Advances in
BOTANICAL RESEARCH incorporating Advances in Plant Pathology
VOLUME 30
Advances in
BOTANICAL RESEARCH incorporating Advances in Plant Pathology Editor-in-Chief J. A. CALLOW
School of Biological Sciences, University of Birmingham, U K
Editorial Board J. H. ANDREWS J. S. HESLOP-HARRISON M. KREIS R. A. LEIGH E. LORD D. G. MANN R. R. SHEWRY I. C. TOMMERUP
University of Wisconsin-Madison, Madison, USA John Znnes Centre, Norwich, UK Universitt de Paris-Sud, Orsay France University of Cambridge, Cambridge, UK University of Califarnia, Riverside, USA Royal Botanic Garden, Edinburgh, UK IACR-Long Ashton Research Station, U K CSIRO, Perth, Australia
Advances in
BOTANICAL RESEARCH incorporating Advances in Plant Pathology Series editor
J. A. CALLOW School of Biological Sciences, University of Birmingham, Birmingham, UK
VOLUME 30
1999
ACADEMIC PRESS San Diego London Boston New York Sydney Tokyo Toronto
This book is printed on acid-free paper. Copyright
0 1999 by ACADEMIC PRESS
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Typeset by Gray Publishing, Tunbridge Wells, Kent Printed in Great Britain by MPG Books Limited, Bodmin, Cornwall
99 00 01 02 03 04 MP 9 8 7 6 5 4 3 2 1
CONTENTS CONTRIBUTORS TO VOLUME 30 ......................................................
ix
CONTENTS OF VOLUMES 18-29 ........................................................
xi
PREFACE ...............................................................................................
xxi
Nitrate and Ammonium Nutrition of Plants: Physiological and Molecular Perspectives
B . G. FORDE and D . T . CLARKSON I . Introduction ............................................................................................... I1. Nitrogen Nutrition in its Natural Context ................................................ 111. Physiology of NO; Uptake ....................................................................... A . Energetics............................................................................................ B. Kinetics .............................. ..................................................... C . Regulation ............................................................... D . Relationships Between NO; Ions and the Hydraul Roots .................................................................................................. IV . Molecular Genetics of NO; Trans t in Microorganisms ...................... A. Prokaryotes .... ....................................................... B. Lower Eukary ............................................................. V . Molecular Genetics of NO; Transport in Plants ...................................... A . NO; Transport Mutants ............ ................................................... B. NO; Transporter Genes ..................................................................... C . Relating the Molecular Genetics to the Kinetic Data ....................... VI . Physiology of NH: Uptake ....................................................................... A. Background ........................................................................................ B . Kinetics of NH: Uptake .................................................................... VII . Molecular Genetics of NH: Transport ..................................................... A . Prokaryotes ......................................................................................... B . Lower Eukaryotes .................................................. C . Higher Plants ...................................................................................... VIII . Efflux of NO; and NH: ........................................................................... A . NO; Efflux ......................................................................................... B. Ammonium Efflux .............................................................................. C . Net Uptake of NO; and NH: Along the Root Length ................... IX . Regulation by the Shoot of NO; and NH: Uptake by Roots ................ A. Evidence for Shoot-derived Signals .................................................... B. Nature of the Shoot-derived Signals ........................................... X . Concluding Remarks: Looking Back and Looking Forward .................... Acknowledgements..................................................................................... References ..................................................................................................
2 3 5 5
5 11 19 21 21 24 28 28 30 38 40 40 44 49 49 50 51 52 52 57 58 61 61 63 67 69 69
vi
CONTENTS
Secondary Metabolites in Plant-Insect Interactions: Dynamic Systems of Induced and Adaptive Responses J . A . PICKETT. D. W. M . SMILEY and C . M . WOODCOCK I . Introduction ............................................................. I1. Feeding and Other Contact Interactions Involving A . Plant Defence by Toxic Mechanisms ................................................. B. Disruption of Insect Feeding by Non-toxic Mechanisms .................. C . Sequestration of Toxicants by Insects ................................................ I11. Insect Interactions With Plants Using Volatile Signals ............................. A . Location of Host Plants ..................................................................... B . Avoidance of Unsuitable Plants ......................................................... IV . Plant Interactions With Predatory and Parasitic Insects ........................... V . Interactions Between Secondary Metabolites and Insect Pheromone Systems...................................................................................... VI . Future Prospects ........................................................................................ Acknowledgements..................................................................................... References ...........................................................................................
92 93 93 97 99 100 100 102 103 105 105 107 107
Biosynthesis and Metabolism of Caffeine and Related Purine Alkaloids in Plants
H . ASHIHARA and A. CROZIER I . Introduction ............................................................................................... I1. Occurrence of Purine Alkaloids in Plants .................................................. A. Camellia .............................................................................................. B. Coffea ................................................................................................. C . Ilex ................................... ....................................................... D . Theobroma and Herrania .................................................................... E . Paullinia .............................................................................................. I11. Purine Nucleotide Metabolism in Higher Plants ....................................... A. Nucleotide Pool in Plant Cells and Tissues........................................ B. Biosynthesis of Purine Ribonucleotides ........................................ C . Catabolism of Purine Nucleotides ...................................................... D . Metabolism of Purine Bases and Nucleosides in Plants .................... E . Other Plant-specific Purine Pathways ................................................ IV . Biosynthesis of Purine Alkaloids ............................................................... A . Methylation of the Purine Ring ......................................................... B. Enzymes Involved in Methylation Steps ............ C . Caffeine Biosynthesis From Purine Nucleotides D. Purine Alkaloid Biosynthesis in Theobromine-accumulating Plants . E. Physiological Studies on Caffeine Biosynthesis .................................. F. Other Routes ...................................................................................... V. Metabolism of Purine Alkaloids in Plants................................................. A . Catabolism of Caffeine and Related Compounds .............................. B. Diversity of Caffeine Metabolism ...................................................... C . Metabolism of Purine Alkaloids as Xenobiotics in Non-purine Alkaloid-forming Plants .....................................................................
118 120 120 122 122 123 123 123 124 126 135 140 143 143 143 150 157 166 167 172 174 174 175 186
vii
CONTENTS
VI . Biotechnology of Purine Alkaloids ............................................................ A . Caffeine Production and Degradation in Cell and Tissue Cultures ............................................................................ B. Decaffeinated Beverages ..................................................................... VII . Summary .................................................................................................... Acknowledgements..................................................................................... References ..................................................................................................
186 186 188 190 191 191
Arabinogalactan-proteinsin the Multiple Domains of the Plant Cell Surface
M . D . SERPE and E . A . NOTHNAGEL I . Introduction ........................................
...............................................
ellular Spaces ......................... I1. Soluble AGPS in the Cell Wall and E A . Biochemical Characterization ............................................................. B. Expression and Function ....................
I11. Cell Wall AGPs (CW-AGPs) ..................................................................... IV . V.
A . Biochemical Characterization ............. B. Expression and Function .................................................................... Plasma Membrane AGPs (PM-AGPs) ...................................................... A . Biochemical Characterization ............................................................. B. Expression and Function .................................................................... Conclusions and Future Prospects ........................ Acknowledgements..................................................................................... References ..................................................................................................
208 211 211 225 238 240 244 256 257 265 270 274 274
Plant Disease Resistance: Progress in Basic Understanding and Practical Application
N . T. KEEN I . Introduction .............................................................................. I1. Pathogenicity and Virulence Mechanisms .................................................
A . Factors Facilitating Pathogen Entry Into and Movement Through the Host .......................................... B. Preformed Resistance Mechanisms .................................................... C . Toxins and Other Virulence Factors ............................ D . Enzymes and Extracellular Polysac E . Newly Discovered Virulence Factors ................................................. 111. Active Disease Resistance ........... .............. A . The Hypersensitive Respons ce Genes B. Avirulence Genes and Elicitors .......................................................... C. Elicitor Presentation .................. D . Active Oxygen Species ............... E . Signal Transduction ................................... F. Systemic Acquired Resistance ............................................................ G. Similarities of the HR and SAR With Defence in Vertebrates and Insects ................................................................................................. H . Defence Response Genes .................................................................... I . Defensins and Related Factors . ............................
292 293 293 294 295 296 297 299 299 300 302 304 305 307 308 309 31 1
...
Vlll
CONTENTS
IV . Approaches to the Use of New Knowledge in Disease Control ............... A. Transgenic Plants Expressing Foreign Disease Resistance Genes ..... B. Transgenic Plants That Inactivate Toxins .......................................... C . Transgenic Plants Expressing Regulatory or Defence Response Genes .................................................................................................. D . Expression of Pathogen Genes in Plants ............................................ E . Other Rationales ................................................................................. V . Conclusions and Future Directions ........................................................... References ..................................................................................................
311 312 313 313 313 314 315 315
AUTHOR INDEX ....................................................................................
329
SUBJECT INDEX ....................................................................................
353
CONTRIBUTORS TO VOLUME 30
H. ASHIHARA Department of Biology, Faculty of Science, Ochanomizu University, Otsuka, Bunkyo-ku. Tokyo 112-8610, Japan D. T. CLARKSON Department of Plant Sciences, IACR-Long Ashton, University of Bristol, Long Ashton, Bristol BS18 9AF, UK A. CROZIER Plant Products and Human Nutrition Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow GI2 8QQ, U K B. G. FORDE Biochemistry and Physiology Department, IACR-Rothamsted, Harpenden, Hertfordshire AL5 2JQ. UK N. T. KEEN Department of Plant Pathology, University of California, Riverside, C A 92521, USA E. A. NOTHNAGEL Department of Botany and Plant Sciences, University of California, Riverside, CA 92521, USA J. A. PICKETT Department of Biological and Ecological Chemistry, IACRRothamsted, Harpenden, Hertfordshire AL5 2JQ, UK M. D. SERPE Department of Biology, Boise State University, Boise, ID 83725, USA D. W. M. SMILEY Department of Biological and Ecological Chemistry, IACR-Rothamsted, Harpenden, Hertfordshire AL5 25Q, UK C. M. WOODCOCK Department of Biological and Ecological Chemistry, IACR-Rothamsted, Harpenden, Hertfordshire AL5 2JQ, UK
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CONTENTS OF VOLUMES 18-29 Contents of Volume 18 Photosynthesis and Stomata1 Responses to Polluted Air, and the Use of Physiological and Bacterial Responses for Early Detection and Diagnostic Tools
H. SAXE Transport and Metabolism of Carbon and Nitrogen in Legume Nodules J. G. STREETER
Plants and Wind
P. VAN GARDINGEN and J. GRACE Fibre Optic Microprobes and Measurement of the Light Microenvironment within Plant Tissues
T. C. VOGELMANN, G. MARTIN, G. CHEN and D. BUTTRY
Contents of Volume 19 Oligosaccharins S. ALDINGTON and S. C. FRY Are Plant Hormones Involved in Root to Shoot Communication?
M. B. JACKSON Second-Hand Chloroplasts: Evolution of Cryptomonad Algae
G.I. McFADDEN The Gametophyte-Sporophyte Junction in Land Plants
R. LIGRONE, J. G. DUCKETT and K. S. RENZAGLIA
xii
CONTENTS OF VOLUMES 18-29
Contents of Volume 20 Global Photosynthesis and Stomata1 Conductance: Modelling the Controls by Soil and Climate
F. I. WOODWARD and T. M. SMITH
In vivo NMR Studies of Higher Plants and Algae R. G. RATCLIFFE Vegetative and Gametic Development in the Green Alga Chlumydomonas
H. VAN DEN ENDE Salicylic Acid and its Derivatives in Plants: Medicanes, Metabolites and Messenger Molecules
W. S. PIERPOINT
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
CONTENTS OF VOLUMES 18-29
...
Xlll
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
S. SAVARY, L. V. MADDEN, J. C. ZADOKS and H. W. KLEIN-GEBBINCK
Contents of Volume 22 Mutualism and Parasitism: Diversity in Function and Structure in the ‘Arbuscular’ (VA) Mycorrhizal Symbiosis E A. 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
xiv
CONTENTS OF VOLUMES 18-29
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
Sensitivity of Indexing Procedures for Viruses and Viroids
H. HUTTINGA 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. LEVESQUE and D. M. EAVES Quality Control and Cost Effectiveness of Indexing Procedures
C. SUTULAR
CONTENTS OF VOLUMES 18-29
xv
Contents of Volume 24 Contributions of Population Genetics to Plant Disease Epidemiology and Management
M. G. MILGROOM and W. E. FRY 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
xvi
CONTENTS OF VOLUMES 18-29
Towards an Understanding of the Population Genetics of Plant-Colonizing Bacteria
B. HAUBOLD and P. B. RAINEY 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 Phytophthoru 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
CONTENTS OF VOLUMES 18-29
xvii
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 H + 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
xviii
CONTENTS OF VOLUMES 1&29
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 hybridu
L. COLOMBO, A. VAN TUNEN, H. J. M. DONS and G. C. ANGENENT The Regulation of C4 Photosynthesis
R. C. LEEGOOD Heterogeneity in Stomata1 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. J. HOWE Plant Transposable Elements
R. KUNZE, H. SAEDLER and W.-E. LONNIG
CONTENTS OF VOLUMES 18-29
xix
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 Ozone Impacts on Agricultural: 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
xx
CONTENTS OF VOLUMES 18-29
Mechanisms of Na+ Uptake by Plants A. AMTMANN and D. SANDERS The NaC1-induced Inhibition of Shoot Growth: The Case for Disturbed Nutrition with Special Consideration of Calcium Nutrition D. B. LAZOF and N. BERNSTEIN
PREFACE
Nitrogen is the mineral nutrient needed in greatest quantity by plants and the one that most frequently limits plant growth. Nitrogen can be obtained from the soil as either nitrate or ammonium, and the mechanisms of uptake of these ions have been reviewed many times in the 300 years or so since the absorption of ‘nitre’ by plants was recognized by Mayow (1681). In recent years the subject has received renewed impetus mainly due to progress made in the cloning and characterization of the various transporters. The review by Forde and Clarkson integrates the large body of information on physiological mechanisms with these advances at the molecular level, with particular emphasis on regulation. It has long been accepted that plant-insect interactions involve the coevolutionary development of defensive secondary metabolites in the plant and adaptation to these by insects. The area has received renewed attention in recent times, partly because of the interest in mechanisms underpinning biodiversity, but also because of the potential for using these natural defences, within an integrated pest control strategy, as an alternative to broad-spectrum pesticides. The review by Pickett, Smiley and Woodcock describes the range of mechanisms by which secondary metabolites act, with particular emphasis on semiochemicals, before considering their potential value in novel crop protection strategies. Purine alkaloids, principally caffeine, theobromine and theophylline, are major components of a range of favourite non-alcoholic beverages and are to be found naturally in over 100 species of plants. The review by Ashihara and Crozier summarizes aspects of general purine alkaloid metabolism before considering recent information on their biosynthesis and degradation. Biotechnological aspects of alternative sources of caffeine production, through cell and tissue culture, and the possibilities for transgenic manipulation of caffeine synthesis are described. The arabinogalactan-proteins are a class of heterogeneous plant proteoglycans with broad taxonomic and anatomical distributions, and a wide range of potential functions, although none of these is, as yet, precisely identified. Serpe and Nothnagel discuss recent data on structure-function inter-relationships with special consideration for the location of AGPs in different domains of the plant cell surface. One of the most rapidly advancing areas in experimental plant science is the study of those processes of molecular communication between microorganisms
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PREFACE
and plants, that initiate either acceptance (compatibility) or rejection (resistance) of a potential parasite. Keen reviews important milestones in the cloning and characterization of pathogen avirulence genes and the matching of complementary host resistance genes before considering how these advances in basic understanding may lead to strategies for practical disease control in the new millennium. As usual the Editor would like to thank all the contributors to this volume for their patience and co-operation in making his task easier. J . A . Callow
Nitrate and Ammonium Nutrition of Plants: Physiological and Molecular Perspectives
BRIAN G. FORDE'* and DAVID T. CLARKSON*
'Biochemistry and Physiology Department. IACR.Rothamsted. Harpenden. Hertfordshire A t 5 2JQ. UK 2Department of Plant Sciences. IACR-Long Ashton. University of Bristol. Long Ashton. Bristol BS18 9AF. UK
I . Introduction ............................................................................................... I1. Nitrogen Nutrition in its Natural Context ................................................ I11. Physiology of NO; Uptake ....................................................................... A. Energetics............................................................................................ B . Kinetics ................................... ....................................................... C . Regulation .......................................................................................... D . Relationships Between NO; Ions and the Hydraulic Properties of Roots ........................................ ..................................................... IV . Molecular Genetics of NO; Transport in Microorganisms ...................... A . Prokaryotes ............................... ................................... B . Lower Eukaryotes .............................................................................. V . Molecular Genetics of NO; Transport in Plants ...................................... A. NO; Transport Mutants .................................................................... B . NO; Transporter Genes ..................................................................... C . Relating the Molecular Genetics to the Kinetic Data ....................... VI . Physiology of NH: Uptake ...... ......................................................... A . Background ........................................................................................ B . Kinetics of NH: Uptake .................................................................... VII . Molecular Genetics of NH: Transport ............... ........... A . Prokaryotes ................................................... ........... B . Lower Eukaryotes .............................................................................. C . Higher Plants ......................................... .................................. VIII . Efflux of NO; and NH: .............................. ................................... A. NO; EMux ......................................................................................... B. Ammonium Efflux .................................................................. C . Net Uptake of NO; and NH: Along the Root Length ...................
2 3 5 5 5 11 19 21 21 24 28 28 30 38 40 40 44 49 49 50 51 52 52 57 58
*Author to whom correspondence should be addressed: Fax: +44 1582 763010; email:
[email protected] Advances in Botanical Research Vol. 30 incorporating Advances in Plant Pathology
ISBN 0-12-005930-4
Copyright 8 1999 Academic Press All rights of reproduction in any form reserved
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B. G . FORDE and D . T. CLARKSON
IX. Regulation by the Shoot of NO7 and NH: Uptake by Roots ................ A. Evidence for Shoot-derived Signals .................................................... B. Nature of the Shoot-derived Signals .................................................. X. Concluding Remarks: Looking Back and Looking Forward .................... Acknowledgements ..................................................................................... References ..................................................................................................
61 61 63 67 69 69
Nitrogen is the mineral nutrient that plants need in the greatest quantities and the one that most frequently limits plant growth and crop yields. Most plants get their nitrogen ( N ) from the soil as either nitrate or ammonium, with some species showing a strong preference for one ionic form over the other. The uptake of nitrate and ammonium ions by roots involves a complex set of membrane transport systems that includes both high- and lowaffinity transporters; net uptake rates can also be strongly influenced by the rate at which these ions efflux from root cells. Here we review our current picture of the mechanisms responsiblefor the uptake and efflux of nitrate and ammonium, attempting to integrate the large body of physiological data with the recent advances in the molecular biology of nitrate and ammonium transporters in bacteria and algae as well as in higher plants. We also review what is known at the physiological and molecular levels about the regulation of the N uptake systems, a process which involves both positive signals from soil nitrate or ammonium and feedback inhibitory signals that are generated by the plant’s internal N status.
I. INTRODUCTION The absorption and assimilation of mineral N by higher plants has been reviewed many times, so it is not our purpose here to go over well-worked ground, but rather to take stock of the latest advances and see how far they help us in understanding processes. Of particular interest has been the recent cloning and characterization of several families of NO, and NH: transporters, which has already shed new light on the structure and regulation of the transport systems. Because progress on the molecular front has inevitably been more rapid in microorganisms than in higher plants, and in several cases is leading the way in advances in our understanding of the plant N transport systems, we include a discussion of the molecular genetics of NO, and NH: transporters in bacteria, fungi and algae. We do not attempt to review the extensive literature on the structure and regulation of the enzymes of the NO; and NH: assimilatory pathways. A series of excellent reviews on these topics has been published in recent years (Crawford et al., 1992; Crawford and Arst, 1993; Marzluf, 1993; Meyer et al., 1993; Wray, 1993; Hoff et al., 1994; Crawford, 1995; Omata, 1995; Daniel-Vedele and Caboche, 1996; Huber et al., 1996; Lam et al., 1996).
NITRATE AND AMMONIUM NUTRITION OF PLANTS
3
We preface the new material by a short introductory background in which we try to set inorganic N nutrition in its context in nature. The literature cited in this background is mostly of a general kind, much of it already summarized in reviews appearing during the last two decades (Haynes and Goh, 1978; Pate, 1980; Clarkson et al., 1986; Haynes, 1986; Jackson et al., 1986; Bloom, 1988; Glass, 1988; Larsson and Ingemarsson, 1989; Larsson, 1994; Touraine et al., 1994; Glass and Siddiqi, 1995; Glass et al., 1997; von Wiren et al., 1997; Daniel-Vedele et al., 1998).
