Planta 190, 297-304. Heiss, S., Schafer, H. J., Haag-Kerwer, A. and Rausch, T. (1999). Cloning sulfur assimilation genes of Brassica juncea L.: cadmium differentially affects the expression of a putative low-affinity sulfate transporter and isoforms of ATP sulfurylase and APS reductase. Plant Molecular Biology 39, 847-857. Hell, R. (1997). Molecular physiology of plant sulfur metabolism. Planta 202, 138-148. Hell, R. and Bogdanova, N. (1995). Characterisation of a full-length eDNA encoding serine acetyltransferase from Arabidopsis thaliana. [X80938] (PGR95-108). Plant Physiology 109, 1498. Hell, R., Schuster, G. and Gruissem, W. (1993). An 0-acetylserine (thiol) lyase eDNA from spinach. Plant Physiology 102, 1057-1058. Hell, R., Bork, C., Bogdanova, N., Frolov, I. and Hauschild, R. (1994). Isolation and characterization of two cDNAs encoding for compartment specific isoforms of 0-acetylserine (thiol) lyase from Arabidopsis thaliana. FEBS Letters 351, 257-262. Hell, R., Schwenn, and Bork, C. (1997). Light and sulfur sources modulate mRNA levels of several genes of sulfate assimilation. In "Sulfur Metabolism in Higher Plants" (W. J. Cram., L. J. DeKok, I. Stulen, C. Brunold and H. Rennenberg, eds}, pp. 181-185. Backhuys, Leiden. Hesse, H. and Altmann, T. (1995). Molecular cloning of a cysteine synthase eDNA fromArabidopsis thaliana. Plant Physiology 108, 851-852. Hesse, H. and Hofgen, R. (1998). Isolation of cDNAs encoding cytosolic (Accession No. AF044172) and plastidic (Accession No. AF044173) cysteine synthase isoforms from Solanum tuberosum (PGR98-057). Plant Physiology 116, 1604. Hesse, H., Lipke, J., Altmann, T. and Hofgen, R. (1999). Molecular cloning and analyses of mitochondrial and plastidic isoforms of cysteine synthase (0acetylserine( thiol)lyase) fromArabidopsis thaliana .Amino Acids 16, 113-131. Hinnebush, A. G. (1992). General and pathway-specific regulatory mechanisms controlling the synthesis of amino acid biosynthetic enzymes in Saccharomyces cerevisiae. In "The Molecular Biology and Cellular Biology of the Yeast Saccharomyces. Gene Expression" (E. W. Jones, J. R. Pringle and J. R. Broach, eds ), pp. 319-414. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Hirai, M. Y., Fujiwara, T., Chino, M. and Naito, S. (1995). Effects of sulfate concentrations on the expression of a soybean seed storage protein gene and its reversibility in transgenicArabidopsis thaliana. Plant and Cell Physiology 36, 1331-1339. Hoff, T., Truong, H.-N. and Caboche, M. (1994). The use of mutants and transgenic plants to study nitrate assimilation. Plant Cell and Environment 17, 489-506. Holmgren, A. (1989). Thioredoxin and glutaredoxin systems. Journal of Biological Chemistry 264, 13963-13966. Howarth, J. R., Roberts, M. A. and Wray, J. L. (1997). Cysteine biosynthesis in higher plants: a new member of the Arabidopsis thaliana serine

214

M. J. HAWKESFORD andJ. L. WRAY

acetyltransferase small gene-family obtained by functional complementaion of an Escherichia coli cysteine auxotroph. Biochimica et Biophysica Acta 1350, 123-127. ldeguchi, T., Akashi, T., Onda, Y. and Rase, T. (1995). eDNA cloning and functional expression of ferredoxin-dependent sulfite reductase from maize in E. coli cells. In: "Research in Photosynthesis: From Light to Biosphere", Volume II (Mathis, P. ed.), pp. 713-716. Kluwer, Amsterdam. lshitani, M., Xiong, L., Stevenson, B. and Zhu, J.-K. (1997). Genetic analysis of osmotic and cold stress signal transduction in Arabidopsis: Interactions and convergence of abscisic acid-dependent and abscisic acid-independent pathways. Plant Cell9, 1935-1949. Iwasaki, T., Yamaguchi-Shinozaki, K. and Shinozaki, K. (1995). Identification of a cis-regulatory region of a gene of Arabidopsis thaliana whose induction by dehydration is mediated by abscisic acid and requires protein synthesis. Molecular and General Genetics 247, 391-398. Jacobson, E. S. and Metzenberg, R. L. (1977). Control of arylsulfatase in a serine auxotroph of Neurospora. Journal of Bacteriology 130, 1397-1398. Jain, A. and Leustek, T. (1994 ). A eDNA clone for 5'-adenylylphosphosulfate kinase fromArabidopsis thaliana. Plant Physiology 105, 771-772. Kaeser, H. and Burns, J. A. (1973). The control of flux. Symposium of the Society of Experimental Biology 21, 65-104. Kaiser, G., Martinoia, E., Schroppelmeier, G. and Heber, U. (1989). Activetransport of sulfate into the vacuole of plant-cells provides halotolerance and can detoxify S02• Journal of Plant Physiology 133, 756-763. Kanno, N., Nagahisa, E., Sato, M. and Sato, Y. (1996). Adenosine 5'-phosphosulfate sulfotransferase from the marine macroalga Porphyra yezoensis Ueda (Rhodophyta); stabilization, purification and properties. Planta 198, 440-446. Kao, C.-Y., Cocciolone, S.M., Vasil, I. K. and McCarty, D. R. (1996). Localization and interaction of the cis-acting elements for abscisic acid, VIVIPAROUS and light-activation of the Cl gene of maize. Plant CellS, 1171-1179. Karmoker,J. L., Clarkson, D. T.,Saker,L. R., Rooney,J. M. andPurves,J. V. (1991). Sulfate deprivation depresses the transport of nitrogen to the xylem and the hydraulic conductivity of barley (Hordeum vulgare L.) roots. Planta 185, 269-278. Kertesz, M. A., Leisinger, T. and Cook, A. M. (1993). Proteins induced by sulfate limitation in Escherichia coli, Pseudomonas putida and Staphylococcus aureus. Journal of Bacteriology 175, 1187-1190. Ketter, J. S., Jarai, G., Fu, Y. H. and Marzluf, G. A. (1991). Nucleotide sequence, messenger RNA stability, and DNA recognition elements of cys-14, the structural gene for sulfate permease-II in Neurospora crassa. Biochemistry 30, 1780-1787. Kim, H., Hirai, M. Y., Hayashi, H., Chino, M., Naito, S. and Fujiwara, T. (1999). Role of 0-acetyl-L-serine in the coordinated regulation of the expression of a soybean seed storage-protein gene by sulfur and nitrogen nutrition. Planta 209, 282-289. Kleppinger-Sparace, K. F. and Mudd, J. B. (1990). Biosynthesis of sulfoquinovosyldiacylglycerol in higher plants- use of adenosine-5'-phosphosulfate and adenosine-3'-phosphate 5' -phosphosulfate as precursors. Plant Physiology 93, 256-263. Klonus, D., Hofgen, R., Willmitzer, L. and Riesmeier, J. W. (1994). Isolation and characterization of two clones encoding ATP sulfurylases from potato by complementation of a yeast mutant. Plant Journal 6, 105-112.

MOLECULAR GENETICS OF SULPHATE ASSIMILATION

215

Klonus, D., Riesmeier, J. W. and Willmitzer, L. (1995). A eDNA clone for an ATP sulfurylase fromArabidopsis thaliana. Plant Physiology 107, 653-654. Kocsy, G., Brunner, M., Ruegsegger, A., Stamp, P. and Brunold, C. (1996). Glutathione synthesis in maize genotypes with different sensitivity to chilling. Planta 198, 365-370. Kopriva, S., Muheim, R., Koprivova, A., Trachsel, N., Catalano, C., Suter, M. and Brunold, C. (1999). Light regulation of assimilatory sulphate reduction in Arabidopsis thaliana. Plant Joumal20, 37-44. Koorneef, M., Reuling, G. and Karssen, C. M. (1984). The isolation and characterization of abscisic acid insensitive mutants of Arabidopsis thaliana. Physiologia Plantarum 61, 377-383. Kouchi, H. and Hata, S. (1993). Isolation and characterization of novel nodulin cDNAs representing genes expressed at early stages of soybean nodule development. Molecular and General Genetics 238, 106-119. Kredich, N. M. (1992). The molecular basis for positive regulation of cys promoters in Salmonella typhimurium and Escherichia coli. Molecular Microbiology 6, 2747-2753. Kredich N. M. (1993). Gene regulation of sulfur assimilation. In "Sulfur Nutrition and Assimilation in Higher Plants" (De Kok, L. J ., Stulen, I., Rennenberg, H., Brunold, C., Rauser, W.E., eds), pp. 37-47. SPB Academic Publishers, The Hague. Kredich, N. M. and Tomkins, G. M. (1966). The enzymatic synthesis of L-cysteine in Escherichia coli and Salmonella typhimurium. Journal of Biological Chemistry 241, 4955-4965. Kuske, C. R., Hill, K. K., Guzman, E., Jackson, P. J. (1996). Subcellular location of 0-acetylserine sulfhydrylase isoenzymes in cell cultures and plant tissues of Datura innoxia Mill. Plant Physiology 112, 659-667. Lappartient, A. G., Vidmar, J. J., Leustek, T., Glass, A. D. M. and Touraine, B. (1999). Inter-organ signaling in plants: regulation of ATP sulfurylase and sulfate transporter genes expression in roots mediated by phloemtranslocated compound. Plant Joumal18, 89-95. Lea, P. J. and Forde, B. G. (1994). The use of mutants and transgenic plants to study amino acid metabolism. Plant Cell and Environment 17, 541-556. Lee, R. B. (1982). Selectivity and kinetics of ion uptake by barley plants following nutrient deficiency.Annals of Botany 50, 429-449. Lee, S. and Leustek, T. (1998). APS kinase from Arabidopsis thaliana: genomic organization, expression and kinetic analysis of the recombinant enzyme. Biochemical and Biophysical Research Communications 247, 171-175. Lee, S. M. and Leustek, T. (1999). The affect of cadmium on sulfate assimilation enzymes in Brassica juncea. Plant Science 141, 201-207. Leggett, J. E. and Epstein, E. (1956). Kinetics of sulfate absorption by barley roots. Plant Physiology 31, 222-226. Lessard, P. A., Allen, R. D., Bernier, F., Crispino, J.D., Fujiwara, T.and Beachey, R. N. (1991). Multiple nuclear factors interact with upstream sequences of differentially regulated S-conglycinin genes. Plant Molecular Biology 16, 397-413. Leung, J. and Giraudat, J. (1998). Abscisic acid signal transduction. Annual Review of Plant Physiology and Plant Molecular Biology 49, 199-222. Leung, J., Bouvier-Durand, M., Morris, P.-C., Guerrier, D., Chefdor, F. and Giraudat, J. (1994). Arabidopsis ABA-response gene ABIJ: features of a calcium-modulated protein phosphatase. Science 264, 1448-1452.

216

M. J. HAWKESFORD and J. L. WRAY

Leustek, T. (1996). Molecular genetics of sulfate assimilation in plants. Physiologia Plantarum 97, 411-419. Leustek, T. and Saito, K. (1999). Sulfate transport and assimilation in plants. Plant Physiology 120, 637-643. Leustek, T., Murillo, M. and Cervantes, M. (1994). Cloning of a eDNA encoding ATP sulfurylase from Arabidopsis thaliana by functional expression in Saccharomyces cerevisiae. Plant Physiology 105, 897-902. Li, Q. and Marzluf, G. A. (1996). Determination of the Neurospora crassa CYS3 sulfur regulatory protein consensus DNA-binding site: amino-acid substitutions in the CYS3 bZip domain that alter DNA-binding specificity. Current Genetics 30, 298-304. Logan, H. M., Cathala, N., Grignon, C. and Davidian, J.-C. (1996). Cloning of a eDNA encoded by a member of the Arabidopsis thaliana ATP sulfurylase multigene family. Expression studies in yeast and in relation to plant sulfur nutrition. Journal of Biological Chemistry 271, 12227-12233. Lunn, J. E., Droux, M., Martin, J. and Douce, R. (1990). Localization of ATP sulphurylase and 0-acetylserine (thiol) lyase in spinach leaves. Plant Physiology 94, 1345-1352. Madamanchi, N. R. and Alscher, R. G. (1991). Metabolic bases for differences in sensitivity of two pea cultivars to sulfur dioxide. Plant Physiology 91, 88-93. Maruyama, A., Ishizawa, K., Takagi, T. and Esashi, Y. (1998). Cytosoliccyanoalanine synthase activity attributed to cysteine synthase in cocklebur seeds. Purification and characterization of cytosolic cysteine synthases. Plant and Cell Physiology 39, 671-680. Marzluf, G. A. (1997). Molecular genetics of sulfur assimilation in filamentous fungi and yeast. Annual Review of Microbiology 51, 73-96. May, M. J. and Leaver, C. J. (1993). Oxidative stimulation of glutathione synthesis in Arabidopsis thaliana suspension cultures. Plant Physiology 103, 621-627. May, M. J., Hammond-Kosack, K. and Jones, J. D. G. (1996). Involvement of reactive oxygen species, glutathione metabolism and lipid peroxidation in the Cf-gene-dependent defense response of tomato cotyledons induced by racespecific elicitors of Cladosporium fluvum. Plant Physiology 10, 1367-1379. May, M. J., Vemoux, T., Leaver, C., van Montagu, M. and Inze, D. (1998). Glutathione homeostasis in plants: implications for environmental sensing and plant development. Journal of Experimental Botany 49, 649-667. McGrath, S. P. and Zhao, F. J. (1996). Sulfur uptake, yield responses and the interactions between nitrogen and sulfur in winter oilseed rape (Brassica napus ). Journal ofAgricultural Science 126, 53-62. McGrath, S. P., Zhao, F. J. and Withers, P. J. A. (1996). Development of sulphur deficiency in crops and its treatment. Proceedings of the Fertiliser Society, No. 379. The Fertiliser Society, Peterborough. Meyer, K., Leube, M.P. and Grill, E. (1994). A protein phosphatase 2C involved in ABA signal transduction inArabidopsis thaliana. Science 264, 1452-1455. Molvig, L., Tabe, L. M., Eggum, B. 0., Moore, A. E., Craig, S., Spencer, D. and Higgins, T. J. V. (1997). Enhanced methionine levels and increased nutritive value of seeds of transgenic lupins (Lupinus angustifolius L) expressing a sunflower seed albumin gene. Proceedings of the National Academy of Sciences of the USA 94, 8393-8398. Mourioux, D. and Douce, R. (1979). Transport du sulfate a travers la double membrane limitante, ou enveloppe, des chloroplastes d'epinard. Biochimie 61, 1283-1292.

MOLECULAR GENETICS OF SULPHATE ASSIMILATION

217

Muller, M. and Knudsen, S. (1993). The nitrogen response of a barley C-hordein promoter is controlled by positive and negative regulation of the GCN4 and endosperm box. Plant Journal 4, 343-355. Murguia, J. R., Belles, J. M. and Serrano, R. (1995). A salt-sensitive 3'(2'),5'bisphosphate nucleotidase involved in sulfate activation. Science 267,232-234. Murguia, J. R., Belles, J. M. and Serrano, R. (1996). The yeastHAL2 nucleotidase is an in vivo target of salt toxicity. Journal of Biological Chemistry 271, 29029-29033. Murillo, M. and Leustek, T. (1995). Adenosine-5'-triphosphate sulfurylase from Arabidopsis thaliana and Escherichia coli are functionally equivalent but structurally and kinetically divergent. Nucleotide sequence of two adenosine5'-triphosphate sulfurylase cDNAs fromArabidopsis thaliana and analysis of a recombinant enzyme. Archives of Biochemistry and Biophysics 323, 195-204. Murillo, M., Foglia, R., Diller, A., Lee, S. and Leustek, T. {1995). Serine acetyl transferase from Arabidopsis thaliana can functionally complement the cysteine requirement of a cysE mutant strain of Escherichia coli. Cellular and Molecular Biology Research 41, 425-433. Nakamura, K. and Tamura, G. (1990). Isolation of serine acetyltransferase complexed with cysteine synthase from Allium tuberosum. Agricultural and Biological Chemistry 54, 649-656. Nakamura, T., Koizumi, N. and Sano, H. (1997). Isolation of a novel cysteine synthase eDNA (AB003041) fromArabidopsis thaliana. Plant Physiology 114, 747. Nakamura, T., Yamaguchi, Y. and Sano, H. (1999). Four rice genes encoding cysteine synthase: isolation and differential responses to sulfur, nitrogen and light. Gene 229, 155-61. Neuenschwander, U., Suter, M. and Brunold, C. (1991). Regulation of sulfate assimilation by light and 0-acetyl-L-serine in Lemna minor L. Plant Physiology 97, 253-258. Ng, A. Y.-N., Blomstedt, C. K., Gianello, R., Hamill, J.D., Neale, A. D. and Gaff, D. F. (1996). Isolation and characterisation of a lowly expressed eDNA from the resurrection grass Sporobolus stapfianus with homology to eukaryotic sulfate transporter proteins (Accession No. X96761 ). (PGR96-032). Plant Physiology 111, 651. Nicholas, K. B., Nicholas, H. B. Jr, and Deerfield, D. W. II (1997). GeneDoc: analysis and visualization of genetic variation. EMBNEW News 4, 14. Nieto-Sotelo, J. and Ho, T.-H. D. (1986). Effect of heat shock on the metabolism of glutathione in maize roots. Plant Physiology 82, 1031-1035. Noctor, G., Strohm, M., Jouanin, L., Kunert, K.-J., Foyer, C. H. and Rennenberg, H. (1996). Synthesis of glutathione in leaves of transgenic poplar overexpressing 1-glutamylcysteine synthetase. Plant Physiology 112, 1071-1078. Noji, M., Murakoshi, I. and Saito, K. (1994). Molecular cloning of a cysteine synthase eDNA from Citrullus vulgaris (watermelon) by genetic complementation in an Escherichia coli Cys- auxotroph. Molecular and General Genetics 244, 57-66. Noji, M., Inoue, K., Kimura, N., Gouda, A. and Saito, K. (1998). Isoform-dependent differences in feedback regulation and subcellular localization of serine acetyltransferase involved in cysteine biosyntheis from Arabidopsis thaliana. Journal of Biological Chemistry 273, 32739-32745. Nussbaum, S., Schmutz, D. and Brunold, C. {1988). Regulation of assimilatory sulfate reduction by cadmium in Zea mays L. Plant Physiology 88, 1407-1410. Ono, A., Izawa, T., Chua, N.-H. and Shimamoto, K. (1996). The rab16B promoter of rice contains two distinct abscisic acid-responsive elements. Plant Physiology 112, 483-491.

218

M. J. HAWKESFORD and J. L. WRAY

Page, R. D. M. (1996). Tree View: An application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences 12, 357-358. Paietta, J. V. (1990). Molecular cloning and analysis of the scon-2 negative regulatory gene of Neurospora crassa. Molecular and Cellular Biology 10, 5207-5214. Peng, Z. and Verma, D.P. S. (1995). A rice HAL2-like gene encodes a Ca2+ -sensitive 3'(2'),5'-diphosphonucleoside 3'(2') phosphohydrolase and complements yeast met22 and Escherichia coli cysQ mutations. Journal of Biological Chemistry 270, 29105-29110. Pickardt, T., Saalbach, 1., Waddell, D., Meixner, M., Miintz, K. and Schieder, 0. (1995). Seed-specific expression of the 2S albumin gene from brazil nut (Bertholletia excelsa) in transgenic Vicia narbonensis. Molecular Breeding 1, 295-301. Pilon-Smits, E. A H., Hwang, S. B., Lytle, C. M., Zhu, Y. L., Tai, J. C., Bravo, R. C., Chen, Y. C., Leustek, T. and Terry, N. (1999). Overexpression of ATP sulfurylase in Indian mustard leads to increased selenate uptake, reduction and tolerance. Plant Physiology 119, 123-132. Prior, A., Uhrig, J. F., Heins, L., Wiesmann, A., Lillig, C. H., Stoltze, C., Soli, D. and Schwenn, J. D. (1999). Structural and kinetic properties of adenylyl sulfate reductase from Catharanthus roseus cell cultures. Biochimica et Biophysica Acta 1430, 25-38. Quintero, F. J., Garciadeblas, B. and Rodriguez-Navarro, A (1996). The SALJ gene ofArabidopsis, encoding an enzyme with 3'(2'),5'-bisphosphate nucleotidase and inositol polyphosphate !-phosphatase activities, increases salt tolerance in yeast. Plant CellS, 529-537. Ravanel, S., Gakiere, B., Job, D. and Douce, R. (1998). The specific features of methionine biosynthesis and metabolism in plants. Proceedings of the National Academy of Sciences of the USA 95, 7805-7812. Rennenberg, H., Schmitz, K. and Bergmann, L. (1979). Long-distance transport of sulfur in Nicotiana tabacum. Planta 147, 57-62. Rennenberg, H., Kemper, 0. and Thoene, B. (1989). Recovery of sulfate transport into heterotrophic tobacco cells from inhibition by reduced glutathione. Physiologia Plantarum 76, 271-276. Renosto, F., Patel, H. C., Martin, R. L., Thomassian, C., Zimmerman, G. and Segel, I. H. (1993). ATP sulfurylase from higher plants. Kinetic and structural characterization of the chloroplast and cytosol enzymes from spinach leaf. Archives of Biochemistry and Biophysics 307, 272-285. Reuveny, Z., Dougall, D. K. and Trinity, P.M. (1980). Regulatory coupling of nitrate and sulfate assimilation pathways in cultured tobacco cells. Proceedings of the National Academy of Sciences of the USA 77, 6670-6672. Roberts, M. A and Wray, J. L. (1996). Cloning and characterisation of an Arabidopsis thaliana eDNA clone encoding an organellar isoform of serine acetyltransferase. Plant Molecular Biology 30, 1041-1049. Robinson, N. J., Tommey, A M., Kuske, C. R. and Jackson, P. J. (1993). Plant metallothioneins. Biochemical Journal 295, 1-10. Rock, C. D. and Zeevaart, J. A (1991). The aba mutant ofArabidopsis thaliana is impaired in epoxy-carotenoid biosynthesis. Proceedings of the National Academy of Sciences of the USA 88, 7496-7499. Rolland, N., Droux, M. and Douce, R. (1992). Subcellular distribution of 0acetylserine (thiol) lyase in cauliflower (Brassica oleracea L.) inflorescence. Plant Physiology 98, 927-935.

MOLECULAR GENETICS OF SULPHATE ASSIMILATION

219

Rolland, N., Droux, M., Lebrun, M. and Douce, R. (1993). 0-Acetylserine (thiol) lyase from spinach (Spinacia oleracea L.) leaf: eDNA cloning, characterization and expression in Escherichia coli of the chloroplast isoform. Archives of Biochemistry and Biophysics 300, 213-222. Rolland, N., Ruffet, M.-L., Job, D., Douce, R. and Droux, M. (1996). Spinach chloroplast 0-acetylserine (thiol) lyase exhibits two catalytically nonequivalent pyridoxal-5' -phosphate-containing active sites. European Journal of Biochemistry 236, 272-282. Romer, S., d'Harlingue, A, Camara, B., Schantz, R. and Kuntz, M. (1992). Cysteine synthase from Capsicum annuum chromoplasts. Characterization and eDNA cloning of an up-regulated enzyme during fruit development. Journal of Biological Chemistry 267, 17966-17970. Ruegsegger, A, Schmutz, D. and Brunold, C. (1990). Regulation of glutathione synthesis by cadmium in Pisum sativum. L. Plant Physiology 93, 1579-1584. Ruffet, M.-L., Droux, M. and Douce, R. (1994). Purification and kinetic properties of serine acetyltransferase free of 0-acetylserine (thiol) lyase from spinach chloroplasts. Plant Physiology 104, 597-604. Ruffet, M.-L., Lebrun, M., Droux, M. and Douce, R. (1995). Subcellular distribution of serine acetyltransferase from Pisum sativum and characterization of an Arabidopsis thaliana putative cytosolic isoform. European Journal of Biochemistry 227, 500-509. Saalbach, 1., Pickardt, T., Machemehl, F., Saalbach, G., Schieder, 0. and Muntz, K. (1994). A chimeric gene encoding the methionine-rich 2S albumin ofthe brazil nut (Bertholletia excelsa H.B.K.) is stably expressed and inherited in transgenic grain legumes. Molecular and General Genetics 242, 226-236. Saier, M. H., Eng, B. H., Fard, S., Garg, J., Haggerty, D. A, Hutchinson, W. J., Jack, D. L., Lai, E. C., Liu, H. J., Nusinew, D.P., Omar, AM., Pao, S. S., Paulsen, I. T., Quan, J. A, Sliwinski, M., Tseng, T. T., Wachi, S. and Young, G. B. (1999). Phylogenetic characterization of novel transport protein families revealed by genome analyses. Biochimica et Biophysica Acta Reviews on Biomembranes 1422, 1-56. Saito, K., Miura, N., Yamazaki, M., Hirano, H. and Murakoshi, I. (1992). Molecular cloning and bacterial expression of eDNA encoding a plant cysteine synthase. Proceedings of the National Academy of Sciences of the USA 89, 8078-8082. Saito, K., Tatsuguchi, K., Murakoshi, I. and Hirano, H. (1993). eDNA cloning and expression of cysteine synthase B localized in chloroplasts of Spinacia oleracea. FEBS Letters 324, 247-252. Saito, K., Kurasawa, M., Tatsuguchi, K., Takagi, Y. and Murakoshi, I. (1994a). Modulation of cysteine biosynthesis in chloroplasts of transgenic tobacco overexpressing cysteine synthase (0-acetylserine(thiol)-lyase ). Plant Physiology 106, 887-895. Saito, K., Tatsuguchi, K., Takagi, Y. and Murakoshi, I. (1994b). Isolation and characterization of eDNA that encodes a putative mitochondrion-localizing isoform of cysteine synthase (0-acetylserine(thiol)-lyase) from Spinacia oleracea. Journal of Biological Chemistry 269, 28187-28192. Saito K., Yokoyama, H., Noji, M. and Murokoshi, I. (1995). Molecular cloning and characterization of a plant serine acetyltransferase playing a regulatory role in cysteine biosynthesis from watermelon. Journal of Biological Chemistry 270, 16321-16326. Saito, K., Inoue, K., Fukushima, R. and Noji, M. (1997). Genomic structure and expression analyses of serine acetyltransferase gene in Citrullus vulgaris (watermelon). Gene 189, 57-63.

