Advances in Botanical Research, Volume 33

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

Author: J. A. Callow

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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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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


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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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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


XV

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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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


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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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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


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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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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


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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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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

6


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


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).

10


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


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


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


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.

18


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

FOLIAR ENDOPHYTES

19

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

20


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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21

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,

22


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).

24


(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,

FOLIAR ENDOPHYTES

25

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).

26


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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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

28


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

FOLIAR ENDOPHYTES

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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.

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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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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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D. KOLLER

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.

PLANTS IN SEARCH OF SUNLIGHT

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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

38

D. KOLLER

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


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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D. KOLLER

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.


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

42

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


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

44

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


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

46

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).


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).)


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).


51

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

52

D. KOLLER

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


53

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,

54

D. KOLLER

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,


55

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

56

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,


57

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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D. KOLLER

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


59

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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D. KOLLER

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


61

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


63

(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).


65

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


67

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


69

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


71

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).

72

D. KOLLER

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


73

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

74

D. KOLLER

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.


75

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.


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


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

VE

+BO

... 0 0>

(1}

VE ( 2}

+7

0

+60

-10

+50

-20

+40

-30 ·.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(~LlJTEDKHLSFTRRYGGSNNNSSSSNALLKEPLLSVEK

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 ofgene : expression :

:

I

'

''

-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

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