11. NITROGEN NUTRITION IN ITS NATURAL CONTEXT Of all the mineral nutrients, N is required in the largest quantities for the construction and maintenance of plant cells. Protein contains on average about 12% N by weight, and the whole dry matter of herbaceous plants typically contains from 1.5 to 4.5% N. With the exception of plants that develop Nfixing root nodules and insectivorous plants, the N found in the plant is normally absorbed through the roots, primarily in an inorganic form as NO, or NH:. The amino-N released by proteolysis from decaying plant or animal matter is first degraded to NH:. In soils or waters with a neutral to basic pH, the NH; then becomes oxidized to NO; by several steps, each depending on specific types of microorganisms. This conversion process can cope with relatively large amounts of NH:, as much analysis of agricultural systems has shown: within just a few weeks after the addition of an NH; fertilizer the free NH: concentration in the soil solution is diminished to a very low level (de Willigen, 1986). However, because nitrifying bacteria do not successfully colonize acidic soils, most of the N released from the turnover of organic matter in such soils may remain as NH;. Because the research literature has been so dominated by the mineral nutrition of agricultural plants of temperate or subtropical origin, N nutrition is often discussed as if NO: were the only significant source of N. More ecologically minded workers, especially those with interests in the huge areas of acidic soils in the higher and lower latitudes, take a different view and emphasize our comparative ignorance of the factors regulating NH: nutrition (Alexander, 1983). Mineral N is the only plant nutrient that is available in significant quantities in both a cationic and an anionic form, and plant roots have transport mechanisms for absorbing both NO; and NH: from the soil. Under some circumstances amino acids may provide another important source of N for plants. These are abundant in certain soils (0.01-0.1% of the dry weight), especially in the organic layers of acid soils where mineralization is slow (Alexander, 1983). Furthermore, the N transferred from fungi to plants in ectomycorrhizal associations is principally in the form of amino acids (Martin and Botton, 1987), and the same may be true of endomycorrhizas (Smith and
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B. G . FORDE and D. T.CLARKSON
Read, 1997). The role of amino acids as a N source for plants and the existence of amino acid uptake systems in plant roots are reviewed in more detail elsewhere (Frommer et al., 1994b; Glass and Siddiqi, 1995). An appreciation of the preferences that different species may have for particular N sources can have important ecological and practical implications (Kronzucker et al., 1997). In the soils of a mature forest, NH; is the predominant source of N, but after disturbance (such as occurs when the trees are cut down) the microbial ecology changes and NO, becomes the main N source. Late-successional species, which are adapted to growth in undisturbed NHi-containing soils, may then be at a competitive disadvantage over those adapted to NO;. In a recent study on white spruce (Picea glauca) it was found that the capacity of this conifer to absorb and process NO; was markedly inferior to its ability to use NH; (Kronzucker et al., 1997), and similar results were obtained with Jack pine (Pinus banksiuna) (Lavoie et ul., 1992). It has been suggested that this reduced capacity to use NO; could account for the difficulties in replanting conifer species on disturbed forest sites (Kronzucker et al., 1997). There is a marked difference in the ionic mobilities of NO; and NH; in most soils. Nitrate makes very few interactions with either the organic matter or the clay/sesquioxidecomponents of the soil and consequently is very mobile (diffusion coefficient N104cm2s-*) (Nye and Tinker, 1977). By contrast, NH: can interact strongly with the lattice of clay minerals, mimicking K + in some respects, and with a similarly low mobility (Cameron and Haynes, 1986). By analogy with K + , it is suggested that the mobility of NH; in soil is 50- to 500fold less than that of NO;, and more than 1000-fold slower than its diffusion in a simple aqueous solution. In the case of NO;, the ions absorbed by the root can be replaced rapidly by diffusion from the bulk soil solution into the rhizosphere. Thus, the PO;] at the root surface will not be much lower than in the bulk soil solution (Jungk, 1996). With NH;, however, its relatively slow diffusion towards the root surface, coupled with its rapid absorption, can create a zone in which the [NH;] is much lower than in the bulk soil solution. Roots have both high- and low-affinity transport systems for the uptake of NH; and NO; from the soil (Sections 1II.B and V1.B). For the reasons just mentioned, in the case of NHZ it is the high-affinity uptake system that is likely to be the more important under most soil conditions, even in acidic soils where the “Hi] in the bulk soil solution can be high. In natural soils, the PO;] is typically < 1 mM (Falkengren-Grerup and Lakkenborg-Kristensen, 1994), and again a high-affinity system would be likely to contribute most to NO; uptake. In well-fertilized agricultural soils, the situation is more complex. The [NO;] measured in such soils at different depths and over the growing season of a winter wheat crop was in the range 0.5-6mM (Gregory et al., 1979; Barraclough, 1986), so that the relative importance of the high- and lowaffinity systems would be expected to differ between different parts of the root system and also to vary with time.
NITRATE A N D AMMONIUM NUTRITION OF PLANTS
5
111. PHYSIOLOGY OF NO, UPTAKE A. ENERGETICS
It is well established that NO; uptake from the soil is an energy-requiring process (Clarkson, 1986). The proportion of root respiration allocated to ion uptake, of which NO; uptake is by far the most costly, has been estimated to be between 25 and 50% for a group of 24 herbaceous species (Lambers et al., 1996). Based on thermodynamic considerations and estimates of the cytosolic [NO;] in root cells, it has been proposed that NO; uptake across the plasma membrane (PM) would be energy-dependent over almost the whole range of [NO;] encountered in the soil (Zhen et al., 1991; Glass et al., 1992; Walker et al., 1995). As explained in the following section, it is now generally accepted that both high- and low-affinity NO; transport across the PM is coupled to the movement of H + down its free-energy gradient, and is therefore dependent on metabolism to supply ATP to the PM H+-ATPase. B. KINETICS
In common with most ion uptake processes, plots of NO; transport velocity vs external concentration yield curves that may be resolved into two or more phases. The kinetic data indicate that there are at least three NO; uptake systems in plant roots: a constitutive low-affinity transport system (LATS) and two high-affinity systems (HATS), one inducible (the iHATS) and the other constitutive (the CHATS)(Aslam et al., 1992; Glass and Siddiqi, 1995). The K , values estimated for the HATS are in the range 7-1 10 pM and the V,,, values in the range 2-9 pmol g-' fresh weight h-'; the corresponding values for the LATS are 170-25 000 pM and 8-700 pmol g-' fresh weight h-' (Peuke and Kaiser, 1996). However, in many of the early studies the net uptake of NO; was monitored by following depletion of NO; from the external medium. Net uptake is the difference between influx and efflux, the latter occurring via a separate mechanism (Section VIII.A), yet the two have often been treated as if they were a single process. The literature abounds with values of K , and V,,, determined in this way. As pointed out by Lee and Drew (1986), under conditions where simultaneous efflux is occurring, the use of net uptake rates would be expected to produce higher K,,, values (and lower V,, values) than those generated using influx data. Kinetic studies that have used 36C1-labelled chlorate (36C10;) should also be treated with caution as ClO; is not only toxic but is also an unreliable analogue of NO; (McClure et al., 1986; Siddiqi et al., 1992; Touraine and Glass, 1997). As discussed in the following sections, some of the most reliable kinetic data on NO; influx have come from short-term measurements of l3NO? or "NO; uptake.
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B. G. FORDE and D. T. CLARKSON
I . The Inducible High-affinity Uptake System (iHA TS) At external NO; concentrations ([NO;],,,) below 1mM, and in roots that have been exposed to NO; for more than a few hours, it is the iHATS that is the most important system for absorbing NO;. Nitrate influx through the iHATS can be measured directly by short-term uptake of 13N03 (Lee and Drew, 1986; Ingemarsson et al., 1987; Siddiqi et al., 1989) or 15NO; (Clarkson et al., 1996). Because efflux of the tracer increases as the cytosolic pool fills with labelled NO;, it is only during the early stages of its filling that it is possible to measure influx reliably. Experiments using tracer washout have indicated that, with one exception (Macklon et al., 1990), the half-time for tracer exchange with the cytoplasmic pool is around 3-7min (Lee and Clarkson, 1986; Siddiqi et al., 1989, 1990). These estimates are compatible with influxes in the order of 5 pmol NO; g-' root fresh weight h-', and a cytoplasmic pool of about 0.4pmol NO; g-' root (on the assumption that cytoplasm is 10% of the root mass). In species of the Brassicae, where net uptake rates as high as 35 pmol g-' fresh weight h-' have been measured by depletion of solutions containing initially 300 pM NO; (Lain6 et al., 1993), the half-times for cytoplasmic exchange may be even shorter. These very high net uptake rates were correlated with species having a large shoot:root ratio. The influx of NO; into cells deprived of NO; for some hours leads to a transient depolarization of the membrane potential across the PM (McClure et al., 1990a; Glass et al., 1992). In barley roots (Glass et al., 1992) and in fronds of Lemna gibba (Ullrich and Novacky, 1981), the extent of depolarization was dependent on the [NO;],,,, whilst in maize it was only the subsequent hyperpolarization that was related to the [NO;],,, (McClure et al., 1990a). These observations, together with the increased transport activity seen at acidic pH (Ullrich and Novacky, 1981; McClure et al., 1990b), are compatible with a H + co-transport mechanism where more than one H + moves across the membrane with each NO; ion (Meharg and Blatt, 1995; Mistrik and Ullrich, 1996). There has been an expectation that such a transport would lead to acidification of the cytoplasm, but the evidence for this is confusing and, as a consequence, the determination of the stoichiometry of H + :NO; has been difficult. Measurement of internal pH using intracellular electrodes inserted into root hairs of Limnobium stoloniferum indicated that the cytoplasm alkalized, rather than acidified, during NO; influx (Ullrich and Novacky, 1990). This was attributed to the activity of nitrate reductase (NR), but this was not actually tested. An alternative approach is to measure the pH close to the root surface using pH microelectrodes; here an alkalization is to be expected as protons cross the PM with the NO;. Using this approach with roots of L. stoloniferum a consistent stoichiometry of 1H :1NO; was measured, but intracellular electrodes revealed the characteristic depolarization of the membrane potential. A stoichiometry of 1:l is electrically neutral and it is assumed that the surface pH measurement underestimates the H + influx by at least 50% +
NITRATE AND AMMONIUM NUTRITION OF PLANTS
7
(Mistrik and Ullrich, 1996). These authors point out that the pH of a solution or compartment is determined solely by differences in the concentrations of strong cations and strong anions, as explained by quantitative acid-base theory (Stewart, 1983). Because the dissociation of water is so low, fluxes of H + and OH- into a compartment cannot, of themselves, change the pH. Such changes will be dependent on compensatory or parallel changes in the distribution of strong ions, the so-called Strong Ion Difference (SID). In practice, such movements will always occur and so pH measurements rarely reveal the stoichiometry at the H + co-transport sites. Undeterred by this, a recent study of NO, uptake in wheat made use of the observation that net H + secretion by roots decreased sharply when NO, was reintroduced into NO;-depleted cultures (Busch and Bottger, 1997). The kinetics of the decline in H + efflux, and the minimum value reached before the membrane depolarization increased H + pumping activity, were used to screen for varietal differences in NO; uptake. Analysis of current-voltage curves, using voltage clamp techniques and with [NO;],,, and pH,,, as variables, has been used to test a number of kinetic models for Hf/NO; co-transport in root hairs of Arabidopsis thaliana (Meharg and Blatt, 1995). The analysis revealed a voltage-dependence of the current carried during NO; uptake, and a charge-coupling stoichiometry of 2( +):lNO, was indicated. External pH influenced the inherent kinetics of the transport current in such a way as to indicate that the two positive charges were carried by protons. Meharg and Blatt went on to propose a kinetic cycle model of the transport mechanism. The main feature of the model is that the kinetic cycle is rate-limited by the transition of the accessibility of negatively charged sites on the unloaded transporter from the inside of the cell to the outside (in molecular terms this is best thought of as some re-orientation or relaxation of the transporter protein). This process will be driven faster as the membrane potential becomes more negative. The best model (Fig. 1) predicts that NO, and H + dissociate very rapidly from the transporter on the cytoplasmic side of the membrane, but that NO; release will be slowed down, possibly to a rate-limiting extent, as the cytosolic NO; concentration (~O;]c,,t) increases (this may explain allosteric-like regulation, see Section III.C.5). Finally, in this tour de force, it is predicted that ions bind to the transporter in the order H + , NO,, H + , and that the binding of NO; is absolutely dependent on the binding of the first H + , thus explaining the profound pH,,,-dependence of the transport. This analysis (in which neither cellular pH nor NO; fluxes were measured directly) gives us what is our most detailed and satisfying picture of the iHATS so far. A similar approach was applied to NO; uptake by the fungal hyphae of Neurospora crassa, and again the results are best described by a 2H /NO; co-transport, but a different binding order of NO; and the two H + ions is predicted (Blatt et al., 1997). +
8
B. G. FORDE and D. T. CLARKSON
As discussed in Section V.B.2, a family of NO;-inducible genes (the NRT2 family), which appears to specify at least one component of the iHATS, has recently been cloned from a number of plant species. 2. The Low-aflinity Uptake System (LATS) At high [NO;],,, ( > 1mM) it is the LATS rather than the HATS that is quantitatively the most important for NO, uptake. The non-saturable behaviour of NO; uptake in the low-affinity range (the rate increasing linearly between 5 and 100 mM), coupled with the fact that it showed a small response to treatment with metabolic inhibitors, had led to the previous supposition that it occurs by passive diffusion (Glass et af.,1990; Siddiqi et al., 1990). However, microelectrodemeasurements of the [NO,],,, and the electrical potential across the PM in barley root cells have indicated that, at 3-5mM, the [NO;],,, is greater than could be achieved by a passive transport process, even at high [NO,],,, (Zhen et af., 1991). Consistent with these results, subsequent electrophysiological studies with barley roots led to the conclusion that the
IN
OUT net -ve charge re-orientates
DRIVEN BY MEMBRANE POTENTIAL
H+ NO;
CONCENTRATION-
DEPENDENT
"?i N2
uncharged re-orientates
/
Fig. 1. A reaction kinetic model of a 2H+:NO; co-transporter present in root hairs of Arabidopsis. The various states of the transporter are designated by N,; all steps are envisaged as being reversible in some circumstances. The forward direction is driven by the effect of the membrane potential on the folding of the polypeptide when it has a net negative charge and the subsequent binding of H + which neutralizes the negative charge. The order in which the ions are released at the inner face of the PM is unknown. Simplified and modified from Fig. 12 in Meharg and Blatt (1995) with kind permission of the authors.
NITRATE AND AMMONIUM NUTRITION OF PLANTS
9
LATS, like the IHATS, is a H+-mediated system operating with a H+:NO; stoichiometry of 2 1 (Glass et ul., 1992). In barley, the kinetic data indicate that the LATS is a more or less constitutive system: it was present in roots that had not been induced by pretreatment with NO; and its activity increased by only 80% after induction, whereas the iHATS was inducible by 20-%-fold (Glass et ul., 1992). However the NRTZ (CHLZ) gene from Arubidopsis appears to encode a NOT-inducible, low-affinity H+/NO; co-transporter (Tsay et ul., 1993; Huang et ul., 1996), indicating that the LATS may also have an inducible component (Section V.C. 1). 3. Constitutive High-qffmity NO; Uptake (cHATS) There is now quite strong genetic and physiological evidence that the highaffinity NO, uptake system has at least two genetically distinct components with differing affinities for NO; and differing patterns of NO; regulation. The CHATSis present in roots that have never been treated with NO;, while the iHATS is present only in NO;-treated roots. Of the two systems, the CHATS has the higher affinity for NO;, but has a very low capacity for NO; uptake. In barley, the CHATSis reported to have a Km for nitrate of 6 2 0 pM,while the same studies estimated the Km of the iHATS to be in the range 13-79 pM (Lee and Drew, 1986; Siddiqi et ul., 1990; Aslam et ul., 1992). Reported values for the V , of the CHATS in non-induced barley roots are from 0.13 to 1.3pmolg-' fresh weight h-' (Lee and Drew, 1986; Behl et ul., 1988; Siddiqi et ul., 1990, Aslam et ul., 1992), while the Vmaxof the iHATS was generally at least an order of magnitude greater. As discussed in Section V.A.2, the isolation of an Arubidopsis mutant that is specifically defective in the CHATS (Wang and Crawford, 1996) suggests that the two components of the HATS are encoded by different genes. In barley and spruce it has been shown that the activity of the CHATSis stimulated by NO; treatment: the difference to the iHATS is that the CHATSis expressed in the absence of NO; and is much less strongly induced by its presence (about three-fold) (Aslam et ul., 1992; Kronzucker et ul., 1995a). The view is often advanced that the function of a constitutive NO; transport system is to allow external NO;, when it does become available, to be absorbed in sufficient amounts to induce the iHATS and the rest of the NO; assimilatory pathway. An alternative view is that passive uptake through an anion channel, although allowing the accumulation of only micromolar concentrations of NO; in the cytosol, could still be sufficient to induce the iHATS (Miller and Smith, 1996); a NO;-permeable channel that could serve this purpose has been identified in the PM of wheat protoplasts (Skerrett and Tyerman, 1994). On the other hand, if there were a NO; receptor on the outer surface the PM (Section III.C.l), the actual transport of NO; into the cell would not be required for induction of the IHATS.
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B. G. FORDE and D. T.CLARKSON
A final point relating to the CHATS is to note the difficulty in establishing that it is truly constitutive, i.e. that its basal level of activity in non-induced roots is truly independent of NO; induction. This is because significant amounts of NO; have been found in seeds of some species (Ngambi et al., 1981; Mack and Tischner, 1990), including barley (A. J. Miller, personal communication) which has been used extensively in these studies. So that even in seedlings that have never been deliberately supplied with NO;, the NO; in the seed might move symplastically into the young root axis, or leak into the external medium and partially induce the transport system. However, the finding that the iHATS is not induced under these circumstances (Aslam et al., 1992) would suggest that either the CHATS is much more sensitive than the iHATS to induction by low concentrations of NO;, or it is indeed constitutively expressed. 4. NO; Transport at the Tonoplast Because of the size of the vacuolar compartment (-90% of the cell volume), and its capacity to accumulate high [NO,], movement of NO; across the tonoplast membrane plays an important part in the process of NO; absorption by roots. Until fairly recently it has not been clear whether movement of NO; into the vacuole is an active or a passive process. With the accurate measurements of [NO,],,, and the tonoplast membrane potential made possible using double- and triple-barrelled NO;-selective microelectrodes, this question could be resolved (Zhen et al., 1991; Walker et al., 1995). Based on values of 4mM for the [NO,],,,, and 1CL20mV for the tonoplast membrane potential, it was calculated that passive transport across the tonoplast would produce an equilibrium PO,] in the vacuole of only 6-9mM (Miller and Smith, 1992, 1996). Nitrate concentrations in vacuoles are very commonly greater than this. For example, values of 39 and 26mM in barley and maize, respectively, were found when the cells were grown with lOmM NO, in the nutrient solution (Miller and Smith, 1996). Therefore, some form of energetic coupling is called for. Because the free-energy gradient of H + is directed into the cytoplasm, any coupling with H + would have to be in an antiport mode. A H +/NO; exchanger would, of course, be highly electrogenic, the vacuole gaining two net negative charges for each NO; ion entering it. The actual mechanism of active transport into the vacuole is unknown. The corollary of the above findings is that unloading of the vacuolar NO; pool should be a passive process (at least down to vacuolar concentrations of 6-9mM, assuming a cytosolic [NO,] of 4mM). There is evidence from a number of studies that the tonoplast possesses strong anion channel activity (Pope and Leigh, 1987; Tyerman, 1992), so that remobilization of NO; from the vacuole may involve anion channels whose activity is modulated in accordance with changing demands from the cytosol (e.g. as a result of an interruption in the NO; supply from the soil).