220

M. J. HAWKESFORD and J. L. WRAY

Sandal, N. N. and Marcker, K. A (1994). Similarities between a soybean nodulin, Neurospora crassa sulphate permease II and a putative human tumour suppressor. Trends in Biochemical Sciences 19, 19. Schafer, H. J., Haag-Kerwer, A and Rausch, T. (1998). eDNA cloning and expression analysis of genes encoding GSH synthesis in roots of the heavymetal accumulator Brassica juncea L.: evidence for Cd-induction of a putative mitochondrial1-glutamylcysteine synthetase isoform. Plant Molecular Biology 37,87-97. Schiffmann, S. and Schwenn, J. D. (1998). Isolation of eDNA clones encoding adenosine-5'-phosphosulfate kinase (EC 2.7.1.25) from Catharanthus roseus (Accession No. AF044285) and an isoform (akn2) from Arabidopsis (Accession No. AF043351). Plant Physiology 117, 1125. Schmidt, A (1968). "Untersuchungen zum Mechanismus der Photosynthetischen Sulfat-reduktion Isolierter Chloroplasten". Thesis, University of Gottingen. Schmidt, A (1973). Sulfate reduction in a cell-free system of Chlorella. The ferredoxin-dependent reduction of a protein-bound intermediate by thiosulfonate reductase. Archive fUr Mikrobiology 93, 29-52. Schmidt, A., and Jager, K. (1992). Open questions about sulfur metabolism in plants. Annual Review of Plant Physiology and Plant Molecular Biology 43, 325-349. Schuster, S., Dandekar, T. and Fell, D. A (1999). Detection of elementary flux modes in biochemical pathways: a promising tool for pathway analysis and metabolic engineering. Trends in Biotechnology 17, 53-60. Schwenn, J. D. (1986). Sulfate assimilation in higher plants- a thioredoxindependent PAPS reductase from spinach leaves. Zeitschrift fUr Naturforschung c 44, 504-508. Schwenn, J. D. (1994). Photosynthetic sulfate reduction. Zeitschrift fUr Naturforschung C 49, 531-539. Schwenn, J. D. and Schrick, U. (1987). PAPS reductase from Escherichia coli: Characterization of the enzyme as a probe for thioredoxins. Zeitschrift fUr Naturforschung C 42, 93-102. Sen Gupta, A., Alscher, R. G. and McCune, D. (1991 ). Response of photosynthesis and cellular antioxidants to ozone in Populus leaves. Plant Physiology 96, 650-655. Setya, A., Murillo, M., and Leustek, T. (1996). Sulfate reduction in higher plants - molecular evidence for a novel 5'-adenylylsulfate reductase. Proceedings of the National Academy of Sciences of the USA 93, 13383-13388. Shen, Q. X. and Ho, T. H. D. (1995). Functional dissection of an abscisic acid (ABA)-inducible gene reveals two independent ABA-responsive complexes each containing a G-box and a novel cis-acting element. Plant Ce//7, 285-307. Shen, Q. X., Zhang, P. H. and Ho, T. H. D. (1996). Molecular nature of ABA response complexes: composite promoter units that are necessary and sufficient for ABA induction of gene expression in barley. Plant Cell 8, 1107-1119. Shewry, P. R. and Tatham, A S. (1997). Disulphide bonds in wheat gluten proteins. Journal of Cereal Science 25, 207-227. Siegel, L. M. (1975). Biochemistry of the sulfur cycle. In "Metabolism of Sulfur Compounds" (D. M. Greenberg, ed.), pp. 217-286. Academic Press, New York. Smith, F. W., Baling, P.M., Hawkesford, M. J. and Clarkson, D. T. (1995a). Plant members of a family of sulfate transporters reveal functional subtypes. Proceedings of the National Academy of Sciences of the USA 92, 9373-9377.

MOLECULAR GENETICS OF SULPHATE ASSIMILATION

221

Smith, F. W., Hawkesford, M. J., Prosser, I. M. and Clarkson, D. T. (1995b). Isolation of a eDNA from Saccharomyces cerevisiae that encodes a high affinity sulphate transporter at the plasma membrane. Molecular and General Genetics 247,709-715. Smith, F. W., Hawkesford, M. J., Baling, P.M., Clarkson, D. T., Vandenberg, P. J., Belcher, A. and Warrilow, A. G. S. (1997). Regulation of expression of a eDNA from barley roots encoding a high affinity sulphate transporter. Plant Joumal12, 875-884. Smith, I. K. and Thompson, J. F. (1969). The synthesis of 0-acetylserine by extracts prepared from higher plants. Biochemical and Biophysical Research Communications 35, 939-945. Smith, I. K., Kendall, A. C., Keys, A. J., Turner, J. C. and Lea, P. J. (1985). The regulation of the biosynthesis of glutathione in leaves of barley (Hordeum vulgare L.). Plant Science 41, 11-17. Strohm, M., Jouanin, L., Kunert, K.-J., Provost, C., Polle, A., Foyer, C. H. and Rennenberg, H. (1995). Regulation of glutathione synthesis in leaves of transformed poplar (Populus tremula x P. alba) overexpressing glutathione synthetase. Plant Joumal7, 141-145. Sunarpi and Anderson, J. W. (1995). Mobilization of sulfur in soybean cotyledons during germination. Physiologia Plantarum 94, 143-150. Sunarpi and Anderson, J. W. (1996a). Distribution and redistribution of sulfur supplied as [S-35)sulfate to roots during vegetative growth of soybean. Plant Physiology 110, 1151-1157. Sunarpi and Anderson, J. W. (1996b ). Effect of sulfur nutrition on the redistribution of sulfur in vegetative soybean plants. Plant Physiology 112, 623-631. Sunarpi and Anderson, J. W. (1997a). Allocation of S in generative growth of soybean. Plant Physiology 114, 687-693. Sunarpi and Anderson, J. W. (1997b). Inhibition of sulphur redistribution into new leaves of vegetative soybean by excision of the maturing leaf. Physiologia Plantarum 99, 538-545. Syers, J. K., Skinner, R. J. and Curtin, D. (1987). Soil and fertiliser sulphur in U.K. agriculture. Proceedings ofthe Fertiliser Society, No. 264. The Fertiliser Society, London. Takahashi, H. and Saito, K. (1996). Subcellular localization of spinach cysteine synthase isoforms and regulation of their gene expression by nitrogen and sulfur. Plant Physiology 112, 273-280. Takahashi, H., Sasakura, N., Noji, M. and Saito, K. (1996). Isolation and characterization of a eDNA encoding a sulfate transporter from Arabidopsis thaliana. FEBS Letters 392, 95-99. Takahashi, H., Yamazaki, M., Sasakura, N., Watanabe, A., Leustek, T., Engler, J.D., van Montagu, M., and Saito, K. (1997). Regulation of sulfur assimilation in higher plants: a sulfate transporter induced in sulfate-starved roots plays a central role in Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the USA 94, 11102-11107. Takahashi H., Asanuma, W. and Saito, K. (1999a). Cloning of anArabidopsis eDNA encoding a chloroplast localizing sulphate transporter isoform. Journal of Experimental Botany 50, 1713-1714. Takahashi H., Sasakura, N., Kimura, A., Watanabe, A. and Saito, K. (1999b). Identification of two leaf-specific sulfate transporters in Arabidopsis thaliana (Accession No. AB012048 and AB004060) (PGR99-154). Plant Physiology 121,686.

222

M. J. HAWKESFORD and J. L. WRAY

Thomas, D. and Surdin-Kerjan, Y. (1997). Metabolism of sulfur amino acids in Saccharomyces cerevisiae. Microbiology and Molecular Biology Reviews 61, 503-532. Thomas, D., Rothstein, R., Rosenberg, N. and Serdin-Kerjan, Y. (1988). SAM2 encodes the second methionine S-adenosyl transferase in Saccharomyces cerevisiae: physiology and regulation of both enzymes. Molecular and Cellular Biology 8, 5132-5139. Thompson, J.D., Gibson, T. J., Plewniak, F., Jeanmougin, F. and Higgins, D. G. (1997). The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25, 4876-4882. Tsang, M. L. and Schiff, J. A. (1976). Properties of enzyme fraction A from Chiarella and copurification of 3'(2'),5'-bisphosphonucleoside 3'(2')-phosphohydrolase, adenosine 5'-phosphosulfate sulfohydrolase and adenosine-5'-phosphosulfate cyclase activities. European Journal of Biochemistry 65, 113-121. Uchimiya, H., Kidou, S., Shimazaki, T., Aotsuka, S., Takamatsu, S., Nishi, R., Hashimoto, H., Matsubayashi, Y., Kidou, N., Umeda, M. and Kato, A. (1992). Random sequencing of eDNA libraries reveals a variety of expressed genes in cultured cells of rice (Oryza sativa L. ). Plant Journal 2, 1005-1009. Varin, L. and Ibrahim, R. K. (1992). Novel flavonol3-sulfotransferase. Purification, kinetic properties and amino acid sequence. Journal of Biological Chemistry 267, 1858-1863. Varin, L., Deluca, V., Ibrahim, R. K. and Brisson, N. (1992). Molecular characterization of two plant flavonol sulfotransferases. Proceedings of the National Academy of Sciences of the USA 89, 1286-1290. Vasil, V., Marcotte, W. R. Jr, Rosenkrans, L., Cocciolone, S. M., Vasil, I. K., Quatrano, R. S. and McCarty, D. R. (1995). Overlap of Viviparous (VP1) and abscisic acid response elements in the Em promoter: G-box elements are sufficient but not necessary for VP1 transactivation. Plant Cell7, 1511-1518. Vidmar, J. J., Schjoerring, J. K., Touraine, B. and Glass, A. D. M. (1999). Regulation of the hvstl gene encoding a high-affinity sulfate transporter from Hordeum vulgare. Plant Molecular Biology 40, 883-892. Warrilow, A. G. S. and Hawkesford, M. J. (1998). Separation, subcellular location and influence of sulphur nutrition on isoforms of cysteine synthase in spinach. Journal of Experimental Botany 49, 1625-1636. Wray, J. L., Campbell, E. 1., Roberts, M. A. and Gutierrez-Marcos, J. F. (1998). Redefining reductive sulfate assimilation in higher plants: a role for APS reductase, a new member of the thioredoxin superfamily? Chemica-Biological Interactions 109, 153-167. Wrigley, C. W., Du Cros, D. L., Fullington, J. G. and Kasarda, D. D. (1984). Changes in polypeptide composition and grain quality due to sulfur deficiency in wheat. Journal of Cereal Science 2, 15-24. Yamaguchi, Y., Nakamura, T., Harada, E., Koizumi, N. and Sano, H. (1997). Isolation and characterization of a eDNA encoding a sulfate transporter from Arabidopsis thaliana. Plant Physiology 113, 1463. Yamaguchi, Y., Nakamura, T., Harada, E., Koizumi, N. and Sano, H. (1999). Differential accumulation of transcripts encoding sulfur assimilation enzymes upon sulfur and/or nitrogen deprivation in Arabidopsis thaliana. Bioscience, Biotechnology and Biochemistry 63, 762-766. Yildiz, F. H., Davies, J.P. and Grossman, A. (1996). Sulfur availability and the SACJ gene control adenosine triphosphate sulfurylase gene expression in Chlamydomonas reinhardtii. Plant Physiology 112, 669-675.

MOLECULAR GENETICS OF SULPHATE ASSIMILATION

223

Yonekura-Sakakibara, K., Ashikari, T., Tanaka, Y., Kusumi, T. and Hase, T. (1998). Molecular characterization of tobacco sulfite reductase: enzyme purification, gene cloning and gene expression analysis. Journal of Biochemistry 124, 615-621. Youssefian, S., Nakamura, M. and Sano, H. (1993). Tobacco plants transformed with the 0-acetylserine(thiol)lyase gene of wheat are resistant to toxic levels of hydrogen sulphide gas. Plant Journal 4, 759-769. Zenk, M. H. (1996). Heavy metal detoxification in higher plants- a review. Gene 179,21-30. Zhao, F. J., Evans, E. J., Bilsborrow, P. E. and Syers, J. K. (1993). Sulfur uptake and distribution in double and single low varieties of oilseed rape (Brassica napus L ). Plant and Soi/150, 69-76. Zhao, F. J., McGrath, S. P., Crosland, A. R. and Salmon, S. E. (1995). Changes in the sulfur status of British wheat-grain in the last decade and its geographical distribution. Journal of the Science of Food and Agriculture 68, 507-514. Zhao, F. J., Hawkesford, M. J. and McGrath, S. P. (1999a). Sulphur assimilation and effects on yield and quality of wheat. Journal of Cereal Science 30, 1-17. Zhao, F. J., Salmon, S. E., Withers, P. J. A., Monaghan, J. M., Evans, E. J., Shewry, P. R. and McGrath, S. P. (1999b). Variation in the breadmaking quality and rheological properties of wheat in relation to sulphur nutrition under field conditions. Journal of Cereal Science. 30, 19-31.

Pathogenicity, Host-specificity, and Population Biology of Tapesia spp., Causal Agents of Eyes pot Disease of Cereals

JOHN A. LUCAS, 1 PAULS. DYER2 and TIMOTHY D. MURRAY3 1

JACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol BS41 9AF, UK 2 Microbiology Division, School of Biological Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK 3 Department of Plant Pathology, Washington State University, Pullman, WA 99164-6430, USA

I. II.

Introduction ................ .... ............. .......................... .. ........... ....... ..................... The Pathogens ........................ ................................ .... ......... ....... ............. ....... A. Isolate Variation..................................................................................... B. Molecular Analysis of Eyespot Populations........................................ III. The Sexual Stage ................................................ ................... .... ................ .... . A. Nature of the Breeding System............................................................. B. Population Biology and Taxonomy of Tapesia Species...................... C. Significance of the Sexual Stage in the Field....................................... D. Current Research Utilizing the Sexual System of Tapesia Species...................................................................................................... E. Geographic Variation in Populations.................................................. IV. Pathogenicity................................................................................................... A. Spore Dispersal and Adhesion.............................................................. B. The Infection Process............................................................................. C. Infection Plaques ..... ......... ........................ .... .. .... ......... .... .. .. .... ..... .. .... .. .. D. Tissue Colonization................................................................................ E. Pathogenicity Factors............................................................................. V. Host Range of Tapesia Species..................................................................... VI. Host Resistance to Tapesia Species .. .. .. .. .................... ...... .................... .... ... VII. Future Prospects............................................................................................. Acknowledgements........................................................................................ References....................................................................................................... Advances in Botanical Research Vol 33 incorporating Advances in Plant Pathology ISBN 11-12-005933-9

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Eyespot is an important stem-base disease of cereal crops in temperate regions. Cultural, genetic and molecular criteria have been used to separate the fungi responsible into two species, Tapesia yallundae (previously W-type) and T. acuformis (previously R-type). Discovery of the apothecial sexual stage (teleomorph) ofTapesia yallundae on straw stubble was a key factor in this change for a pathogen previously believed to be asexual (anamorph: Pseudocercosporella herpotrichoides). Sexual reproduction is controlled by a two-allele heterothallic system in both species, although mating appears to be rare in T. acuformis. Infection of cereal hosts is achieved by formation of multicellular plaques, and the colonization process is described. The host range includes wild grass species as well as small-grain cereals, and new genetic sources of resistance to the disease have been identified in Triticum species and wild relatives. Recent advances in the molecular genetics of the pathogens will aid analysis of pathogenic variation in eyespot.

I.

INTRODUCTION

Eyespot, caused by the fungus Pseudocercosporella herpotrichoides (teleomorph Tapesia yallundae and Tapesia acufonnis ), is one of the complex of diseases which infect the stem base of temperate cereals, causing premature ripening of grain, and predisposing the crop to lodging. The disease occurs in most of the major wheat-growing areas of the world, but is especially prevalent in cool, wet regions such as north-west Europe, the north-west USA and New Zealand. Eyespot has been reported from Africa (Morocco, South Africa, particularly the Western Cape, and Tunisia), Australia (New South Wales, South Australia, Victoria, and Tasmania), Europe (Austria, Belgium, Bulgaria, Denmark, Finland, France, Germany, Great Britain, Greece, Hungary, Italy, Netherlands, Norway, Poland, Romania, Sweden, Switzerland, USSR, and Yugoslavia), Japan and North America (Ontario and British Columbia, Canada; Idaho, New York, Oregon, and Washington, USA) (Anonymous, 1981; Furuya and Matsumoto, 1996). In the USA, eyespot is often described by the alternative name of strawbreaker footrot. There are no reports from South America, despite apparently favourable conditions for the disease in Argentina and the southern states of Brazil. The economic importance of cereal eyespot has been difficult to determine due to the problem of accurate diagnosis of the disease in the presence of other stem-base pathogens, and the absence, until recently, of fully effective chemical treatments for the disease. There is no doubt, however, that it is potentially one of the most damaging diseases of winter cereals, especially wheat (Polley and Thomas, 1991 ). Over the past 15 years there has been a gradual but sustained increase in research on eyespot disease, prompted initially by the development of resistance to the previously effective methyl benzimidazole (MBC) fungicides (Cavelier and Leroux, 1983; Brown et al., 1984, King and Griffin,

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1985), and subsequently by advances in understanding of the genetics and molecular genetics of the pathogens. Important new insights have been gained concerning the taxonomy of the pathogen( s) involved, the pathogen life cycle, infection processes, population biology, and control of the disease. This has radically altered perceptions of the disease, and also established eyespot as a model pathosystem for addressing questions on the population biology and genetics of a mainly trashborne fungus which survives for part of the infection cycle as a saprophyte on crop residues. Previous reviews (Fitt et al., 1988, 1990; Lucas, 1995) have been mainly concerned with the epidemiology and management of cereal eyespot disease. The current review focuses on the biology and genetics of the pathogen( s), with the main emphasis on pathogenicity, host-specificity, and population structure. Progress in identifying sources of genetic resistance to eyespot is included. The relevance of advances in understanding of these topics to the diagnosis and control of the disease is also considered.

II. THE PATHOGENS A.

ISOLATE VARIATION

A fungus associated with eyespot disease symptoms on wheat was first described as Cercosporella herpotrichoides Fron (Sprague, 1936). This was later renamed Pseudocercosporella herpotrichoides (Fron) Deighton (Deighton, 1973). However, studies of collections of field isolates suggested that more than one type of fungus was associated with eyespot lesions, and various schemes were proposed to accommodate this biological diversity. A variety of criteria were used, leading to several classification systems which were approximately, but not exactly, equivalent (Table I). Until recently, this was a potential source of confusion. The most widely adopted scheme separated isolates into two main groups, the W-type and R-type, on the basis of their differential pathogenicity to wheat and certain cultivars of rye TABLE I Classification schemes used for the two main types of P. herpotrichoides observed in samples from the field Terminology/nomenclature used

Main criteria used

W-type N FIE

R-type L S/F

P. herpotrichoides var. herpotrichoides

P. herpotrichoides var. acuformis

Pathogenicity to rye Growth rate Growth rate and colony morphology Spore morphology

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in infection tests; R-type isolates cause significant lesions on both wheat and rye, whereas W-type isolates infect wheat but have only a limited capacity to infect rye (Lange-de la Camp, 1966). These pathotypes also differ in growth rate and colony morphology in culture, with R-types forming slow-growing, feathery-edged colonies, and W-types forming faster-growing, even-edged colonies (Hollins et al., 1985). In France, Cavelier et al. (1987) used growth rates to classify isolates as either slow (L) or fast (N) growing types, whereas Nicholson et al. (1991b) used the terms slow/feathery (SF) and fast/even (FIE) to describe these contrasts in cultural behaviour. A more formal taxonomic system was proposed by Nirenberg (1981) who subdivided the fungus into two varieties, P. herpotrichoides var. herpotrichoides and P. herpotrichoides var. acuformis on the basis of spore and colony morphology. None of these schemes proved entirely satisfactory, for both practical and biological reasons. Conventional pathogenicity tests, based on inoculation of wheat and rye seedlings, are time-consuming, taking 10-12 weeks to complete, and can give variable results (Scott and Hollins, 1980; Creighton, 1989). Furthermore, additional pathotypes, the C- and S-types, have been described, based on differences in pathogenicity to the wild grass hosts couch, Elymus repens, and Aegilops squarrosa (Scott and Hollins, 1980; Cunningham, 1981). Growth rates and colony morphologies are easier to assess, but do not always correlate with pathogenicity (see below), most likely due to cultural instability in a proportion ofR-type, S!F isolates (Julian et al., 1994a). Spore morphologies have proved difficult to interpret, due to variation dependent on cultural conditions. Alternative biochemical and molecular markers were therefore sought to clarify the extent of variation in field populations of the eyespot pathogen. B.

MOLECULAR ANALYSIS OF EYES POT POPULATIONS

Julian and Lucas (1990) analysed 101 isolates from the UK, France, Germany, Denmark, New Zealand, South Africa and the USA, using isozyme markers by comparison with cultural characters. A proportion of isolates were also tested for pathogenicity to wheat, rye and barley hosts. Seven out of 16 enzymes detected were differential, whereas polymorphisms in esterase, glutamate dehydrogenase, malate dehydrogenase, glucose phosphate isomerase, and malic enzyme clearly differentiated between the W- and R-pathotypes. Isozyme patterns also discriminated two related species, P. anguioides and P. aestiva. Most isolates fell into two groups which largely correlated with the FE and SF morphological types. However, there were some exceptions in the form of fast-growing isolates which gave R-type isozyme patterns. Morphologically variant sectors from R-type parent cultures retained the original pattern. There was no evidence within

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pathotypes for any variation due to differences in geographical origin. Priestley et al. (1992) compared esterase patterns obtained from a collection of isolates with some DNA markers (see below). Isozymes again separated P. herpotrichoides from P. anguioides and P. aestiva, and discriminated between the W- and R-pathotypes, as well as the C-type. Isolates classified by Nirenberg (1981) asP. herpotrichoides var. herpotrichoides and var. acufomis on the basis of morphological criteria were shown to be similar to the W- and R-pathotypes. a-Esterase isozyme patterns were used by Campbell et al. (1996) in an analysis of 89 isolates from wheat, barley and grass hosts in South Africa. All the isolates clustered together with electromorphs similar to FE (W-type) isolates from other countries, and distinctly different from SF (R-type) isolates. This study, and subsequent surveys (P. W. Crous, personal communication) suggest that only one type of the fungus is associated with eyespot disease in South Africa. DNA-based markers have proved to be of particular value in resolving questions of pathotype identity and diversity in Pseudocercosporella on graminaceous hosts. Nicholson et al. (1991b) used restriction fragment length polymorphisms (RFLPs) of total DNA and rONA to separate isolates into two main groups, which in most cases coincided with the FE and SF classification; once again a few morphological variants were encountered. This analysis was subsequently extended to RFLPs of mitochondrial (mtDNA) and rONA of 64 isolates of diverse geographical origin (Nicholson et al., 1993). R-pathotype isolates gave one mtDNA profile, whereas W -pathotypes had a second profile, which was also found inC and S-type isolates. Considerable polymorphism in rONA profiles was found among the isolates examined, especially within theW-types. On the basis of the mtDNA results the authors concluded that the two main forms of P. herpotrichoides might be genetically separated. Thomas et al. (1992) constructed a partial genomic library from a W -type isolate and derived probes which were hybridized to DNA from a collection of isolates. Most of the probes appeared to be single copy sequences and distinguished between the two pathotypes. The W-types again gave a higher degree of polymorphism than the R-types. The authors concluded that the sensitivity and specificity of DNA markers might enable detection of specific types of the pathogen in the field, without the need for isolation into culture. A parallel approach was adopted by Frei and Wenzel ( 1993) who used probes based on repetitive genomic clones and cluster analysis to differentiate isolates. Takeuchi and Kuninaga (1994) used a different method to estimate genetic relatedness based on the reassociation kinetics of nuclear DNA. Their study included FE and SF isolates from Japan, North America, Europe, South Africa and New Zealand, as well as P. anguioides and P. aestiva. Results indicated high relatedness (~ 82%) within the two morphological types, but much lower values (24-34%) between them. Both

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groups showed little DNA relatedness to either P. aestiva or P. anguioides. The authors concluded that P. herpotrichoides may consist of two separate genomic species. In a subsequent study (Takeuchi and Kuninaga, 1996), RFLPs of mitochondrial DNA from the different pathotypes and species were analysed to estimate relatedness. The nucleotide sequence divergence of mtDNA between FE and SF isolates was shown to be greater than that between fungal isolates from the same taxa. The authors concluded that there are no close relationships among the different types and species of Pseudocercosporella from graminaceous hosts, and that the FE and SF types are genetically distinct taxonomic species. The reliability of random amplified polymorphic DNAs (RAPDs) as molecular markers has been questioned (Jones et al., 1997), but in the case of eyespot, given careful choice of primers and well-defined amplification conditions, they have proved to be a convenient and robust method for detecting variation and discriminating between the eyespot pathogens. Furthermore, pathotype-specific amplification products from RAPD analysis have provided the basis for the development of molecular diagnostic techniques for eyespot (Nicholson et al., 1994), and, more recently, quantitation of the pathogens using competitive PCR (Nicholson et al., 1997). In an initial study, Nicholson and Rezanoor (1994) used ten random 10mer oligonucleotide primers to generate RAPD profiles from a collection of 23 pathogen isolates representative of typical W-and R-types, as well as the less well characterized C-and S-types. Cluster analysis of haplotypes based on all amplification products, or only the major bands, distinguished two main groups, one of which contained only R-types. The C-types separated as a distinct subgroup within the overall W-type cluster, butS-types could not be separated within this group. A high level of variability was observed within the W- C- and S-type cluster, and the authors speculated that this might reflect the occurrence of sexual reproduction in this group (see below). The relative simplicity of the RAPD technique made it applicable to much larger samples. For instance, in a survey of eyespot populations from the UK, France and Germany, Papaikonomou and Lucas (1994) used three primers (OPA-10, OPB-04 and OPB-11; Operon Technologies) diagnostic for theW- and R-pathotypes, to generate RAPD profiles from more than 500 isolates. Each isolate was also assessed for growth rate and colony morphology. Around 23% of the isolates screened were W-type, and the large majority of these had typical fast growth rates and even-edged colonies (FIE). The R-type isolates sampled were morphologically more diverse, and more than 10% had intermediate or fast growth rates indistinguishable from W-types. This confirmed that cultural morphology alone is not a reliable way of classifying isolates. The most likely explanation for the occurrence of morphologically variant R-types is cultural instability in a proportion of isolates which form faster-growing sectors on agar. These can retain their variant morphology when serially subcultured via mycelial transfer or

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spores, or after passage through straw or a host plant (Julian et al., 1994a). Interestingly, in a few cases, reversion to the parental morphology is observed; the mechanistic basis for this morphological switch is not known. An alternative polymerase chain reaction (PCR)-based approach to isolate characterization was used by Poupard et at. (1993) who amplified internal transcribed spacer (ITS) regions of rDNA genes from the different pathotypes and then compared the sequences. They found a high degree of homology within a pathotype (only one nucleotide change in ITS1 or ITS2), and relatively close homology between the pathotypes. Nevertheless there was sufficient sequence divergence (six nucleotides in ITS1 and three in ITS2) to discriminate between the two types, and to permit design of primers specific for W- orR-type isolates. PCR amplification of a ribosomal DNA fragment combined with restriction enzyme digestion was subsequently used to compare sixty isolates from different geographic regions (Gac et al., 1996). Two distinct types were again identified, and although there was an overall correlation between the PCR-based assay and the morphological criteria proposed by Nirenberg (1981), the molecular assay was faster and more accurate in classifying isolates with overlapping characters. Overall, these various biochemical and molecular studies provided strong, but not definitive, evidence that the W- and R-types of Pseudocercosporalla herpotrichoides are genetically separated and represent distinct species. Final confirmation, however, depended on evidence obtained from studies on genetic crosses between isolates of the different pathotypes.

III. THESEXUALST AGE Since the first published description (Fran, 1912), the fungi causing eyespot disease were known only to reproduce by asexual means, with P. herpotrichoides included in the Deuteromycotina. It was therefore a surprise when Wallwork (1987) discovered that, following incubation on moist sand, apothecia (the sexual reproductive structures of Discomycete fungi) developed on straw infected with eyespot disease collected from Y allunda Flat in Southern Australia. Ascospores were obtained from these apothecia and were shown to produce vegetative colonies and conidia characteristic of P. herpotrichoides, confirming that the pathogen was undergoing sexual reproduction. The teleomorph was later named Tapesia yallundae (Wallwork and Spooner, 1988). Following this first report, apothecia ofT. yallundae were subsequently detected at field sites in New Zealand (Sanderson and King, 1988), many European countries (Hunter, 1989; Moreau et at., 1989; King, 1990; Nicholson et al., 1991a; Cavelier, 1994; Sindberg et al., 1994; Dyer and Lucas, 1995) and South Africa (Robbertse, et al., 1994). This widespread occurrence of the sexual stage (Fig. 1) suggested that sexual reproduction may be an intrinsic part of the life cycle of eyespot

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fungi (Fig. 2), a fact that had most likely not been recognized previously because the sexual stage had not yet been described. The discovery that eyespot fungi were capable of undergoing sexual reproduction raised several questions relating to the nature of breeding systems, population biology and taxonomy of P. herpotrichoides, and the significance of the sexual stage in the field. A.

NATURE OF THE BREEDING SYSTEM

Ascomycete fungi reproduce sexually by homothallic or heterothallic means (Dyer eta/., 1992). A preliminary investigation by Nicholson eta/. (1991a) suggested that recombination had occurred during the sexual cycle of T. yallundae, indicating that outbreeding was possible. But the major advance came in 1993 when Dyer et al. devised a protocol to induce the sexual cycle of T. yallundae under laboratory conditions, involving the use of sterile barley straw incubated at 10°C under continuous white light. By using this protocol it was demonstrated that apothecia only formed when sexually compatible pairs of isolates were present, and that recombination of molecular markers occurred during the sexual cycle, i.e. T. yallundae exhibits a two-allele heterothallic mating system (Dyer eta/., 1993). The two mating types were designatedMATJ-J andMATJ-2 in accordance with Yoder eta!. (1986). This result was later confirmed by Robbertse et al. (1994) and Moreau and Maraite (1995).

Fig. 1. Apothecia of Tapesia yallundae on overwintered wheat stubble.

TAPESIA SPP. AND EYESPOT DISEASE OF CEREALS

+

Conidia produced on infected debris

MATl-2

I

Lesion development and penetration of successive leaf sheaths

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234

J. A. LUCAS, P. S. DYER and T. D. MURRAY B.

POPULATION BIOLOGY AND TAXONOMY OF TAPES/A SPECIES

A major question arising from the discovery of the Tapesia teleomorph was whether all of the groupings ofP. herpotrichoides (W-and R-types; Nand L forms etc.) were able to undergo sexual reproduction, or if the sexual cycle was restricted to certain types. All of the initial field reports of Tapesia apothecia described ascospore discharge that gave rise to even-edged, fastgrowing colonies characteristic of the W-type (var. herpotrichoides) (Sanderson and King, 1988; Hunter, 1989; Moreau eta/., 1989; Nicholson et a/., 1991a). However, there was one unconfirmed report of the detection of apothecia (with morphology similar to those of the W-type) in Germany which yielded slow growing, feathery-edged colonies characteristic of the Rtype (var. acufonnis) (King, 1990). Conclusive evidence for sexual reproduction in the R-type was finally obtained when Dyer eta/. (1994b) detected similar apothecia in England discharging ascospores which produced feathery-edged colonies, with RAPD molecular markers used to verify that offspring were of the R-type grouping. Sexual crosses were then set up to establish whether the various groupings of P. herpotrichoides were interfertile, and therefore of the same biological species, or infertile thus representing different species (Gardner et al., 1991 ). Nicholson et al. (1995) used the in vitro protocol of Dyer et a/. (1993) to demonstrate that the W-, C- and S-types of P. herpotrichoides were interfertile and therefore of the same species. In contrast, apothecia failed to develop in any crosses with R-type isolates, even between R-type isolates derived from the ascospore offspring of Dyer et al. (1994b). Then in 1996, Dyer et al. and Moreau and Maraite simultaneously reported techniques to induce the sexual cycle of the R-type (var. acufonnis) ofP. herpotrichoides in vitro. These protocols were used to show that the fungus exhibits a two-allele heterothallic mating system, requiring the presence of sexually compatible isolates for sex to occur, with recombination of molecular and classical markers observed during the sexual cycle (Dyer eta/., 1996; Moreau and Maraite, 1996). Dyer et al. further demonstrated that R-type isolates failed to cross with isolates of the W/CIS grouping under conditions allowing sexual reproduction in the R grouping. This provided the first conclusive evidence that the two groups are indeed separate species since they form distinct breeding groups (Gardner et al., 1991). The names T. yallundae and T. acufonnis were accepted for the W/C/S and R-type groupings, respectively, in accordance with a proposal by Robbertse et al. (1995). Both primary (mating ability, molecular markers) and secondary (morphology, pathogenicity) criteria were then described to distinguish the two species (Dyer et al., 1996). Finally, the authors advocated the use of the holomorph names T. yallundae and T. acufonnis in future scientific usage, rather than the Pseudocercosporella (Ramulispora) anamorphs, in accordance with standard botanical nomenclature (Greuter et al., 1988). It is noteworthy that despite

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being separate species T. yallundae and T. acuformis produce apothecia that appear to be morphologically identical. A similar situation has been observed with the genetically incompatible mating groups of Gibberella fujikuroi (Leslie, 1991) and Nectria haematococca (Van Etten and Kistler, 1988), indicating that genes involved in ascocarp production are conserved despite evolutionary divergence. C. SIGNIFICANCE OF THE SEXUAL STAGE IN THE FIELD

The sexual stage of Tapesia is potentially of great importance in the field for a number of reasons. Ascospores have been shown to be forcibly ejected from apothecia of T. yallundae and may therefore become airborne (Sanderson and King, 1988) and provide a long-range wind dispersed inoculum capable of infecting cereal crops (Daniels et al., 1995; Dyer and Lucas, 1995). This contrasts with conidia which are splash dispersed over short distances (Soleimani et al., 1996). In addition, the sexual cycle can lead to greater variation within the pathogen population, with novel genotypes produced among the ascospore offspring as a result of recombination (Dyer et al., 1993, 1996; Moreau and Maraite, 1996). This provides the pathogen

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J. A. LUCAS, P. S. DYER and T. D. MURRAY

with the genetic flexibility to respond to selection pressures leading, for instance, to the possible appearance of ascospore isolates with increased resistance to fungicides (Fig. 3). The significance of the sexual stage will clearly depend on the extent to which apothecia are able to develop and discharge ascospores under field conditions. Dyer et al. (1994a) studied factors affecting the production of apothecia ofT. yallundae on inoculated wheat crops and discovered that they were absent from green (living) tissue and instead formed on infected straw stubble that was left after harvest. There was a notable seasonal variation in production of apothecia, with maximum numbers produced in early spring, although mature apothecia were detected over a nine month period after harvest. It was later shown that numbers of apothecia also varied according to the severity of infection of the preceding crop (Bateman et al., 1995). The finding that straw stubble (exposed to light) is required for the sexual cycle may help to explain why the teleomorph was not identified before 1987, because it was previously a common agricultural practice to burn straw stubble after harvest (Jenkyn et al., 1994). However, environmental objections, together with the implementation of the European Union setaside policy (Ansell and Tranter, 1992), have since led to a greatly increased area of standing straw stubble, thereby raising the possibility of increased sexual reproduction. In a pioneer survey, King (1991) detected Tapesia apothecia on 1-2% of stems at nine out of37 stubble sites (wheat and barley) in Germany. A later survey by Dyer and Lucas (1995) obtained comparable results, with apothecia of T. yallundae detected at 20 out of 45 stubble sites (mainly wheat) in England in early spring. Apothecia were present on less than 3% of stems at most locations, although they were found on 15-32% of stems at four sites. A recent survey of stubble sites in New Zealand has revealed similar results, with apothecia present at 25% of sites but on no more than 6% of stems (Dyer and Bradshaw, unpublished results). Taken as a whole, these surveys suggest that the sexual cycle is not likely to provide a major source of inoculum at most field locations. However, sexual reproduction is likely to be important in maintaining genetic diversity within the pathogen population, with the added potential of long-range dispersal. Although much is now known about the occurrence of T. yallundae apothecia in the field, some questions remain unresolved. In particular it is unclear to what extent the sexual stage may occur on wild grasses, which could provide a significant source of inoculum. In his original paper Wallwork (1987) reported the development of apothecia of Tapesia on wheat straw, but also observed their production on Bromus diandrus and barley grass (Hordeum leporium) straw. More recently, apothecia of T. yallundae have been detected on Yorkshire Fog (Holcus lanatus) in New Zealand (Dyer and Sheridan, unpublished results). Meanwhile it is unclear whether a similar pattern of seasonal development of apothecia, as seen on wheat (Dyer et al., 1994a), occurs on other cereals, although current

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fieldwork involving barley appears to support a similar pattern (Dyer and Bateman, unpublished results). In addition, apothecia of Tapesia have not yet been detected in the field in America, despite the discovery that isolates of both mating-types of T. yallundae are present in field populations in the Pacific NorthWest (Murray and Douhan, unpublished results). Finally, there have only been three reports of apothecia of T. acuformis being detected in the field (King, 1990; Dyer et al., 1994b; Moreau and Maraite, 1996). This is particularly surprising as recent field surveys have suggested that T. acuformis was the predominant pathogen for eyespot disease in the UK and parts of northern Europe in the early to mid-1990s (Polley and Turner, 1995; Birchmore and Daniels, personal communication). There are various potential explanations for the seemingly low occurrence of apothecia ofT. acuformis. It is possible that the majority offield isolates are ofthe same mating-type, so sexual reproduction cannot occur, as seen with N haematococca mating population I (Snyder et al., 1975). Alternatively, sexual reproduction may only be triggered by an unusual set of environmental conditions rarely encountered in the field. Finally, most field isolates may be of low sexual fertility, or even sterile, as observed with Magnaporthe grisea (Leung and Taga, 1988). Evidence for the latter explanation is that it is much harder to induce the sexual cycle ofT. acuformis in vitro compared with that ofT. yallundae (Dyer et al., 1996; Moreau and Maraite, 1996). Also, attempts to produce the sexual stage in vivo on artificially inoculated sites have either failed or yielded very few apothecia (Dyer et al., 1994a; Dyer and Bateman, unpublished results). Thus, T. acuformis may reproduce almost exclusively by asexual means, which may explain why a lower degree of genetic variation has been detected in isolates of T. acuformis compared with those of T. yallundae (Nicholson and Rezanoor, 1994). D.

CURRENT RESEARCH UTILIZING THE SEXUAL SYSTEM OF TAPESIA SPECIES

It is now possible to manipulate the sexual cycle ofT. ya!lundae to determine

the genetic basis of differential pathogenicity and ho:J specificity, e.g. how many genes confer theW-, C-or S-pathotype (Nicholson et al., 1995). The sexual cycle is also being used to determine the nature of resistance to the demethylation inhibitor (DMI) fungicide prochloraz, with initial results indicating both a mono- and a polygenic component (Dyer et al., 1998). The effect of sexual reproduction on variation within populations is being assessed by comparison of known asexual and sexual field sites in Europe and South Africa, and it may be possible to test whether increased population diversity correlates with greater risk of pathogen evolution. Other research is focused on cloning the mating-type genes governing sexual compatibility (Dyer et al., 1997). Such genes will provide useful molecular

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J. A. LUCAS, P. S. DYER and T. D. MURRAY

markers with which to determine the field distribution of complementary mating-types ofT. yallundae and T. acuformis. E.

GEOGRAPHIC VARIATION IN POPULATIONS

The discovery of the sexual stage and confirmation that eyespot disease is caused by two distinct species has provided a new perspective with which to interpret worldwide field data, with differences and shifts in populations of T. yallundae and T. acuformis evident. For instance, T. yallundae seems to be the only form of eyespot disease present in South Africa, with the apparent absence ofT. acuformis (Campbell et al., 1996). Also, the South African population has remained sensitive to MBC fungicides (Robbertse et a/., 1996) whereas resistance is widespread in New Zealand and European populations (Birchmore et al., 1987; Fitt, 1992) and in the Pacific North West of the USA (Murray, 1996). In Europe and New Zealand, isolates of both species are often found at the same field sites although the relative proportion on cereal crops varies year on year. Leroux and Gredt (1997) monitored the population structure of eight field plots in France from 1985 to 1995 and found that T. acuformis constituted 24-65% of isolates present over this period, and that 23-56% of stems had lesions from which both T. acuformis and T. yallundae could be isolated. However, recent surveys suggest that T. acuformis has been the more prevalent species during the early to mid-1990s. This may be partly due to the increased use of azole fungicides which preferentially select for T. acuformis and reduce sexual reproduction in T. yallundae (Bateman, 1993; Bateman et a/., 1995). However, this population shift may be reversed if newly introduced fungicides, such as cyprodinil, preferentially select for T. yallundae. Other significant changes in population structure may arise from the appearance and spread of field isolates resistant to DMI fungicides, as seen in parts of Northern France (Leroux and Gredt, 1997).

IV.

PATHOGENICITY

A. SPORE DISPERSAL AND ADHESION

Although ascospores have been shown to be infective (Daniels et al., 1995), it is likely that the predominant form of inoculum in the field is conidia formed on straw persisting from a previous crop. These needle-shaped spores are produced under wet conditions and are splash-dispersed over short distances (Soleimani et al., 1996). Conidia are flexible rather than rigid and adopt the shape of the surface on which they impact, wrapping around trichomes or following the topography of host epidermal cells (Daniels et al., 1991). They stick more readily to hydrophobic than to hydrophilic surfaces. Each spore is enveloped in an extracellular matrix (ECM) of mucilaginous

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material that also aids adhesion, protects against desiccation, and may serve as a scaffold for the localization of enzymes or other proteins secreted during the early stages of development on the host surface. In electron micrographs the ECM around spores and germ tubes initially has a fibrous appearance which becomes more electron dense with time, possibly due to the accumulation of protein. B. THE INFECTION PROCESS

Early studies using light microscopy (e.g. Defosse, 1966) described spore germination, germ tube elongation, and the formation of appressoria on the surface of the host. Multicellular melanized structures are subsequently formed on leaf sheaths. Aggregations of these infection plaques are clearly visible under a low power dissecting microscope, and are responsible for the dark 'pupil' seen in mature eyespot lesions. Bateman and Taylor (1976) showed that the host coleoptile is important in the establishment of infection as it is a highly susceptible tissue and provides a base from which the pathogen can spread onto and penetrate leaf sheaths. Under conditions of high humidity, germinating conidia can establish a substantial mycelial network on the surface of the coleoptile, and further sporulation may take place on this tissue (Soulie et al., 1985). Such microcycle conidiation can often be observed on inoculated, detached coleoptiles. Glynne (1952) reported that spores may form on the coleoptile and leaf sheaths of young wheat plants before macroscopic symptoms are evident, and such spores can presumably contribute to the spread of infection. Although these studies provided a general description of the early infection process, an important limitation is that they did not discriminate between the two pathotypes (now separate species) of the eyespot pathogen. The first comparative report of early pathogenesis was provided by Daniels et al. (1991) who used a combination of light and electron microscopy to study the infection of susceptible wheat seedlings by four isolates of each species. Spores adhering to the coleoptile surface usually germinated at one or both poles to produce germ tubes which, in the case of T. yallundae, extended along the grooves over the anticlinal walls between epidermal cells. Germ tubes ofT. acuformis did not exhibit this orientated growth. Extending germ tubes of T. yallundae formed swollen appressorium-like structures at intervals along the anticlinal cell wall grooves. In cases where the germ tubes grew at an angle to the orientation of epidermal cells, appressoria were only formed over cell-cell junctions. The signal triggering this morphogenetic response has not been identified although it does not appear to be simply topographical, as it does not occur on plastic replicas of the host surface. In T. acuformis, spores sometimes formed multiple lateral germ tubes, and appressoria were usually produced at the tips of short hyphae that were randomly placed on the host surface. The difference in appressorial

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J. A. LUCAS, P. S. DYER and T. D. MURRAY

positioning between the two species determines the subsequent pattern of tissue penetration. In T. yallundae the penetration hyphae entered the anticlinal cell walls and grew intramurally as far as the next cell layer, where a network of branching hyphae colonized both anticlinal and periclinal walls. With T. acuformis, penetration was directly into epidermal cells with hyphae traversing cell walls into adjacent cell layers. A further difference between the species was a greater proliferation of surface mycelium in T. acuformis, with the formation of ribbon-like runner hyphae through the aggregation of individual hyphae into cord-like structures. C.

INFECTION PLAQUES

The most distinctive infection structure produced by the eyespot fungi is the multicellular plaque formed on leaf sheaths. Infection plaques are produced by branching and aggregation of hyphae, followed by differentiation of cells to form a plate of pseudoparenchyma-like tissue. Within the plaque, cells in contact with the host surface elongate and secrete extracellular matrix to produce an '0-ring' seal similar to that observed in appressoria formed by other fungi, for instance the rice blast pathogen Magnaporthe grisea (Howard and Valent, 1996). These cells are the subsequent source of infection hyphae which penetrate the host cuticle and epidermal cell wall. At the same time, cell wall thickening and pigmentation takes place, most likely due to the deposition of melanin. Infection plaques ofT. yallundae and T. acuformis can be distinguished on the basis of morphology, with T. yallundae forming loose, asymmetrical plaques and T. acuformis more compact, circular plaques of closely associated cells (Daniels eta/., 1991 ). Typically, plaques are formed in the longitudinal grooves overlying vascular traces in the leaf sheath. The principal role of the infection plaque is penetration of the host, with the closely attached central cells acting as a compound appressorium. These highly differentiated structures may also perform other functions. Cells at the periphery of plaques secrete a mucilaginous matrix which may protect against desiccation, and also produce adventitious hyphae which extend across the surface of the leaf sheath to initiate new foci of infection (Daniels eta/., 1991 ). Furthermore the thickened, sclerenchyma-like tissue persists on the surface of straw after host senescence and is therefore likely to be important in perennation of the pathogen. Relatively little is known about the induction of infection plaques, or the processes involved in their differentiation. Morphologically similar, pigmented aggregations of cells may be produced in culture, especially at points of mechanical obstruction, such as contact with a solid surface, or when nutrients are limiting (Deacon, 1973). On the host plant, plaques differentiate in the confined space between leaf sheaths. They may also be produced on the base of Petri dishes, beneath the agar (Fig. 4(C) and (D)) or when the fungus extends between sheets of inert material (P. Bowyer,

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Fig. 4. Infection plaques formed by Tapesia acuformis. (A) Plaque formed on surface of leaf sheath. Bar = 20 ftm. (B) Undersurface of plaque lifted from plant surface showing central appressorial cells with '0-rings' and infection hyphae protruding. Bar= 20 ftm. (C) Plaque formed on plastic surface (base of Petri dish). Ba = 20 ftm. (D) Impression left by plaque on plastic surface after removal. Note outline of adhesive rings and dents made by emerging infection pegs. Bar = 10 f1m. Scanning electron micrographs courtesy of Alison Daniels, AgrEvo UK Limited.

personal communication). This suggests that physical cues might trigger plaque induction. Positioning of plaques over vascular tissues might be due to either topographical signals or differences in host exudates at these sites. The structural similarity of cells acting as appressoria within plaques suggests that at least some of the signal and response pathways might be conserved between these compound structures and the simple appressoria formed by other pathogens (Dean, 1997). There are also likely to be some similarities in the penetration process. The appressorial cells within plaques establish an intimate contact with the host cuticle, with tight adhesion and possible localization of enzymes within the matrix seal (Daniels et al., 1991 ). Penetration occurs by formation of an infection hypha at the cell tip, which punctures the host cell wall. Simultaneous penetration by multiple appressorial cells occurs from the under surface of the plaque (Fig. 4(B) ), leading to a sieve-like appearance of

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J. A. LUCAS, P. S. DYER and T. D. MURRAY

the corresponding host surface. The mechanism of penetration is not known, although localized erosion of the cuticle and alterations in the ultrastructure of the cell wall around the infection hypha implicates the action of hydrolytic enzymes. There is also evidence, however, for the generation of physical force during penetration, as inert surfaces beneath infection plaques are pitted at sites of infection hypha formation (Fig 4(0)). This again suggests parallels with other pathogens such as Magnaporthe (Howard et al., 1991; Money and Howard, 1996). Melanin is known to play an important role in appressorial function, both in foliar pathogens like Magnaporthe, Colletotrichum (Perpetua et al., 1996) and Alternaria (Takano et al., 1997), and root pathogens such as Gaeumannomyces (Money et al., 1998). Pigment mutants have been isolated in Tapesia (Perry and Hocart, 1998) and these may be affected in both melanin biosynthesis pathways and pathogenicity. D. TISSUE COLONIZATION

Following penetration of coleoptiles, hyphae ofT. yallundae grow within cell walls, predominantly in the middle lamella, whereas T. acuformis shows a more random pattern of tissue invasion (Daniels et al., 1991 ). On leaf sheaths, due to the positioning of infection plaques over vascular traces, initial establishment occurs in cells adjacent to vascular tissues. Subsequently the pathogens grow through the leaf sheath to emerge on the inner surface, where runner hyphae initiate infection of the next, underlying leaf sheath. With pathogenic isolates on a susceptible host this sequential colonization and penetration of leaf sheaths eventually leads to infection of the stem, whereas with less pathogenic isolates, infection is arrested at an earlier stage, with fewer leaf sheaths colonized. Comparatively little information is available on the host resistance mechanisms involved, although deposition of osmiophilic material in cell walls has been observed during infection (Soulie et al., 1985) and beneath infection plaques (Daniels et al., 1991 ). Murray and Bruehl (1983) described formation of lignified cell wall appositions at penetration sites in epidermal cells of eyespot resistant wheat cultivars. Subsequently, Murray and Ye (1986) reported that lignified papillae, stain halos, and a hypersensitive reaction occurred in the six varieties they studied. More papillae occurred in the leaf sheaths of resistant than susceptible varieties, and fewer successful penetrations by the pathogen occurred when papillae were present. The hypersensitive reaction occurred only at sites where papillae formed and, when present, reduced the number of successful penetrations below that at sites where only papillae were present. Infection from ascospores follows a broadly similar pattern to infection from conidia, at least in the case of T. yallundae. In the only microscopic study conducted to date, Daniels et a/. (1995) allowed apothecia of T. yallundae to discharge ascospores onto the stem base of wheat seedlings, and

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followed events from spore adhesion to lesion formation using scanning electron microscopy. Ascospores germinated to form extending superficial hyphae which aligned in grooves between epidermal cells. Penetration occurred from simple appressoria, and growth within coleoptile tissues was largely intramural. At 10-17 days after ascospore discharge, infection of leaf sheaths was seen, with formation of multicellular infection plaques morphologically similar to those produced after infection by conidial inoculum. This coincided with early symptoms of leaf sheath browning. Later, typical eyespot lesions were formed on stem bases, and re-isolates from such lesions were confirmed by RAPD fingerprinting to be T. yallundae. Recombination of RAPD bands, indicating genetic reassortment, was also demonstrated for most of the re-isolates. In contrast to the confirmed infectivity of ascopores reported by Daniels et al. (1995), Frei and Gindrat (1995) were unable to achieve infection of wheat and barley stems with ascopores of T. yallundae. E.