NITRATE AND AMMONIUM NUTRITION OF PLANTS
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C. REGULATION
The pathway of NO; uptake and assimilation is a highly regulated one, and its regulation has received much attention in bacteria, algae and fungi (Fernandez and Chrdenas, 1989; Marzluf, 1993; Omata, 1995; Lin and Stewart, 1998), as well as in higher plants (Crawford and Arst, 1993; Hoff et al., 1994; DanielVedele and Caboche, 1996; Huber et al., 1996). The enzymes NR and nitrite reductase (NiR) have been the most closely studied because they can be readily assayed and because specific antibodies and cDNA probes have been available for some time. Nitrate transport on the other hand is more difficult to assay, and until recently the relevant probes were not available to study their expression at the mRNA and protein levels. The work on NR has established that transcription of its structural genes is subject to both substrate induction and feedback repression (Daniel-Vedele and Caboche, 1996), and that the activity of the enzyme itself is reversibly inactivated by phosphorylation and binding of an inhibitory protein (Huber et al., 1996). In view of the highly regulated nature of NR, the question arises whether this is the key regulatory step in the pathway and whether the changes that are seen in rates of NO; uptake in response to changing N nutrition are driven by changes in NR activity. In algae it has been established that this is not the case and that the NO; uptake step is at least as highly regulated as NR: in Chlamydomonas the NO; transporter genes are induced by NO, and repressed by NH; (Quesada and Fernandez, 1994), and in both Chlorella and Chlamydomonas the NO; uptake system is more rapidly inhibited by NH; than is NR itself (Pistorius et al., 1978; Florencio and Vega, 1982). In higher plants the available evidence supports a similar conclusion. Using NR-deficient barley mutants it was shown that the NO; uptake system was induced independently of NR (Warner and Huffaker, 1989), and higher-plant NO; transporter genes have been demonstrated to be both NO; -inducible (Tsay et al., 1993; Trueman et al., 1996a; Quesada et al., 1997; Amarasinghe et al., 1998) and feedback-repressible (Quesada et al., 1997; Amarasinghe et al., 1998) at the mRNA level. The separate regulation of NO; uptake and NR in higher plants is perhaps not surprising when one considers that reduction by NR in the root is only one of four possible fates of the absorbed NO; (the others being efflux, storage in the vacuole and xylem-mediated transport to the shoot). 1. Induction by NO; and Speculations on the Putative NO; Receptor The uptake of NO; is regulated very differently from that of other ions, in that the pathway is induced by its substrate: in the absence of a supply of external NO; the potential for transport activity declines. In contrast, the high-affinity transporters for SO,, inorganic phosphate, K + and NH; are stimulated, in a nutrient-specific manner, by a period of nutrient deprivation (Clarkson and Liittge, 1990). In maize roots not previously exposed to NO5 the rate of 13NO;
12
B. G . FORDE and D. T.CLARKSON
influx increased 2.5-fold after exposure for 6 h to l00pM NO,, after which there was no further increase (Hole et al., 1990). Low concentrations of NO; are also able to induce the NO, uptake system, and with similar kinetics to those seen when the same concentration of NO, is used, indicating that it is not due to oxidation of the NO; to NO; (Aslam et al., 1993). Evidence obtained using inhibitors of RNA and protein synthesis has indicated that induction involves the synthesis of new transporter protein (Hole et al., 1990; Aslam et al., 1993; Siebrecht et al., 1995). Molecular studies have confirmed that the abundance of mRNAs for both low-affinity (NRTZ) and high-affinity (NRT2) NO, transporter genes (Sections V.B. 1 and V.B.2.) increases rapidly when N-starved roots are treated with NO; (Tsay et al., 1993; Trueman et al., 1996a; Quesada et al., 1997; Amarasinghe et al., 1998; Krapp et al., 1998). Using antiserum against a barley NRT2 polypeptide, it has been shown that there is a corresponding increase in the amount of cross-reacting protein (M. Hansen, S. Dunn and B. G. Forde, unpublished observations). Studies of the expression of the genes for NR and for the NRT2 high-affinity NO; transporter in roots of Lotus japonicus showed them to be co-ordinately regulated during both induction and de-induction (D. T. Clarkson, I. M. Prosser, A. S. Smyth, I. Onyeocha and B. G. Forde, unpublished results). Where and how is the NO; signal perceived? In Chlamydomonas there is evidence that the [NO,],,, is important for induction. When Chlamydomonas cells were engineered so that they constitutively over-expressed NR, the NO; transporter genes were no longer NO,-inducible (Navarro et al., 1996). The authors proposed that the high levels of NR activity decreased the [NO;],,, below the concentration required to induce transcription of the transporter genes. The available evidence for barley favours a NO, receptor on the external surface of the PM. In barley roots, a NO, (or NO;) concentration of only 10pM was sufficient to obtain almost complete induction of the iHATS (Aslam et al., 1993). Even very short pulses of NO, (e.g. 70pM for 5min) applied to barley seedlings were able to induce the iHATS: the NO; uptake capacity began to increase within 60 min of the pulse and reached a maximum after 4 h, after which it rapidly decayed (Tischner et al., 1993). These results suggest that it is external NO; that is being sensed and show that transporter activity is labile in the absence of the NO, signal. The idea that NO; is sensed externally is also supported by the observation that the abundance of NR mRNA falls dramatically within just 30min of withdrawing external NO, from barley plants, even though tissue PO;] did not change significantly over the same period (although xylem [NO,] did decline rapidly) (Sueyoshi et al., 1995). Similarly, Siddiqi and colleagues found that removal of external NO; led to a reduction in activity of the influx system, even though tissue [NO;] remained high (Siddiqi et al., 1989). In the latter case the authors proposed that the influx system was responding to rapid changes in [NO,],,,. However, the observations of Miller and colleagues with NO;-
NITRATE AND AMMONIUM NUTRITION OF PLANTS
13
selective microelectrodes (Zhen et al., 1991; van der Leij et al., 1998) suggest that the cytosolic NO; pool in barley roots is strongly buffered by eMux from the vacuole when external NO; is withdrawn (Section III.C.5). Therefore, the regulatory mechanism is either very sensitive to small changes in vacuolar or cytosolic NO; concentrations or is responding to the much larger changes in “0,lext. What is true of barley may not hold for all plant species. In L.japonicus there is evidence that it is the internal NO; pool to which the NO;-regulated genes are responsive. When NO; was withdrawn from roots of L. japonicus there was no decline in the abundance of the mRNAs for NR and the NRT2 highpffinity NO; transporter until 48 h later (D. T. Clarkson, I. M. Prosser, A. S. Smyth, I. Onyeocha and B. G. Forde, unpublished results). In Escherichia coli, NO; is sensed externally by means of a signal transduction pathway belonging to the family of two-component signalling systems (Cavicchioli et al., 1996). Homologues of this family have been found in plants, some of which are responsible for transducing the signals from ethylene and cytokinin (Wurgler-Murphy and Saito, 1997). Evidence that phosphorylation reactions (and Ca2+ ions) are involved in NO; signalling in maize leaves has been obtained using inhibitors (Sakakibara et al., 1997), but no direct evidence for a two-component signalling pathway for NO, has yet been obtained in plants. An alternative candidate for an extracellular NO, receptor in plants is the PM-bound form of NR (or PM-NR). This protein, which has been identified in Chlorella (Tischner et al., 1989; Stohr et al., 1993) and in higher plants (Ward et al., 1988; Demarco et al., 1994; Klobus et al., 1994; Barr et al., 1995; Chen and Wang, 1995), cross-reacts with antiserum raised against cytosolic NR, but is smaller and quite distinct from the cytosolic form and is covalently linked to the outer surface of the PM by a glycosyl-phosphatidylinositol (GPI) anchor (Stohr et al., 1995a; Kunze et al., 1997). Its function in higher plants is unknown, but in Chlorella it appears to have a role as a blue-light sensor, mediating the stimulatory effect of blue light on NO; uptake in these cells (Stohr et al., 1995b). Many cell-surface molecules that transmit intracellular signals in human T lymphocytes are GPI-anchored, and as they lack a cytoplasmic domain they are thought to transmit their signals by interacting with other cell-surface proteins that do span the PM (Resta and Thompson, 1997). Thus, if PM-NR is a NO, receptor, it is likely to operate in conjunction with a transmembrane component that has yet to be identified. 2. Inhibition by NHZ Many studies have shown that the presence of NH,f in the nutrient solution can inhibit NO, uptake (e.g. Haynes and Goh, 1978; Rufty et al., 1982; Breteler and Siegerist, 1984; Lee and Drew, 1989; Siddiqi et al., 1990; Touraine and Glass, 1997). The reverse effect, a stimulation of NO, uptake by low concentrations of NH:, has also been reported (e.g. Bloom and Sukrapanna,
14
B. G . FORDE and D. T.CLARKSON
1990), and this has been attributed to the acidification of the medium that is associated with NH; utilization, and the consequently increased H gradient across the PM to drive NO, influx (Smart and Bloom, 1998). However, at a high concentration of NH; ( 5 mM) this beneficial effect was no longer evident and the inhibitory effect dominated. The inhibitory effect of NHZ on NO, influx can be both short and long term. In barley roots, NH; in the external medium caused a rapid inhibition of 13NO; uptake (within 3 min) and an equally rapid reversal of inhibition when it was removed (Lee and Drew, 1989). Because the inhibitory effect increased with the logarithm of the pH;Iext, the authors suggested that it was due to depolarization of the membrane potential in the PM. Consistent with this, direct measurements on tomato and barley root hairs found that NH,f did cause a temporary membrane depolarization, and that uptake of inorganic phosphate was inhibited as well as NO; (Ayling, 1993). Thus, the very rapid effects of NH; on NO; influx seem to be rather non-specific, and because the membrane potential gradually reverts to normal (presumably due to activation of the H + pump), this component of NH; inhibition should also be short-lived (< 3C60min). Longer-term inhibitory effects of NH; on NO, uptake are probably due, at least in part, to feedback regulation from amino acids or other products of NH; assimilation rather than NH; itself (see the next section). In support of this it has been found that treating roots with methionine sulphoximine (MSX), an inhibitor of the first step in the pathway of NH; assimilation, can relieve NH; inhibition of net NO, uptake (Breteler and Siegerist, 1984; Lee et al., 1992), although in one study it did not (de la Haba et al., 1990). There are also reports suggesting direct effects of the external and internal NH,f pools on NO, uptake. A direct effect of [NH;Iext on NO; uptake was suggested by experiments in which NH; inhibited the induction of the NO; uptake system by NO, (or NO;), but only if the NH; was present in the external medium when the inducers were added (Aslam et al., 1996a). Loading of the barley roots with NH; prior to induction raised root pH;] from 1.4 to 30mM, but if NH; was absent from the external solution during induction itself no inhibitory effect was observed. The authors speculate that all of the tissue NH; might be sequestered in the vacuole, leaving cytosolic NH,f concentrations low after removal of the external source of NH;. However, observations discussed in Section VI.A.3 make this seem unlikely. An alternative possibility is that the [NH;],,, somehow has a negative effect on induction of NO; uptake. However, it should be noted that in these experiments NO; uptake was measured by depletion of the external solution (Aslam et al., 1996a), so that the effect of the NH; could have been to stimulate NO; efflux, rather than inhibit influx, as has been reported to occur (Deane-Drummond and Glass, 1983; Deane-Drummond, 1985; Aslam et al., 1994). +
NITRATE AND AMMONIUM NUTRITION OF PLANTS
15
The longer-term inhibitory effect of NH; on NO, uptake by sunflower plants was attributed to a decreased rate of NO; reduction (de la Haba et al., 1990): the NH; treatment appeared to have a direct inhibitory effect on root NR activity and this was accompanied by a slight increase in the root [NO;].
3. Feedback Inhibition by Amino Acids It is universally agreed that the iHATS for NO; is subject to negative feedback regulation by some product(s) that monitors the N status of the tissue. When feedback regulation of an enzyme occurs at the transcriptional level it is normally referred to as ‘feedback repression’ and when it is at the level of allosteric modulation of enzyme activity as ‘feedback inhibition’. In the case of NO; transport both of these mechanisms could be operating, so our use of the term feedback inhibition in the following discussion is for convenience and should not be taken to imply that only one kind of regulation is involved. Feedback inhibition of the iHATS is relieved under conditions where the N status of the plant is diminished, such as when the N supply is insufficient to meet the demands from growth or N storage. Two basic methods have been used to investigate this phenomenon (see Fig. 2). In the first (Case I), the plants are deprived of N for several days to reduce their overall N status before challenging them with NO; and measuring influx rates. The experiments of Lee and Drew showed that the period of N starvation stimulated influx by the iHATS, but that the full extent to which the system had been derepressed was only apparent after the N-starved plants had been given a 5-h pre-treatment with NO; to re-induce the iHATS (Lee and Drew, 1986). Similar results have been reported by others (e.g. Siddiqi et al., 1989, 1990; Hole et al., 1990). However, in recent experiments with perennial ryegrass (Lolium perenne), the V,, for net NO; uptake decreased continuously over a 12-day period of NOT-deprivation, with no evidence that feedback inhibition had diminished, even though the demand for N would have been acute (the wild-type plants in this experiment increased in size throughout the 12-day period and their % N declined) (Bakken et al., 1997). In the second type of experiment (Case 2), the addition rate of NO, is set at a value that is lower than the potential relative growth rate (e.g. Mattsson et al., 1991), or the plants receive a very low [NO:] that is continuously maintained (e.g. Clement et al., 1978; Macduff and Jackson, 1992) (Fig. 2). In such circumstances the [NO;] is sufficient to keep the system induced through the positive effect on transcription, but the negative effect of the feedback inhibitor(s) is low. Thus, demand is high but the system is able to make a kinetic response because some NO, is present. A further complication arises if comparisons are to be made between plants that are growing at different rates due to the N-supply regime. As Ingestad and Lund showed some time ago (Ingestad and Lund, 1979), when growth processes slow down, a steady state with N supply can be established in which NO; uptake will also slow down. This suggests that the influence of demand
16
B. G . FORDE and D. T. CLARKSON
0
PM
out
Soil I
in
Amino N from shoot
Vacuolar NO; pool
I
I
F I1
assimilation
@ mRNA
indiictinn
~
@
reoression
induction
Fig. 2. Model for the regulation of the iHATS for NO; transport. For simplicity the model indicates only transcriptional controls, but other regulatory mechanisms could also be involved (Section 1II.C). Two alternative signalling pathways for NO; induction are shown; the evidence favouring each is discussed in Section 1II.C.1. The model helps to illustrate the differences between two types of experimental procedure that have been used to study feedback regulation of the iHATS (Section III.C.3). CASE 1: plants are grown on medium containing NO;, and the NO; is then removed. The flux of NO; through the iHATS transporter then ceases and the flux through the assimilatory pathway declines, accompanied by a decrease in the size of the amino-N pool. Metabolite repression of transporter gene expression is thus relieved. The cytosolic NO; pool is replenished for a while from the vacuolar NO; pool, but the flux across the tonoplast is substantially slower than across the PM. Thus, the positive effect of NO; on expression of the transporter genes is maintained until the vacuolar NO; pool is depleted, at which time they will be switched off and the physiological activity of the iHATS will begin to decline. (Note that in barley, the evidence discussed in Section 1II.C.1 favours an external NO; receptor, and in this situation the activity of the iHATS might be expected to decline more rapidly after withdrawal of external NO; .) Because the transporter genes have been switched off (de-induced), a short period of re-induction with NO; is required before the extent to which the influx system has been derepressed can be fully assessed. CASE 2: plants are kept on a continuous, subadequate NO; supply. Here the inductive effect of NO; is maintained, but the amino-N pool is small and feedback repression is weak. Thus, continuously high levels of gene expression and iHATS activity are expected.
on transport rates (i.e. Case 2) is most likely to be seen in transitional states where the plant is moved from one supply state to another. As reviewed elsewhere, studies with a variety of plant species have shown that supplying amino acids exogenously to roots leads to a reduction in the rate of NO; uptake, while conditions that decrease the amino acid content of the
NITRATE AND AMMONIUM NUTRITION OF PLANTS
17
root can stimulate NO; uptake (Atilio and Causin, 1996). In the filamentous fungi, glutamine is thought to be a key metabolite repressor in N-regulatory circuits (Marzluf, 1993), but its role in feedback regulation of NO; uptake in plants is still not clear. In a study of young soybean (Glycine max) plants, NO; uptake was inhibited by a number of externally supplied amino acids (Muller and Touraine, 1992), an effect which was subsequently shown to operate on NO; influx and not on efflux (Muller et al., 1995). However, in these experiments the most potent inhibitor of NO; uptake was not glutamine, but arginine (Muller and Touraine, 1992). Similarly, when the effect of externally applied amino acids on NO; uptake by maize cell suspension cultures was investigated (Padgett and Leonard, 1996), a correlation was found between the size of the total amino acid pool and the rate of NO; uptake, but there was no correlation with respect to the pool of glutamine nor any other individual amino acid. While transamination may quickly lead to accumulation of glutamine, it is hard to argue that glutamine is the key regulatory compound when other amino acids produce greater effects. Nevertheless, the interpretation of such experiments can be difficult as the relevant amino acid pool may be localized to one compartment of the cell (such as the cytosol, which is < 10% of the cell volume) and it may be restricted to particular tissues within the root. There is now evidence that feedback inhibition of NO, influx operates at least partly through an effect on synthesis of the transporter. Addition of 5 mM glutamine or NH&l to NO;-grown Nicotiana plumbaginifolia plants led to a rapid decline in the abundance of the mRNA for the NRT2 high-affinity NO; transporter (Quesada et al., 1997; Krapp et al., 1998). The effect, which was seen within 3 h of the treatment, was more pronounced with glutamine than with NHZ, again suggesting that it was the products of NHZ assimilation rather than NH: itself that was responsible. Similar repressive effects of NH,f treatment on the accumulation of NRT2 mRNA have been seen in both L. japonicus (D. T. Clarkson, I. M. Prosser, A. S. Smyth, I. Onyeocha and B. G. Forde, unpublished results) and soybean (Amarasinghe et al., 1998). In the L. japonicus experiments it was found that the NH,f treatment had no effect on the "Hi] in the root, but did produce small increases in the glutamine and asparagine pools. A N . plumbaginifolia mutant (meal 15), selected for resistance to methylamine (a toxic analogue of NH:), appears to be altered in the feedback regulation of NO; uptake (Godon et al., 1996). Whereas NO; uptake in the wild type was inhibited by methylamine (possibly after conversion to methylglutamine), NO; uptake by the mutant was found to be less sensitive to the inhibitory effect. The reduced inhibition of NO; uptake by methylamine was correlated with a diminished repression of the NRT2 gene at the mRNA level (Krapp et al., 1998). Only the sensitivity to methylamine was affected by the mutation: NO; uptake in the mutant was still susceptible to inhibition by NHZ .
18
B. G . FORDE and D. T.CLARKSON
Two regulatory mutants (Nrgl and Nrg2) of Chlamydomonas have also been isolated in which the NO, transporter and assimilatory genes are no longer subject to negative regulation by NHZ (or its derivatives) (Prieto et al., 1996). These mutants are particularly promising because the corresponding genes were generated by insertional mutagenesis, so their cloning should be relatively straightforward. 4 . Feedback Inhibition b y Internal NO; Studies with an NR-deficient barley mutant provided evidence that the internal pool of NO; itself has an inhibitory effect on NO; influx (King et al., 1993). The mutant (narla;nar7w) showed strong feedback inhibition of NO; influx within about 5days of exposure to a low concentration of NO;, despite the greatly reduced flux of N into reduced N products. A corollary of this result is that any treatment that leads to an increase in the internal NO; pool could have an indirect effect on NO; influx. For example, if NR activity were inhibited by NH;, as was reported for sunflower (de la Haba et al., 1990), this could play a part in the inhibitory effect of NHZ on NO; uptake (Section III.C.2). It would be of particular interest to monitor changes in cytosolic and vacuolar NO, pools within the roots and shoots of NRdeficient and wild-type plants during the time-course of induction and feedback inhibition to establish whether there are any correlations with changes in the rate of NO, influx. 5. Regulation of the Cytosolic NO; Pool
Early attempts to estimate [NO,],,, by tracer washout studies with 13N gave values in the range 1-35 mM, depending on [NO;],,, (Lee and Clarkson, 1986; Siddiqi et al., 1991). Such results implied that [NO;],,, is not strongly regulated (Devienne et al., 1994a, b). A very different picture has emerged from direct measurements of NO; ion activity using NO; -selective microelectrodes inserted into the cytosol of cortical and epidermal cells of barley roots (Zhen et al., 1991; Walker et al., 1995; Miller and Smith, 1996). The critical refinement of this technique was the construction of multibarrelled electrodes which are able to report simultaneously on the membrane potential, the pH and the NO; activity. The pH electrode indicates whether the tip is in the cytosol (pH -7) or in the vacuole (PH 5-6). When barley was grown in nutrient solution at pH 6 , with NO; concentrations from 0.1 to lOmM, all of the cytosolic recordings were grouped around a common mean of 4 mM, whereas the [NO;],,, varied with the external concentration. It has been shown that the [NO;],,, is maintained for many hours after external NO; has been withdrawn, the vacuolar store acting as a buffer (van der Leij et al., 1998). The apparent homeostasis for cytosolic NO; raises intriguing questions about how the [NO;],,, could be maintained despite rapid changes in the rate of flux (e.g. when external NO; is withdrawn or when the rate of assimilation declines). The implication is that the activity of the NO, transporters in the
NITRATE AND AMMONlUM NUTRITION OF PLANTS
19
PM and the tonoplast should be sensitive to relatively small deviations in the so that influx from the external or vacuolar pools is rapidly inhibited when the [NO&,,, rises and stimulated when it falls. This is consistent with the evidence discussed above that NO; influx is subject to feedback regulation by the internal NO; pool (King et al., 1993). A challenge for the future will be to is monitored and how this is establish the mechanism by which the (NO;], transmitted into changes in activities of the transporters.
(NO;],
D. RELATIONSHIPS BETWEEN NO- IONS AND THE HYDRAULIC PROPERTIES dF ROOTS
I . Eflect of N Deficiency on Hy&aulic Conductance in Roots Nutrient deficiencies, including N-deficiency, have long been known to cause reductions in stomata1conductance. It was the pioneering work of Radin and colleagues that related these events to a major decrease in the hydraulic conductance (&)of roots (Radin and Ackerson, 1981; Radin and Eidenbock, 1984) and of the PMs of individual root cells (Radin and Matthews, 1989). There is also a long series of reports that the presence of NO; in the bathing medium of excised root systems stimulates exudation of xylem sap (Minsnall, 1964, 1968; Ezeta and Jackson, 1975; Triplett ez al., 1980). A feature, common to all these experiments, was that seedlings were given pre-treatments with water or KCl, which supported small volume flows (J,) of sap; the addition of KN03 to the bathing medium increased J, after a number of hours. In a re-examination of this phenomenon (Barthes et al., 1995) it was found that increases in Jv were unrelated to the osmolarity of the sap: the assumption had been made in some earlier work that the more rapid deposition of NO; (and of K+) in the xylem would provide an increased osmotic driving force, In fact, although solute fluxes into the xylem were and hence a greater .Iv. higher in NO;-treated roots than in Cl--treated roots, the water fluxes were greater still, showing that the & of the root system was one of the major parameters affected by the presence of NO;. This result is predicted by a number of studies which showed that deprivation of NO;, SO:- or inorganic phosphate caused similar, major reductions ( ~ 7 5 8 0 % )in hydraulic conductance within a few days (Radin, 1990, Karmoker er al., 1991; Carvajal et al., 1996). While the conductances declined, there were no obvious signs of disturbed metabolism or growth, and the effects in wheat seedlings were reversed in a few hours when either NO; or inorganic phosphate was resupplied. In searching for an explanation for the effect of NO;, many of the authors above were unaware that precisely the same phenomena may be observed during episodes of P or S deprivation. In a further paper (Barthes et al., 1996) it was shown that the stimulatory effect of NO? on J , in excised maize roots can be largely blocked by pre-treating roots with tungstate or MSX, which demonstrably inhibited NR and glutamine synthetase (GS),respectively.
20
B. G . FORDE and D. T.CLARKSON
Moreover, the negative effect of tungstate treatment on J , could be reversed by supplying urea as a source of NH;. These results indicate that it is not NO, itself that acts as a signal to regulate hydraulic conductance, and suggests that there is some interaction between downstream products of N assimilation and the root L,. In a further development of this work, NO, nutrition has been shown to have a far greater impact on the hydraulic conductivity measured using an osmotic driving force (root pressure exudation) than with an imposed hydrostatic pressure (Hoarau et al., 1996). This is explained by the fact that in the former conditions a large proportion of water crosses the root via the transcellular pathway, which involves serial passage across cortical cell membranes, whilst under hydrostatic pressure much of the cellular pathway is by-passed in the cortical apoplast (see Steudle and Frensch, 1996). Changes in the L, may lead to a variety of physiological changes in the plant, culminating in the closure of stomata and a greater allocation of photosynthates to root growth (Chapin et al., 1988), but the relationship between these events is still unclear. It has been suggested that they may be part of a generalized response to nutrient deficiency, mitigating the effects of an inadequate nutrient supply by slowing leaf development and channelling resources to root growth in advance of a major nutritional crises (Chapin et al., 1988; Chapin, 1990).
2. Role of Aquaporins It has become clear that PMs in plant roots have abundant water channels, in the form of aquaporins (Kammerloher et al., 1994), and that these may account for a large fraction of the total water conductance of these membranes (Steudle and Henzler, 1995). Evidence to the contrary was recently reported from a study of the water permeability of isolated membrane vesicles from cultured tobacco cells (Maurel et al., 1997), but this may relate to the rather special circumstances in which such autotrophic cells are cultured; in the Murashige and Skoog medium used, PM water fluxes would be very small in comparison with, say, root tissue. The unexplained effect of nutrient anion deprivation on root and PM L, may be due to some interaction with water channel function (Carvajal et al., 1996). There is evidence that a putative PM aquaporin may need to be phosphorylated for it to function, and a membrane-associated Ca2+dependent protein kinase may be involved in this (Johansson et al., 1996). If this is a plausible way of altering the L, of the PM, then there must also be an appropriate protein phosphatase awaiting discovery. The kinase and the phosphatase would then be key components of the regulatory mechanism that controls the opening of the aquaporins in response to changes in nutrient availability.
NITRATE AND AMMONIUM NUTRITION OF PLANTS
21
IV. MOLECULAR GENETICS OF NO; TRANSPORT IN MICROORGANISMS A. PROKARYOTES
Nitrate is an important N source for many bacteria, but some bacteria also use the NO, ion as an alternative electron acceptor in energy generation (the process of NO; respiration) (Lin and Stewart, 1998). Both processes involve transmembrane fluxes of NO; and/or NO;, and the advantages of prokaryotic systems for molecular genetics have meant that much progress has been made in recent years in identifying and characterizing the transporters responsible for these fluxes. Because there is significant overlap between transporters of NO, and NO; (some transporters carry both anions), both types will be considered in this section. The NO;/NO; transporters so far identified in prokaryotes belong to three distinct groups: the ABC type, the NarK type and the NirCFocA-FdhC type. As we will see, only NarK and its homologues are related to NO, transporters so far found in plants.
I . ABC-type NO, and NO, Transporters Amongst prokaryotes, the process of NO; uptake has been most closely studied in the cyanobacteria, particularly the unicellular Synechococcus spp. (Lara et al., 1993a, b; Omata, 1995). Synechococcus has an active NO, transport system with a K , for NO; of 1 pM (Rodriguez et al., 1992). Based on its ability to competitively inhibit NO; uptake, NO; appears to be transported by the same carrier system. The high-affinity NO,/NO; permease of Synechococcus sp. strain PCC7942 (formerly Anacystis nidulans R2) is encoded by the nrtABCD gene cluster, located in the same operon as narB and nirA, the structural genes for NR and NiR. The nrtABCD genes, which are regulated by N availability (Suzuki et al., 1995), encode a multicomponent permease belonging to the ABC (ATP-binding cassette) superfamily of transporters. The ABC transporters are a large and diverse group of proteins that share a conserved ATP-binding domain and transport a wide range of different types of substrate (Higgins, 1992; Hediger, 1994). The bacterial import systems are sometimes referred to as ‘binding protein-dependent transport systems’ because of their dependence on substrate-binding proteins in the periplasm (the compartment between the inner and outer membranes of Gram-negative bacteria). In addition to the periplasmic-binding protein, a typical bacterial ABC transporter consists of four subunits: two hydrophobic subunits with six membrane-spanning segments each, and two hydrophilic subunits located on the cytoplasmic face of the membrane. The first two form the pore through which the substrate traverses the membrane and are believed to determine substrate-specificity, while the second two bind ATP and couple ATPhydrolysis to the transport process. In prokaryotes the subunits are usually encoded by four separate genes within the same operon.
22
B. G. FORDE and D. T.CLARKSON
Based on sequence comparisons with other ABC transporters the likely functions of the components of the Synechococcus NO; transporter can be deduced (Omata, 1995). The nrtB gene is predicted to encode the hydrophobic subunits (presumed to function as a homodimer), and nrtC and nrtD to encode the ATPase subunits (see Fig. 3). The nrtA gene encodes a hydrophilic 48-kDa polypeptide with a potential signal peptide at its N-terminus (Omata, 1991), which is the precursor for an abundant 45-kDa membrane-bound lipoprotein (Maeda and Omata, 1997). It was proposed that NrtA is the periplasmic substrate-binding protein for the NO;/NO; transporter (Omata, 1999, and this hypothesis was subsequently substantiated by experiments showing that the NrtA protein is able to bind NO; (and NO;) with a h g h affinity ( K , = 0.3 pM) (Maeda and Omata, 1997).
Fig. 3. Model for the structure of the multicomponent ABC-type NO;/NO; transporter from the cyanobacterium Synechococcus sp. PCC7942. The hydrophobic NrtB subunit is presumed to function as a dimer, which serves as the membranespanning component of the transporter. The NrtC and NrtD subunits, containing the ATP-binding domains, are on the cytoplasmic face of the membrane. The NrtA subunit is a 45-kDa lipoprotein thought to be the equivalent of the periplasmic substratebinding protein in other ABC transporters; it has a high affinity for NO; and NO; and is anchored to the membrane. In addition to its ATP-binding domain, the NrtC subunit has an additional NrtA-like regulatory domain with a role in mediating feedback inhibition. The model is based on that presented by Omata (1995).
NITRATE AND AMMONIUM NUTRITION OF PLANTS
23
The NrtC polypeptide has some interesting features and may have an important regulatory function: whilst the 30-kDa NrtD polypeptide is an apparently typical example of an ATP-binding subunit, the 72-kDa NrtC polypeptide is an unusual mosaic consisting of an N-terminal domain with 58% identity to NrtD, and a C-terminal domain with 30% identity to NrtA (Omata et al., 1993; Omata, 1995). The C-terminal domain could be deleted without affecting the NO; or NO; uptake activity of the transporter (Kobayashi et al., 1997), but the modified transporter was no longer subject to feedback inhibition by NH:. Studies on a closely related Synechococcus species (PCC6301) showed that assimilation of the NH; is required for its inhibitory effect on NO, transport, suggesting that glutamine or a metabolite of glutamine is involved (Lara et al., 1987). The NrtA-like domain seems to have an important role in this feedback inhibition of the NO;/NO; transporter. The molecular mechanisms responsible are unclear, although there is some evidence that phosphorylation-dephosphorylation reactions may be involved (Rodriguez et al., 1994a). In ABC transporters the driving force for substrate translocation across the membrane is believed to come from ATP hydrolysis (Higgins, 1992). However, studies of NO; uptake in Synechococcus sp PCC7942 found a stringent requirement for Naf ions in the external medium (Rodriguez et al., 1992), leading to the suggestion that NO; transport in this organism is driven by the energy of the Naf gradient across the PM. A detailed kinetic model has been proposed in which NaN03 is the substrate for the transporter and Naf additionally behaves as an activator (Rodriguez et al., 1994b). The [NO,],,, used in these experiments (20 pM) is in the range where disruption of the nrtA gene eliminates NO; uptake (Omata et al., 1989), indicating that only the ABC-type transporter is active under these conditions. If the NO; transporter in this cyanobacterium is energized by the Na+ gradient, and not by ATP hydrolysis, it would represent an entirely novel mechanism for an ABC transporter. It remains to be seen how the apparent conflict between the molecular and the physiological data on NO, transport in Synecococcus can be resolved. 2. The NarK Family of NO; and NO; Transporters The E. coli narK gene (narKEc) was first identified by virtue of its location within the same cluster as the narGHZ genes encoding the major form of respiratory NR (Lin and Stewart, 1998). Nucleotide sequencing showed it to encode a 50-kDa hydrophobic protein with 12 putative transmembrane domains (Noji et al., 1989). Although initially thought to be a NO;/NO; antiporter (Demoss and Hsu, 1991), later work using I3NO; and a NO;sensitive fluorophore suggested that it is a NO; extrusion system (Rowe et al., 1994). E. coli has a second narK-related gene, narU, which is able to complement a narK mutant (Bonnefoy and Demoss, 1994), and the narK gene from Bacillus subtilis (narKBs)was likewise able to complement the E. coli narK
24
B. G . FORDE and D. T. CLARKSON
mutant (Ramos et al., 1993), indicating that these members of the narK family can also function as NO, efflux systems. Two other narK homologues may specify NO, influx systems: the B. subtilis nasA gene, which is located in the same gene cluster as the genes for assimilatory NR and NiR (Ogasawara et al., 1995; Ogawa et al., 1995), and the Staphylococcus carnosus narT gene, a mutation in which causes a defect in NO, uptake without causing NO; accumulation (Fast et al., 1996). However, it is not yet certain that there is a clear distinction between the NO; influx systems and the NO; efflux systems; the possibility that some members of the bacterial NarK family can perform both functions does not seem to have been fully tested. As discussed in Section V.B.3, NarK and its bacterial homologues are related to a family of high-affinity NO; transporters found in fungi, algae and higher plants. 3. NO; Transporters of the NirC-FocA-FdhC Family The E. coli nir operon includes three genes (nirB, nirD and cysG) that are necessary and sufficient for synthesis of the major NADH-dependent NiR in this species (Peakman et al., 1990; Harborne et al., 1992). A fourth gene in the same operon (nirc) encodes a 29-kDa hydrophobic polypeptide that has homology to bacterial formate transporters (Suppmann and Sawers, 1994). As members of this family are small and generally predicted to have no more than six transmembrane domains, they probably function as dimers (Suppmann and Sawers, 1994). Circumstantial evidence suggests that nirC is likely to encode a NO, influx system. Recent studies in Chlamydomonas indicate that the Narl gene, which is related to the NiRC-FocA-FdhC family, may encode a chloroplast NO, transporter (E. Fernandez, personal communication). B. LOWER EUKARYOTES
There is good evidence that NO; uptake in fungi (Eddy and Hopkins, 1985; Downey and Gedeon, 1994; Blatt et al., 1997) and in green algae (Aparicio et al., 1994) is driven, as in higher plants, by the H + gradient. Consequently, information on the molecular biology of NO; transporters in lower eukaryotes is particularly relevant to plant NO; transport and has already led to the cloning of one family of high-affinity NO; transporters in higher plants (the NRT2 family, see Section V.B.2). Most of the work on the molecular characterization of NO; transporters in lower eukaryotes has concentrated on the fungus Aspergillus nidulans and the green alga Chlamydomonas reinhardtii. More recently, studies have begun on the molecular genetics of the NO; assimilatory pathway in a NO;-assimilating yeast, Hansenula polymorpha. As in bacteria, the identification of the
NITRATE AND AMMONIUM NUTRITION OF PLANTS
25
transporter genes in lower eukaryotes has been greatly aided by the clustering in the genome of many of the genes associated with NO; assimilation. 1. Aspergillus nidulans The molecular biology and genetics of NO, assimilation has been extensively investigated in A . nidulans (Johnstone et al., 1990), and it was from this filamentous fungus that the first cloning and sequencing of a NO, transporter gene was reported (Unkles et al., 1991). The crnA mutants of A . nidulans were isolated as an unusual class of chlorate-resistant mutants that were able to grow on NO; as a sole N source (Tomsett and Cove, 1979). The crnA gene is located within a cluster of genes associated with NO; assimilation, adjacent to the structural genes for NiR and NR (niaD and niiA, respectively) (Brownlee and Arst, 1983; Johnstone et al., 1990). It was initially reported that crnA encoded a hydrophobic polypeptide with 483 amino acids and 10 predicted transmembrane domains (Unkles et al., 1991), but a correction to the C-terminal part of the published sequence showed that the CRNA protein has, in fact, 507 amino acids (Unkles et al., 1995) and, based on its hydropathy profile, 12 transmembrane domains (Trueman et al., 1996b). Until recently the only evidence that crnA encodes a NO, transporter was the genetic evidence cited above. However, it has now been demonstrated that when a full-length crnA mRNA is injected into Xenopus oocytes it is able to direct the synthesis of a functional H -t-dependent NO, transport system that has a stoichiometry of 2H+/NO; and a K, for NO; of 2.5 pM (J.-J. Zhou, L. J. Trueman, K. J. Boorer, B. G. Forde and A. J. Miller, unpublished results). There are several lines of evidence suggesting that CRNA is not the only NO, transporter in A . nidulans. First, crnA mutants are only defective in NO, uptake during the first 16 h of conidiospore germination, and not in mature mycelia (Brownlee and Arst, 1983), indicating that other NO, uptake systems exist and that expression of these is developmentally regulated. Secondly, Brownlee and Arst found no simple Michaelis-Menten relationship between [NO,] and the rate of NO; uptake (Brownlee and Arst, 1983), and recent uptake studies with 13NO; found evidence of biphasic kinetics similar to those seen in plant roots, with a high-affinity system saturating at -100pM and a low-affinity system that is linear up to at least lOmM (A. D. M. Glass, personal communication). With the finding that homologues of crnA also exist in higher plants (the NRT2 genes, see Section V.B.2), it now seems that NO; transport systems in A . nidulans have even more in common with those in higher plants than might once have been thought. 2. Chlamydomonas reinhardtii Largely through the work of Fernandez and colleagues, the green alga Chlamydomonas reinhardtii is proving to be an extremely useful model system for plant NO; transport. As was the case with A . nidulans, the first step
26
B. G . FORDE and D . T. CLARKSON
towards identifying the genes for the high-affinity NO, transport system in this alga was the cloning and mapping of a cluster of genes involved in NO, assimilation (Quesada et al., 1993). Five genes were identified within a 32-kb region of the Chlamydomonas genome, one of which (Nitl) is the structural gene for NR, while the other four (Narl, Nar2, Nrt2;Z and Nrt2;2) appear to be involved directly or indirectly in the uptake of NO, and NO;. The nucleotide sequences of a full-length Nrt2;l cDNA clone and a partial Nrt2;2 cDNA clone showed them to be closely related to each other and to encode polypeptides homologous to the A . nidulans CRNA NO, transporter (Quesada et al., 1994). The 59-kDa NRT2; 1 polypeptide is about 31 % identical to CRNA and, like CRNA, is predicted to possess 12 membrane-spanning domains (Quesada et al., 1994). However, as will be seen below, the Chlamydomonas NO, transporters are rather more similar in sequence and membrane topology to their higher-plant homologues than to CRNA (Sections V.B.3 and V.B.4). A Chlamydomonas mutant lacking all three NO; transporter genes (Nar2, Nrt2;I and Nrt2;2) was unable to use NO, as sole N source, and uptake was restored only if Nar2 was reintroduced along with either Nrt2;l or Nrt2;l (Quesada et al., 1994). More detailed studies established that the Nar2/Nrt2;2encoded system (System 1) efficiently transports both NO; and NO; (K, for NO; = 1.6 pM), while the Nar2/Nrt2;2-encoded system (System 2) is specific for NO; ( K , for NO; = 11pM) (Galvan et al., 1996). In addition, Chlamydomonas has a further high-affinity NO; transport system (System 3). System 3 is encoded by another member of the Chlamydomonas NRT2 gene family, NRT2;3, which is not located within the same gene cluster (Quesada et al., 1998). A summary of the molecular genetics of the NO; and NO; transport systems in Chlamydomonas is presented in Fig. 4. The requirement for Nur2 in addition to the NRT2 genes that specify the conventional hydrophobic polypeptides is intriguing. The precise function of the NAR2 protein is still unknown, but formal possibilities include it being an essential component of the membrane transporter itself, having a positive role in controlling the transcription of the Nrt2 genes or perhaps being involved in the processing and targeting of the NRT2 proteins to the PM. There is preliminary evidence from expression studies in Xenopus oocytes that the second of these alternatives can be eliminated: while no NO; transporter activity was obtained when oocytes were injected with Nrt2;I mRNA on its own, a NO;-dependent inward current was detected when Nrt2;l and Nar2 mRNAs were co-injected (A. J. Miller, personal communication). The high-affinity NO,/NO; transport systems in Chlamydomonas may belong to a growing class of membrane transporters that are composed of at least two types of subunit, one catalytic subunit containing the conventional six to 12 transmembrane domains and another accessory or regulatory subunit which is either absolutely required for transport function or modulates the activity of the catalytic subunit. Two of the best-characterized examples of
NITRATE AND AMMONIUM NUTRITION OF PLANTS
?
Svstem 1 NO;-specific
t
27
NO;-specific transporter
t
Fig. 4. Molecular genetics of the transport systems for NO; and NO; in Chlamydomonas. Chlamydomonas has three systems responsible for uptake of NO; and NO?, all with a high affinity for their substrates (Galvan et al., 1996). System 1 is bispecific and has a very high affinity and high capacity for both NO; and NO,; System 2 is specific for NO; and has a lower affinity and lower capacity for its substrate than System 1; System 3 is a very high-affinity transporter specific for NO;. Systems 1 and 2 are encoded by two closely related genes, NRT2;l and NRT2;2, belonging to the NNP family, but in each case the Nar2 gene is also required for expression of a functional transport system; the precise role of the Nar2 gene product is unknown. The NRT2;S gene which encodes a component of System 3 is homologous to the other NRT2 genes but is not part of the same gene cluster; the structure and function of the NarS gene that is closely linked to it have not yet been established (E. Fernandez, personal communication).
such transport systems are the mammalian Na+ pump, consisting of a catalytic a subunit and a regulatory subunit (Rossier et al., 1987), and the mammalian cardiac K + channel IKs,which consists of a large subunit with six transmembrane domains and a smaller subunit (IsK) with one transmembrane domain (Barhanin et al., 1996). Further information on the structure and function of NAR2 will be of great interest and, as will be discussed in Section V.B.2, may also be very relevant to our understanding of high-affinity NO, transporters in plants. 3. Hansenula polymorpha In the past few years the budding yeast S. cerevisiae has emerged as a powerful tool for cloning and characterizing genes for plant membrane transporters (Frommer and Ninnemann, 1995). Its usefulness stems from the ready availability of a wide range of transport mutants and the necessary techniques and vectors to allow the expression of foreign genes. Unfortunately, because S. cerevisiae does not use NO, as a N source it is unsuitable for the cloning and characterization of plant NO; transporters.