PATHOGENICITY FACTORS

The factors aiding colonization of host tissues have not been conclusively identified, although cell wall-degrading enzymes are likely to play a part. When grown on wheat cell walls as a nutrient substrate, P. herpotrichoides produces a sequence of extracellular enzymes, especially hemicellulases (Cooper et al., 1988). The main activities were arabanase, xylanase and laminarinase. Much lower levels of cellulase and pectolytic activities were detected. Regulation of arabanase and laminarinase appeared to be constitutive. These authors concluded that the predominance of hemicellulases is most likely related to the chemistry of the monocotyledon cell wall. Mbwaga et al. (1997) assayed several enzymes in extracts from young wheat plants infected with the eyespot pathogens, and found that activities of pectin methylesterase (PME), polygalacturonase, pectin lyase, cellulase, xylanase and arabanase were higher in infected than in healthy control plants. The earliest increases were in PME, xylanase and arabanase, especially in seedlings inoculated with an isolate ofT. yallundae. However, significant activity was also found in extracts from healthy seedlings and the authors concluded that it was not possible to attribute the changes solely to production of enzymes by the pathogen. There is a need for a comparative analysis ofT. yallundae and T. acuformis and their relative enzymic capabilities. However, the diversity of cell walldegrading enzymes produced by plant pathogenic fungi, including different isoenzymes, substrate specificities, and complex interactions between activities, means that analysis of their relative importance in pathogenicity will depend on the development of efficient methods for the targeted disruption of both individual genes and combinations of genes encoding such enzymes.

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Similarly, little is known about the types and sources of nutrients utilized by the eyespot pathogens during host tissue colonization. The most likely carbon sources are sugars released from cell walls, although the early pattern of vascular colonization of leaf sheaths (Daniels et al., 1991) indicates that the pathogen might access solutes translocated in phloem tissues. In more advanced infections there is evidence of extensive digestion of cell walls and destruction of vascular tissues, especially the phloem (Murray and Bruehl, 1983). This observation might explain the premature ripening (whitehead symptoms) seen in mature eyespot-infected plants, as well as the stem weakening which leads to collapse (lodging) in advanced infections. Recently, Coff eta/. (1998) showed that the plant protein plastocyanin is progressively degraded in eyespot-infected stems, and used quantitative measurement of the protein as an estimate of disease severity. Under controlled conditions, at temperatures favourable for infection (10-15°C), development of visible eyespot lesions takes 6-12 weeks, depending on the isolate and host cultivar used. However, microscopic observations (Murray and Ye, 1986; A. Daniels, personal communication) reveal that infection of the coleoptile and more than one leaf sheath occurs more rapidly than this, indicating that the pathogen undergoes a period of asymptomatic growth within the host. Hence there appears to be a biotrophic phase of host colonization prior to cell necrosis and visible browning of tissues. Indeed, recent PCR analyses have been able to detect and quantify pathogen presence before symptoms become visible (Nicholson eta/., 1997). Rates of host colonization may differ between the two species. In inoculation tests using wheat under controlled conditions, isolates ofT. yallundae often give higher infection scores than T. acuformis, due to a more rapid progression through the leaf sheaths. Poupard et a/. (1994) used an ELISA test to compare colonization rates between the two species under glasshouse conditions and confirmed that T. acuformis was slower to penetrate the coleoptile and leaf sheaths. In field plots inoculated with the two species, T. yallundae generally became established on stems more rapidly than T. acuformis, with more severe symptoms evident at Zadoks growth stage 71 (Goulds and Fitt, 1991). However, by harvest there was little difference in eyespot incidence or severity between the species in two out of the three growing seasons when experiments were carried out. In field tests using their ELISA assay, Poupard et al. (1994) also observed that at ripening both species gave similar infection scores.

V.

HOST RANGE OF TAPES/A SPECIES

Early workers did not differentiate between Tapesia yallundae and T. acuformis. Consequently, it is not known whether the host ranges of these fungi are the same or different. The important cultivated small grain hosts of

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the eyespot pathogens include bread wheat (Triticum aestivum L. em Thell.), barley (Hordeum vulgare L.), oats (Avena sativa L.), and rye (Secale cereale L.). Many wild and cultivated grasses are also hosts, including: Aegilops sp., Agropyron sp., Agrostis sp., Alopecurus myosuroides Huds., Apera spica-venti (L.) Beauv.,Arrhenatherum elatius (L.) Beauv. ex J. & C. Presl., Bromus sp., Cynosurus cristatus L., Dactylis sp., Echinochloa crusgalli (L.) Veauv ., Festuca idahoensis Elmer, Holcus lanatus L., Hordeum leporinum (Link), Koeleria nitida Nutt., Lolium sp., Phalaris canariensis L., Phleum pratense L., Poa sp., Sitanion hystrix (Nutt.) J. C. Sm., and Triticum sp. (Oort, 1936; Sprague, 1936; Sprague and Fischer, 1952; Cunningham, 1965, 1980; Booth and Waller, 1973). A few species of dicotyledonous plants are reported to be hosts including Balsamorhiza sp., Delphinium sp., Lithosperrnum ruderale Douglas ex Lehm., and Lomatium tritematum (Pursh) J. M. Coulter & J. Rose (Anonymous, 1960; Farr et al., 1989). It is doubtful that these dicotyledonous plants are hosts for Tapesia since no pathogenicity data are known for these species. Sprague (1934) reported on the ecological associations of P. herpotrichoides and cited these dicotyledonous plants only as part of a consociation and not as hosts. In a recent survey (Hocart, McNaughton, Nicholson and Ennos, unpublished) samples were collected from barley and wild grasses in a single field in Tayside, Scotland. Isolates of the eyespot pathogens were successfully recovered from the cereal crop, barley, and several species of grass including annual meadow grass (Poa annua ), couch (Elymus repens) and cocksfoot (Dactylis glomerata). The isolates from barley were all T. acuforrnis. Nine isolates obtained from each of the principal hosts were tested for pathogenicity to barley, couch and annual meadow grass under glasshouse conditions. All the isolates colonized meadow grass, but grew without inducing disease symptoms. The barley isolates were highly pathogenic to barley, but also induced symptoms on couch. The isolates from couch gave similar results on this host, but were less pathogenic to barley. Hence, although isolates were able to colonize grass hosts other than their host of origin, some evidence was obtained for host species-adapted groups. Population diversity was also assessed for 91 isolates from this survey using RAPD fingerprinting. Multilocus analysis showed that there were 87 multilocus genotypes among the 91 isolates; only four genotypes were present more than once. The diversity detected suggests that sexual reproduction is occurring, or has occurred, in these populations despite the rare detection of apothecia of T. acuforrnis in the wild.

VI.

HOST RESISTANCE TO TAPES/A SPECIES

Sprague (1936) reported on the susceptibility of a wide range of cereals and grasses toP. herpotrichoides and identifiedAegilops ventricosa and Haynaldia

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villosa (now Dasypyrnm villosum) as potential sources of resistance to eyespot for cultivated wheat. The eyespot resistance gene Pchl was later transferred from Aegilops ventricosa to wheat through a series of complex crosses (Maia, 1967). Vincent et al. (1952) identified the French variety Cappelle Desprez as resistant to eyespot. The eyespot resistance gene Pch2 was later identified in Cappelle Desprez (Law et al., 1976; de Ia Pefia et al., 1996). Pch2 is less effective in controlling eyespot than Pchl, which is widely used in the development of eyespot-resistant varieties (Johnson, 1992). This gene is present on chromosome 70 and intervarietal substitutions for 70 into eyespot susceptible genotypes confer a high level of resistance (J ahier et al., 1989). Although Pchl is very effective, loss in grain yield can still occur in varieties containing this gene when eyespot is severe. Thus, there is a need for new eyespot-resistance genes that can be used alone or in combination with this and other resistance genes. Identification of new eyespot-resistance genes has been difficult due to the quantitative nature of resistance and the variation that occurs in disease development in greenhouse and field tests (Lind et al., 1994). Lind (1992) developed an ELISA test to measure colonization of wheat plants by P. herpotrichoides. Evaluation of varieties for resistance using this test was more reliable after than before anthesis. Lind (1992) concluded that ELISA values obtained from young plants were not useful in predicting adult plant resistance to eyespot. De Ia Peiia and Murray (1994) developed a seedling test for eyespot resistance where 2-week-old plants are inoculated with a (3glucuronidase (GUS)-transformed strain of P. herpotrichoides. Resistance ratings using this test are closely correlated with ratings made on older, fieldgrown plants and are useful in predicting adult-plant resistance. This GUS seedling test has been used to identify new sources of eyespot resistance in wild relatives of wheat, to determine the genetic control of resistance, and to facilitate mapping and tagging of eyespot resistance genes (Murray et al., 1994; Yildirim et al., 1995, 1998; de la Pefia et al., 1996, 1997; Cadle et al., 1997; Figliuolo et al., 1998;). More recently, Nicholson et al. (1997) have developed a competitive PCR assay that allows quantification of individual Tapesia species within plant tissues. All of these tests are based on differential colonization of resistant and susceptible genotypes by the pathogen. The GUS seedling test has been used to identify eyespot-resistant accessions of Triticum tauschii, T. monococcum, T. durnm, T. dicoccoides, T. turgidum, Aegilops markgrafii, A. speltoides, Amblyopyrnm muticum, and Dasypyrnm villosum obtained from various germplasm collections (Yildirim et al., 1995; Cadle et al., 1997; Figliuolo et al., 1998; Murray et al., unpublished). New eyespot resistance genes have been identified in D. villosum and T. tauschii thus far and analysis of additional species is in progress (Murray et al., 1994; Yildirim et al., 1998). Murray et a/. (1994) studied the resistance of D. villosum against T. yallundae using the GUS seedling test. The resistance gene in D. villosum was

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mapped to chromosome 4VL using a series of disomic chromosome addition lines. Segregation in the F2 progeny from a cross between the eyespotsusceptible Yangmai-5 (4V(4D)) substitution line and resistant Chinese Spring disomic addition line of chromosome 4V indicated that a single dominant gene controlled resistance. This gene (Pch3) was located on the distal portion of the long arm of chromosome 4V in a 33-cM interval bracketed by RFLP markersXcdo949 andXbcd588 (Yildirim et al., 1998). Uslu et al. (1998) studied resistance in D. villosum toT. yallundae and T. acuformis using the competitive PCR test developed by Nicholson et al. (1997) to measure colonization in a series of Dasypyrum chromosome addition lines in Chinese Spring. They found that lines containing chromosomes 1V, 2V, 4V, and possibly 3V were significantly more resistant to T. yallundae than Chinese Spring, the susceptible control. In contrast, only lines containing chromosomes 1V, 2V, 5V and possibly 3V were significantly more resistant to T. acuformis than Chinese Spring. The fact that the 4V addition line was not more resistant to T. acuformis than the susceptible control is significant because it indicates that resistance to the two pathogens may be conferred by different genes, which would be important for variety development programmes. Genetic analysis of resistance in T. tauschii using the GUS seedling test revealed the same single dominant gene for eyespot resistance in two different resistant accessions (Cadle, 1997). This gene was not linked to RFLP markers that are linked with Pchl on chromosome 7DL, nor to markers linked with Pch3 on chromosome 4VL. Therefore, this resistance gene is not allelic to Pchl or to Pch3 and represents a previously unknown gene. Assefa and Fehrmann (1998) evaluated the resistance of 194 accessions from seven Aegilops species to several wheat diseases, including eyespot. Around 10% of the accessions were resistant to eyespot. Many of the accessions of Triticum (Aegilops) tauschii exhibited resistance to more than one of the diseases tested, including leaf and stem rust, and Septoria tritici blotch. The authors concluded that since T. tauschii is the donor of the D genome in hexaploid wheat, this species can be readily exploited in the transfer of resistance genes for bread wheat improvement.

VII. FUTURE PROSPECTS Research findings over the past decade have provided significant new perspectives on eyespot disease of cereals. Confirmation that the disease can be caused by two different species T. yallundae and T. acuformis has important implications for disease management. Future chemical control, plant breeding and disease monitoring programmes will need to screen against and distinguish between both species. The differential sensitivity of

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the two species (originally pathotypes) to some fungicides, such as most sterol demethylation inhibitors (DMis) is well documented (Bateman, 1990; Leroux and Gredt, 1997). Although the recently introduced anilinopyrimidines, such as cyprodinil, appear to be equally active against both T yallundae and T. acuformis (Leroux and Gredt, 1997), effective use of fungicides, and especially implementation of fungicide-resistance strategies, will depend on a clear understanding of the genetics and population biology of the two species. For instance, the apparent absence of sexual crossing between the species (Nicholson et al., 1995; Dyer et al., 1996) suggests that they represent completely separate gene pools, although somatic hybrids have been produced under laboratory conditions (Di San Lio et al., 1994). The eyespot pathogens have already proved their ability to adapt to selection pressures, as shown by the evolution of resistance to MBC (Hocart et al., 1990), and later DMI fungicides (Cavelier et al., 1994: Julian et al., 1994b; Leroux et al., 1994; Leroux and Gredt, 1997). The best prospect for sustainable control of eyespot disease therefore lies in the use of integrated control measures, combining varietal resistance with fungicides used according to predictions of disease risk. Progress in the identification and selection of novel sources of host resistance (see above) indicates that breeders will have additional genes to deploy against the disease. Efforts to introgress these new resistance genes into cultivated wheat for use in variety improvement programmes are in progress. Questions regarding the effectiveness of these genes in hexaploid wheat and their effect when used in combination with other eyespot-resistance genes have not yet been resolved. Likewise, questions about the effectiveness of these new resistance genes against T. yallundae and T. acuformis and the possibility of pathogenic specialization remain to be answered. These uncertainties aside, the discovery of new resistance genes offers the promise of improved genetic control of eyespot as well as a broader genetic base to exploit in variety improvement programmes. There is a need, however, for more fundamental research on the genetic control of host range in the eyespot pathogens, not only to clarify how cereal hosts resist infection, but also to indicate the likely durability of novel resistance genes. Conversely, more work on the sexual stage of the pathogens is required to estimate the extent to which recombination can extend variation in the pathogen population, and whether this alone can increase disease risk. Furthermore, there are still a number of questions to be answered concerning the role of the sexual stage in eyespot epidemics, such as the relative importance of ascospore inoculum, and the influence of the sexual cycle on the population dynamics and genetics of the pathogens. Molecular genetic approaches are now becoming available for the Tapesia species, especially T. yallundae, and will aid the identification of factors determining the pathogenicity and host specificity of these fungi. Transformation of T. yallundae was first reported by Blakemore et al.

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(1989) and modifications of this protocol are now efficient enough to obtain frequencies allowing routine gene disruption, complementation and insertional mutagenesis (E. Mueller and P. Bowyer, personal communication). Analysis of mutants reduced in pathogenicity should identify the key processes involved in infection. Preliminary studies on pigment mutants produced by UV mutagenesis (Perry and Hocart, 1998) have suggested that melanin may play a role in pathogenicity. The reporter genes GUS (de la Pefta and Murray, 1994) and green fluorescent protein (GFP, P. Bowyer, unpublished) have both been expressed in transformed strains. Such reporters can be used to visualize and quantify the pathogen in planta but will also be of value for expression studies with specific genes. PCR-based cloning using degenerate primers has already isolated several genes from T. yallundae, including ornithine decarboxylase (ODC) (Mueller et al., 1998) and a mating type gene (Dyer et al., 1997). The availability of sexually fertile strains of opposite mating type, along with procedures for the recovery of ascospore progeny, means that genetic analysis can also be used to confirm correlations between specific genes and traits such as pathogenicity or resistance to fungicides. Ultimately such studies should identify novel targets for chemical intervention in the infection process. Molecular genetic approaches can also aid understanding of resistance to known targets; for example, PCR amplification of the ;"\tubulin gene has identified the specific point mutations leading to single amino acid changes in this protein associated with different resistance phenotypes to MBC and N-phenylcarbamate fungicides in Tapesia species (Albertini et al., 1999). As well as clarifying structure-activity relationships, such genetic analysis is potentially of great value in identifying and mapping the incidence of particular fungicide resistance alleles in the pathogen population. An alternative, and to date neglected target, is pathogen reproduction. By utilizing genomic information emerging from Ascomycete genetic models such as Aspergillus, it should be possible to identify genes involved in spore formation and sexual differentiation. Once the physiological and molecular basis of sporulation is known it may be feasible to design compounds able to interfere with spore production and hence to prevent dispersal of the pathogen. This is especially important because changes in management of crops, such as earlier sowing of winter cereals (favouring infection) and creation of large areas of standing straw stubble (allowing the sexual cycle to occur) may lead to increased severity of eyespot disease in future years. ACKNOWLEDGEMENTS The authors thank Alison Daniels and Richard Birchmore (AgrEvo UK Ltd), Mark Hocart (University of Edinburgh) and Paul Bowyer (IACR-

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Long Ashton) for providing unpublished data or Figures. PSD thanks the Biotechnology and Biological Sciences Research Council of the United Kingdom (BBSRC) for a University Research Fellowship. JAL thanks Valerie Topps for help in compiling the references. IACR-Long Ashton receives grant-aided support from BBSRC.

REFERENCES Albertini, C., Gredt, M. and Leroux, P. {1999). Mutations of the beta-tubulin gene associated with different phenotypes of benzimidazole resistance in the cereal eyespot fungi Tapesia yallundae and Tapesia acuformis. Pesticide Biochemistry and Physiology 64, 17-31. Anonymous (1960). "Index of Plant Diseases in the United States". USDA Agriculture Handbook No. 165. Anonymous (1981). "Distribution Maps of Plant Diseases. Pseudocercosporella herpotrichoides (Fran) Deighton". Commonwealth Mycological Institute. Map No. 74, 4th edition. Ansell, D. J. and Tranter, R. B. (1992). "Set-aside: Theory and in Practice". University of Reading, Department of Agricultural Economics and Management Centre for Agricultural Strategy. Assefa, S. and Fehrmann, H. (1998). Resistance inAegilops species against leaf rust, stem rust, Septaria tritici blotch, eyespot and powdery mildew of wheat. Journal of Plant Disease and Protection 105, 624-631. Bateman, G. L. (1990). Comparison of the effects of prochloraz and flusilazole on foot rot diseases and on populations of the eyespot fungus Pseudocercosporella herpotrichoides in winter wheat. Zeitschrift fur Pflanzenkrankheiten und Pflanzenschutz 91, 508-516. Bateman, G. L. (1993). Development of disease symptoms and fungal pathogens on shoot bases in continuous winter-wheat and effects of fungicides. Plant Pathology 42, 595-608. Bateman, G. L. and Taylor, G. S. (1976). Significance of the coleoptile in establishment of seedling infection on wheat by Pseudocercosporella herpotrichoides. Transactions of the British Mycological Society 67, 513-514. Bateman, G. L., Dyer, P. S. and Manzhula, L. (1995). Development of apothecia of Tapesia yallundae in contrasting populations selected by fungicides. European Journal of Plant Pathology 101, 695-699. Birchmore, R. J., Buckley, E. S., Browning, S. and Russell, P. E. ( 1987). Sensitivity to prochloraz and carbendazim of New Zealand isolates of Pseudocercosporella spp. Australasian Plant Pathology 16, 66-67. Blakemore, E. J. A., Dobson, M. J., Hocart, M. J., Lucas, J. A. and Peberdy, J. F. (1989). Transformation of Pseudocercosporella herpotrichoides using two heterologous genes. Current Genetics 16, 177-180. Booth, C. and Waller, J. M. (1973). Pseudocercosporella herpotrichoides. CMI Descriptions of Pathogenic Fungi and Bacteria No. 386. Brown, M. C., Taylor, G. S. and Epton, H. A. S. (1984). Carbendazim resistance in the eyespot pathogen Pseudocercosporella herpotrichoides. Plant Pathology 33, 101-111. Cadle, M. M. (1997). Identification and genetic characterization of resistance to Pseudocercosporella herpotrichoides in diploid wheat relatives Triticum monococcum and T. tauschii. MSc Thesis, Washington State University.

TAPES/A SPP. AND EYESPOT DISEASE OF CEREALS

251

Cadle, M. M., Murray, T. D. and Jones, S. S. (1997). Identification of resistance to Pseudocercosporella herpotrichoides in Triticum monococcum. Plant Disease 81, 1181-1186. Campbell, G. F., Janse, B. J. H., Marais, G. F. and Crous, P. W. (1996). Only one species of Ramulispora is associated with eyespot disease of wheat in South Africa. South African Journal of Science 92, 29-33. Cavelier, N. (1994). Pourra-t-on jamais en finir avec le pietin-verse? Premiere observation en France de Ia forme sexuee du champignon. Phytoma 462, 17. Cavelier, N. and Leroux, P. (1983). La resistance du pietin-verse aux benzimidazoles et aux thiophanates. Perspectives Agricoles 68, 33-35. Cavelier, N., Rousseau, M. and Le Page, D. (1987). Variabilite de Pseudocercosporella herpotrichoides, agent du pietin-verse des cereales: comportement in vivo de deux types d'isolats et d'une population en melange. Zeitschrift fur Pflanzenkrankheiten und Pflanzenschutz 94, 590-599. Cavelier, N., Pineau, C. and Prunier, M. (1994). Characteristics of Pseudocercosporella herpotrichoides isolates resistant to prochloraz. In "Fungicide Resistance. BCPC Monograph No. 60" (S. Heaney, D. Slawson, D. W. Hollomon, M. Smith, P. E. Russell and D. W. Parry, eds), pp. 67-70. British Crop Protection Council, Farnham. Coff, C., Poupard, P., Xiao, Q., Graner, A and Lind, V. (1998). Sequence of a plastocyanin eDNA from wheat and the use of the gene product to determine serologically tissue degradation after infection by Pseudocercosporella herpotrichoides. Joumal of Phytopathology 146, 11-17. Cooper, R. M., Longman, D., Campbell, A, Henry, M. and Lees, P. E. (1988). Enzymic adaptation of cereal pathogens to the monocotyledonous primary wall. Physiological and Molecular Plant Pathology 32, 33-47. Creighton, N. (1989). Identification of W-type and R-type isolates of Pseudocercosporella herpotrichoides. Plant Pathology 38, 484-493. Cunningham, P. C. (1965). Cercosporella herpotrichoides Fron on gramineous hosts in Ireland. Nature 207, 1414-1415. Cunningham, P. C. (1980). Studies of host specificity of indigenous isolates of Pseudocercosporella herpotrichoides.Annals ofApplied Biology 94,301 ( abstr.). Cunningham, P. C. (1981) Occurrence, role and pathogenic traits of a distinct pathotype of Pseudocercosporella herpotrichoides. Transactions of the British Mycological Society 76, 3-15. Daniels, A., Lucas, J. A and Perberdy, J. F. (1991). Morphology and ultrastructure of W and R pathotypes of Pseudocercosporella herpotrichoides on wheat seedlings. Mycological Research 95, 385-397. Daniels, A, Papaikonomou, M.P., Dyer, P. S. and Lucas J. A (1995). Infection of wheat seedlings by ascospores of Tapesia yallundae: morphology of the infection process and evidence for recombination. Phytopathology 85, 918-927. Deacon, J. W. ( 1973 ). Pseudoparenchyma produced by Cercosporella herpotrichoides in culture. Transactions of the British Mycological Society 60, 537-545. Dean, R. A (1997). Signal pathways and appressorium morphogenesis. Annual Review of Phytopathology 35, 211-234. Defosse, L. (1966). Les premiers stages de !'infection du Cercosporella herpotrichoides Fron sur froment, orge, seigl et avoine. Bulletin du Recherche Agronomie de Gembloux 1, 561-569. Deighton, F. C. (1973). Studies on Cercosporella and allied genera IV. Cercosporella Sacc., Pseudocercosporella gen nov. and Pseudocercosporidium gen nov. Mycological Papers 133, 1-62.