28
B. G. FORDE and D. T. CLARKSON
H . polymorpha, on the other hand, is a methylotrophic yeast which does have a NO; assimilatory pathway and for which expression vectors and efficient transformation protocols are also available (Gellisen and Hollenberg, 1997). A H . polymorpha homologue of the A . nidulans crnA gene (YNTI)gene, is located within a gene cluster that includes the structural genes for NR and NiR (Avila et al., 1995; PCrez et al., 1996). A yntl mutant was unable to grow on NO, as sole N source (even at 5 mM) and showed a marked deficiency in the capacity to take up NO; at 0.25 or 1mM (Perez et al., 1996). These results suggest that H . polymorpha has no low-affinity NO, uptake system and may only have one crnA-type gene specifying the high-affinity system. It therefore seems that NO, uptake in H . polymorpha is much less complex than in A . nidulans (Section IV.B.l). As discussed in Section V.B.2, the yntl mutant is now being used for the cloning and functional characterization of plant NO; transporter genes.
V. MOLECULAR GENETICS OF NO, TRANSPORT IN PLANTS A. NO? TRANSPORT MUTANTS
The genetic approach that has proved so powerful in gaining access to NO; transporter genes in bacteria, fungi and algae, has met with more limited success in vascular plants, not just because of the greater size of their genomes (and the unfortunate lack of clustering of genes associated with the NO; assimilatory pathway), but also because of the difficulty in obtaining the necessary mutants. The most commonly used approach for identifying mutants of the NO; assimilatory pathway has been to screen for chlorate resistance. Chlorate (Clog), an analogue of NO;, is highly toxic to plants when converted by NR to chlorite, so that resistance to chlorate can be achieved either by a defect in NO; uptake or by a deficiency in NR activity. However, it is only in Arabidopsis that this approach has produced NO; uptake mutants, so that other more laborious approaches have been needed to obtain uptake mutants in other plant species. I . Low-affinity Uptake Mutants A mutant isolated at high frequency in an early screening for chlorate resistance in Arabidopsis was found to be defective in chlorate uptake (Oostindier-Braaksma, 1970; Oostindier-Braaksma and Feenstra, 1973). This mutant (originally B1 or chll) was subsequently characterized in a series of papers by Doddema, Feenstra and colleagues (Doddema et al., 1978; Doddema and Otten, 1979; Doddema and Telkamp, 1979; Scholten and Feenstra, 1986a, b). The chll mutant was reported to be defective only in the low-affinity NO; uptake system (Doddema and Telkamp, 1979), although there were also pleiotropic effects on uptake of K + and C1- ions (Scholten and Feenstra,
NITRATE AND AMMONIUM NUTRITION OF PLANTS
29
1986b), a complication that has still to be explained. The cloning and characterization of the NRTl (CHLl) gene responsible for the defect in NO; and chlorate uptake is described in Section V.B. 1. Conflicting data about the phenotype of chll-5, an NRTl deletion mutant, have recently emerged (Huang et al., 1996; Touraine and Glass, 1997). Huang and colleagues found that when the chll-5 mutant seedlings were grown on medium containing KN03 they showed a 33% reduction in net NO; uptake rate, measured over a 24-h period, and an even greater reduction in net uptake over an 18-day period. On the other hand, Touraine and Glass (1997) were unable to detect any effect of the mutation on net NO; uptake over a 17-day period, nor on NO; influx, measured for a 10-min period with 13NO;. However, both groups found that the mutant was defective in NO; uptake when continuously grown on NH4N03. Touraine and Glass explained their results by proposing the existence of a second LATS that was able to compensate for the chll-5 deficiency in the KN03-grown plants and that growth on NH4N03down-regulated this second LATS sufficiently to unmask the effects of the chll-5 mutation. On slightly different grounds Huang and colleagues also proposed a second LATS in Arabidopsis (see also Section V.C. 1). The reasons for the discrepancies between the results obtained by the two groups are unclear, but given the evident sensitivity of the different components of the LATS to the previous N nutrition of the plants, they could be due to differences in the growth conditions used. 2. High-affinity Uptake Mutants To isolate a high-affinity NO; uptake mutant from Arabidopsis, Wang and Crawford (1996) devised a novel selection protocol in which an M2 population of partially NR-deficient (nia2) seedlings was screened for resistance to 0.1 mM chlorate in the absence of NO;. Using this approach they were able to identify two chlorate-resistant mutants that had normal levels of NR activity (Wang and Crawford, 1996). Both mutants mapped to the same locus (originally designated NRT2, but since renamed CHL8 to avoid confusion with members of the crnA-related NRT2 gene family; N. Crawford, personal communication). The phenotype of the ch18 mutant is consistent with a defect in the constitutive high-affinity NO, uptake system, as might be expected when there was no NO; present to induce the iHATS during the screening for chlorate resistance. When NO; uptake was assayed in NHi-grown seedlings at pH 7, the ch18 mutant was unable to absorb NO; concentrations < 0.5mM for the first 30min after exposure to NO;, but showed wild-type uptake rates in the low-affinity range ( > 2.5 mM). (For reasons that are unclear, the effect was less pronounced at lower pH.) High-affinity NO; transport activity began to appear after 30min of NO; treatment, but even after prolonged exposure to NO; it remained much lower than in the controls, indicating that the same high-affinity uptake system also contributes to NO; uptake in fully NO;induced roots.
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B. G. FORDE and D. T. CLARKSON
High-affinity NO; uptake mutants have been isolated in barley (Wallsgrove et al., 1989) and rice (Hasegawa, 1996) using the labour-intensive strategy of assaying the NO, uptake rates of individual M2 seedlings from 250 pM KN03. From a screen of 40 000 barley seedlings, a total of 45 uptake-deficient mutants were recovered (Wallsgrove et al., 1989), but because of instability in the phenotype these were not characterized further. From a similar screen of 35 000 M2 rice seedlings, only one uptake-deficient mutant (NUE5O) was identified (Hasegawa, 1996). The rate of NO, uptake in this mutant was 5 0 4 0 % of the wild type (on 250pM K N 0 3 ) and the seedlings had increased resistance to chlorate. It is interesting that, like the Arabidopsis chll mutant, NUE5O also showed a defect in K + uptake. B. NO, TRANSPORTER GENES
Early approaches to cloning higher-plant NO; transporters began by trying to identify root membrane proteins that increased in abundance after NO; treatment (McClure et al., 1987; Dhugga et al., 1988) and similar studies have been reported more recently (Ni and Beevers, 1994; Ageorges et al., 1996). However, although NO;-induced synthesis of a subset of polypeptides was detected in each case (summarized in Glass and Siddiqi, 1995), there was no evidence that any were components of NO, transporters, and the corresponding genes have not been cloned. Antiserum against the mammalian Band 3 anion exchanger was shown to cross-react with a number of polypeptides from maize root membrane preparations (Rosenberg et al., 1997), but whether any of these were NO;-inducible was not reported. While a biochemical approach may yet prove useful if sensitive microsequencing techniques can be applied to the NO; -inducible polypeptides resolved on two-dimensional (2-D) polyacrylamide gels, the NO; transporter genes so far isolated from higher plants have been identified through molecular genetic approaches. 1 . NRTl Genes The NRTl (CHLI) gene responsible for the NO; uptake deficiency in the Arabidopsis chll mutant (Section V.A. l), was cloned by T-DNA tagging (Tsay et al., 1993). The tagged N R T l gene was found to encode a hydrophobic 65kDa protein with 12 putative membrane-spanning domains (Tsay et al., 1993). Evidence that the NRTl gene product is a low-affinity NO; transporter was obtained by expressing the gene in Xenopus oocytes and demonstrating a NO; and pH-dependent membrane depolarization, indicating the activity of a H dependent NO; uptake system (Tsay et al., 1993). The K , for NO; of the NRTl transporter expressed in oocytes has been estimated as 8.5 mM (Huang et al., 1996), consistent with the phenotype of the chll mutant (Section V.A. 1). + -
NITRATE AND AMMONIUM NUTRITION OF PLANTS
31
What is known about NRTl expression in Arabidopsis is consistent with a role in the uptake of NO; from the soil. It is expressed mainly in roots and is rapidly induced by NO;, with mRNA abundance reaching a maximum about 2 h after NO, treatment of plants previously grown without NO, (Tsay et al., 1993). In situ hybridizations with roots of Arabidopsis seedlings indicate that close to the root tip it is expressed mainly in the epidermal cell layer, while further back from the tip its expression shifts to the cortex and endodermis (Huang et al., 1996). No expression was detected within the vascular region, apparently ruling out any role in long-distance transport. Two NRTl homologues have been cloned from oil-seed rape (Brassica napus) (Muldin and Ingemarsson, 1995), and the properties of one of these (BnNRTI;2) have been studied in Xenopus oocytes (Zhou et al., 1998). The amino acid sequence of BnNRT1;2 is 91% identical to Arabidopsis NRT1, and like its Arabidopsis homologue its expression in roots is rapidly and strongly induced by NO;. Surprisingly, when expressed in oocytes, BnNRT1;2 was found to be an effective transporter not only of NO; but also of basic amino acids, particularly histidine; other amino acids and dipeptides tested did not elicit significant currents. The inward cation currents obtained with both types of substrate would be consistent with a H+-coupled system, but the pH optima for the two substrates were quite different. Although another member of the PTR family can transport histidine (Frommer et al., 1994b), this is the first reported example of a transporter able to carry both amino acids and NO;. How the BnNRT1;2 transporter can mobilize two such different types of substrate and whether its amino acid transport functions are significant in planta, remain to be established. Given the strong similarity between the BnNRT1;2 and the Arabidopsis NRTl sequences, it would be surprising if their functional properties were markedly different. Two NRTI homologues (LeNRT1;I and LeNRT1;2) have also been isolated from tomato and found to be differentially regulated (Lauter et al., 1996). Both are expressed mainly or exclusively in roots, but LeNRT1;I is expressed constitutively in the presence or absence of NO; or NHZ, while expression of LeNRT1;2 is transiently induced by NO;. From fractionation experiments performed on aeroponically grown plants with large root hairs, it was concluded that the NO;-inducible gene, LeNRT1;2, is expressed preferentially or exclusively in root hairs, while LeNRT1;I is expressed both in root hairs and in the main part of the root (Lauter et al., 1996). The NRTl transporters belong to a rapidly growing group of transporters known as the PTR family, most of which catalyse the movement of small peptides (Steiner et al., 1995). The Arabidopsis peptide transporters, AtPTR2-A and AtPTRZB, are 25 and 39% identical, respectively, to the Arabidopsis NRTl transporter (Song et al., 1996). Despite the apparent difference in the substrate specificity of NRTl and other members of the PTR family, phylogenetic analysis establishes it as a bona fide member of the peptide transport family, being more closely related to AtPTR2-B than AtPTR2-B is to
32
B. G. FORDE and D. T. CLARKSON
AtPTR2-A (Steiner et al., 1995). Furthermore, a peptide sequence motif (F-YX-X-I-N-X-G-S-L) that was identified as being distinctive of this group of peptide transporters is equally well conserved in NRTl (Steiner et al., 1995). With this in mind, it will clearly be important to investigate the transport properties of NRTl in more detail and to establish whether any other members of the PTR family can transport NO;. A putative chloroplast NO; transporter (NiTR1) recently cloned from cucumber (Genbank accession No. 269370) also belongs to the PTR family, but is only distantly related to NRTl. The evolution of the PTR family, and its relationship to the NRT2 high-affinity NO; transporters will be discussed further in Section V.B.3. 2. NRT2 Genes The first higher-plant members of the NRT2 family of NO; transporters were isolated from barley by virtue of their homology to the A . nidulans crnA gene (Trueman et al., 1996a). The two barley NRT2 cDNAs were found to be very similar, encoding polypeptides of 55 kDa which are 92% identical in amino acid sequence. Additional NR T2 cDNAs have subsequently been cloned from Nicotiana plumbaginifolia (Quesada et al., 1997), from soybean (Amarasinghe et al., 1998), and from Arabidopsis and L. japonicus (H. Zhang, I. Onyeocha and B. G. Forde, unpublished results) demonstrating that the gene family is widespread in higher plants. In barley it has been estimated that there could be as many as 10 NRT2 genes (Trueman et al., 1996a), but the dicots analysed up to now have much smaller gene families. In Arabidopsis there are just two NRT2 genes, but only one of these (AtNRT2;l) seems to be expressed at significant levels in roots (H. Zhang and B. G. Forde, unpublished results). Attempts to demonstrate NO; transporter activity with the barley, N. plumbaginifolia or L. japonicus NRT2 genes expressed in Xenopus oocytes have been unsuccessful (J.-J. Zhou, F. J. Theodoulou, L. J. Trueman, A. J. Miller and B. G. Forde, unpublished results). By analogy with the NRT2 transporters in Chlamydomonas (Section IV.B.2), the failure to obtain functional expression of the higher-plant NRT2 transporters could be due to the absence, in the oocytes, of a component needed for the correct processing or functional activation of the catalytic NRT2 polypeptide. Until the homologue of the Chlamydomonas Nar2 gene has been cloned from higher plants it may not be possible to reconstitute the higher-plant NRT2 transport system in oocytes. Direct experimental evidence that higher-plant NR T2 genes do encode highaffinity NO; transporters has been obtained by expressing two barley NRT2 cDNAs (HvNRT2A and H v N R T ~ Bformerly , BCHl and BCH2) in the yntl NO;-deficient mutant of H . polymorpha (N. Brito, L. J. Trueman, J. Siverio and B. G. Forde, unpublished results). Complementation of the NO; uptake deficiency was obtained at low external WO,] (< lOOpM). However, the activity of the barley NRT2 transporters was much lower than that of the native YNTl transporter expressed under the control of the same promoter, perhaps supporting the idea that a second gene product is needed for full
NITRATE AND AMMONIUM NUTRITION OF PLANTS
33
activity. A similar situation may apply to the Arabidopsis inorganic phosphate transporters, which when expressed in apho84 mutant of S. cerevisiae catalysed either zero (Smith et al., 1997) or very low rates (Muchhal et al., 1996) of phosphate uptake. The Arabidopsis phosphate transporters are homologous to the yeast PH084 polypeptide, which appears to be part of a multisubunit complex (Bun-Ya et al., 1992, 1996; Yompakdee et al., 1996). Thus, the highaffinity transporters for both NO, and phosphate may prove to be much more complex and difficult to dissect than has so far been the case for other types of membrane transporters in plants. 3. Phylogenetic Relationships It has been noted that the plant and algal NRT2 transporters, the A . nidulans CRNA transporter and the E. coli NarK transporter are part of a much larger superfamily of membrane transporters known as the Major Facilitator Superfamily (MFS) (Trueman et al., 1996a). This family has recently been designated the NNP (Nitrate-Nitrite Porter) family (Pa0 et al., 1998). Members of the MFS generally have 12 transmembrane domains and are characterized by two conserved sequence motifs in the vicinity of transmembrane domains 2-4 (Henderson, 1991; Baldwin, 1993). Well-known examples of MFS transporters include the mammalian facilitative sugar transporters, bacterial H - and Na +-dependent sugar transporters and bacterial antibiotic efflux pumps, and phylogenetic analysis has shown that the transporters can be grouped into a number of subfamilies or clusters which correspond to their transport functions (Marger and Saier, 1993). The NNP family are fairly typical members of the MFS, having the 12 predicted transmembrane domains and the two consensus motifs in the expected locations (Trueman et al., 1996a). As seen in Fig. 5, the NNP family forms a distinct cluster within the MFS. Figure 6 shows the phylogenetic relationships between 10 members of the NNP cluster. These relationships more or less reflect what would be expected from the phylogeny of the species from which the genes were isolated, suggesting that the higher-plant genes are the direct homologues of the A . nidulans crnA and Chlamydomonas NRT2 genes. It also suggests that an ancestral NO,/NO; transporter arose early in evolution, even before the divergence of prokaryotes and eukaryotes. It was noted that NarK, CRNA and the NRT2 transporters share a highly conserved sequence (A-G-(w,l)-GN-M-G), located within transmembrane domain 5 , leading to the suggestion that this motif may be involved in substrate recognition (Trueman et al., 1996a). Examination of the sequences of the PTR family of peptide and NO; transporters indicates that they too belong to the MFS: Fig. 7 shows an alignment of peptide sequences of members of the PTR family with those of representatives of the six previously identified clusters of the MFS (see Fig. 5 and Trueman et al., 1996a). The two regions of the MFS superfamily that contain the signature motifs for this superfamily (Henderson, 1991; Baldwin, +
34
B. G. FORDE and D. T. CLARKSON
DtpT.1 AtNRT1
\
\
I
BicA
1
,NorA
// F ~~
KgtP
CRNA
4
HvNRT2A
HUP-1
Gtr-2
Gal-2
UhpT
PgtP Fig. 5 . Phylogenetic tree showing the relationship between representative members of seven subfamilies of the MFS superfamily. The more strongly conserved N-terminal halves of the protein sequences were aligned using PILEUP (Devreux et al., 1984), and the phylogenetic analysis was carried out using the PHYLIP package (Felsenstein, 1991) as described previously (Trueman et al., 1996a). The seven clusters correspond to functional groups: cluster 1 contains bacterial drug-resistance proteins; cluster 2, facilitated sugar transporters; cluster 3, facilitators for Krebs cycle intermediates; cluster 4, phosphate-phosphate ester antiporters; cluster 5, H +/oligosaccharide symporters; cluster 6, NO; and NO, transporters of the NNP family (Marger and Saier, 1993; Trueman et al., 1996a). As discussed in the text, cluster 7 contains the PTR family of oligopeptide transporters, a family that also includes the Arubidopsis low-affinity NO; transporter (AtNRTl). Only selected diverse members of each cluster were used in the analysis. The accession codes for the amino acid sequences are as follows (SWISSPROT codes where available, otherwise Genbank): NorA, NORA-STAUU; BicA, BCR-ECOLI; Lacy, LACY-KLEPN; CitA, CIU2-ECOLI; KgtP, KGTP-ECOLI; Hup- 1, HUP1-CHLKE; Gtr-2, GTR2-MOUSE; Gal-2, GAL2-YEAST; PgtP, PGTP-SALTY; UhpT, UHPT-ECOLI; HvNRT2.1, U34198; CRNA, CRNA-EMENI; NarKEc, NARK-ECOLI; NasA, NASA-BACSU; AtNRTl, CHLl-ARATH; DtpT- 1, DTPT-LACLA; AtPTR2A, PTZA-ARATH. Further details about the transporters of clusters 1-5 and the phylogeny of the MFS superfamily can be found in the review by Marger and Saier (1993). An updated phylogenetic analysis of the MFS, in which over 300 transporters are classified into 18 families, has recently been published (Pa0 et al., 1998).
NITRATE AND AMMONIUM NUTRITION OF PLANTS
35
Fig. 6. Phylogenetic tree for the N N P family of NO; and NO; transporters. The sequences were aligned and the tree constructed as described in the legend to Fig. 5. HvNRT2A and HvNRT2B (U34198 and U34290; barley NRT2 gene products); NpNRT2A (YO8210; N . plurnbaginifolia NRT2 gene product); NRT2; 1 (225438; Chlamydomonas NRT2 gene product); CRNA, (CRNA-EMENI, A . nidulans highaffinity NO; transporter); NarKEc(NARK-ECOLI; E. coli NO, efflux system); NarU (NARU-ECOLI; E. coli NarK homologue); NarKB, (NARK-BACSU; B. subtilis NarK homologue); NasA (NASA-BACSU; B. subtilis NarK homologue).
1993) lie in the vicinity of the short hydrophilic loop between transmembrane domains 2 and 3, and within and immediately following the hydrophilic loop between transmembrane domains 3 and 4. The alignments show that there are significant similarities in both these regions between members of the PTR family and the previously established members of the M F S (Fig. 7). As shown
B. G . FORDE and D. T. CLARKSON
36
MFS Cluster
A
B
Consensus:
(V,I, L,M)-(V,I,L,M)-x-x-x-x-(V,I,L,M)-x- (G/A)- (V,I,L,M)-x-x-G
NITRATE AND AMMONIUM NUTRITION OF PLANTS
37
by the phylogenetic tree in Fig. 5 , the PTR family forms a distinct seventh cluster within the MFS. If, as this analysis suggests, the NRTl and NRT2 NO; transporters do have a common ancestry, they may also have related structures and mechanisms of action. 4 . Structural Comparisons Between Members of the N N P Family and Identification of Possible Regulatory Domains Despite the strong sequence homologies between the plant NRT2 transporters and their fungal counterparts, there are some distinct differences in their predicted membrane topologies (Trueman et al., 1996a). The two fungal transporters, CRNA and YNTl, are each predicted to have a large hydrophilic central loop of approximately 90 amino acids between transmembrane domains 6 and 7, while the corresponding loop in the Chlamydomonas and higher-plant NRT2 transporters is only about 30 residues. As if to compensate, the algal and plant members of the family have a highly charged C-terminal domain of about 70 amino acids which is absent in the fungal sequences. As both the central loop and C-terminal domains are predicted to be on the cytoplasmic side of the PM (based on comparisons with other members of the MFS, for which this has been established experimentally), it has been suggested that these hydrophilic domains may have a regulatory role (Trueman et al., 1996a, b). In support of this hypothesis, evidence has been obtained that the Cterminal domain of the higher-plant NRT2 transporter is not required for its transport activity: a deletion mutant of the barley HvNRT2A transporter, lacking the C-terminal domain, had similar NO; uptake activity to the fulllength polypeptide when expressed in H . polymorpha (N. Brito, L. J. Trueman, J. Siverio and B. G. Forde, unpublished results).
Fig. 7. Alignment showing sequence conservation between the PTR family and selected members of the MFS superfamily. Sequences from eight members of the PTR family were aligned with conserved regions of representatives from the other six clusters of the MFS superfamily using PILEUP (Devreux et al., 1984). The sequences are clustered according to their membership of the seven clusters of the MFS superfamily as indicated. Two regions of each polypeptide sequence were chosen for comparison. (A) The region around the loop joining transmembrane domains 2 and 3 that contains the first of two previously identified MFS signature motifs (D/N-R-x-G-R-R/K) (Henderson, 1991). (B) The region around the start of transmembrane domain 4 that contains the second of the MFS signature motifs (I-x-x-R-x-x-x-G-x-x-x-G). Positions that are conserved between the PTR family (cluster 7 of the MFS) and the representatives of at least five of the other six clusters are highlighted (white on a black background), and other conserved residues are indicated by a grey background. Core consensus motifs, which differ from those originally proposed by Henderson on the basis of a less diverse collection of sequences, are shown below each alignment. The accession codes for the majority of the sequences are in the legends to Figs 5 and 6. SWISSPROT codes for the additional sequences are: ScPTR2, PTR2-YEAST; CaPTR2, PTR2-CANAL; YhiP, YHIP-ECOLI; PepT- 1, PETl-RABIT.
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B. G. FORDE and D. T. CLARKSON
The mammalian facilitative glucose transporters, amongst the most intensively studied members of the MFS, have a large central loop as well as a C-terminal hydrophilic tail (Mueckler, 1994). The C-termini of GLUTl and GLUT4 have been shown to be involved in regulating glucose transport, both directly and indirectly: directly, through intermolecular interactions that can modulate intrinsic glucose transport activity in response to changing physiological conditions (Dauterive et al., 1996), and indirectly through a role in determining the subcellular distribution of the transporters (Jhun et al., 1992; Rampal et al., 1995). Other studies have indicated the existence in adipocytes of proteins that bind to the C-terminus and the central loop of GLUTl and GLUT4, and which are thought to be involved in regulation of transporter activity (Liu et al., 1995; Shi et al., 1995). Clearly, an important research goal for the future will be to determine whether the cytosolic domains of CRNA and the NRT2 transporters are involved in the allosteric regulation of NO; influx, perhaps mediating feedback inhibition by cytosolic NO; or N metabolites (Section 1II.C). C. RELATING THE MOLECULAR GENETICS TO THE KINETIC DATA
As set out in Section III.B, kinetic studies on a range of higher plant species have led to the conclusion that there are at least three NO, uptake systems: a constitutive high-affinity system (CHATS), an inducible high-affinity system (iHATS) and a constitutive low-affinity system (LATS). To what extent can we relate these kinetic data to the information we now have about the genes? I . Low-aflinity Uptake Systems At least one component of the LATS appears to be specified by the Arabidopsis NRTl gene. This conclusion is based on the phenotype of the chll mutant (Section V.A.l) and the functional properties of the NRTl gene product expressed in oocytes (Section V.B.l). However, RNA gel blots show NRTl to be strongly induced by NO; (Tsay et al., 1993), suggesting that it specifies a previously unrecognized inducible LATS. The existence of two differentially regulated LATS would be consistent with the leaky and conditional nature of the phenotype of the chll mutant: as we saw in Section V.A.1, the severity of the chll phenotype depends on the N nutrition of the plants prior to the experiment, and under certain nutritional conditions the rate of NO; uptake by the mutant is the same as the wild type. These observations would be most easily explained if Arabidopsis has more than one low-affinity NO; transporter and if the alternative system(s) is regulated by N source in a different way to N R T l (Huang et al., 1996; Touraine and Glass, 1997). Consistent with this, the two tomato N R T l genes were found to be differentially regulated: one being induced by NO; and the other expressed constitutively (Lauter et al., 1996). Huang and colleagues have
NITRATE AND AMMONIUM NUTRITION OF PLANTS
39
identified a candidate for a second component of the LATS from the Arabidopsis expressed sequence tag (EST) database. This gene (NLTI) is related to NRTZ (but only distantly), and functional analysis in Xenopus oocytes indicates that it also encodes a NO; transporter (unpublished results of N. C. Huang and Y.-F Tsay cited in Huang et al., 1996). In addition, the Arabidopsis genome-sequencing project has also uncovered a gene on chromosome 2 that is very closely related to NRTZ (Genbank accession No. 2760834). The NRTl transporters remain an enigmatic group, belonging as they do to a family of peptide transporters and, at least in the case of BnNRT1;2, apparently having the ability to transport amino acids as well as NO, (Section V.B.l). 2. High-affinity Uptake Systems Although their contribution to high-affinity NO, uptake has not yet been properly assessed in planta, the current evidence suggests that NRT2 genes encode at least one component of the iHATS (Section V.B.2). The Chlamydomonas members of the family have a K , for NO; of 2-11 p M (Galvin et al., 1996), which is lower than most estimates for the iHATS (Section III.B.I), but this could reflect a difference between algae and multicellular plants. The kinetic evidence that the iHATS and the CHATS are distinct transport systems (Section III.B.3) has been supported by the isolation of the Arabidopsis ch18 mutant that is selectively impaired in the CHATS (Section V.A.2). Nevertheless, it is still possible that the iHATS and the CHATSare specified by different members of the same gene family rather than by entirely unrelated genes. In barley there are estimated to be at least seven members of the NRT2 gene family (Trueman et al., 1996a), and prolonged exposures of Northern blots showed that NRT2 mRNAs are present in roots of barley seedlings that have never been exposed to external NO; (L. J. Trueman and B. G. Forde, unpublished results), so it cannot be excluded that one or more members of the NRT2 family are responsible for NO; transport activity in uninduced roots. Table I summarizes for Arabidopsis, the species for which the information is at present most complete, the current state of knowledge on the relationship between known NO; transporter genes and the NO, uptake systems identified from kinetic studies. Five genes have now been implicated in controlling NO, uptake in this species, and it seems very likely that more await discovery.
40
B. G. FORDE and D. T. CLARKSON
TABLE I Arabidopsis genes implicated in controlling NO; uptake and their tentative assignment to NO; transport systems identified from kinetic studies Gene
NO: uptake system
N R TI (CHLI)
inducible, low affinity
NTLl
?, low affinity
AtNRT2;l
inducible, high affinity
At N R T2;2
?
CHL8a
constitutive, high affinity
Evidence
Reference
phenotype of chll mutant; functional expression in oocytes; NO;-inducibility of mRNA in roots EST with homology to NRTI; functional expression in oocytes; possible candidate for constitutive LATS homology to NRT2 high-affinity transporters; NO;-inducibility of mRNA in roots homology to NRT2 high-affinity transporters (but expressed at very low levels) phenotype of ch18" mutant
Doddema and Telkamp (1979); Tsay et al. (1993); Huang et al. (1996) Huang et al. (1996)
H. Zhang and B. G. Forde (unpublished results) H. Zhang and B. G. Forde (unpublished results) Wang and Crawford (1996)
aOriginally designated NRT2/nrt2 in Wang and Crawford (1996), but renamed to avoid confusion with crnA homologues (N. Crawford, personal communication).
VI. PHYSIOLOGY OF NH: UPTAKE A. BACKGROUND
The uptake of NH; has been studied less intensively than that of NO;. Of the world's major crop plants, only rice frequently receives NHZ as its principal source of inorganic N, but it is clear that many forest species adapted to acidic or waterlogged soils may show a preference for NH: ions, even to the point of growing rather poorly with NO; as the sole source of N (see Section I1 and also Lavoie et al., 1992 and references therein). It is also clear that many species that normally use NO, also have an efficient system(s) for absorbing NH; which is constitutively expressed at high levels. Indeed, where such species are presented with a mixed NO;/NH; source, NH; is absorbed more rapidly, for example in perennial ryegrass (Clarkson et al., 1986) and barley (Macduff and Jackson, 199 1).
NITRATE AND AMMONIUM NUTRITION OF PLANTS
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1. Dffusive Uptake of NH3? One source of uncertainty that has complicated our understanding of NH: uptake relates to the possibility that there is a flux of ammonia (NH3) across the PM from external solutions where NH: and NH3 are in a pH-dependent equilibrium. The pKa for NH3 in aqueous solution is 9.25, so that below pH 7 only 1% is in the unprotonated form, and it is only in very alkaline conditions that it represents a major fraction of the total ‘ammonium’ (NH3 + NHZ) pool. It has sometimes been suggested that NH3 may diffuse across membranes faster than NH: is transported. Studies with Lemna did indicate that the unprotonated form was being absorbed at significant rates when the [NHzIext was > 0.1 mM and the high-affinity transport system had become saturated (Ullrich et al., 1984). If this phenomenon occurred generally it would be expected that ‘ammonium’ uptake would increase sharply on moving from mildly acidic to basic solutions, i.e. as the abundance of NH3 rises. This effect has been looked for, but not found, in studies of rice (Wang et al., 1993a), Chlorella (Schlee and Komor, 1986) and Chara corallina (Deane-Drummond, 1984). Indeed, in rice, the influx of ‘ammonium’ due to the low-affinity transport system (i.e. at [NH:Iex, > 1 mM) was 25-35% lower at pH 7.5-9.0 than at pH 6.0, despite the abundance of NH3 increasing more than 300-fold over this pH range (Wang et al., 1993a). In Typha latifolia, a wetland species which may frequently encounter NH: as its principal N source, the V,,, for ammonium uptake was largely unaffected in the pH range 3.5-8.0, and the apparent K, did not change in the pH range 5.CL8.0 (Dyhr-Jensen and Brix, 1996). On the other hand, some evidence favouring NH3 diffusion into roots has recently been reported (Kosegarten et al., 1997). In root hair cells of rice and maize, the addition of 2mM ‘ammonium’ to a previously N-free bathing solution resulted in a transient alkalization of the cytosol, followed by a prolonged decline to the original value over a period of about 50min. The response was dependent on the pHext, there being no response at pH 5 (where NH3 is essentially absent) and an increasing transient peak as the pH was increased above pH 7 (i.e. as the [NH31extincreased). The alkalization was less when GS was inhibited with MSX. It was concluded that alkalization was the combined effect of: (a) a rapid diffusion of NH3 across the PM and its subsequent protonation within the cytosol; and (b) a contribution from H + consumption in the GS-GOGAT cycle. While these results are clearly compatible with diffusive uptake of NH3, they do not establish the quantitative significance of the process as a contribution to ‘ammonium’ uptake. The experiments were performed in N-starved conditions, when the cytosolic ‘ammonium’ concentration would have been low, so that under alkaline conditions the diffusion gradient for NH3 may well have been directed inwards. The provision of an external N supply, either of NO; or NH;, would quickly increase the cytosolic ‘ammonium’ concentration.
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Therefore, particularly in neutral or acidic soil conditions, it is more likely that the gradient for NH3 diffusion will be outwardly directed (see also Section VIII.B), so that net NH3 uptake could not occur. This would be consistent with the observations made by others that ‘ammonium’ uptake is accompanied by a rapid depolarization of the PM (Ayling, 1993; Wang et al., 1994; Hermann and Felle, 1995), the effect being explained by large net inflows of positive charge, carried either by NHZ alone or NHZ + H + . In summary, most ‘ammonium’ absorbed by roots probably enters in the protonated form, with diffusion of NH3 occurring primarily in the outward direction. The low- and high-affinity transport systems responsible for NH: uptake in plants are discussed in Sections V1.B and VI1.C. 2. Thermodynamic Considerations As a monovalent cation, NH; can diffuse across the PM in response to the inside-negative electric PD. However, the extent to which this actually happens will depend on the membrane potential and on the [NH;] inside and outside the cell. An unexceptional membrane potential of, say, -120mV would produce by diffusion alone a [NH4+lCyt100-fold greater than the [NH;],,,. Estimates of [NH,+],,, are few, but usually lie within the range 1-40mM (Lee and Ratcliffe, 1991; Wang et al., 1993b). Consequently, when the pH:] at the root surface is > 10 pM and the cytosolic concentration is at the lower end of the observed range, NH; transport could occur by diffusion through a channel (Ullrich et al., 1984; Wang et al., 1994). Conversely, if the [NH4+],,, is < 10 pM and the [NH4flCyt remains at 2 1 mM, there would be a need for some kind of energetically coupled high-affinity transport system (HATS), such as a H + co-transporter. The need for an active transport system would clearly be extended to much higher [NH4+Iextunder conditions where the [NH4flCytwas at the upper end of the observed range. The AtAMT1 high-affinity NH; transporter cloned from Arabidopsis, which is expressed in roots and is thought to couple NH; uptake to the H + gradient (Ninnemann et al., 1994), appears to fulfil the requirements for a HATS for NHZ (Section VI1.C). Thermodynamic considerations have often been advanced to explain the apparent preferential absorption of NH4f from mixtures with NO; (e.g. Clarkson et al., 1986). In reality, the difference in energy cost between the uptake of the two ions is less than might be expected. Because most NH; ions are assimilated in roots, and 1 mole of H + is produced for each mole of NH; assimilated, there is an additional cost (in ATP) to pump these protons out of the cell via the PM H +-ATPase. Nevertheless, experiments with barley indicated that their apparent preference for NH,f over NO, did become more pronounced under conditions where energy metabolism was limited (i.e. at low temperature) (Macduff and Jackson, 1991).
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3. Compartmentation of Intracellular NH; At present there is no published method for measuring intracellular [NH;] directly using an ion-selective microelectrode; neither are there fluorescent imaging techniques. An NHt-sensing microelectrode has been produced, but has been used only to measure the extracellular [Mi]close to the root surface (Henriksen et al., 1990, 1992). Because K + ions interfere significantly with the electrode response, it is necessary to know precisely the K + activity near the electrode tip; it is probably this that has prevented its intracellular use. In vivo 14N-NMR can be exploited to separate cytoplasmic and vacuolar compartments (Lee and Ratcliffe, 1991). The method depends on the pH dependence of two properties of the I4NH4+ signal; viz. its spin-lattice relaxation time, and the exchange of protons between water and NH;. The latter has a marked effect on the line shape of the 'H and the I4NH4f signals. The principles are well explained for non-specialists by the authors (Lee and Ratcliffe, 1991) and, in view of the power of this technique, it is surprising that it has not been used more frequently. Its major limitation is uncertainty in the analysis of line shapes; this predominates over other sources of error in calculating the partitioning of NH; to cytoplasm and vacuole. Applying the method to root tips and to 2-cm root segments cut from the apical lOcm of maize primary roots, Lee and Ratcliffe found no detectable cytoplasmic NH; in roots pre-treated with 1.5mM NH4N03, but an equivalent NH; supply from (NH4)2S04 (NH; alone) resulted in a [NH4fIyyt between 3 and 8 mM (and a [NH&,, of 15 mM). The very low [NH4+lCy, in NH4N03-grown roots was consistent with a GS activity that was six times higher than needed to match the rate of NHZ influx across the PM. When GS activity was blocked with MSX, there was an enormous increase in [NH4+lCy, (to 90mM), but little change in [NH&. The authors were intrigued why [NH4flCytshould be so much greater in roots that received NH,f as sole N source, and concluded that there must be a lower efficiency of NHZ assimilation in these circumstances (Lee and Ratcliffe, 1991). This may be why it is so often reported that plants grow better on mixed NHt/NO; sources (for earlier literature see Haynes and Goh, 1978). Given that there is usually a small positive membrane potential between the cytoplasm and the vacuole, the greater [NHt] found by Lee and Ratcliffe in the vacuole cannot be in thermodynamic equilibrium with the N H t pool in the cytoplasm. Transport into the vacuole must then either be energized (perhaps making use of the H + gradient in antiport mode), or the acidic vacuole must act as a trap for cytoplasmic NH3 diffusing across the tonoplast. NH3 diffusing in this way would become protonated and retained if the intrinsic tonoplast permeability to NH: was low. Determining the kinetics of isotope exchange between I3N-loaded roots and the external medium has proved to be a useful alternative approach to estimating the [NH$],,,. Because of the short half-life of I3N (about 10 min) no information about exchanges across the tonoplast can be obtained, and
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quantities in this compartment are derived by subtraction of quantities in the cytoplasm and extracellular space from the total NH; measured by independent chemical means. This method has been applied by Glass and colleagues to both rice (Wang et al., 1993b) and white spruce (Kronzucker et al., 1995b). In the rice experiments, the plants were supplied with NH&l at concentrations that limited growth (0.02 mM), were optimal (0.1 mM) or were supra-optimal (1 mM). Plants equilibrated with these concentrations were then loaded with 1 3 ~ 4 Catl the same concentrations and subsequently transferred to unlabelled solutions, after which exchange of tracer with 14NH; in the external medium was measured. In steady-state conditions, a higher rate of [NH;] supply was correlated with a higher [NH;lCy, and an increased rate of NH; efflux (as a percentage of influx). For instance, the [NH;],,, in the supraoptimal treatment was 38mM, which was 10-fold higher than in growthlimiting conditions. By contrast, the [NH;Iv,, in the supra-optimal treatment was only 6mM, just two-fold higher than in growth-limiting conditions. In earlier work it was shown that the tissue [NH$] increased rapidly from -1 mM to > 30mM when NO;-grown wheat roots were transferred to NH; medium (Morgan and Jackson, 1988). A report that the cytoplasm of maize root tips contained < lOpM NH: (Roberts and Pang, 1992) may have been erroneous because the NMR spectra used to make this estimate were collected over a period when there was no NH: in the medium bathing the excised root tips; NH; within the tissue is usually assimilated quickly through GS. The variability in the [NH&, observed in both rice (Wang et al., 1993b) and spruce (Kronzucker et al., 1995b) suggests that, despite the existence of an efflux mechanism (Section VII1.B) and an NHi-assimilating enzyme of highaffinity, the cytosolic NH; pool is not as strictly regulated as the cytosolic NO, pool would appear to be (Section III.C.5). It is not clear why there should be this difference. B. KINETICS OF NH: UPTAKE
I . High-afJinity Uptake In a number of herbaceous and woody species a saturable HATS for NH; has been found that shows a clear dependence on metabolism (Glass, 1988; Glass et al., 1997). The use of inhibitors to collapse the H f gradient across the PM of rice roots caused a > 80% decline in NH; uptake by the HATS, but had a smaller effect on the LATS (Wang et al., 1993a). Estimates of the K , for the HATS fall within the range 17-188 pM (Ullrich et al., 1984; Wang et al., 1993a; Kronzucker et al., 1996). In rice, its kinetic properties were found to be variable according to the intensity of the NH$ supply to which the plants had been acclimated (Wang et al., 1993a): in plants adapted to growth-limiting supplies of NH$ the V,,, was higher and the K,,, for NH$ much lower than in plants adapted to an abundant supply of NH;.