252

J. A. LUCAS, P. S. DYER and T. D. MURRAY

de Ia Pefia, R.C. and Murray, T. D. (1994). Identifying wheat genotypes resistant to eyespot disease with a ~-glucuronidase-transformed strain of Pseudocercosporella herpotrichoides. Phytopathology 84, 972-977. de Ia Pefia, R. C., Murray, T. D. and Jones, S. S. (1996). Linkage relations among eyespot resistance gene Pch2, endopeptidase Ep-Alb, and RFLP marker Xpsrl21 on chromosome 7A of wheat. Plant Breeding 115, 273-275. de Ia Pefia, R. C., Murray, T. D. and Jones, S. S. (1997). Identification of an RFLP interval containingPch2 on chromosome 7AL in wheat. Genome 40, 249-252. Di San Lio, G. M., Hocart, M. J., Lucas, J. A and Peberdy, J. F. (1994) Overcoming vegetative incompatibility within and between pathotypes of Pseudocercosporella herpotrichoides by protoplast fusion. Mycological Research 98, 653-659. Dyer, P. S. and Lucas, J. A (1995). Incidence of apothecia of Tapesia yallundae at set-aside sites in England and sensitivity of the ascospore offspring to the fungicides benomyl and prochloraz. Plant Pathology 44, 796-804. Dyer, P. S., Ingram, D. S. and Johnstone, K. (1992). The control of sexual morphogenesis in the Ascomycotina. Biological Reviews 61, 421-458. Dyer, P. S., Nicholson, P., Rezanoor, H. N., Lucas, J. A and Peberdy, J. F. (1993). Two-allele heterothallism in Tapesia yallundae, the teleomorph of the cereal eyespot pathogen Pseudocercosporella herpotrichoides. Physiological and Molecular Plant Pathology 43, 403-414. Dyer, P. S., Bateman, G. L., Lucas, J. A and Peberdy, J. F. (1994a) Seasonal development of apothecia of the cereal eyespot pathogen Tapesia yallundae on straw stubble in the UK. Annals ofApplied Biology 125, 489-500. Dyer, P. S., Papaikonomou, M.P., Lucas, J. A and Peberdy, J. F. (1994b ). Isolation of R-type progeny of Tapesia yallundae from apothecia on wheat stubble in England. Plant Pathology 43, 1039-1044. Dyer, P. S., Nicholson, P., Lucas, J. A and Peberdy, J. F. (1996). Tapesia acufonnis as a causal agent of eyespot disease of cereals and evidence for a heterothallic mating system using molecular markers. Mycological Research 100, 1219-1226. Dyer, P. S., Bowyer, P., Lucas, J. A and Peberdy, J. F. (1997). Cloning of a putative mating-type gene from the Discomycete fungus Tapesia yallundae. In "Program, Nineteenth Fungal Genetics Conference", p. 49. Asilomar, University of California. Dyer, P. S., Lucas, J. A and Peberdy, J. F. (1998). Genetics of resistance to sterol demethylation (DMI) fungicides in the cereal eyespot pathogen Tapesia yallundae. In "Seventh International Congress of Plant Pathology", abstract.5.5.26. British Society for Plant Pathology, Edinburgh. Farr, D. F., Bills, G. F., Chamuris, G. P. and Rossman, A Y. (1989). "Fungi on Plants and Plant Products in the United States". APS Press, St Paul, MN. Figliuolo, G., Jones, S. S., Murray, T. D. and Spagnoletti Zeuli, P. L. (1998). Characterization of tetraploid wheat germplasm for resistance to Pseudocercosporella herpotrichoides, cause of eyespot disease. Journal of Genetic Resources and Crop Evolution 44, 47-56. Fitt, B. D. L. (1992). Eyespot of cereals. In "Plant Diseases of International Importance: Volume I, Diseases of Cereals and Pulses" (U. S. Singh, A N. Mukhopadhyo, J. Kumar and H. S. Chaube, eds), pp. 336-354. Prentice-Hall, Englewood Cliffs, NJ. Fitt, B. D. L., Goulds, A and Polley, R. W. (1988). Eyespot (Pseudocercosporella herpotrichoides) epidemiology in relation to prediction of disease severity and yield loss in winter wheat: a review. Plant Pathology 37, 311-328.

TAPES/A SPP. AND EYESPOT DISEASE OF CEREALS

253

Fitt, B. D. L., Goulds, A., Hollins, T. W. and Jones, D. R. (1990). Strategies for control of eyespot (Pseudocercosporella herpotrichoides) in UK winter wheat and winter barley. Annals of Applied Biology 117, 473--486. Frei, P. and Gindrat, D. (1995). Morphological diversity in cultivation, carbendazim sensitivity and pathogenicity of Tapesia yallundae ( anamorph, Pseudocercosporella herpotrichoides ). Canadian Journal of Botany 73, 1379-1384. Frei, U. and Wenzel, G. (1993). Differentiation and diagnosis of Pseudocercosporella herpotrichoides (Fron) Deighton with genomic DNA probes. Journal of Phytopathology 139, 239-237. Fron, M.G. (1912). Contribution a I' etude dele maladie du 'piet noir des cereales' ou 'maladie du pietin'. Annales de la Science Agronomique, France. IV, 1. Furuya, H. and Matsumoto, T. (1996). Development of eyespot disease in winter wheat in a north-western region of Honshu Island, Japan. Annals of the Phytopathological Society of Japan 62, 194-198. Gac, M. L., Montfort, F., Cavelier, N. and Sailland, A. (1996). Comparative study of morphological, cultural and molecular markers for the characterization of Pseudocercosporella herpotrichoides isolates. European Journal of Plant Pathology 102, 325-337. Gardner, E. J., Simmons, M. J. and Snustad, D.P. (1991 ). "Principles of Genetics", 8th edition. Wiley, New York. Glynne, M. D. (1952). Production of spores by Cercosporella herpotrichoides. Transactions of the British Mycological Society 36, 46-51. Goulds, A. and Fitt, B. D. L. (1991) The development of eyespot on stems of winter wheat and winter barley in crops inoculated with W-type orR-type isolates of Pseudocercosporella herpotrichoides. Journal of Plant Diseases and Protection 98, 490-502.

Greuter, W., Burdet, H. M., Chaloner, W. G., Demoulin, V., Grolle, R., Hawksworth, D. L., Nicolson, D. H., Silva, P.C., Stafleu, F. A., Voss, E. G. and McNeill, J. (1988). "International Code of Botanical Nomenclature, Adopted by the Fourteenth International Botanical Congress, Berlin, July-August 1987 (Regnum Vegetabile 118)". Koeltz Scientific Books, Konigstein. Hocart, M. 1., Lucas, J. A. and Peberdy, J. F. (1990). Resistance to fungicides in field isolates and laboratory induced mutants of Pseudocercosporella herpotrichoides. Mycological Research 94, 9-17. Hollins, T. W., Scott, P.R. and Paine, J. R. (1985) Morphology, benomyl resistance and pathogenicity to wheat and rye of isolates of Pseudocercosporella herpotrichoides. Plant Pathology 34, 369-379. Howard, R. J. and Valent, B. (1996). Breaking and entering: host penetration by the fungal rice blast pathogen Magnaporthe grisea. Annual Review of Microbiology 50,491-512. Howard, R. J., Ferrari, M.A., Roach, D. A., and Money, N. P. (1991). Penetration of hard substrates by a fungus employing enormous turgor pressures. Proceedings of the National Academy of Sciences of the USA 88, 11281-11284. Hunter, T. (1989). Occurrence of Tapesia yallundae, the teleomorph of Pseudocercosporella herpotrichoides, on unharvested wheat culms in England. Plant Pathology 38, 598-603. Jahier, J., Tanguy, A.M. and Doussinault, G. (1989). Analysis of the level of eyespot resistance due to genes transferred to wheat from Aegilops ventricosa. Euphytica 44, 55-59. Jenkyn, J. F., Gutteridge, R. J. and Jalaluddin, M. (1994). Straw disposal and cereal diseases. In "Ecology of Plant Pathogens" (J.P. Blakeman and B. Williamson. eds), pp. 285-300. CAB International, Wallingford.

254

J. A. LUCAS, P. S. DYER and T. D. MURRAY

Johnson, R. (1992). Past, present and future opportunities in breeding for disease resistance, with examples from wheat. Euphytica 63, 3-22. Jones, C. J., Edwards, K. J., Castaglione, S., Winfield, M. 0., Sala, F., van de Wiel, C., Bredemeijer, G., Vosman, B., Matthes, M., Daly, A., Brettschneider, R., Bettini, P., Buiatti, M., Maestri, E., Malcevschi, A., Marmiroli, N., Aert, R., Volckaert, G., Rueda, J., Linacero, R., Vazquez, A. and Karp, A. (1997). Reproducibility testing of RAPD, AFLP and SSR markers in plants by a network of European laboratories. Molecular Breeding 3, 381-390. Julian, A. M. and Lucas, J. A. (1990). Isozyme polymorphism in pathotypes of Pseudocercosporel/a herpotrichoides and related species from cereals. Plant Pathology 39, 178-190. Julian, A.M., Hardy, J. E. and Lucas, J. A. (1994a). Cultural variability in the cereal eyespot pathogen Pseudocercosporella herpotrichoides. Mycological Research 98, 396-402. Julian, A. M., Hardy, J. E. and Lucas, J. A. (1994b). The induction and characterisation of isolates of Pseudocercosporella herpotrichoides with altered sensitivity to the fungicide prochloraz. Pesticide Science 41, 121-128. King, A. C. (1990). First record of Tapesia yallundae as the teleomorph of Pseudocercosporella herpotrichoides var. acufonnis, and its occurrence in the field in the Federal Republic of Germany. Plant Pathology 39, 44-49. King, A. C. (1991 ). Obsetvations of Tapesia yallundae and the cultural phenotypes of their progeny. Plant Pathology 40, 367-373. King, J. E. and Griffin, M. J. (1985). Sutvey of benomyl resistance in Pseudocercosporella herpotrichoides on winter wheat and barley in England and Wales in 1983. Plant Pathology 34, 272-283. Lange-de Ia Camp, M. (1966). Die Wirkungsweise von Cercosporella herpotrichoides Fron., dem Erreger der Halmbruchkrankheit des Getreides. II. Agressivitat des Erregers. Phytopathologische Zeitschrift 56, 155-190. Law, C. N., Scott, P.R., Worland, A. J. and Hollins, T. W. (1976). The inheritance of resistance to eyespot (Cercosporella herpotrichoides) in wheat. Genetic Research Cambridge 25, 73-79. Leroux, P. and Gredt, M. (1997). Evolution of fungicide resistance in the cereal eyespot fungi Tapesia yallundae and Tapesia acufonnis in France. Pesticide Science 51, 321-327. Leroux, P., Marchegay, P., Migeon, J. L. and Maumene, C. (1994). Resistance to inhibitors of sterol C-14 demethylation in the cereal eyespot fungus Pseudocercosporella herpotrichoides. In "Fungicide Resistance, BCPC Monograph No. 60" (S. Heaney, D. Slawson, D. W. Hollomon, M. Smith, P. E. Russell and D. W. Parry, eds), pp. 117-120. British Crop Protection Council, Farnham. Leslie, J. F. (1991). Mating populations in Gibberella fujikuroi (Fusarium section Liseola). Phytopathology 81, 1058-1060. Leung, H. and Taga, M. (1988). Magnaporthe grisea (Pyricularia species), the blast fungus. Advances in Plant Pathology 6, 175-188. Lind, V. (1992). Measuring the severity of eyespot disease caused by Pseudocercosporella herpotrichoides (Fran) Deighton in wheat cultivars at different growth stages. Plant Breeding 108, 202-209. Lind, V., Ziichner, S., Spanakakis, A. and Thiele, A. (1994). Analyses of interactions of wheat cultivars in tests for resistance to Pseudocercosporella herpotrichoides. Plant Breeding 113, 272-280. Lucas, J. A. (1995). Cereal management and the epidemiology of stem-base diseases. In "A Vital Role for Fungicides in Cereal Production" (H. G. Hewitt, D.

TAPES/A SPP. AND EYESPOT DISEASE OF CEREALS

255

Tyson, D. W. Hollomon, J. M. Smith, W. P. Davies and K. R. Dixon, eds), pp. 95-104. BIOS Scientific, Oxford. Maia, N. (1967). Obtention de bles tendres resistants au pietin-verse par croisements interspecifiques bles xAegilops. Comptes Rendus de l'Academie d'Agriculture de France 53, 149-154. Mbwaga, A.M., Menke, G. and Grossman, F. (1997). Investigations on the activity of cell wall-degrading enzymes in young wheat plants after infection with Pseudocercosporella herpotrichoides (Fron) Deighton. Journal of Phytopathology 145, 123-130. Money, N. P. and Howard, R. J. (1996). Confirmation of a link between fungal pigmentation, turgor pressure, and pathogenicity using a new method of turgor measurement. Fungal Genetics and Biology 20, 217-227. Money, N. P., Caesar-Tonthat, T.-C., Frederick, B. and Henson, J. M. (1998). Melanin synthesis is associated with changes in hyphopodial turgor. permeability, and wall rigidity in Gaeumannomyces graminis var. graminis. Fungal Genetics and Biology 24, 240-251. Moreau, J. M. and Maraite, H. (1995). Bipolar heterothallism in Tapesia yallundae for the two varieties of the anamorph Pseudocerco,\porella herpotrichoides. Mycological Research 99, 76-80. Moreau, J. M. and Maraite, H. (1996). Evidence for a heterothallic mating system in Tapesia acuformis using benomyl sensitivity and esterase isoenzyme profiles. Mycological Research 100, 1227-1236. Moreau, J. M., Van Schingen, J. C. and Maraite, H. (1989). Detection of Tapesia yallundae, the teleomorph of Pseudocercosporella herpotrichoides on wheat stubbles in Belgium. Mededlingen van de Faculteit Landbouwwetenschappen Rijksuniversiteit (Gent) 54, 555-559. Mueller, E., Bailey, A., Renwick, A. and Bowyer, P. (1998). Cloning and functional characterization of ornithine decarboxylase in Tapesia yallundae. In "Seventh International Congress of Plant Pathology VII". Abstract 1.10.16. British Society for Plant Pathology, Edinburgh. Murray, T. D. (1996). Resistance to benzimidazole fungicides in the cereal eyespot pathogen Pseudocercosporella herpotrichoides in the Pacific NorthWest. Plant Disease 80, 19-23. Murray, T. D. and Bruehl, G. W. ( 1983). Role of the hypodermis and secondary cell wall thickening in basal stem internodes in resistance to strawbreaker foot rot in winter wheat. Phytopathology 73, 261- 268. Murray, T. D. and Ye, H. (1986). Papilla formation and hypersensitivity at penetration sites and resistance to Pseudocercosporella herpotrichoides in winter wheat. Phytopathology 76, 737-744. Murray, T. D., de la Pefta, R. C., Yildirim, A. and Jones, S. S. ( 1994). A new source of resistance to Pseudocercosporella herpotrichoides, cause of eyespot disease of wheat, located on chromosome 4V of Dasypyrum villosum. Plant Breeding 113. 281-286. Nicholson, P. and Rezanoor, H. N. (1994). The usc of random amplified polymorphic DNA to identify pathotype and detect variation in Pseudocercosporella herpotrichoides. Mycological Research 98, 13-21. Nicholson, P, Rezanoor, H. N. and Hollins, T. W. (1991a). Occurrence of Tapesia yallundae apothecia on field and laboratory inoculated material and evidence for recombination between isolates. Plant Pathology 40, 626-634. Nicholson, P., Hollins, T. W., Rezanoor, H. N. and Anamthawat-Jonsson, K. (1991b). A comparison of cultural, morphological and DNA markers for the classification of Pseudocercosporella herpotrichoides. Plant Pathology 40, 584-594.

256

J. A LUCAS, P. S. DYER and T. D. MURRAY

Nicholson, P., Rezanoor, H. N. and Hollins, T. W. (1993). Classification of a worldwide collection of isolates of Pseudocercosporella herpotrichoides by RFLP analysis of mitochondrial and ribosomal DNA and host range. Plant Pathology 42, 58-66. Nicholson, P., Rezanoor, H. N. and Hollins, T. W. (1994). The identification of a pathotype-specific DNA probe for the R-type of Pseudocercosporella herpotrichoides. Plant Pathology 43, 694-700. Nicholson, P., Dyer, P. S., Rezanoor, H. N., Lucas, J. A, Walkowaik-Cagara, I. and Hollins, T. W. (1995). Mating relationships between different pathotypes of Tapesia yal/undae, the teleomorph of the eyespot pathogen Pseudocercosporella herpotrichoides. Mycological Research 99, 457-465. Nicholson, P., Rezanoor, H. N., Simpson, D. R. and Joyce, D. (1997). Differentiation and quantification of the cereal eyespot fungi Tapesia yal/undae and Tapesia acuformis using a PCR assay. Plant Pathology 46, 842-856. Nirenberg, H. I. (1981). Differenzierung der erreger der Halmbruchkrankheit. I. Morphologic. Zeitschrift fUr Pflanzenkrankheiten und Pflanzenschutz 88, 241-248. Oort, A J. P. (1936). De oogvlekkenziekte van de granen, veroorzaakt door Cercosporel/a herpotrichoides Fron. Tijdschr. Plziekt. 42, 179-234 (English summary). Papaikonomou, M. and Lucas, J. A (1994). RAPD analysis of 515 eyespot isolates from field sites in Europe. "Brighton Crop Protection Conference Proceedings- Pests and Diseases", pp. 481-486. BCPC, Farnham. Perpetua, N. S., Kubo, Y., Yasuda, N., Takano, Y. and Furusawa, I. (1996). Cloning and characterization of a melanin biosynthetic THRJ reductase gene essential for appressorial penetration of Colletotrichum lagenarium. Molecular Plant-Microbe Interactions 9, 323-329. Perry, K. E., and Hocart, M. J. (1998). A role for melanins in pathogenicity of the eyespot fungus? In "Seventh International Congress of Plant Pathology", Abstract 1.8.39. British Society for Plant Pathology, Edinburgh. Polley, R. W. and Thomas, M. R. (1991). Surveys of diseases of winter wheat in England and Wales, 1976-1988.Annals ofApplied Biology 119, 1-20. Polley, R. W. and Turner, J. A (1995). Surveys of stem base diseases and fusarium ear diseases in winter wheat in England, Wales and Scotland, 1989-1990. Annals ofApplied Biology 126, 45-59. Poupard, P., Simonet, P., Cavelier, N. and Bardin, R. (1993). Molecular characterization of Pseudocercosporel/a herpotrichoides isolates by amplification of ribosomal DNA internal transcribed spacers. Plant Pathology 42, 873-881. Poupard, P., Grare, S., Cavelier, N. and Lind, V. (1994). Development of Pseudocercosporella herpotrichoides (Fron) Deighton var. herpotrichoides and var. acuformis on wheat plants measured by ELISA Journal of Phytopathology 140, 301-311. Priestley, R. A, Dewey, F. M., Nicholson, P. and Rezanoor, H. N. (1992). Comparison of isoenzyme and DNA markers for differentiating W -pathotype, R-pathotype and C-pathotype of Pseudocercosporella herpotrichoides. Plant Pathology 41, 591-599. Robbertse, B., Campbell, G. F. and Crous, P. W. (1995). Revision of Pseudocercosporella-like species causing eyespot disease of wheat. South African Journal of Botany 61, 43-48. Robbertse, B., Crous, P. W. and Holz, B. (1994). Tapesia yallundae collected from wheat stubble in South Africa. Mycopathologia 125, 23-28.

TAPES/A SPP. AND EYESPOT DISEASE OF CEREALS

257

Robbertse, B., Holz, G. and Crous, P. W. (1996). Sensitivity of South African Ramulispora herpotrichoides isolates to carbendazim and ergosterol biosynthesis inhibitors. Plant Pathology 45, 270-275. Sanderson, F. R. and King, A. C. (1988). Field occurrence of Tapesia yallundae, the teleomorph of Pseudocercosporella herpotrichoides. Australasian Plant Pathology 17,20-21. Scott, P. and Hollins, T. W. (1980). Pathogenic variation in Pseudocercosporella herpotrichoides.Annals ofApplied Biology 94, 297-300. Sindberg, S., Munk, L. and Schulz, H. (1994). Tapesia yallundae, the teleomorph of Pseudocercosporella herpotrichoides observed for the first time in Denmark. Zeitschrift fUr Pflanzenkrankheiten und Pflanzenschutz 101, 316-320. Snyder, W. C., Georgopoulos, S. G., Webster, R. K. and Smith, S. N. (1975). Sexuality and genetic behaviour in the fungus Hypomyces (Fusarium) solani f. sp. cucurbitae. Hilgardia 43, 161-185. Soleimani, M. J., Deadman, M. L. and McCartney, H. A. (1996). Splash dispersal of Pseudocercosporella herpotrichoides spores in wheat monocrop and wheat-clover bicrop canopies from simulated rain. Plant Pathology 45, 1065-1070. Soulie, M.-C., Vian, B., Guillot-Salomon, T. and Curie, M. (1985). Host-parasite interactions during infection by Cercosporella-herpotrichoides agent of eyespot: morphology of the parasite and wall ultrastructure of susceptible and resistant hosts. Canadian Journal of Botany 63, 851-858. Sprague, R. ( 1934 ). The association of Cercosporella herpotrichoides with the Festuca consociation. Phytopathology 24, 669-676. Sprague, R. (1936). Relative susceptibility of certain species of Gramineae to Cercosporella herpotrichoides. Journal ofAgricultural Research 53, 659-670. Sprague, R. and Fischer, G. W. (1952). Check list of the diseases of grasses and cereals in the western United States and Alaska. In "State College of Washington, Circular 194". Takano, Y., Kubo, Y., Kawamura, C., Tsuge, T. and Furusawa, I. (1997). The Alternaria alternata melanin biosynthesis gene restores appressorial melanization and penetration of cellulose membranes in the melanindeficient albino mutant of Colletotrichum lagenarium. Fungal Genetics and Biology 21, 131-140. Takeuchi, T. and Kuninaga, S. (1994) Genetic-relationships in Pseudocercosporella herpotrichoides determined from DNA relatedness. Mycological Research 98, 1059-1064. Takeuchi, T. and Kuninaga, S. (1996). Determination of relationships in Pseudocercosporella herpotrichoides by analysis of mitochondrial DNA. Mycological Research 100, 693-701. Thomas, D., Maraite, H. and Boutry, M. (1992). Identification of rye-type and wheat-type of Pseudocercosporella herpotrichoides with DNA probes. Journal of General Microbiology 138, 2305-2309. Uslu, E., Miller, T. E., Rezanoor, N.H. and Nicholson, P. (1998). Resistance of Dasypyrum villosum to the cereal eyespot pathogens Tapesia yallundae and Tapesia acuformis. Euphytica 103, 203-209. Van Etten, H. D. and Kistler, H. C. (1988). Nectria haematococca mating populations I and VI. Advances in Plant Pathology 6, 175-188. Vincent, A., Panchet, J. and Koller, J. (1952). Recherche de varietes de bles tendres peu sensibles au pietin-verse: resultats preliminaires. Annates Amelioration de Plantes 2, 459-472. Wallwork, H. (1987). A Tapesia teleomorph for Pseudocercosporella herpotrichoides, the cause of eyespot of wheat. Australasian Plant Pathology 16, 92-93.

258

J. A. LUCAS, P. S. DYER and T. D. MURRAY

Wallwork, H. and Spooner, B. (1988). Tapesia yallundae- the teleomorph of Pseudocercosporella herpotrichoides. Transactions of the British Mycological Society 91, 703-705. Yildirim, A, Jones, S. S., Murray, T. D., Cox, T. S. and Line, R. F. (1995). Resistance to stripe rust and eyespot diseases of wheat in Triticum tauschii. Plant Disease 79, 1230-1236. Yildirim, A, Jones, S. S. and Murray, T. D. (1998). Mapping a gene conferring resistance to Pseudocercosporella herpotrichoides on chromosome 4V of Dasypyrum villosum in a wheat background. Genome 41, 1-6. Yoder, 0. C., Valent, B. and Chumley, F. (1986). Genetic nomenclature and practice for plant pathogenic fungi. Phytopathology 16, 383-385.

AUTHOR INDEX

Numbers in italics refer to pages o n which full refere nces are listed

B

A Abe, H. 205, 208 Acker, G. 3, 34 Adiputra, I. G . K. 182, 208 Aert, R. 230, 254 Agosti, R. D. 47, 124 Ahlich, K. 6, 29 A hmad, M. 72, 123 Ainsworth, C. 171,209 Akashi, T. 173, 214 Akita, S. 142, 157 Albertini, C. 249, 250 Alexandre, J . 56, 115 Alikhan, Q. R. 7, 9, 30 Allaway, W. G. 62, 63, 118 Allen, G. J. 54, 56, 115, 125 Allen, R. D. 202,216 Allen, R . J. 202,208 Alscher, R. G. 197, 216,220 Alston, A. M . 153, I 56 Altmann, T. 168, 188, 189, 194, 213 Amancio, S. 195, 196,208,210 Anamthawat-Jonsson, K. 228, 229, 255 Anderson, C. J . 143,154 A nderson , J. W.l82,208,211,221 Anse ll, D. J. 236,250 Anson, A. E. 5. 28, 31 Aotsuka, S. 173, 222 Applewhite, P. B. 45, 62, 127 Archer, R . .148, 154 Arihara, J. 135, 154 Arrillaga, I. 207, 208 Artis, P. 109, 116 Arz, H. E. 169, 185, 208 Asanuma, W. 165, 175, 180, 222 Asard, H . 70, I 15 Ashikari, T. 171, 202, 223 Aslund, F. 186, 209 Asprey, G. F. 47, I26 Assefa, S. 247, 250 Assman,S. M.41, 55, 102,115,122, 123 Awazuhara, M. 202, 211 Aylor, D. E. 12, 29

Bailey, A. 249, 255 Bailey, P. H. J. 141, 154 Bairoch, A. 164, 208 Baker, C. J. 151, 154 Baii, J. 152,156 Ball, N. G. 68, 80, 115 Balian~. C. L. 71, 115 Banks, J. R. 143, 144, 146, 149, 155 Barbier-Brygoo, H . 61 , 129, 179, 2ll Bardin, R . 231 , 256 Bare, C. 51, 125 Barklund, P. 5, 13, 29 Ba rley, K. P. 1968 135, 157 Barroso, C. 168, 189, 194, 196, 197,204,

208, 212 Bassett, E. N. 25, 29 Bateman, G . L. 236,237, 238, 239, 248,

250, 252 Batschauer, A. 72, I23 Beachey, R. N. 202,208, 2I I , 216 Beck, D. L. 135, 154 Becker, D. 52, /20 Begg, J. E. 41, 74, 75, 98, Ill, 115, I22 Belcher, A. 163, 170. 181 , 194, 195, 196,

221 Belles, J. M. 187,207, 212.217 Bennett, A. B. 40, 131 Benschop, M.64, 116 Berendt, U. 186,209 Berg, V. S. 91, 92, 96, 97. 101, 102, Ill ,

115, 117, 131 Bergfeld , R. 67, 125 Bergmann, L. 1979 183, 2I8 Bernasconi, P. 7 1, I I 7 Bernier, F. 202,208, 216 Bernstein, M. E. 5, 6. 12, 17, 18, 29 Berry, P. 151, 154 Bertauche, N . 198,212 Bethier, S. 148, 149,156 Bettini, P 230, 254 Sewell, M.A. 54, 56, 125 Bialczyk, J. 51. 103, I I5

260

AUTHOR INDEX

Bick, J. A. 160, 186, 209 Bilderback, D. E. 65,115 Bills, G. F. 3, 29, 245, 252 Bilsborrow, P. E. 183, 223 Birchmore, R. J. 237, 238, 250 Bi~egger.~.3,6, 18,20,21,25,30 Bjorkman, 0 . 37, 59, 63, 98, 11zz Buckley, N. G. 10, 33 Buiatti, M. 230, 254 Biinning, E. 42, 116 Burdet, H. M. 234, 253 Burkholder, P. K. 59, 63, 116, 117 Burns, J. A. 1973 191,214 Bush, H. 54, 62, 20 Butin, H. 7, 26, 31 Byers, M. 162, 210

c

Caboche,M. 196,214 Cabral, D. 13, 30 Cadle, M. M. 246, 247, 250, 251 Caesar-Tontbat, T.-C. 242,255 Calhoun, L.A. 21, 22,30 Callan, B. E. 2, 3, 24, 27, 28,30 Camara, B. 173, 183,219 Camattari, G. 51, 119 Cambridge, M. 182, 194,212 Campalans, A. 204,210 Campbell, A. 243, 251 Campbell, D. J. 143, 154 Campbell, E. I. 166, 167, 185, 186, 187, 188, 193, 194,202,212,222 Campbell, G. F. 229, 234, 238,251,256 Campbell, N. A. 44, 47, 50,117 Carroll, F. E. 3, 4, 6, 7, 10, 11 , 12, 13, 17, 18, 19,20,30,33 Carroll, G . 2, 3, 4, 5, 6, 7, 8, 11, 12, 13, 14, 16, 17, 18, 19,2~29,30,32,33 Cashmore, A. R. 72,123 Castaglione, S. 230, 254 Castle, S. L. 162,210 Catalano, C. 196,215 Cathala, N. 166, 171, 192, 193, 205,212,213, 216 Caubergs, R.J. 70, 115 Causier, B. E. 53, 124 Cavelier, N. 227, 228,231, 244, 248,251,253, 256 Cejudo,F.J. 189,197, 198,204,208,212 Cerana, R. 51, 119 Cerkauskas, R. F. 2, 11 , 23, 24, 26, 33 Cervantes, M. 165, 183, 206,216 Chaloner, W. G. 234, 253 Chamuris, G. P. 245, 252 Cbanway, C. P. 22, 33 Chapela, I. H. 2, 7, 8, 13, 26, 30, 34 Chefdor, F. 204, 216 Chen, Y. C. 167, 186, 192, 206, 209, 210, 218 Cbino,M. 162,194,196,202,211,213,215

Cho, M. H. 67, 117 Cbory, J. 103, 126 Chouquet,I. 148,154 Christie, J. M. 71, 72, 117 Chua, N.-H. 204, 218 Chumley, F. 232, 258 Churchill, K. A. 180, 210 Clare, R. 151, ]54 Clark, C. L. 3, 21, 30 Clarkson, D. T. 163, 170, 175, 180, 181, 182,194,195,196,208,210,212,214, 221 Clay, K.. 3, 20, 21, 30, 34 Cleland, R. E. 37, 104, 129 Cocciolone, S. M. 204, 214, 222 Coff, C. 244, 251 Cohen,M.86,12J Coillot, L. 47, 124 Colcombet, J. 179,211 Colombo, R. 51,119 Cook, A.M. 195,214 Cooper, R. M. 243, 251 Cosgrove, D. J . 37, 66, 67, 117, 128 Cote, G. G. 53, 54, 56, 60, 61, 62, 95, 102, 117,121,125, 127 Coulter, K. R. 200, 210 Coutinho, T. A. 25, 26, 34 Coutts, M.P. 141 , 142, 143, 144, 147, 148, 149,150, 152,154,156 Covitz 173 Cox, T. S. 246, 258 Craig, S. 162,217 Crain, R. C. 53, 54, 56, 60, 61, 62, 95, 117, 121,122,125,127 Creighton, N. 228,251 Crispino, J.D. 202,216 Cronlund, S. L. 94, 117 Crook,M.J. 142,143, 144,146,147,148, 149,150,152,154,155 Crosbie, T. M. 135,154 Crosland, A. R. 162,223 Crous,P. W.229,231,232,234,238,251, 256,257 Cunningham, P. C. 1965. 228, 245, 251 Curie, M. 239, 242, 257 Currah, R. S. 22, 31 Curtin, D. 160, 221

D d'Harlingue, A. 173, 183,219 Daly, A. 230, 254 Dandekar, T. 192, 220 Daniels, A, 235,237,238,239,240,241, 242,243,244,251 Darrah, L. L. 135,154 DaiWin, C. 37, 42, 43, 45, 58, 59, 110, 117 DatWin, F. 37, 42, 43, 45, 58, 110, 117 Da~en, H. H. A. 62,116

261

AUTHOR INDEX Davidian, J. C. 163, 166, 171, 180, 181, 182, 192,193,194,205,210,212,213,216 Davies, J. M. 55, 117 Davies, J.P. 202,203,210,223 Davis, G. 67, 122 Dawut, L. 72,123 Dayanandan, P. 37, 117 de Ia Peiia, R. C. 246, 249, 252, 255 de Vrije, T. 55, 125 Deacon,J. VV. 240,251 Deadman, M. L. 235, 238, 257 Dean, R. A. 241,251 Deerfield, D. VV. II 176,217 Defosse, L. 239, 251 Deighton, F. C. 227,251 Deluca, V. 185, 222 Demmig-Adams, B. 37,115 Demoulin, V. 234, 253 Department of the Environment, Transport and the Regions 161,210 Dessureault, M. 4, 16, 17,32 Dewey, F. M . 229, 256 Dexter, A. R. 153,156 Dhindsa, R. S. 197,211 Di San Lio, G . M. 248, 252 Dichtl, B. 207,211 Dietz, K. J. 180, 211 Digby,J.64,65,119 Diller, A. 167, 188,217 Diogo, E. 195, 196,208,210 Dittmer, H. J. 155 Oilton, R. A. 197,211 Dobranic, J . K. 7, 9, 30 Dobson, M . J. 248,250 Dolk, H . E. 1931 69, 117 Dominguez, J. R. 189,212 Donahue, R. 91, 92, 97, 102, 117 Donaubauer, E. 7, 26, 31 Donovan, L. S. 135, 155 Dort, J. B. V. 64,116 Dorworth, C. E. 2, 3, 24, 27, 28,30 Douce, R. .160, 167, 173, 180, 188, 191, 192, 193,211,216,217,218,219 Dougall, D. K. 195,218 Doussinault, G. 246, 253 Dreyfuss, M. 3, 6, 25, 31, 33 Drou~ M. 167,173,188,191,192,193,211, 216,219 Du Cros, D. L. 162,222 Dubetz, S. 98, 111, 117 Duparque, A. 142, 155 Dutton, L. 72, 123 Dyer, P. S. 231,232,234, 235,236, 237, 248, 249, 250, 251 ,252, 256

E Eating, P. M. 163, 170, 175, 181, 183, 194, 195,196,221 Eastham J. 111, 126

Eckerskorn, C. 180, 211 Edwards, K. J. 230, 254 Edwards, R. 197, 211 Eggum, B. 0. 162, 217 Ebert, D. L. 81, 89, 118 Ehleringer, J. 42, 69, 74, 98, 101, 102, 109, 110,111,112,118,119,124,130 Ehrenstein, G. 51, 125 Ellis, R. J. 103, I 18 Elzenga, J. T. M. 95, 104, 118, 128 Elzenga, T. M. 105, 116 Eng, B. H . 164,219 Engler, J . 0.165, 221 Ennos, A. R. 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 146, 147, 148, 149, 150,151,152, 153, 154,155 Epstein, E. 1956 163, 216 Epton, H. A. S. 227,250 Erath, F. 94,118 Ernst, VV. H. 0. 180, 211 Esashi, Y. 191, 216 Espinosa-Garda, F. J. 7, 9, 23,31 Evans,E.J. 162,183,223 Evans,L. T.62,63,118 Evans, M. L. 67, 125

F Fard, S. 164, 219 Farr, D. F. 245, 252 Fehrmann, H. 247, 250 Fell, D. 191, 192,211, 220 Pelle, H. H. 55, 123 Fenn, P. 25, 29 Fernando, A. A. 22,31 Ferrari, M. A. 242, 253 Field, C. 108, 131 Figliuolo, G. 246, 252 Filner, P. 70, 127 Findlay, J. A. 21, 22,30 Firbas, F. 46, 120 Firn, R. D. 40, 42, 64, 65 ,118, 119 Fischer, C. 47, 52, 53, 124 Fischer, G. W. 245, 257 Fischer, K. 180, 211 Fischer-Schliebs, E. 55,118 Fisher, D. B. 162, 183, 209 Fisher, F. J. F . 81, 89, 118 Fisher, P. J. 4, 5, 6, 8, 11, 18, 28, 31, 32,33 Fisher, P.M. 81, 118 Pitt, B. D . L. 9, 11, 12, 31, 227,238, 244, 252,253 Fitter, A. H. 138, 140, 141, 144, 148, 152, 155, /56 Fitzgerald, M.A. 182, 211 Flach, D. 44, 50,119,124, 129 Fleurat-Lessard, P. 54, 61, 127 Fliigge, U. I. 180, 211 Foglia, R. 167, 188,217 Fondeville, J. C. 62, 63, 118

262

AUTHOR INDEX

Forde, B. G. 196, 215 Foroni, C. 170, 171, 194, 195, 201, 209 Forseth, I. 74, 94, 98, 109, 110, 111, 112,117,

118,119 Foster, R. G. 59, 127 Fouere,A. 142,155 Fournier,~.

Giraudat, J. 198, 204,212,216 Giromini, L. 51,119 Gisbert, C. 207,208 Gisselmann, G. 167, 169, 185, 187, 208,

209

148,154

Fox, D. 51,125 Foyer, C. H. 197, 217, 221 Frachisse, J. ~. 179,211 Frampton, C.~. 10,34 Frankland, B. 37, 119 Franssen,J. ~.64,65,119 Frederick, B. 242, 255 Frei, P. 243, 253 Frei, U. 229, 253 Freudling, C. 50, 52, 119 Friedrich, J. W. 196,211 Frolov,I. 168,188,192,194,213 Fron, ~.G. 231,253 Fu, Q.A. 101, 102, 111, 112, 119 Fu, I'. H. 163,200,211,214 Fujiwara, T. 162, 194, 196,202,211,213,215,

216 Fukuda, S. 72, 126 Fukushima, R. 173, 201, 220 Fullington, J. G. 162,211,222 Furuhashi, A. 202, 211 Furusawa,I.242,256,257 Furuya,H.226,253 Furuya,~.

103,126 Futai, K. 4, 5, 7, 8, 12, 16, 17, 18, 22, 31,32

G Gac, ~. L. 231,253 Gaff, D. F. 164, 173, 175, 217 Gage, D. A. 162, 183, 209 Gakiere, B. 160,218 Galen, C. 73, 129 Galston, A. W. 44, 45, 59, 62, 63, 120, 127 Gange, A. C. 22, 26, 27,31 Garber, R. C. 44,117 Garber, R. G. 47, 50, 117 Garciadeblas, B. 187, 207, 218 Gardner, E. J. 234, 253 Garg, J. 164, 219 Gartner, B. L. 148, 155 Gaxiola, R. 207, 212 Geballe, G. T. 45, 62, 127 Geiger, H. H. 135,156 Georgopoulos, S. G. 237, 257 Gianello, R. 164, 173, 175, 217 Gibson, T. J. 178, 222 Gilbert, S. ~. 182, 194,212 Gilboa, S. 92, 97,128 Gilkes, A.F. 196 Gil-Mascarell, R. 187, 207,208,212 Gilroy, S. 54, 119 Gindrat, D. 243, 253

Glaser, H.-U. 207, 212 Glass, A. D.~. 170, 182, 195,215,222 Gleed, D. 64, 119 Glendening, T. M. 185, 212 Glynne, ~.D. 239, 253 Goday, A. 204, 210 Godfrey, ~. J. S. 10, 34 Goh, C.-H 101, 119 Goldschmidt, E. E. 187, 212 Gomez, L.A. 61, 63, 119 Goodman, A.M. 144, 148, 152, 153,155 Gorton, H. L. 42, 46, 103, 119, 120 Gosti, F. 198, 212 Gotoh, K. 90, 99, 102, 111, 127 Gotor, C. 168, 189, 194, 196, 197, 198,

204,208,212 Gottwald, J. 67, 124 Gouda, A. 188, 193, 201,218 Goulds, A. 227, 244, 252, 253 Grabov, A. 50,116 Gradmann, D. 50, 52, 119 Graf, F. 6, 18, 31 Graner, A. 244, 251 Grare, S. 244, 256 Gredt,~.238,248,249,250,254

Greenman, J. ~. 98, 102, 110,131 Greuter, W. 234, 253 Griffin, J. 151, 154 Griffin, M. J. 227, 254 Griffith, G. S. 4, 14, 19, 24, 25, 26,30 Grignon, C. 163, 166, 182, 192, 193, 205,

210,213,216 Grill, E. 204, 217 Grimshaw, C. 142, 146, 147, 155 Grolle, R. 234, 253 Gross, A. 180, 211 Grossman, A. 202,203,210,223 Grossman, F. 243, 255 Gruissem, W. 173, 213

Guern,J.6l,129, 179,211 Guerrier, D. 204, 216 Guggino, S. E. 59, 63, 127 Guillot-Salomon, T. 239, 242, 257 Guiltinan, M. J. 204,212 Guri, A. 197,212 Gutierrez-Alcala, G. 189, 212 Gutierrez-Marcos, J. F. 166, 167, 185, 186, 187,188,193,194,202,212,222 Gutteridge, R. J. 236,253 Guzman, E. 188,215

H Haag-Kerwer, A. 170, 171, 184, 185, 197,

213,220

263

AUTH OR INDEX Haberland!, G. 68, 80, 120, 147, 155 H able, W. 67, 124 H ader, D-.P. 38, 120 Haggerty, D. A. 164,219 Hagiwara, S. 54, 62, 106, 127, 128 H agmann, L. 13,30 Hall, A. E. 98, 109, 112, 128 Hallauer, A. R. 135, 156 H allma nn , J. 20, 31 H a lmschlagcr. Yon E. 7, 26, 31 H amili, J . D.164, 172,175, 21 7 H ammond, S.D. 111, 118 Hammond-Kosack, K. 197, 216 H ampp, R . 47, 49, 94, 118, 124, 180, 212 Hangarter, R . P. 71, 103, 104, 123, 126 H anson, A. D. 162, 183, 209 Harada, E. 165, 193, 194, 195, 196, 202,223 H arder, R . 46, 120 H ardie, D. G . 203, 212 H arper, J. F. 55, 126 Harrison, K. R. 182,209 H art, J. W. 65, 70, 120,/27 Hase, T. 171, 202. 214,223 H asegawa, K. 37, 64, 116 Hashimoto. H. 173,222 Hata, K. 4, 5, 7, 8, 12, 16, 17, 18, 22,31, 32 H ata,S. 170,215 Hatzfeld, Y. 166, 171 , 192,205,213 H aupt, W. 38. 67, 116, 120 Hauschild, R . 168, 188, 192, 194,213 H averkamp, T . 167, 186, 187, 209 H awkesford, M . J. 163, 164, 170, 191,209, 210,212,213.220, 221,222,223,224 Hawksworth, D. L. 234, 253 Hayami, J. 103, 120 Hayashi , H . 196,202,211,215 H ayes, A. J. 17,32 Hebard. F. V. 37, 117 Heber, U. 180,214 H ecker, D. 56, 123 Hedrich. R . 52. 54, 62, 120 Heins. L. 171. 185.218 Heiss. S. 170, 171, 184,185,197,213 Helander. M . J. 3, 9. 14, IS, 16, 17, 32 Helander, M . L. 3, 7, 15, 17, 32 Hell, R. 160, 167, 168, 187, 188, 192, 193, 194, 202,209, 213 H endricks, S. B. 62, 63, 118 Henry, M . 243. 251 He nson, J. M. 242,255 Herbert, T. J . 98, 120 H crschbach, C. 162, 183,209 Hesse, H . 168, 172, 188,189, 194, 213 H e therington, A. M . 54, 56, 101, 124 Heuchelin.S. I01 , 102,1 11,115 H iggins. D. G . 178.222 Higgins, T . J. Y . 162, 217 Hill, K. K. 188.2/5 Hillman. W . S. 59, 63, 120, 122

H innebush, A. G. 199, 213 H irai, M. Y. 162, 194, 196, 202, 2/J, 213. 215 H irano, H . 170, 189,206,219 Ho, T. H . D. 197,204,217,220 Hocart, M. J. 242, 245, 248, 249, 250. 252. 253,256 Hoff, T. 196, 214 Hofgen, R. 168, 171. 184, 188, 189, 194, 213,215 Ho hloch, C. 54, 60, 62, 124 H o1denrieder, 0 . 20, 32 Hollingdale, J. 8 1, 89, I 18 Hollins, T . W. 227,228,229,230,231, 232, 234,237,246,248, 253,254. 255, 256,257 Holmes, M.G. 59. 120 Holmgren, A. 186, 214 Holz, B. 231, 232, 238, 256. 257 Honda, C. 202, 211 Horemans, N. 70, IJ5 Horwitz, B. A . 72, 120 H osokawa, D. 205, 208 Howard, R. J. 240 , 242, 253, 255 Howarth, J. R. 168, 188, 193, 20 1,214 Hsiao, T. C. 92, 98, 101 , 102, 111 , //5 Hunter, T. 231.234, 253 Hutchinson, W. J. 164. 219 Hwang, J. U . 55, 120 Hwang, S. B. 192, 206.218

I

Ibrahim, R. K. 185, 207, 222 !chid a, A ..M. 50, 51 , 56. 94. 123 1deguchi, T. 171,214 Iglesias, A. 49, 120 li no, M. 66, 7 1, 120, 123 lmaizum i, T. 72, 126 Inoue, K. 173, 188, 193, 201,218,220 Inoue, Y. 142, 156 lnze, D . 197, 198, 216 Irvine. R. F. 55. 125 Irving, M.S. 44, 46. 47. 49, 50, 51. 89. 94. 96, 120 lshitani, M. 203,204, 214 lshizawa, K. 191,2/6 lsnard. A. D. 199.209 !wasa , K. 5 1.125 Iwasaki, T. 205, 208,214 lzawa, T . 204. 218

J

Jack, D. L. 164, 219 Jackson, P. J . 188,191,215, 219 Jacobso n, E. S. 200, 214 Jaffe, M. J. 62. 120 Jager, K. 160, 185, 187.220 Jahier, J. 246, 253 Ja in, A. 169, 214

264

AUTHOR INDEX

Jalaluddin, M. 236, 253 Janoudi, A.-K. 71, 120 Janse, B. J. H. 229, 238, 251 Jarai, G. 163, 214 Jeanmougin, F. 178, 222 Jcnkyn, J. F.236,253 Job, D. 160,188,193,211,218,219 Johnson, J. A. 7, 9, 17, 30,32 Johnson, R. 246, 254 Johnsson, A. 101, 128 Jones, C. J. 230,254 Jones, D. R. 227, 253 Jones, J.D. G. 191,216 Jones,S.S.246,241,251,252,255,25 8

Jones, W. N. 68, 121 Jorns, M.S. 12,123 Jouanin, L 197,217,221 Journeu, R. D. 44, 129 Joyce,D.230,244,246,247,256 Jui, P. 135, 155 Julian, A. M. 228, 231, 248, 254 Jurik, T. W. 69, 109,121,131

Kim,H.53,54,56,60,61,62,95,121 , 196, 2fJ2,2ll,215

Kim, J. 55, 120 Kim, S.-T. 72,123 Kimura, A. 175, 222 Kimura, N. 188, 193, 2{)1, 218 King, A. C. 231,234, 235, 236, 237,254, 257

King,J. E.221,254 Kinoshita, T. 53, 54, 101,119, 121, 128 Kirkwood, I. A. 15, 32 Kistler, H. C. 235, 257 Kiyosawa, K. 46, 121 Kjellberg, B. 73, 113, 121 Klein, W. H. 59, 120 Kleppinger.Sparace, K. F. 185,215 Kloek, M. 135, 155 Klonus, D. 166, 171, 183, 184,215 Knudsen, S. 202, 217 Kocsy, G. 197,215 Koinuma, K. 142,156 Koizumi, N. 165, 168, 193, 194, 195, 196, 202,217,223

K

Kaeser, H. 191, 214 Kado, R. T. 56,115 Kadota, A. 67, 103, 120, 125 Kagawa, T.68, 104,106,121 Kaiser, G. 180, 214 Kalkuhl, A. 54, 60, 62, 124 Kanegae, T. 72,126 Kanno, N. 186, 214 Kao, C.-Y. 204, 214 Karlsson, S. 73, 113, 121 Karmoker, J. L. 196, 214 Karp, A. 230, 254 Karsscn, C. M. 64,116, 198,215 Karun-Neumann, G. 61,122 Kasarda, D. D. 162, 211, 222 Kato, A. 142, 175,156,222 Kaubnan, P.B.37, 117 Kawamura, C. 242, 257 Kawashima, R. 98, 111, 121 Kehr, R. D. 2, 4, 5, 11, 14, 16, 17, 23, 24, 25, 32

Kelliher, F. M. 10, 34 Kemper, 0 . 182, 218 Kendall, A. C. 197,221 Kendrick, R. E. 61, 62, 11,115,116,129 Kertesz, M. A. 195, 214 Kestensson, I. 73, 113, 121 Ketter, J. S. 163,214 Kevan, P. G. 73, 113, 121 Kevern, T. C. 135, 156 Keys, A. J. 197, 221 Khurana, J. P. 71, 121 Kidou, N. 173,222 Kidou, S. 173, 222 Kigel, J. 104, 121

Koller, D. 43, 44, 45, 47, 48, 74, 76, 77, 78, 79, 81, 82, 83, 85, 86, 87, 88, 89, 92, 95,96,97,99, 101,102,103, 104, 106, 109,110,111,120,121,122,127,128, 130 Koller, J. 246, 257 Konjevic, R. 71, 120 Koorneef, M. 198,215 Kopriva, S. 196,215 Koprivova, A. 196, 215 Kouchi, H. 170, 215 Koukkari, W. L. 59, 63,120, 122

Kowalski, T. 2, 4, 5, 6, 7, 11, 13, 14, 16, 17, 18,23,24,25,2~29,32

Kredich, N.M. 192, 198, 199,215 Kricl W.-M. Kubo, Y. 242, 256, 257 Kubota, M. 102, 126 Kunert, K.-J. 197,217,221 Kuninaga,S.229,230,257 Kuntz, M. 173, 183, 219 Kurasawa, M. 191, 206, 219 Kushibi.ki, H. 1979 142,156 Kuske, C. R. 188, 197,215,219 Kusumi, T. 171, 202,223 Kuzmanoff, K. M. 67, 125

L Laflamme, G. 4, 16, 17, 32 Lagarias, J. C. 72,126 Lai, E. C. 164, 219 Lam, S.-L. 69, 80, 122 Lambers, H. 182, 194, 212