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This dual effect on the kinetic parameters strongly resembles that reported for the effects of K + supply on the HATS for K + (e.g. Glass, 1976; Drew et al., 1984). Because in both rice (Wang et al., 1993b) and spruce (Kronzucker et al., 1995b), the [NH;lCyt rises substantially as the external "Hi] increases, allosteric regulation of the transporter by the cytosolic NH; pool is a distinct possibility. The HATS in rice appeared insensitive to changes in the pH of the external medium in the range pH 4.5-9 (Wang et al., 1993a), which would appear to contradict the notion of a H+-coupled transport system. However, it is possible that the necessary H + gradients were maintained close to the PM due to the activity of the H+-ATPase extruding H + into the confined space of the apoplast (Sentenac and Grignon, 1985; Grignon and Sentenac, 1991). The constitutive level of expression of the HATS for NH: is much more significant than is the case for HATS for NO;, and the degree to which it is stimulated by the presence of its substrate is much lower. Evidence that the HATS for NH: is expressed even in the absence of its substrate has come from studies on cereals (Morgan and Jackson, 1988; Wang et al., 1993b; Mack and Tischner, 1994) and white spruce (Kronzucker et al., 1995b, 1996); uptake rates with a V,, of around 4-6 pmol g-' fresh weight h-' are found in roots that have never been exposed to exogenous NH:. A relatively modest increase in HATS is generally observed several hours after N-starved roots are resupplied with NH;, and this is followed by a decline (Goyal and Huffaker, 1986; Morgan and Jackson, 1988; Mack and Tischner, 1994; Kronzucker et al., 1996). Whether this stimulation is a true induction, involving an synthesis of new transporter protein, or reflects some kind of activation of pre-existing transporters, remains to be seen. The down-regulation of influx that occurs on prolonged exposure to NH; could suggest that the activity of the uptake system is responsive to the internal N status. Consistent with this, activity of the HATS in wheat and oat roots increased 5-10-fold when seedlings were deprived of an external N source for 6 days (Morgan and Jackson, 1988). A negative correlation has been reported between intracellular levels of glutamine or asparagine and the rate of NH: influx by maize roots (Lee et al., 1992). On the other hand, a striking negative correlation between I3NH4+influx and cytosolic "Hi] and tissue [NH;] was reported in rice roots (Wang et al., 1993b), suggesting that NH; itself might have a regulatory role. Taken together these observations are consistent with a picture in which the HATS for NHZ, while not requiring external NH; for its expression, is nevertheless stimulated by its presence and subject to repression by a downstream N pool. The LeAMTl gene from tomato, which encodes a highaffinity NH; transporter (Section VII.C), was found to be expressed at similar levels in N-deprived plants and in plants resupplied with NH; (Lauter et al., 1996); only addition of NO, led to a decrease in the abundance of the LeAMTI mRNA. These results demonstrate that the LeAMTl gene is not
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NHi-inducible, but because only low rates of N supply and relatively short periods were used in the starvation-resupply experiments, its ability to respond to feedback repression may not have been fully tested. It is also important to note that LeAMTl is specific to root hairs, so that there are likely to be other NH: transporter genes expressed elsewhere in the root that could be regulated in a different way. As discussed in Section VII.B.l, the MEP genes that encode NH: transporters in S . cerevisiae are subject to N catabolite repression. Although there is known to be competition between K + and NH: uptake by roots (Section VI.B.3), major quantitative interactions between K and NH: in the high-affinity range would seem unlikely in view of the evidence that the NH: transporters from S. cerevisiae (Marini et al., 1994) and Arabidopsis (Ninnemann et al., 1994) and the HKTl high-affinity transporter of K f from wheat (Schachtman and Schroeder, 1994) are highly selective and have little capacity for transporting the other cation. There is some confusion in the earlier literature on this point. For instance, studies on the HATS for NH: in maize roots showed that its K , for NHQ doubled, and its V,,, decreased by 25%, when 200pM K + was present in the uptake medium (Vale et al., 1988a). As discussed below (Section VI.B.3), the concentration range over which the high-affinity systems dominate the uptake processes for NH: and K + varies with the nutritional status of the experimental plants. In conditions where the plants are of high K or N status it is expected that the respective HATS would be repressed, so even from quite dilute solutions the LATS would become significant; this might lead to interactions between the two ions being misinterpreted as competition for the HATS. +
2. Low-affinity Uptake At [NH:Iex, above about 0.5 mM, I3NH; influx displays a linear relationship with concentration (Wang et al., 1993a; Kronzucker et al., 1996), suggesting that low-affinity uptake occurs by diffusion. As already discussed, the available evidence indicates that the LATS, like the HATS, transports the protonated form of NH: (Section VI.A.l). Thus, despite its diffusive nature, the LATS would still depend on maintenance of the membrane potential to provide the electrical driving force. The energy-dependence of the LATS in rice roots has been demonstrated (Wang et al., 1993a). Unlike the HATS for NH;, the activity of the LATS appears to be insensitive to the N status of the plant (reviewed by Glass et al., 1997). There have been no reports of specific channels for NH: in the PM, and it is likely that low-affinity NH: uptake mainly occurs via the same system as low affinity K + uptake, i.e. K + channels. Studies on rye roots indicate that a voltage-independent K f channel in the PM is permeable to NH,f (White, 1996), and a voltage-dependent, inward-rectifying K + channel (CKCl) with significant permeability to NH,f has been reported in maize coleoptiles (Hedrich et al., 1995). The cloned Arabidopsis inward-rectifying KATl K channel, when expressed in Xenopus oocytes, showed a conductance for NH: +
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that was 30% of its conductance for K + (Schachtman et af., 1992). A related member of the same K + channel family, AKT1, has a similar affinity for NH; when expressed in yeast (Bertl et af., 1997) and is a better candidate for participating in low-affinity NHZ uptake because, unlike KATl, it is strongly expressed in roots (Kochian et af.,1993; Lagarde et af., 1996). It has recently been reported that K + uptake by HAKl, a root-specific member of a new family of high-affinity K + transporters cloned from barley, is strongly inhibited by 5 mM NH; (Santa-Maria et al., 1997), suggesting that this family of active K + transporters might also contribute to low-affinity NH; uptake. The different components of the HATS and LATS for NHZ as currently understood are summarized diagrammatically in Fig. 8.
Fig. 8. Possible mechanisms for high- and low-affinity NH: uptake by roots. The Arubidopsis AMTl transporter (Ninnemann et ul., 1994) is a candidate for the highaffinity NH: uptake system (HATS), which is believed to be H+-coupled, the H f gradient being generated by the PM H+-ATPase. Low-affinity NH: uptake is thought to be mediated primarily by K + channels, such as Arubidopsis AKTl (Bertl et ul., 1997), and possibly by certain high-affinity K + transporters with a low-affinity for N H t , such as the barley HAKl transporter (Section VI.B.2). Even diffusive uptake of NH: through the K + channel, which is often against the chemical gradient, will depend on the membrane potential maintained by the PM-ATPase. As discussed in Section V1.A. 1, diffusion of the uncharged species, NH3, is unlikely to contribute significantly to uptake.
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3. Competitive Interactions Between NH: and K f Uptake As would be expected if K + and NH; share the same ion channels, there is good evidence for competitive interactions between the two ions in their uptake by roots. The nature of these interactions is complex and depends on the concentrations involved and on the N and K status of the plants. The available data, discussed below, are most consistent with a model where: (1) the HATS for K f has a very low affinity for NH: (and vice versa); (2) the LATS for K + (probably consisting of K + channels) is subject to NH; competition; and (3) under high K or N status, the LATS for K+/NH; dominates because the respective HATS (for K + or NH:) is repressed. The first part of this model is supported by the evidence already discussed (Section V1.B. 1) that the respective HATS for K + and NH: are quite specific and are not inhibited by the presence of the other cation, so that competition between the two ions should be restricted to the LATS. In maize, Vale and colleagues found that K + influx from 200 pM KCl could be resolved into NHl-sensitive and NHt-resistant components (Vale et al., 1987, 1988a, b), which, according to the model above, would correspond to the LATS and HATS for K f , respectively. K + uptake is reported to be most sensitive to NH; inhibition in plants with high K status (Scherer et al., 1984; Vale et al., 1988b), consistent with preferential repression of the NHt-resistant component (the HATS for K + ) under these conditions. In line with these observations, studies with barley showed that 5 days of K + starvation led to a 10-fold increase in K + absorption, but had no effect on NH: uptake (Lee and Rudge, 1986); this is the result expected if the HATS for K + , but not the LATS, had been derepressed. NH,f/K' interactions have also been investigated in rice, where both longand short-term effects of mixed nutrition at various NH: and K + supply rates were studied (Wang et al., 1996). Only at low K status was NH: uptake significantly inhibited by K + . Earlier results with rice showed that the HATS for NH: is repressed under these conditions (Wang et al., 1993a, b), so that most NH; uptake would have occurred via the LATS, which is more subject to competition from K + . In barley it was similarly found that competition between the two ions was least under conditions of N starvation, where the HATS for NH; is likely to have been derepressed (Section VI.B.l). Depriving barley plants of N for 3 days was found to produce a similar 2.5fold increase in the uptake of both NH: (from 0.2mM) and K f (from 0.5 mM) (Lee and Rudge, 1986). The stimulation of the K + uptake system by N starvation is difficult to explain, especially if starvation results in derepression of the HATS for NH: which seems to have little affinity for K + (Ninnemann et al., 1994). The difficulty might be resolved if it could be demonstrated that the LATS for NHZ was also derepressed by N starvation, although the available evidence is against this (Glass et al., 1997). Much of the earlier work showing interactions between NH; and K + uptake used relatively high WH:],, where dramatic inhibition of K uptake +
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was noted in the short term (e.g. Rufty et al., 1982). In their experiments, Rufty and colleagues observed that when roots were first challenged with NH: there was a concentration-dependent net eMux of K + (Rufty et al., 1982); when steady net K + uptake was eventually resumed it was at a slower rate than before the NH: treatment started. The authors interpreted these results in terms of a possible K+/NH,f exchange process across the PM. However, in the light of what has been discovered since then (Ayling, 1993), it seems possible that the initial entry of NH: may have depolarized the membrane potential and that K + efflux then occurred via outwardly rectifying K + channels (Section III.C.2). 4. An NH: Channel in Root Nodules It has become possible to isolate relatively pure fractions of the peribacteriod membrane (PBM) that separates the N-fixing bacteroids from the host cytosol in leguminous root nodules. Large fluxes of N occur across this membrane as the products of N2 fixation (NH, and/or NH:) are passed to the host for assimilation by the GS-GOGAT system. Patch-clamping studies on the PBM indicated the presence of two classes of channels: one that saturated with increasing [NH:] (with a K , of 37.5mM) and another that showed a linear dependence on [NH:] (Tyerman et al., 1995). At high ionic concentrations (150 mM), the conductance was non-selective between NH:, K + and Na +,but at 20 mM (the likely "Hi] within the symbiosome) the channel was somewhat selective for NH:. The authors suggest that the channel may be set in one of two states prior to isolation of the PBM. The transport model for this system was envisaged as follows. The H+-pump in the PBM acidifies the symbiosome inner space and the NH3 produced by the nitrogenase reactions is protonated to NH:. In the host tissue, the [NH:] is kept low due to the high GS activity. A combination of the concentration gradient and membrane potential then drives NH,f through the channel. Note that this is formally equivalent to entry of NH: into root cells from the outside solution and may be similar to other passive NH: uniporters from plants. A candidate cDNA clone for the peribacteroid NH: transporter has been cloned from soybean by complementation of the yeast rneplmepd mutant (Kaiser et al., 1998). Surprisingly, the encoded protein of 348 amino acids has only one or two putative membrane-spanning domains and has no homology to previously sequenced membrane transporters.
VII. MOLECULAR GENETICS OF NH,f TRANSPORT A. PROKARYOTES
For most bacteria NH: is the preferred source of N, and in many cases they have been shown to have energy-dependent uptake systems for NH: (Kleiner,
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B. G . FORDE and D. T. CLARKSON
1993). Bacterial NH,f transporters are generally secondary systems which use the membrane potential as the driving force for active transport, although a Kf/NH; antiport system has been reported in E. coli (Jayakumar et al., 1985). There has been surprisingly little work on the molecular biology of NH; transport in prokaryotes. One E. coli gene (amtA) was isolated by complementation of an NH; uptake mutant and found to encode a soluble protein that was initially thought to represent a regulatory component of the NH: uptake system (Fabiny et al., 1991). However it was later discovered that amtA is identical to the cysQ gene required for cysteine synthesis in aerobically grown E. coli (Neuwald et al., 1992), so the role of amtA/cysQ in NH,f transport is unclear. A structural gene for a bacterial NH,f transporter was first cloned from Corynebacterium glutamicum (Siewe et al., 1996). The C. glutamicum amt gene encodes a hydrophobic polypeptide of 452 amino acids with 11 putative membrane-spanning regions. Disruption of the amt gene produced a mutant which had only 5% of the methylamine uptake activity of the wild type. (Because of the difficulty of studying the transport of NH,f itself, [I4C]methylamine is frequently used as a convenient radiolabelled analogue.) From the pH optimum for uptake and the level of accumulation of methylamine in the bacterial cells at equilibrium, it was deduced that methylamine is taken up in its protonated form via an uniport mechanism that is driven by the membrane potential. Genome-sequencing projects have revealed amt-related genes in a variety of other bacterial species (reviewed in Marini ef al., 1997), but these have not been functionally characterized. B. LOWER EUKARYOTES
1. Saccharomyces cerevisiae S. cerevisiae has three NH; transport systems, encoded by the MEPl, MEP2 and MEP3 genes which all belong to the same family as the C. glutamicum amt gene (Marini et al., 1994, 1997). MEPl and MEP2 encode high-affinity uptake systems (K, = 5-10pM and 1-2pM, respectively), while MEP3 encodes a low-affinity system ( K , = 1.4-2.1 mM). The properties of the MEP gene products were studied using 14C-methylamine as substrate, after establishing that NH: was the only cation that appreciably competed with methylamine for uptake (Marini et al., 1994). Significantly, K f ions had no effect on methylamine uptake, even when present in 10-fold excess. A triple deletion mutant (meplA mep2A mep3A) was unable to grow on media containing less than 5mM NH; (Marini et al., 1997). The same triple mutant was used to demonstrate the importance of the NH,f permeases even for yeast cells growing on other N sources, where their role is to retrieve ammonium that would otherwise be lost to the medium by excretion (Marini et al., 1997).
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The MEP genes are regulated by the N source: a good N source (such as glutamine or asparagine) represses all three genes, while a poor one (such as proline or low concentrations of NH;) derepresses them (Marini et al., 1994, 1997). Intriguingly, it has recently been reported that MEP2 also serves as an ammonium sensor whose role, under conditions of N starvation, is to trigger differentiation of the diploid yeast cells into a filamentous pseudohyphal growth form (Lorenz and Heitman, 1998). 2. Chlamydomonas reinhardtii In Chlamydomonas there appear to be two high-affinity NH; uptake systems with properties remarkably similar to the MEPl and MEP2 systems in yeast: a high-capacity constitutive system with a K, for NH; of 27 pM, and a lowercapacity NHl-repressible system with a K, of 7.5pM (Franco et al., 1988). Although mutants defective in the latter system have been isolated (Franco et al., 1987), the corresponding gene has not yet been cloned. C. HIGHER PLANTS
A high-affinity NH; transporter (AtAMTZ) was cloned from Arabidopsis by complementation of the S . cerevisiae meplmep2 mutant (Ninnemann et al., 1994). AtAMTl encodes a 53-kDa polypeptide with an estimated nine to 12 transmembrane domains and 30% identity to the yeast MEPZ gene product. AtAMT1 differs from MEPl in having a 21-residue hydrophobic domain at its N-terminus, which may be involved in membrane targeting. The biochemical properties of AtAMT1 expressed in S. cerevisiae have been studied in detail (Ninnemann et al., 1994). The pH optimum for methylamine uptake was 7 (with very low rates of uptake at pH 9), and the requirement for glucose and its sensitivity to protonophores and inhibitors of the H + pump indicate that it is an active uptake system driven by the H + gradient. The K, for methylamine was 65 pM and the Ki for NH; was wlOpM, values that are in the same range as those reported for the HATS for NH; in plant roots (Section VI.B.1). Like MEPl, the AtAMT1 transporter was insensitive to inhibition by K + . The data therefore suggest that AtAMT1 is a high-affinity NHi-specific H +/NH4+co-transport system. This would be intensely electrogenic, leading to strong depolarization of the membrane potential, similar to what has been proposed for a high-affinity K + transporter (Maathuis and Sanders, 1993; Schachtman and Schroeder, 1994). A tomato homologue of AtAMTZ has been cloned from a root hair cDNA library and shown to be able to complement the yeast mepZmep2 mutant (Lauter et al., 1996). This gene (LeAMTZ) is expressed specifically in roots (not detectably in stems and leaves) and within the root its expression is highest in
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root hairs. As discussed in Section VI.B.l, this gene appeared to be expressed more or less constitutively under all conditions of N nutrition tested. The expression of LeAMTI specifically in roots is consistent with a role in high-affinity NH; uptake from the soil. However, AtAMTl is expressed in both roots and leaves (and to a much lesser extent in stems). This suggests that the function of AtAMT1 may include the recovery of NH3 effluxing from the cytoplasm into the extracellular space, where it will become protonated to form NH;. In roots such scavenging is likely to be particularly important under Nlimiting conditions; in leaves the same process may enable the recovery of photorespiratory NH3 from the mesophyll apoplast, although in conditions where the external [NH:] is > l00pM the principal mechanism for uptake could be by diffusion through NH; -conducting channels. As discussed in Section VI.B.2, cloning of K + channels and high-affinity K + transporters is providing evidence that these could be important for lowaffinity NHZ uptake.
VIII.
EFFLUX OF NO, AND NH; A. NO; EFFLUX
The efflux of NO, from root cells can play a significant role in determining the net rate of NO, uptake, for example when NH; is available as an alternative N source (Deane-Drummond, 1985; Aslam et al., 1994), at high ~ O , ] , , , (Teyker et al., 1988; ter Steege, 1996) or under conditions of stress (Macduff and Jackson, 1992; Wieneke, 1995). In some circumstances as much as 80% of the NO; influx can be simultaneously dissipated by efflux (e.g. DeaneDrummond, 1985; Oscarson et al., 1987; ter Steege, 1996). This appears to be a wasteful process and one that is unique to NO, with respect to the scale on which it occurs. Other anions are effluxed from roots but never to the same extent. I . Effect of Physical Perturbation Nitrate efflux can be observed in physically undisturbed roots growing in steady-state conditions, but it can also be transiently enhanced by physical disturbance of the roots to the extent that there can be an actual net loss of NO; from the tissue for several minutes (Aslam et al., 1996b). The disturbance need not be severe in order to bring this about. Simply transferring a group of plants from one beaker of solution to another, or the use of excessively vigorous aeration may be enough to provoke a large efflux. Efflux of NO; across the PM should be energetically downhill and could therefore occur through anion channels. A plausible sequence of events leading to disturbance-induced NO, efflux starts with depolarization of the membrane potential (Gronewald et al., 1979), which stimulates Ca2+ influx through
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voltage-sensitive channels (Rincon and Hanson, 1986), which triggers the opening of efflux channels for ISf and anions (Tyerman, 1992). Transgenic plants expressing aequorin, a bioluminescent Ca2+ indicator, have provided dramatic evidence that touch or movement of the plant can induce rapid increases in [Ca2+],,, (for review see Trewavas and Knight, 1994). The effects of physical disturbance have been quantitatively documented in excised maize roots, where transient and sometimes permanent changes in the rate of exudation were observed when the roots were moved from one solution to another of similar composition (Miller, 1987). We have introduced this point simply to illustrate that one's view of the quantitative significance of efflux in controlling net uptake of NO; may be influenced strongly by which species is used, in particular the physical rigidity of the root system and its branches, the experimental treatment of the plants and the time-scale over which observations are made (Bloom and Sukrapanna, 1990). The last of these factors is particularly relevant to those who work with radioactive I3N: such experiments are necessarily of short duration (the halflife of 13Nis about 10 min) and, unless care is taken, perturbation effects will be confounded with the steady-state efflux process. This should be of greatest concern in compartmental analysis where roots loaded with 13N0, may be transferred between different containers, or have the solution drained from the container in which they are mounted, at frequent intervals. It is obvious that, if such treatment were to provoke an exceptional efflux of NO,, the measured half-time and the rate constant for cytoplasmic exchange might be seriously distorted and might lead to spurious estimates of the steady-state cytoplasmic pool size. This possibility was not considered in earlier papers (e.g. Lee and Clarkson, 1986). The estimates of [NO;],,, made using ion-selective microelectrodes (-4 mM in barley; Miller and Smith, 1996) are generally lower than those determined by tracer exchange, and were not affected by increases in the [NO,],,, (Zhen et al., 1991). Working with the same species, but using compartmental tracer analysis, other workers reported [NO,],,, varying from 1 to 37 mM depending on the mO,]ext (Siddiqi et al., 1991). It is possible that the latter results were affected by increased efflux from the cytosol resulting from mechanically stimulated opening of NOT-conducting channels in the PM or the tonoplast. Looked at from another angle, one might regard the undisturbed plant itself as a laboratory artefact whose sensitivity to mechanical disturbance is greatly enhanced by its equanimity. There is evidence, in other studies of mechanical perturbations, that repeated stimulation leads to a refractory state (Trewavas and Knight, 1994). This has been seen in the touch-induced increase in [Ca2+],,, which was seen after the first stimulation of a still plant but became progressively damped with repeated stimulations (M. Knight, personal communication). This may be related to the inactivation of ion channels. In the brackish water alga, Nitellopsis obtusa, a surge of cytosolic Ca2+ may open C1- channels but they quickly become inactivated and do not seem to recover
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when the [Ca2+],,, falls (Kataev et al., 1984; Tyerman, 1992). If there is anything in this idea, the less careful researchers, whose plant material is frequently moved around or whose roots are disturbed by vigorous aeration, may escape the problems that have appeared in other work. 2. Measurements of Efflux Experiments with spinach (Spinacia oleracea) show that the rate of NO; efflux depends both on the external [NO;] and on the relative growth rate (RGR) of the plants. The results of ter Steege (1996) show that in plants with a RGR of 0.17day-', and supplied with 0.8mM NO;, the efflux was 46% of the influx. In plants growing at 0.27day-', under the same conditions, the efflux fell to 8% of influx. A 10-fold rise in the [NO;],,, to 8mM increased these efflux values to 75 and 50% of influx, respectively. In these experiments care was taken to minimize physical disturbance of the plants and efflux was calculated as the difference between 13NO; influx, measured over 2.5 or 5min and net uptake of "NO; measured over 2 h, the estimates of the two rates thus being made on the same set of plants. Changes in the specific activity of 15N or I3N in the external medium have also been used to measure efflux. Teyker and colleagues, working with maize, observed a marked decrease in the specific activity of 15N in the labelling medium as 14NO; effluxed from the roots (Teyker et al., 1988). They showed that efflux, as a proportion of influx, was relatively large ( > 50% in two maize inbred lines) in plants receiving abundant NO; in the external medium ( ~ 7 . 5 m MNO;), but that absolute and relative values of efflux decreased markedly if the plants were deprived of NO; for various periods of time before 15NO; was supplied. They found a linear, inverse correlation between the efflux of 14NO; from the roots and the tissue [NO;]. A rather similar technical approach was adopted in work with pea (Oscarson et al., 1987) and Lemna (Ingemarsson et al., 1987). In these experiments, net uptake was monitored by NO; depletion and influx by the decrease in the radioactivity of the solution. Because of the efflux of I4NO; from tissues, the radioactivity decreased more rapidly than the total [NO;], so that the specific activity of 13Nin the external solution decreased. The changes were monitored continuously by pumping solution from the uptake vessels through a cuvette in a spectrophotometer and past a Geiger-Muller tube. In N-sufficient pea roots, efflux was 47% of influx, but this fell to values of 5-17% in plants whose growth was rate-limited by their N supply when grown by the Ingestad nutrient-addition-rate system. Therefore, in most respects these results are compatible with those obtained with maize (Teyker et al., 1988), but in pea there was an anomalous result with plants that had exhausted their daily dose of NO; some hours before being challenged with a l3N-labe1led solution of 11 mM NO;. In such plants, there was a large influx of I3N but a massive efflux of I4NO; of between 75-89% of the influx. This result appears to be counter-intuitive and the authors suggest that, at the [NO;],,, used, the plants
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were in a state analogous to the compensation point of photosynthesisrespiration. While this may have been true in that instance, compensation points are likely to be at much lower [NO;],,, in other species grown in other kinds of experimental set-up. When cultured in flowing nutrient solutions, rapidly growing plants of perennial ryegrass (Clarkson et al., 1986, 1992), barley (Macduff and Hopper, 1986) or oil-seed rape (Macduff et al., 1987) can absorb sufficient N from solutions in the 5-20 pM range. 3. The Role of Efflux in the Control of Net NO; Uptake From the above discussion it is evident that under conditions where NO; influx exceeds the demand for N by the plant, NO; efflux rises steeply. This may occur naturally if the growth of the plant is slowed down, for example by reductions in light intensity or temperature, or experimentally by increasing the [NO;],,,. However, it can only be in very rare circumstances in nature that plants are confronted with excessive supplies of NO;, particularly given the large capacity of the vacuole for NO; storage. Thus, the efflux mechanism may have evolved for a reason other than the disposal of excess NO;. What might this be? In nearly all studies of anion channels they are reported to be highly permeable to both NO; and C1- (for references see Tyerman, 1992), and at high concentrations of the two anions there appears to be a marked interaction between their fluxes (Cram, 1973). Thus, it is plausible that the welldocumented efflux of NO; from the roots of crop species could be a modern manifestation of a more ancient mechanism for turgor regulation and dealing with excess C1-. Opinions have differed sharply about the significance of efflux in the regulation of net NO; uptake. On the one hand, Deane-Drummond has placed efflux as the principal control, pointing out that it is subject to metabolic regulation in both Chara (Deane-Drummond, 1985) and pea (DeaneDrummond, 1986). On the other hand, Lee and Drew found that NO; influx in barley was stimulated by N demand and inhibited by NH:, while efflux remained unaffected (Lee and Drew, 1986). Similarly, Muller and colleagues found that that NO; influx but not efflux was inhibited by exogenously supplied amino acids (Muller et al., 1995). As with so many differences of opinion, the correct interpretation probably rests between these two positions. Efflux might be thought of as an over-spill mechanism that is stimulated when there is an imbalance between NO; uptake and the demand for NO;; under steady-state conditions the influx rate is well-adjusted to the plant’s requirements, so efflux is minimal. It is possible that NO; itself is the factor that controls the gating of the anion channels involved, perhaps as part of the mechanism that regulates [NO;],,, (Section III.C.5). By analogy, cytoplasmic C1- has been shown to have a direct effect on the gating of outwardly rectifying anion channels of the PM of Arabidopsis callus tissue (Lew, 1991).