Laoe,S.J.28,32 Lang, A. P. G. 75, 122, 128 Lange-de la Camp, M. 228, 254

265

AUTHOR INDEX Langenheim, J. H. 7, 9, 23,31 Langley, D. 28, 32 Lappartient, A. G. 182, 195,215 Lassalles, J.P. 56, 115 Lavanchy, P. 186,209 Law, C. N. 246, 254 LePage, D. 228,25/ Lea, P.J. 196, 197, 215,221 Leaver, C. 197, 198, 216 Lebrun.~. 167,173,188,192,193,219 Lechowski, Z. 51, 103, 115 Lee, R. B. 163, 181 , 215 Lee, S. 167, 169, 185, 188, 194, 197, 215,217 Lee, Y.51, 53, 54, 55, 60, 62,/20, 122, 123, 127 Lees, P. E. 243, 251 Legault, D. 4, 16, 17,32 Leggett, J . E. 163, 216 Leisinger, T. 195, 214 Leopold, A. C. 69, 80, 122 Leroux, P.227, 238, 248,249,250, 251,254 Leshem, Y. Y. 77, 122 Leslie, J. F. 235,254 Lessard, P. A. 202, 208, 216 Leube.~. P.204,217

Leuchtmann, A. 8, 19, 33 Leung, H. 237, 254 Leung,J. 198,204,212,216 Leustek, T. 160, 165, 166, 167, 169, 182, 183, 185, 186, 187, 188, 192, 194, 195, 197, 205, 206,209,210,214,215,216, 217, 218, 220, 221 Levitan, I. 76, 77, 78, 110, 121, 122 Lew, R. R. 62, 122 Lewis, B. D. 67,122 Lewis, D. H. 11, 32 Lewis, G. J. 149, 154 Lewis, M. 196,208 Li, c. J. 162, 183, 209 Li, J . 55, 123 Li, Q. .216 Lillig, C. H. 173, 185, 218 Lin, C. 72, 123 Linacero, R. 230, 254 Lind, V. 244, 246, 251, 254, 256 Line, R. F. 246, 258 Linnemeyer, P. A. 105, 116 Lipke,J. 168,188, 189,194, 213 Liscum, E. 71, 104, 117, 123 Lister, G. R. 81, 89, 118 Liu, H. J. 164, 219 Liu, Y. J. 71, 123 Loeb, B. M. 22, 33 Logan, H. ~. 166, 171,193,205, 212,216 Lommel, C. 55 , 60,123 Lonergan, T. A. 59, 63, 127 Longman, D. 243, 251 Lopez-Coronado, J. ~- 187, 207,212 Lottspeich, F. 180,211

Lucas, J.A.227,228,230, 231, 235,236, 250,251 , 252, 253,254,256 Ludlow, M. M. 90, 91, 92, 94, 97, 98, 99, 102, 110, 111,123,128 Lunn, J.E. 188, 191 , 192, 216 Liittge, U. 55, 118 Lytle, C. ~- 192, 206,218

M ~a, L.-G. 67, 123 ~aathuis, F. J. M. 50, 51, 56, ~acDonald,l. R. 65, 120

94, 123

Machemehl, F. 162, 219 MacRobbie, E. A. C. 44, 50, 55, 57, 123 Madamanchi, N. R .. 197, 216 Maeshima, M. 55, 123 ~aestri, E. 230, 254 Magan, N. 15, 32 Maia, N. 246, 255 Malcevschi, A. 230, 254 Malhotra, K. 72, 123 Mancinelli, A. L. 103, 123 Mannix, D. G. 200, 211 Manolson, M. F. 56, 123 Manzhula, L. 236, 238, 250 Marais, G. F. 229, 238, 251 Maraite, H. 229,231 , 232, 234, 235, 237, 255,257 Marchegay,P.248,254 Marcker, K. A. 163, 220 Marcotte, W. R. 204, 212, 222 Mariaux, J .-B. 55, I 18 Marm iroli, N. 230, 254 Marquez, S. 131 Martin, F . 148, 149, 156 Martin, J.i 88,191, 192, 193,211,216 Martin, R. L. 184, 218 ~artinoia, E. 55, 123, 180,214 Martiny-Baron, G . 56, 123 Maruyama, A. 191, 216 Marzluf, G. A. 163, 198, 200, 210, 21J , 214, 216 ~asuda, Y. 89, 121 ~atsubayashi, Y. 173, 222 Matsui, A. 202, 211 ~atsumoto, T. 226, 253 Matsuura-Endo, C. 55, 123 Mattheck C. 144, 147, 149, 156 Matthes, M. 230, 254 ~aumene, C. 248, 254 ~aurel, C. 57, 129 ~ay,M. J . 197,198,216 Mayer, A. ~- 37, 124 Mayer, W.-E. 44, 47, 49, 50, 52, 53, 54, 60, 62, 94, 118, 124, 129 Mbwaga, A. M. 243, 255 ~c~nsh, M. R . 54, 56, 101, 124 McBride, R.P. 17, 32

266

AUTHOR INDEX

McCartney, H. A. 9, 11, 12, 31,235,238,257 McCarty, D. R. 204, 214, 222 McCune, D. 197,220 McCutcheon, T. L. 8, 32 McGrath, S. P. 160, 161, 162, 182, 209, 210, 216,223 Mcintyre, G. I. 65, 69, 124 Mcleod, A. R. 15, 32 McNaughton 245 McNeill, J. 234, 253 Meixner, M. 162, 218 Me lchinger, A. E. 135, 156 Menke,G.243,255 Mermall, V. 67, 124 Messeguer, R. 204, 210 Metzenberg, R. L. 1977 200,214 Meyer, K. 204, 217 Meyer, W.-E. 50, 52, Il9 Meyer, W .$.92,124 Migeon, J . L. 248,254 Millar, C. S. 5, 9, 32 Miller, D. 67, 124 Miller, J . A. 21, 22, 30 Miller, J.D. 3, 8, 21, 30,34 Miller, T . E. 247, 257 Millet, B. 47,124 Millner, P. A. 53, /24 Mischke, C. 51, 125 Miskelly, D. M. 162, 211 Misra, R. K. 153, 156 Miura, N. 173,206,219 Mohr, H . 103,124 Molvig, L. 162,217 Monaghan, J. M . 162,223 Money,N. P.242,253,255 Mon tesinos, C. 207, 208 Montfort, F. 231,253 Mo ntrichard, F. 207,212 Mooney, H. A. 42, 108, 109, 110,124, 131 Moore, A. E. 162,217 Mooseker, M.S.67,J24 Moran, N. 44, 49, 50, 51, 52, 53, 54, 62,122, 124,125,127 Moreau,J. M.23 1,232,234,235,237,255 Moreau,M.52,129 Moreno, V. 207, 208 Morris, P.-C. 198, 204, 212, 216 Morse, M. J .42,43, 47,49, 54,59,62, 125, 127 Mourioux, D. 180,217 Moyssct, L. 50, 61,125 Mudd, J . B. 185,215 Mueller, E. 249, 255 Muheim, R. 196,215 Murr,S.54,56,115, 125 Mulkey, T. J . 67, 125 Muller, E. 3, 4, 6, 10, 12, 13, 16, 18, 19, 30, 33,34 Muller, M. 202, 217

Mullins, C. E. 152, 156 Munk, L. 231,257 Munnik, T. 55, 125 Miintz, K. 162, 218, 219 Murakoshi, I. 173, 189, 191, 193,206,218, 219,220 Murata, T. 67,125 Murguia, J. R. 187, 207, 217 Murillo, M. 165, 166, 167, 183, 185, 186, 187,188,206,216,217,220 Murray, T. D. 237, 238, 242, 244, 246, 249, 251,252,255,258 Musgrave, A. 55, 125

N Nagahisa, E. 186, 214 Nagpal, P. 103, /26 Naito, S. 162, 194, 196, 202,211,213,215 Nakamura, K. 192, 217 Nakamura, M. 173, 191,206,223 Nakamura, T. 165,168, 173,193,194,195, 196,202,217,223 Nakanishi, Y. 55, 123 Nass, H . G. 135,156 Neale, A. D. 164, 173, 175,217 Neenan,M. 135,150,156 Negbi, M. 65, 125 Neuenschwander, U. 195, 196, 206,217 Neuvonen,S.3, 7,9, 14, 15, 16,11,32 Ng, A Y.-N. 164, 170, 175, 217 Nicholas, H. B. Jr. 176,217 Nicholas, K. B. 176,217 Nicholl, B. C. 148, 149, 150,156 Nicholls, C. F. 135, 155 Nicholson, P. 228, 229, 230, 231, 232,234, 235, 237, 244, 245, 246, 247, 248, 252, 255,256,257 Nick, P. 67, 97, 125 Nicolson, D. H. 234, 253 Nieto-Sotelo, J. 197, 217 Niklas, K. J., 147, 149, 156 Nirenberg, H. I. 228,229,231,256 Nishi, R. 173,222 Nishimura, M. 54, 121 Nishimura, N. 53,128 Nishizakj, Y. 94, 95, 102, 107, 125, 126 Noble, H . M. 28, 32 Noctor, G. 197, 217 Noguchi, K. 202, 2ll Noji, M. 165, 170, 188, 193,201,218,220, 221 Nozue, K. 71, 72,117, 126 Nuccio, M. L. 162, 183, 209 Nusinew, D. P. 164, 219 Nussbaum, S. 197,218

0 Ohiwa, T . 67, 126 Okamoto, H. 72,126

267

AUTHOR INDEX Oku, T. 101, 119 Omar, A. M. 164, 219 Onda, Y. 173,214 Ono, A. 204,218 Oort, A. J.P. 245,256 Oosterhuis D. 111, 126 OttoneHo, S. 170, 171, 194, 195,201,209 Owens,J. C. 1976135,156 Owttrim, G. W. 174, 209

p Page, R. D. M. 178,218 Pages, M.204,210 Paietta,J. V . 200,211,218 Paine, J. R. 228, 253 Palmer, J. H. 47,126 Palmer, J. M . 70, 126 Pao,S.S. 164, 219 Papaikonomou, M. P.230, 234, 235, 237, 238, 239,242,243,251, 252,256 Parcy, F. 198, 212 Parks, B. M. 103,/26 Patel, H. C. 184, 218 Paulsen, I. T. 164,219 Peberdy, J. F. 232, 234, 235, 236, 237, 240, 241,242,244,248,249, 250,251,252, 253,256

Pei, Z.-M. 51, 52, 55, 126, 130 Pellerin, S. t 42, /55 Peng, Z. 187, 207, 2/8 Perry, K. E. 242, 249, 256 Petrini, L. E. 4, 6, 18, 19, 31,33 Petrini, 0 . 2, 3, 4, 5, 6, 7, 8, 10, 11, 13, 15, 16, 17, 18, 19,25,26,28,30,31,32,33,34 Petrucco,S. l70, 171,194, 195, 201,209 Pfanz, H . 180, 21J Pfeffer, W. 58, 126, 135, 156 Pickard!, T. 162, 218,219 Pilon-Smits, E. A. H. 192,206,218 Pimenta, M. J. 162, 183,209 Pineau, C. 248, 251 Pleasants J. M. 69, 109, 121,131 Plewniak, F. 178,222 Poff, K. L. 70, 71,/21, 127 Poljakoff-Mayber, A. 37,124 Polle, A. 197, 221 Polley, R. W. 226,227,237,252,256 Ponchet, J . 246, 257 Poole, D. S. 103, 126 Poole, R. J. 56, 123 Post, P. L. 67, 124 Poulton, J. E. 185,212 Poupard, P. 231, 244,251,256 Powell, G. K. 71, IJ7 Powles,S. B.59,63, 111, 112,115,126 Pratt, R. 59, 63, 116, 117 Priestley, R. A. 229, 256 Prins. H . B. A. 95, 104, 118,128 Prior, A.171, 185,186,209,218

Prosser, I. M. 163, 221 Prosser, I. P. 164, 170, 213, 214 Prunier, M ..248, 251 Pruvost, C. 197, 221 Pugh, CJ.J. F. 10, 33 Purves, J. V. 195, 196,210,214

Q

Quail, P. H. 103, 126 Quan, J. A. 164, 219 Quatrano, R. 204, 212, 222 Quinones, M.A. 70, 126 Quintero, F. J. 187, 207,218

R Radosevich, S. R. 71, I 15 Raghavan, V.. 63, 130 Raibekas, A. A. 71, 72, 117, 123 Rajendrudu, G. 74, 98, 109, /26 Raju, M. V. S. 44, 124 Rama Das, V. S. 74, 98, 109, 110, 126, 127 Randall, P. J. 162, 210 Ranjeva, R. 52, 129 Ranta, H. 3, 7, 9, 14, 15, 16, 17,32 Raschke, K. 44, 46, 54, 62, 120,126 Rausch , T. 170,171, 184. 185, 197, 213, 220 Ravanel, S. 160,218 Ray, D. 149, 150,156 Ray, T. S. 65, 129 Read , N. D. 54, 119 Reed, J. W. 103,126 Reed, R. 54, 92, 98, 111, 112, 126, 129 Reonenberg, H. 162, 182, 183, \97, 209, 217,2/8,221

Renosto, F. 184, 218 Renwick, A. 249, 255 Reuling, G. 198, 215 Reuveny, z. 195, 218 Reymond, P. 67, 71,11 7,126 Rezanoor, H. N. 228, 229, 230, 231,232, 234,237,244,246,247,248,252,255, 256,257

Rich, T. C. G. 66, 89, 126 Riesmeier, J. W. 166, 171, 183, 184,2/5 Rinnar, T. 101, 128 Ritchie, R. M. 143, 154 Ritter, S. 44, 46, 47, 48, 49, 50, 51, 85, 89, 92,94, 96,97.99, 102,103,111 , 120, 122, 127

Roach, D. A. 242, 253 Robbertse, B. 231 ,232,234, 238,256,257 Roberts, M.A. 166, 167, 168, 185, 186, 187, 188. 192, 193, 194,201,212,2/4, 219,222

Robertson, D. E. 72, 123 Robinson, N. J. 197,219 Roblin, G. 54. 61, 127 Rock , C. D. 198, 219

268

AUTHOR INDEX

Rodrigues, K. F. 8, 33 Rodriguez, P.L. 187, 207, 212 Rodriguez-Navarro, A. 187,207,218 Roennenberg, T. 59, 127 Rogers, R. R. 135, 156 Roje, S. 162, 183, 209 Rolland, N. 173, 188, 192, 193, 219 Romer, S. 173, 183, 219 Romero, L. C. 189, 197, 198, 204, 208, 212 Rooney, I.~- 196,214 Rosenberg, N. 199, 222 Rosenkrans, L 204, 222 Rossman, A. Y. 245, 252 Rothstein, R. 199,222 Rousseau, ~- 228, 251 Rudiger, W. 67, 127 Rueda, J. 230, 254 Ruegsegger, A. 197,198,215,219 Ruffet, M .-L. 167, 168, 188, 192, 193,211, 219 Ruge, W . A. 94, 118 Russell, P. E. 238, 250 Russell, W. A. 135, 156

s

Saalbach, G. 162,219 Saalbach, I. 162, 218, 219 Sabnis, D. D. 44, 127 Sacriste, S. 148, 149, 156 Saier, M. H. 164, 219 Sailaja, ~- V. 74, 98, 110, 127 Sailland, A. 231, 253 Saito K. 160, 165, 166, 172, 175, 180,186, 188, 189, 191, 193,201, 206, 216, 218, 219,220,221,222 Sajus, P. 188, 192, 193, 2ll Sakai, N. 142, 157 Saker, L. R. 195, 196, 210, 214 Sala, F. 230, 254 Saldlvar-Garcfa, P. 9, 31 Sales, E. 207, 208 Salmon, S. E. 162,223 Salomon, ~ . .67, 71, 72, 117, 127 Sandal, N. N. 163, 220 Sanders, D. 50, 51, 54, 56, 94, 115, 123, 125 Sanderson, F. R. 231, 234, 235, 257 Sankar, A. 72,123 Sano,fi. 165,168,172,191, 193,194,195, 196,202,206,217, 223 Santos, ~. 196,208 Sasakura, N. 165, 175,221, 222 Sato, H. 90, 99, 102, 111,127 Sato, M. 186, 214 Sato, Y. 186, 214 Satter, R. L. 42, 43, 44, 45, 47, 49, 50, 51, 54, 59,60, 62,63, 96, 103,11~120,123, 125,127 Schafer, E. 67, 122, 125 Schafer,fi.J. 173,184,185,197,213,220

Schantz, R. 173, 183,219 Schauf, C. l. 62, 122 Scherer, G. F. E. 56, 123 Schiede~o. 162,218,219 Schiff, J. A. 187, 212, 222 Schiffmann, S. 169, 174, 185, 208, 220 Schjoerring, J. K. 170, 182, 222 Schmidt, A. 160, 183, 185, 187, 220 Schmidt, G. A. 135, 156 Schmidt, w. 70, 127 Schmitz, K. 183, 218 Schmutz, D. 197, 198,218,219 Schneider, M. J. 62, 63, 118 Scboeneweiss, D. F. 23, 24, 33 Schopfer, P. 67, 125 Schrader, L. E. 1978 196, 211 Scbriek, U. 185, 186, 220 Schroeder, J. I.50,51,52,54,55,56,62, 94,123, 126,127,129, 130 Schroppelmeier, G. 180, 214 Schulz, H. 231, 257 Schumacher, VV.46,120 Schuster, G. 173, 213 Schuster, S. 192, 220 Schwartz, A. 77, 78, 82, 87, 88, 89, 92, 97, 104, 109, 110, 111,121, 127,128 Schwenn, J.D. 160, 167, 169, 174, 185, 186,181,202,205,208,209, 213,218, 22() Scope!, A. L. 71, 115 Scott, K. 151, 154 Scott, P. R. 228, 246, 253, 254, 257 Sebald, W. 180,211 Segel, I. H. 184, 218 Sei, H. 70, 130 Sellars, E. K. 250, 154 Sen Gupta, A. 197,220 Serdin-Kerjan, Y. 199, 222 Serlin, B. S. 62, 122 Serrano, E. E. 106, 128 Serrano, R. 187, 207, 208, 212, 217 Setya, A. 167, 185, 186, 187, 220 Shackel, K. A 98, 109, 112, 128 Shak, T. 43, 44, 89, 99, 100, 101, 111, 122, 130 Sharpe, P. J.Ii.44,128 Shaw, R. H. 110,129 Shell, G. S. G. 75, 128 Shen, Q. X. 204, 220 Shen, T. L. 162,183, 209 Sheriff, D. W. 90, 91, 92, 94, 97, 98, 99, 102, 111, 128 Sherwood, M. 14, 17,33 Sherwood-Pike, M. 4, 13, 33 Shewry, P. R. 162, 221 , 223 Sbibagaki, N. 202, 211 Sbibaoka, fl. 69, 73, 78, 128 Shimamoto, K. 204,218 Sbimazaki, K. 53, 54, 101,119, 121, 128

269

AUTHOR INDEX Shimazaki, T . 173,222 Shinozaki, K. 205, 208, 214 Shishido, M. 22, 33 Short, T . W. 67, 126 Shuttleworth, J. E. 70, 116, 128 Sibaoka, T. 59, 63, 130 Sidebotto m, P. J. 28,32 Sieber, T . N. 3, 5, 6, 8, 15, 16, 18, 19, 20, 21, 25, 29, 30, 32, 33 Sieber-Canavesi, F. 5, 6, 8, 16, 18, 19,33 Siegel, L. M. 1975. 187,221 Sikora, R. A. 20, 31 Silva, P.C. 234, 253 Simmons, M. J. 234,253 Simon, E. 50, 61, 63, 119, 125 Simonet, P. 231,256 Simpson, D. R. 230, 244, 246, 247, 256 Sinclair, J. B. 2, 11, 23, 24, 26,33 Sindberg, S. 231, 257 Skinner, R. J. 160, 221 Skrove, D. 101, 128 Sliwinski, M. 164, 219 Smith, A. P. 73, 128 Smith, F. A. 54, 126 Smith, F. W. 163, 164, 170, 175, 181, 182, 194, 195, 196, 213,210,221 Smith, H . 25, 26, 34, 37, 66, 71, 80, 89, 126, 128 Smith, I. K. 188, 197,221 Smith, J. A. C. 57, 129 Smith, K. A. 152, 156 Smith, M. 109, 128 Smith, M. K. 15, 32 Smith, S. N. 237,257 Snow, R. 41,75, 128 Snustad, D . P. 234, 253 Snyder, W. C. 237, 257 Soleimani, M. J . 235,238,257 Sol~-sugraites, L. 50, 125 Soii, D . 171 , 185,218 Sou lie, M.-C. 239, 242, 257 Spagnoletti Zeuli, P. L 246, 252 Spalding, E. P. 66, 67, 117, 122, 128 Spanakakis, A. 246, 254 Spence, R. D . 44, 128 Spencer, D. 162, 217 Spencer-Smith, J . L. 1975 135, 150,156 Spooner, B. 231,258 Spoor, 0. 152, 156 Sprague, R. 227,245,257 Staal, M. 104, 128 Stafleu, F. A. 234, 253 Stamp, P. 197,215 Stanton, M. L. 73, 129 Starracb, N. 44, 50, 119, 124, 129 Steudle, E. 57, 129 Stevens, A. 207, 211 Stevenson, B. 203, 204, 214 Stevenson, K. R. 110, 129

Stockton, M. E. 62, 122 StokesA.l47, 148, 149,152, 156 Stoltze, C. 171, 185,218 Stolzy, L H. 135, 157 Stone,J. K.3, 4,5, 13, 14, 18,30,33,34 Stovall, M. E. 20, 34 Strohm, M . 197, 217,221 Stro ng, D . R. 65, 129 Suh,S.53,55,120,J12 Sun, D.-Y. 67, 123 Sunarpi 182, 221 Surdin,-Kerjan, Y. 163, 198, 199,200,207, 209,212,222 Suske, J. 3, 34 Suter, M. 186,188,195, 196,206,109, 215,217 Sutton, B. C. 3, 4, 6, 10, 12, 13, 18, 19, 30, 31 Swinburne, T. R. 11, 34 Syers, J. K. 160, 183, 221, 223 Sylvester-Bradley, R. 151, 154 Sze, H. 180, 210

T

Tabe, L. M. 162, 217 Taga, M . 237, 254 Tai, J. C. 192,206,218 T akagi, T. 191,216 Takagi, Y . 172, 189, 191,206,219,220 Takahashi H. 164, 165, 175, 180, 182, 189, 193, 194, 202,221, 222 Takamatsu, S. 173, 222 T akano, Y.242,256,257 T akeuchi, T. 229, 230, 257 Tamura, 0. 192,217 Tanada, T.63, 103,129 Tanaka, H. 46, 121 Tanaka, Y.55 , 123, 171,202,223 Tanguy, A. M. 246, 253 Tarczynski, M. C . 162, 183, 209 Tat bam, A. S. 162, 221 Tatsuguchi, K. 173, 189, 191, 206, 219, 220 Taylor, 0 . S. 227,239, 250 Taylorson, R. 37, 119 Tenca, P. L. 170, 171, 194, 195, 201 , 109 Te rashima, K. 142, 157 Terry, N. 192, 206, 218 Tester, M. A. 54,126 Thibaut, B. 148, /54 Thiel, G . 50, 54, 116, 129 Thiele, A. 246, 254 Thoene, B. 182,218 Thomas, D . 198, 199,200, 207, 209,212, 222,229,257 Thomas, M. R. 226, 256 Tbomassian, C. 184, 218 Thomine, S. 61,129, 179,21 1 Thompson, J. D . 178,222 Thompson, J. F. 1969. 188,221

AUTHOR INDEX

270

Thomson, W. W. 44, 117, 129 Thuleau, P. 52, 129 Todd,D.3,5, 10,12,22,26,3 4 Tollervey, D. 207, 211 Tomkins, G. M. 1966 192, 215 Tommcy, A.M. 197,219 Tomos, A. D. 44, 46, 47, 49, SO, 51, 89, 94, 96,120 Torssell, B. W. R. 74, 98, 111, 115 Toti, L. 8, 34 Touraine, B. 170, 182, 195,215,222 Trachsel, N. 196,215 Tranter, R. B. 236, 250 Travis, R. L. 92, 98, 111, 112,126, 129 Trentham, D. R. 54, 116 Tretyn, A. 61, 129 Trewavas, A J. 54, 119 Trinity, P. M. 195, 218 Truong, H.-N. 196, 214 Tsang, M. L. 187,212,222 Tseng, T. T. 164, 219 Tsuda,M. 12, 16,17,32 Tsuge, T. 242, 257 Turner, J. A 237, 256 Turner, J. C. 197,221 Tyreman, S. D. 57,129

u

Uchimiya, H. 173,222 Ugalde, T. D. 182, 211 Uhrig, J. F. 171, 185,218 Ullberg, D. 109, 128 Umeda, M. 173, 222 Urao, T. 205, 208 Uslu, E. 247, 257

v

Valent, B. 232, 240, 253, 258 van de Wiel, C. 230, 254 van Elk, A. G. 104, 128 Van Etten, H. D. 235, 257 van Montagu, M. 165, 197, 198, 216, 221 Van Schingen, J. C. 255 Van Volkeoburgh, E. 37, 95, 104, 105, 116, 118, /28, 129 Vandenberg, P. J. 163, 170, 181, 194, 195, 196,210,221 Vanduijo, B. 61, 129 Vardar, Y. 69, 116 Varin, L. 185, 207, 222 Varlet-Grancher, C. 109, 110, 116, 129 Vartanian, N. 198,212 Vasil, I. K. 204, 214, 222 Vasil, v. 204,222 Vazquez, A. 230, 254 Vega,J.M. 168, 189, 194,197,196,204, 208, 212 Verhoef, K. 23, 34 Verma, D. P. S. 187, 207, 218

Vernoux, T. 197, 198, 216 Vian, B. 239, 242, 257 Vidrnar,J.J. 170,182,195,2 15, 222 Vincent, A. 246, 257 Vince-Prue, D. 37, 129 Viret, 0 . 8, 34 VO<:hting, H. 78, 130 Vogelmaon, T. C. 80, 89, 97, 99, 102, 111, 117,130 Volckaert, G. 230, 254 von Denffer, D. 46, 120 von Sachs, J. 37, 43, 58,130 Vosman, B. 230, 254 Voss, E. G. 234,253 Vredenberg, W.J.62,116

w Wach~ S.

164, 219 Wada, M. 67, 68, 70, 71, 72, 103, 104, 106, /17,120,121, 125,126,130 Waddell, D. 162, 218 Wagner, G. 61, 129 Wainwright, C. M. 42, 102, 111, 130 WalkerS. 92, 111, 124,126 Walldate, P. J. 9, 11, 12, 31 Walkowaik-Cagara, I. 234, 237, 248, 256 Wallace, D. H. 90, 92, 97, 1 11, 112, 130 Waller,J. M.245,250 Wallwork, H. 231, 236, 257, 258 Ward, J. M.S 1,52,55,56,12 6,130 Ward, M. J. 52,129 Warpeha, K. M. F. 70,126 Warrilow, A. G. S. 163, 170, 181, 191 , 194, 195, 196,221,222 Watanabe, A. 165,175,221,2 22 Watanabe,M.3 7, 102,104,126,1 29 Watanabe, S.59,63,130 Webster, R. 162,210 Webster, R. K. 237, 257 Weintraub, M. 1951. 44, 130 Weisenseel, M. H. 67,116 Wenzel, G. 229,253 Werk, K. S. 69, 109,130 Werker, E. 43, 44, 89, 99, 111, 130 Wetherell, D. F. 58,130 Wheatcroft, R. 8, 34 Whitaker, T. 152, 157 Whitehead, D. 10, 34 Whitelam, G. 71, 120 Whitelam, G. C. 66, 89, 126 Whitney, N. J. 3, 8, 17, 21, 22, 30, 32,34 Widler, B. 16, 18, 34 Wiech, E. 44,124 Wien, H. C. 90, 92, 97, 111, 112,130 Wiesmann, A. 171, 185,218 WiWams, C. W. 63, 130 Wil!jams, S. E. 40, 131 Willmitzer, L. 166, 171, 183, 184,215 Wilson, D. 2, 11 , 13, 14, 20, 21, 22, 34

AUTHOR INDEX Wilson, R. 8, 34 Wilson, W . P. 98, 102, llO, 131 Winfield, M. 0. 230, 254 Wingfield, M. J. 25, 26,34 Withers, P. J. A. 161, 162, 216,223 Wolf, A. H. SO, 129 Wookey, P. A.. 6, 18,31 Worland, A. J . 246,254 Wray, J. L. 185, 187, 188, 192, 196,203,212, 213,218,222

Wright, M. 81, 89, liB Wrigley, C. W. 162, 2ll, 222 Wu, H. 44, 128

X

Xiao, Q. 244,251 Xiong,L. 203, 204,214

y Yamaguchi, Y. 165, 168, 173, 193, 194, 195, 196, 202,217,223 Yamaguchi-Shinozaki, K. 205,208,214 Yamaki, T. 73, 128 Yamamiya, K. 102, 126 Yamamoto, R. 89,121 Yamazaki, M. 165, 173,206,219, 221 Yasuda, N. 242,256 Yasumori, M. 202,211 Yates, D. J. 80, 130 Ye. H. 242.244, 255

271

Yeh, K.-C. 72, 126 Yildirim, A. 246, 247, 255, 258 Yildiz, F. 202,203,210, 223 Yin, H. C. 46, 77, 78, 89, 109, Ill, 131 Yoder, 0 . C. 232,258 Yokoyama, H. 173, 193,220 Yonekura-Sakakibara, K. 171,202,223 Young, G. B. 164, 219 Youssefian, S. 173, 191,206,223 Yu, F. 96, 97, 101,131 Yueh, Y. G. 54, 62, 117

z

Zacher!, M. 67, 127 Zamski, E. 45, SO, 65. 125 Ze'evi, 0. 65, 125 Zeevaart, J. A. 198,219 Zeiger, E. 70, 106, 108, 128, 131 Zenk, M. H. 197,223 Zhang, H. 69, 109, 121, 131 Zhang, P. H. 204, 220 Zhao, F. J. 160. 161, 162, 182, 183,209, 216,223

Zhu, J.-K. 203,204, 214 Zhu, Y. L. 192,206,218 Ziegler, I. 1977 180,212 Zimmerman, G. 184,218 Zimmermann, S. 61,129 Zuber, M.S. 135, 154, 156 Zuchner.S.246,254

SUBJECT INDEX

A ABA response complexes (ABRCs) 204 ABA response element (ABRE) 204 ABA responsive gene expression 204-5 Abies 9 Abies alba 6, 8, 15, 16,19 Abies balsamea 8, 18 abscisic acid (ABA) 198 Acerpseudoplatanus (sycamore) 10,26 Acer saccharum 149 Acremonium spp. 5 AcysC1189 adenosine-5-phosphosulphate (APS) 183, 186, 187, 188 Adiantum 68, 104 Adiantum capillus-veneris 72 Adiantum cuneatum 70, 72 Aegilops 245 Aegilops markgrajii 246 Aegilops speltoides 246 Aegilops squarrosa 228 Aegilops ventricosa 246 Aglaia 146 Agropyron sp. 245 Agrostis sp.245 Albizzia 63 Albizzia julibrissin 44, 63 Albizzia lophanta 61 Allium porrum 137 Allium tuberosum 192 Alnus spp. 6, 8 Alopecurus myosuroides 245 Alternaria 242 Alternaria alternata 13, 22 Amblyopyrum muticum 246 Amphiporthe castanea 19, 25 Anthostomella formosa 4, 5 Anthostomella pedemontana 4 Apera spica-venti 245 Apiognomonia quercina 26 APS kinase 185, 187 APS reductase 185, 186, 187, 193, 194, 197 APS sulphotransferase 186, 195 APS1183 APS2,APS3 202 aquaporins (PIP1 )57

Arabidopsis 51, 67, 104, 164, 175, 180, 183, 184, 185, 187, 188, 189, 191, 192, 193, 194,196,198,201,202,203,204,205, 207 Arabidopsis Genome Initiative 201 Arabidopsis thaliana 57, 71, 164, 176 Arrhenatherum elatius 245 Ascomycetes 7, 8 Aspergillus 249 Aspergillus niger 28 AST12 175 AST68 175 AST77175 AST82 175, 180 Asteromella sp. 20 AtCS-C 189 Atcys-3A 189, 198 AtCys-B 189 ATP sulphurylase 183, 185, 187, 193, 194, 195,197,205-6 Aureobasidium apocryptum 26 Aureobasidium pullulans 4, 10 Avena sativa (oats) 245

B Bacillus 22 Balansia 21 Balansia cyperi 20 Balsamorhiza 245 BAP 103-4 Basidiomycetes 8 Betula pubescens var. tortuosa (mountain birch) 7 'biochemical partitioning of resources' 20 blue light 65-7 'Bona fide endophytism'23 Botryosphaeria dothidea 26 Brassica 164 Brassica juncea 184, 205 Brassica napus (oil-seed rape) 144, 163, 183 Bromus 245 Bromus diandrus 236

Cactaceae 58

c

274

SUBJECT INDEX

cadmium pollution 197 Calocedrus decurrens 16 Camarosporium 6 Candida 28 Candida albicans 28 Capparis spinosa 74 Cassia fasciculata 54, 61 Castanea sativa (European chestnut) 6, 19,

20,25 Catharanthus roseus 185

CCCP (carbonylcyanide-mchlorophenylhydrazone) 95, 106, 107 Cenangium 7 Cenangium ferruginosum 4, 12, 26 Ceratopteris richardii 67 Cercosporella herpotrichoides 227 Chamaecyparis 9 Chamaecyparis lawsoniana 16 chemostimulants28 chestnut blight 27 Chlamydomonas reinhardtii 201, 202 Chiarella 187 choline-0-sulphate 200 chromophores 70-1 'circumnutation' 4, 59 Citrullus vulgaris Sat gene 201 Citrus natsudaidai 24 Cladosporium cladosporioides 13 Coelomycetes 7 Colletotrichum 6, 25, 242 Colletotrichum gloeosporioides 24 Colletotrichum phyllachoroides 6 Colpoma quercinum 24, 26 Compositae 58 Coniothyrium 7 Convolvulaceae 58 Crozophora tinctoria 73, 75 Cryphonectria parasitica (chestnut blight) 20, 21,25 cryptochromes 72 Cryptosphaeria populina 26 Cryptosporiopsis 20, 28 Cryptosporiopsis abietina 20, 23 Cucumis sativus 69 Cucumis vulgaris 70 Cupressaceae 8 Cupressus 9 Cyclaneusma minus (autumn needle cast) 4, 25 Cynosurus cristatus 245 Cyperus rotundus 20 cyprodinil 248 Cys B 199 cys14 163 CYS3 protein 200 'cysteine synthase' complex. 192 cysteine synthesis 188-91 Cytospora chrysosperma 26

D Dactylis 245 Dactylis glomerata (cocksfoot) 245 Dasypyrum villosum 245-6, 247

DCMU [N-(3,4-dichlorophenyl)-N'dimethylurea] 95, 105, 106, 107 Delphinium sp. 245 demethylation inhibitors (DMis) 248 diaheliotropic movements (diaheliotropism) 42, 46, 75, 76, 78-9, 102 in flowers 112-13 laminar 109-10 diaphototropic movement (diaphototropism) 42, 81, 83,85 of expanding leaves 109 of growing shoots 108-9 3'(2'),5'-diphosphonucleoside 3'(2')phosphohydrolase(3'phosphonucleotidase) 187 Diplodina castaneae 25 Diplodina salicomiae 5 Discula quercina 26 Discula umbrinella 8 Dolichos lablab 98 Donnan free space (DFS) 50 Drechslera sp.13 Drepanosiphum platanoides 27 Dryas integrifolia 112 Dryas octopetala 6, 19, 113

E Echinochloa crusgalli 245

ELISA 244, 246 Elodea canadensis 95 Elymus repens 245, 228

'end-of-day' (EOD) light 37 endophytes authenticity of endophytic character 10-11 biocontrol of weeds 27-8 as bio-indicators of air pollution 3, 14, 15 colonization 12-19 environmental factors affecting 14-16 species composition and canopy characteristics 16-17 detrimental associations 22-7 diversity of associations 3-8 among fungal species 7-8 interspecific 3-4 intraspecific 5-7 ecology 10-19 geographic and climatic factors 17-19 growth promotion 22 Gymnspermae as host plants 9-10 host interactions 20-38

275

SUBJECT INDEX host plant phenology 10 indirect enhancement of insect colonization 26--7 inhibition of host plant growth 26--7 latent pathogens 22--6 mutualistic associations 20-2 physiology 9 protection from insect herbivory 21-2 resistance to diseases 20-1 sporulation, dispersal and infection 11-12 substrate utilization 20 utilization and manipulation of associations 27-8 EOD ('end-of-day') irradiation 53 Epicoccum 6 Epicoccum nigrum 4 epiphytes, definition 2 Erythrina herbacea 98 Escherichia coli 180, 185, 186, 187, 195, 198,

heliotropic movements (heliotropism) 42, pulvinar 110-11 solar tracking by 72-89 heptelidic acid 21 heptelidic acid chlorohydrin 21 Heterobasidion annosum 20 HIR 103-4 Holcus lanatus (Yorkshire fog) 236, 245 Hordeum leporium (barley grass) 236, 245 Hordeum vulgare (barley) 245 Hormonema dematioides.22 host-neutral endophytes 4 hvstl 163 hydroheptelidic acid. 21 Hyoscyamus spp. 68 Hypoxylon atropunctatum 25 Hypoxylon mammatum 26

I Impatiens glandulifera 146 interspecific diversity 3-4 intraspecific diversity 5-7

201

Eucalyptus spp. 26 Euterpe oleracea 8

J

F Fagopyrum esculentum 103 Fagus grandiflora (American beech) 7 Fagus sylvatica 28 Festuca idahoensis 245 t1avin adenine dinucleotide (FAD) 72 Funaria 67 Fusarium 6, 25 Fusarium oxysporum 20 Fusicoccum quercus 24 FV (fast vacuolar) channels 56

G Gaeumannomyces 242 Gcn4p 199-200 Geniculosporium serpens 5 Gibberella fujikuroi 235 c>-gliadins 162 '"1-gliadins 162 w-gliadins 162 glutaredoxin (CPFC) 186 glutathione 197-8 Glycine max 92, 97, 111-12 Gossypium 111 growth-related movements 40 Guignardia philoprina 13 GUS seedling test 246 Gymnospermae 3, 9-10, 28

H HAL genes 206-?P AP 207 Haynaldia villosa 245-6 Hedera 65, 68 Hedera helix 65 Helianthus 69, 78 Helianthus annuus (sunt1ower) 69, 72, 146

'Javart' disease of European chestnut 25 ]uncus 13 ]uncus bufonius 13 Juniperus occidentalis 16

K Koeleria nitida 245

L Lactuca serriola 69 laminar phototropism 89,99-101 Larix 9 Larix laricina (eastern larch) 7 Lasiodiplodia theobromae 25 Lavatera 44, 78, 80, 85, 86, 89, 109, 111 Lavatera cretica 76, 77, 79, 81, 82, 85, 86, 87,88 Leguminosae 43, 58 Lemna 67 Leptodontium orchidicola 22 Leptostroma 8, 12, 16, 21, 22 Limnanthemum 68 Lithospermum ruderale 245 lodging 141 Lolium sp. 245 Lomatium triternatum 245 Lophodermium 7, 15 Lophodermium piceae l3 Lophodermium pinastri 4, 8, 16 Lophodermium seditiosum 4 Lupinus 44, 89, 102, 110 Lupinus arizonicus 102, 110 Lupinus palaestinus 85, 99, 100 Lupinus succulentus 99 lysophosphatidylcholine (LPC) 53

276

SUBJECf INDEX

M Macrophoma piceae 20 Macroptilium 99 Macroptilium atropurpureum 94 Magnaporthe 242 Magnaporthe grisea 237, 240 Mallotus wrayi 153 Malva 78, 89, 109, 111 Malva neglecta 46, 77, 89 Malvaceae 43 Malvastrum rotundifolium 110 MBC fungicides 226-7,248, 249 Melilotus alba 97 Meloidogyne incognita 20 Meria parkeri 5, 8, 13, 22 MET genes 199-200, 202 Metasequoia 9 methenyltetrahydrofolate 72 methyl benzimidazole (MBC) fungicides 226-7,248,249 Mimosa pudica 42, 44 Monstera 65, 68 Monstera gigantea 65 Mougeotia 62 MYB205 MYC205 mycoherbicides 27

N N-acetylserine 199 nastic movements 42, 45 Nectria haematococca 235, 237 negative phototropism 65 Nephilium 146 Neurospora crassa 163, 200,202 Nicotiana tabacum 202 nitrogen metabolism 195-6

0 0-acetylserine (OAS} 188, 192, 195, 196, 199 0-acetylserine (thiol) lyase (OASTL) 188, 189,191,192-3,194,196,197,206 oak wilt 27 Oenotheraceae 58 Ophiognomonia sp. 13 Opuntia 44 Oritrophium limnophilum 112 ornithine decarboxylase (ODC) 249 Oxalidaceae 43, 58 Oxalis oregana 59, 63, 112 Oxalis regnelii 101

p Papaver radicatum 112 PAPS reductase 185, 186, 187 paraheliotropic movements (paraheliotropism) 42, 102 Parthenocissus 65 PCR assay 231, 244, 249

Periphyllus acericola 27 Pestalotia spp. 6 Pestalotiopsis 22 Pestalotiopsis funerea 7, 9, 23 Pezicula cinnamomea 20, 24, 25 Pezicula sp. 28 Phaeosphaeria junicola 13 Phalaris canariensis 245 phanerogams 3 Phaseolus 46, 53, 54, 92, 94, 96, 99, 101, 103, 107 Phaseolus coccineus 52 Phaseolus multif!orus 91 Phaseolus vulgaris 48, 59, 90, 91, 92, 98, 104 'phellophytes' 2, 5 Phialocephala 7, 12, 16-17, 22 Phialocephala fortinii 22 Phialocephala scopiformis 5 Phleum pratense 245 Phomopsis 19, 22 Phomopsis occulta 23 3'-phosphoadenosine-5'-phosphosulphate (PAPS) 185,186,187,188,207 phospholipase 2 (PLA ) 53 photomorphogenesis fl photonastic pulvinar responses 96-7 photosynthetically active radiation (PAR) 37-8 phototropic movements, growth-mediated 63-72 direct control 64-8 indirect control68-70 leaf movements 90-107 photoreceptors for 70-2 phototropin 71 Phyllosticta 20, 21 Phyllosticta abietis 20 Phyllosticta cryptomeriae 20 Phyllosticta multicomiculata 20 Phyllosticta pseudotsugae 20 Phytoalexin production 20 Phytophthora cactorum 20 Picea 9 Picea abies 5, 8, 15, 20, 142 Picea sitchensis 15, 20 pine blister rust 27 Pinus 4, 8, 9, 17, 25 Pinus banksiana 4, 16 Pinus brutia 22 Pinus densiflora 4, 7, 8, 12, 16, 22 Pinus pinaster (maritime pine) 148 Pinus radiata 10 Pinus resinosa 4, 8, 16 Pinus sylvestris (Scots pine) 4, 15, 26, 28 Pinus thunbergii 4, 7, 8, 12, 16 Pisum sativum 134

277

SUBJECT INDEX plagioheliotropic movement (plagioheliotropism) 42, 75 plant movements adaptive strategies 107-13 adaptations to terrestrial environment 107 biological clock and 59 circadian control of ion fluxes 52-3 concept 38 control of 41-3 de-etiolated seedlings 69-70 diurnal control of ion fluxes 53-5 diurnal movements 107-8 flowers and inflorescences 58 functional analysis 106 generation 38-9 gravity and light 63-4 leaves 58-63, 68-9, 73-7 morphological constraints 97-8 motor for turgor-mediated movements 43-58 nocturnal phase 77-8 operational aspects 46-52 perception of directional light 90-4 photoreceptor genes 71-2 role of cytoskeleton 67-8 of seedlings 64-5 shoot apices 72-3 solar signal perception 78-89 stress and 101-2, 111-12 structural features 43-5, 97 Plectophomella sp.20 Pleospora bjorlingii 5 Pleospora salicomiae 5 Pleuroplaconema sp. 7, 9 Poa 245 Poa annua (couch) 245 Podocarpus 9 polyunsaturated fatty acids (FPFA) 53 Populus tremuloides (aspen) 7 Porphyra yezoensis 186 Potentilla fruticosa 22 PRH proteins 186 prochloraz 237 protein-protein interaction 192-3 Pseudocercosporella (Ramulispora) 230 anamorphs 234 Pseudocercm,porella aestiva 228, 229, 230 Pseudocercosporel/a anguioides 228, 229, 230 Pseudocercosporella herpotrichoides 226, 227, 229,230,231,232,243 Pseudocercosporella herpotrichoides var. acuformis 228, 229 Pseudocercosporella herpotrichoides var. herpotrichoides 228, 229 Pseudotsuga menziesii (Douglas fir) 4, 8, 13, 19 PSOI130 164 pteridophytes 3

pulvinar chloroplasts, role in plant movement 106-7 pulvinar phototropism spectral independence 102-7 in trifoliate leguminous leaves 97-9 Pythium ultimum 20

Q

Quercus 24 Quercus emoryi 13 Quercus garryana 22 Quercus ilex (holly oak) 4 Quercus petraea (sessile oak) 7 Quercus rubra (red oak) 25

R random amplified polymorphic DNA (RAPDs) 8, 230, 243 RD294 203 reverse photonastic movements 42 RFLPs 229, 247 Rhabdocline parkeri 4, 8, 13, 14, 26 Rhizoctonia oryzae 20 Rhizoctonia so/ani 20 Rhizosphaera kalkhoffi 15 Rhytisma acerinum 26 Robinia 61 Robinia pseudoacacia 50, 91 root anchorage adventitious roots 139 coronal and prop root systems 146 costs of 138-9 in crop plants 150-1 experimental study methods 137-8, 142-3 intermediate systems 146-7 mature plants 140-1 mechanics 143-7 misconceptions 134-5 models, use of 147-51 morphology 147-8 numerical models 152-3 plate systems 143-4 resistance to overturning 141-7 resistance to uproots 135-41 root branching 249 single root extraction 135-7 soil properties and 151-2 strengthening only basal areas 138 tap roots 144-6 theory 141-2 using basal root hairs 138 Rubisco (ribulose-! ,5-bisphosphate carboxylase/oxygenase) 194 ( + )-rugulosin 21

s

SAC3 protein 203 Saccharomyces cerevisiae 202, 207

278

SUBJECT INDEX

S-adenosylmethionine (AdoMET) 199 Salicomia perennis 5 Salix glauca 22 Salmonella typhimurium 198 Samanea 46, 52, 54, 60, 95 Samanea saman 47,54 SAR-52 201 SAT1188 Sat-52 188 SAT-A 188 Schizothyrium sp 14 Sclerophoma pythiophila 4 SCON1, SCON2 protein 200 Sec ale cereale (rye) 245 SEF4 201-2 Seiridium juniperi 23 Selaginella 65, 67 Septaria tritici 24 7 Sequoia 6 Sequoia sempervirens (coastal redwood) 7, 9 serine acetyltransferase (SAT) 188, 192-3 SHSTl, SHST2 and SHST3 transporter 163-4 shstl, shst2, shst3 163 Silphium spp 69 Sinapis alba 66 Sitanion hystrix 245 skotonastic movements t 42 skototropism 65 'smart plant' technology. 161 'solar tracking' 42, 98-9 by heliotropism 72-89 solar timekeeping 58-63 pulvinar photoreceptors for 62-3 Sparmannia africana 68 Sphaeropsis sapinea 25 spectral analysis 102-4 Spirogyra 67 Sporobolus 175 Sporobolus stapfiana 164 Sporormiella 6 spruce budworm.21 Stachys sylvatica 75 Stagnospora innumerosa 13 Stagonospora 5 Staphylococcus aureus 28 Stylosanthes 175, 194 Stylosanthes hamata 163 Suaeda fruticosa 6 sulphate activation 183-5 control of flux 191-2 environment sensing 198-207 environmental regulation and interaction 191-207 long distance transport 182-3 reduction of 185-8 reductive pathway of assimiliation 183-91 regulation of expression 180-2

sites of expression 175-9 subcellular transport 179-80 in transgenic plants 200-4 uptake and translocation 163-83 sulphate transporters 163-75 sulphur in agriculture 160-1 crop quality and yield 161-2 status 198-200 sulphite reductase 185, 187 sulphur deficiency 161, 207 sulphur fertilization 161 sulphur metabolism salt tolerance and 206-7 studies on 160-2 sulphur pollution 161 sulphur sinks 162 sulphur supply 193-5 'sun-tracking' 42 SV (slow vacuolar) channel 56 'symptomless endophytes' 3 Synechococcus 180

T Tapesia breeding system 232 geographic variation 238 host range 244-5 host resistance 245-7 infection plaques 240-2 infection process 239-40 isolate variation 227-8 molecular analysis 229-31 pathogenicity 238-44 pathogens 227-31 population biology 234-5 sexual stage 231-8 spore dispersal and adhesion 238-9 taxonomy 234-5 tissue colonization 242-3 Tapesia acuformis 226, 234, 235, 237, 238, 239-40,241,242,243,244,245,247-8 Tapesia livido-fusca 5 Tapesia yallundae 226, 231, 232, 235-8, 239-40,242,243,244,245,246,247-9 Taxus 13 terpenoids. 23 Thaumetopoea pityocampa (pine processionary moth) 22 Thecodiplosis japonensis (pine needle gall midge) 22 thigmonastic movements 42 thigmorphogenic responses 148-9 thioredoxin (CGPC) 186 thiosulphonate reductase 185, 187 Thuja 9 Thuja plicata 16 tonoplast, role in plant movements 55-7 Trichophyton mentagrophytes 28

279

SUBJECT INDEX Trimmatostroma salicis 5 Triticum 245 Triticum aestivum (bread wheat) 146, 245 Triticum dicoccoides 246 Triticum durum 246 Triticum monococcum 246 Triticum (Aegilops) tauschii 164, 246, 247 Triticum turgidum 246 Tropaeolum spp. 68, 80 tropic movements 42 Tryblidiopsis pinastri 5, 13 ttst I, ttst2 164 turgor-related movements 40

u

unilateral excitation 42 U redinales 3 Urtica spp 68

v

Vaccinium myrtillus 28 Vaucheria 67 Vaucheria sessilis 67 vectorial excitation 42 after-effects 86

in laminar heliotropism of pulvinated leaves 78-86 remote phototropic control by 86-9 Vicia 101 Vicia faba 53, 63 Vicia narbonensis 162 VK channels 56

w

water channels, role in plant movements 57-8

water free space (WFS) 47, 51 windthrow 141

X Xanthium strumarium 73, 75 xanthophyll de-epoxidation cycle38 xanthoxin 64 Xylaria spp. 8 Xylariaceae 13, 19 'xylotropic endophytes' 2

z

Zea mays (maize) 146, 194, 195