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Studies with barley roots indicate that efflux is inducible by NO, over much the same time-course as influx (Aslam et al., 1996~).In these experiments the roots were loaded with NO, in conditions where the induction of the iHATS was inhibited by the presence of cycloheximide but the roots could absorb NO, via the LATS. In the presence of the inhibitor the efflux was only 30% of that observed in control roots where an equivalent tissue [NO,] had accumulated during normal induction of the HATS. These experiments seem to tell us that efflux is not a simple function of root [NO;], but the protocols used were complex and there may have been many interacting factors. Regulatory factors that need to be considered include cytoplasmic pH and the membrane potential across the PM, which are now recognized as regulators of both transporters and channels. In a paper concerned with the effects of physiological stress on the efflux of NO; and NH,f in Italian ryegrass (Lolium multzflorum) and white clover (Trifolium repens) (Macduff and Jackson, 1992), it was suggested that measurement of tracer efflux into the external medium may underestimate the extent to which there is cycling of the two ions across the root PMs. It was argued that the tracer appearing in the external medium comes from the epidermis and the peripheral layer(s) of the cortex. In more deeply located cortical cells effluxed ions may be re-absorbed before they can diffuse to the outer medium. The authors speculate that this apparent futile cycling of NO; across the PM may confer the ability to rapidly adjust the symplastic [NO;] so that a diffusion gradient can be maintained between the root periphery and the stele. This idea had its origin in an earlier paper where fine control of cell [NO,] was studied in Chara (Deane-Drummond, 1984). The true scale of efflux, and of the energy loss in this seemingly wasteful cycling, may indeed be very hard to estimate in an organ with many cell layers. This idea bears on the notion that species differ in the efficiency of NO, uptake, as manifested by the share of total respiration for which the uptake process can account, a topic discussed further in the next section. 4 . Influence of Efflux on the Metabolic Cost of NO; Absorption The metabolic costs of ion transport are largely incurred by the activity of the Hf-transporting ATPase in the PM. The separation of charge by the H + ATPase allows the major cations, K + and Ca2+, to move into the cell by diffusion, while the H electrochemical gradient is used principally for the ‘uphill’ transport of NO,. It is clear that eMux represents a kind of slippage in the NO; transport process and that it is wasteful of energy. In an analysis of the respiratory costs of various root functions by Lambers and colleagues, the respiratory cost of ion transport was arrived at by progressively subtracting O2 consumption for growth and maintenance from the total consumption, leaving transport costs as a residual value (Lambers et al., 1991, 1996). We do not attempt to justify this approach here, but merely point out that when rapidly-growing and slow-growing species of grasses were +
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compared it was found that the transport costs of the latter were much greater. If the NO, transport process is less efficient in slow-growing species it could be because the component of NO, efflux in these species is intrinsically larger. Some preliminary results comparing slow-growing Deschampsia ji'exuosa with a rapidly-growing clone of Dactylis glomerata suggest that there is something in this idea. In the slow-growing species, but not the rapidly-growing one, there was a large efflux of NO, during the night; during the day both species had rather low rates of efflux (Lambers et al., 1997). 5 . Loading the Xylem With NO,
Before entering the xylem vessels, NO; within the root symplast must efflux across the PM of cells within the stele. For many years it has been appreciated that this process may be subject to controls that differ from those affecting initial entry into the symplast. It has always been more difficult to find ways of studying xylem loading (see Clarkson, 1993), but a major step in this direction was taken with the development of a method for isolating protoplasts from cells of the xylem parenchyma of barley roots (Wegner and Raschke, 1994). These protoplasts can then be used to look for the presence of PM ion channels (the likely pathway for ion effluxes) and to characterize the circumstances in which they open. Protoplasts have already proved very useful in studying factors involved in turgor changes in stomata1 guard cells and it is interesting that a number of analogous channel conductances were found in the xylem parenchyma cells of the root (Wegner and Raschke, 1994; Wegner et al., 1994). The opening of the outwardly rectifying anion channel responsible for these occurs over the probable range of membrane potentials between the xylem sap and the xylem parenchyma cells, and it has similar conductances to C1- and NO;. At all probable combinations of membrane potential and NO; concentration gradients, movement of NO; into the xylem will occur by diffusion from the root symplast, so that an active transport system for xylem-loading of NO; should not be required. B. AMMONIUM EFFLUX
Ammonium efflux seems to be ubiquitous, but it should be remembered that if the species moving across the membrane is NHZ this efflux is likely to be thermodynamically uphill. However, given a cytoplasmic pH M 7.CL7.8, it is just as likely that it is NH3 which diffuses passively from the cells and is then protonated in the external solution (Section V1.A. l), giving the false impression that it was the ionic form that had crossed the PM. Wang et al. confirmed that the labelled N effluxing from '3NHt-labelled roots was recovered as the NHZ ion in the external solution (Wang et al., 1993b).
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In rice, under steady-state conditions, rates of ammonium efflux were positively correlated with [NH;lCyt (Wang et al., 1993b), and a similar response to increasing [NH4flCytwas seen in white spruce (Kronzucker et al., 1995b). When maize or sorghum roots were first placed in a solution containing "NH; it was possible to measure a decrease in the specific activity of "N in the medium as l4NH4f effluxed from the root (the source of which was most probably from deamination of amino acids) (Feng et al., 1994). Inhibiting GS with MSX greatly increased both [NH4flCyt (Lee and Ratcliffe, 1991) and efflux (Feng et al., 1994). As steady-state concentrations of NH; in the root tissue seem to vary with the supply, it is hard to make a case for efflux regulating the net uptake of NH;. It seems likely that, as in yeast (Section VII.B.l), an important function for high-affinity NH; uptake systems in plant roots is to retrieve NH; lost from epidermal and cortical cells by efflux. Because NH; is generated endogenously by a wide variety of metabolic processes (including NO; assimilation), this role may be important no matter what N source is available to the plant. C. NET UPTAKE OF NO, AND NH; ALONG THE ROOT LENGTH
To understand the processes that may limit N acquisition from the soil it is essential to know whether the uptake of NO; and NH; takes place along the entire root length or is restricted to certain metabolically active root zones. This physiological information may also be helpful for comparing with the results of studies in which the spatial pattern of expression of the transporters is investigated by histochemical immunolocalization and in situ hybridizations. 1. Laboratory Studies Technical complications have made it difficult to obtain reliable estimates of uptake activity along the length of the root. One problem is that NO; and NH; absorbed at one zone of a root may efflux to the external medium from another part of the root, so that the approach of applying tracers locally and building up a map of net uptake over the root surface is fraught with difficulties. In an alternative approach, H + efflux from roots treated with NH; (Marschner and Romheld, 1983) or deprived of NO; (Moorby et al., 1985) has been visualized by colour changes in pH-sensitive dyes incorporated into agar films overlying root surfaces. The results obtained strongly suggest that quite old tissue in cereal and dicot roots (as distant as 50cm from the tips of roots and with heavily thickened endodermae) absorb and contribute some nutrients freely to the xylem (for reviews see Clarkson, 1993, 1996). A better technique, which eliminates the need to separate root segments by physical barriers or to use tracers, makes use of microelectrodes that are selective for either NO; or NH: (Henriksen et al., 1990, 1992). The tip of the
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electrode is sequentially positioned at a number of points in the unstirred layer of solution close to the root surface. Owing to the rapid absorption of the ions, and their slower diffusion from the bulk solution into the unstirred layer, a depletion zone develops around the root. The profile of this zone can be used to calculate the net flux of the ion across a unit of root surface. This unit is defined by reference to the position of the electrode relative to the geometric centre of the root and the root cross-sectional area (Henriksen et al., 1990). The system provides opportunities to study variations of net uptake rates with time at a given position or at different positions along the length of the axis, as well as to record mutual effects of the two ions on one another's uptake. In seminal axes of barley treated with 0.2mM NO; or NH:, this approach was used to show that net uptake of both ions varied both in time and at different positions along the apical 6 cm (Henriksen el al., 1992). Perhaps the most significant observation was of distinct differences in the pattern of net NO; uptake along individual root axes, even though these had been selected for morphological uniformity in the first instance. These patterns did not change with time. Thus, in two of the four examples shown, uptake 1 cm from the root tip was significantly less than at 6cm from the tip, tip values being lO(r200 nmol m-2 s-', while in two others a more or less constant rate of uptake was measured, tip values being in the range 200-300 nmol mP2s-'. The uptake of NH: displayed a general trend of being greatest in the 1-cm zone and becoming less at 6cm from the tip. An ingenious analysis of the "N content of regions of a maize root system made use of the fact that the accumulation rate of "N tracer changes in different ways in the various tissue zones, depending on whether they are exporting or importing label from other parts of the root system (Lazof et al., 1992). Theoretical considerations led the authors to suppose that the initial rate of "N accumulation would rise to a maximum value as the cytoplasmic compartment filled with tracer, but that it would subsequently either slow down in root zones that were exporting labelled N, or continue to rise in zones that were importing labelled N from elsewhere. The authors used 15N03 supplied at either 0.1 or lOmM and found that the influx was least in the 0-5mm apical regions of primary roots and that these zones imported considerable amounts of tracer from other parts of the root during a 15-min uptake period. As NO; is thought not to be translocated to any great extent in phloem, this import may have been due to labelled products of NO; assimilation. In the zone preceding the emergence of lateral roots, the influx was more than 10-fold greater than at the tip when O.lmM NO; was supplied and about 2.5-fold greater when 10 mM NO; was supplied. The influx in the apical zone (0-5 mm) was 13-fold greater at lOmM than at 0.1 mM NO;, while the much smaller increases in other zones suggest that LATS for NO; was quantitatively most significant in the youngest tissue. A study of NO, uptake from soil-grown roots of wheat exposed to agar blocks containing "N, found a fairly uniform value of 580nmolm-2s-'
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(recalculated from authors’ data) over the various elements of the whole root length (Brady et al., 1993). Rather different results were obtained with maize seedlings using both in vitro and in situ techniques to compare NO; uptake rates along the primary root (Reidenbach and Horst, 1997). Net uptake rates by intact roots were assayed under conditions where the HATS should dominate and were found to decline steadily from the root tip towards the basal part of the root. In some treatments, uptake over the apical lOcm was twice that in older regions, the diffetential being greatest where plants were of low N status and at night, especially if grown in shade. However, when the uptake assay was performed on isolated root segments supplied with 0.2mM NO; and 1mM sucrose, this decline was no longer seen. The authors suggested that in the intact plant the rate of NO; uptake in the basal part of the root could have been limited by the availability of carbohydrates, and that the higher NO; uptake activity in the apical root zone was due to the preferential allocation of assimilates to this region. Thus, the evidence suggests that whilst there can be some quantitative differences in uptake activities in different parts of the root, all zones of a cereal root system have the capacity to take up NO;, and probably NH; as well. 2. The Effect of Soil Factors and Implications for Modelling NO; Uptake From Soils Despite the apparent capacity of all parts a mature root system to absorb nutrients, in practice external factors can have a major effect on the actual distribution of nutrient uptake activity within the roots (Marschner, 1995; Jungk, 1996). For example, nutrients are generally unevenly distributed within the soil, even in an agricultural system (Gregory et al., 1979), while a low soil water content (such as might be found in the latter part of the growing season) can also severely restrict nutrient availability: for alfalfa it was shown that under drying soil conditions just 3% of the root system was responsible for more than 60% of the total nutrient uptake (Fox and Lipps, 1960). It is soil factors of this type that may explain why Wiesler and Horst, working with two maize cultivars in the field, found 10-20-fold differences in N uptake rates per unit root length at different soil depths (Wiesler and Horst, 1994). Experiments with flowing nutrient solutions have shown that NO; concentrations at the root surface as low as 20-100pM can sustain maximum growth rates in a range of crop species (Edwards and Barber, 1976; Clement et al., 1978; Steingrobe and Schenk, 1991), and based on models for the movement of NO; in the soil it has been estimated that a [NO;] in the soil solution of 300-400pM would be sufficient to maintain sufficient NO; at the root surface to saturate the uptake system (Barraclough, 1986; P. B. Barraclough, personal communication). A paradox pointed out previously is that cereals and other crops respond to heavy applications of N-fertilizer beyond those needed to produce very high [NO,] in the soil solution (Robinson et al., 1991). A simplifying assumption made in Barraclough’s
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model was that the whole root system participates equally in NO; uptake. Whilst the physiological experiments described above largely support this assumption in relation to the potential of different parts of the root to take up NO;, the paradox can probably be explained in terms of the soil factors that restrict uptake over large parts of the root by limiting the root’s access to nutrients.
IX. REGULATION BY THE SHOOT OF NO, AND NH; UPTAKE BY ROOTS During vegetative growth, the nutrient demand placed on a given element of root length or surface will largely depend on the shoot biomass it supplies and the rate at which leaves and stems are growing. These growth processes determine the rate at which nutrients are unloaded from the root symplast and enter the xylem. The ratio of shoot biomass to root length or root surface area changes dramatically from large values in young seedlings to smaller, relatively stable ones in older plants (Wild and Breeze, 1982; Wild et al., 1987). As the shoot:root ratio falls the flux across the root surface needed to support a given growth rate should also fall. The corollary to this would be that a decrease in shoot:root ratio would be an appropriate adaptive response to the imposition of growth-limiting N supplies, and indeed this is the response most often seen (Ingestad and Lund, 1979; McDonald et al., 1986), although not invariably (see Robinson, 1994). A. EVIDENCE FOR SHOOT-DERIVED SIGNALS
1. Manipulation of Shoot Demand Shoots can be made to grow faster than roots, for some time at least, by keeping the leaves and/or (in monocots) the basal leaf meristems, at an optimal temperature while the roots are kept at an unfavourable one (e.g. Engels and Marschner, 1990; Sattelmacher et al., 1990; Macduff and Jackson, 1991; Clarkson et al., 1992). As the shoot:root ratio increases, so does the demand on the nutrient supply from the root (Wild and Breeze, 1982). There are numerous instances showing that roots can respond to this by a progressive increase in the uptake rate of various nutrients and the rate at which they are transferred to the xylem (see Wild et al., 1987). In maize, the growth of the shoot is controlled largely by the leaf base temperature (LBT), which affects the activity of the leaf meristems (Engels and Marschner, 1990). An experimental system was designed to insulate the shoot base from the root zone where the temperature (RZT) could be controlled at various values. When cooling was first applied, a RZT of 12°C combined with a shoot base temperature (SBT) of 23°C had the predictable effect of severely depressing the uptake of NO; and NH,f and the transport of N to the xylem;
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evidently a metabolic effect on the transport mechanisms (Engels et al., 1992; Engels and Marschner, 1996). At this stage of the experiment the shoot:root fresh weight ratio was approximately 1, but this soon changed as the shoot grew faster than the roots. By 3-5days the ratio had increased to 2 and continued to increase for a further 5-7 days. In parallel there was a doubling in the rate of uptake of both NO; and NH: at 12°C. The authors attributed this result to demand for N overcoming the influence of a temperature unfavourable to ion uptake (Engels et al., 1992). An alternative possibility is that the inhibitory effect of low temperature was overcome by increasing the abundance of the transporters in the PM. This matter could be explored using the newly available molecular probes for transporter mRNAs and polypeptides (Sections V.B and VI1.C). When both SBT and RZT were held at 12"C, there was no shift in the shoot:root ratio and no increase in ion transport rate with time (Engels et a[., 1992; Engels and Marschner, 1996). Therefore the response observed is not an adaptation to life at an unfavourable temperature, but a manifestation of the adjustment of transport rates by demand (Clarkson et al., 1988). 2. Manipulation of the Root's Access to the N Supply When root systems are exposed to localized supplies of inorganic N, they respond in ways that maximize their ability to capture the available N (Robinson, 1994). This has been revealed by a variety of experiments where it is engineered that part of the root system (the donor) is exposed to an abundant NO; (or NH:) supply, while the remainder of the root experiences a lesser (or zero) supply. When a root axis enters such a zone or compartment and becomes a donor, two things usually occur. First, there is a kinetic response during which the net absorption rate of NO; increases markedly in comparison with roots uniformly exposed to NO;, even though the exposure to external NO; is similar and biochemical analysis reveals no difference in the N status of the roots in question (Drew and Saker, 1975; Burns, 1991). Secondly, there is a phase during which growth and lateral branching of the donor root is more vigorous, leading to a greatly increased number and length of lateral roots within the N-rich zone (Drew, 1975; Drew and Saker, 1975; Burns, 1991). The ability of non-uniform N supplies to elicit localized effects on root metabolism and morphology is clear evidence that a root axis (or section of a root axis) can respond to a general demand for N in the plant and not simply to its own N status. A model for how lateral root growth is regulated by the NO; supply, based on studies with Arabidopsis, has recently been put forward (Zhang and Forde, 1998). In Arabidopsis, NO, affects elongation, but not initiation, of lateral roots, and both inhibitory and stimulatory effects are observed, depending on the [NO;],,, and whether the NO; is supplied to the whole root system or just to part of it (Zhang and Forde, 1998). The model proposes that NO; has two opposing effects on lateral root development: a localized stimulatory effect that
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depends on the [NO;],,, at the lateral root tip; and a systemic inhibitory effect that depends on the total amount of NO, absorbed by the root system and its influence on the N status of the plant. It is the balance between these two effects that determines the rate of development of an individual lateral root. The model predicts the existence of an inhibitory signal emanating from the shoot, the intensity of which is related to the N status of the plant. It is possible that the same signal could be responsible for feedback-regulating the NO; transport system in response to the demand for N from the shoot. B. NATURE OF THE SHOOT-DERIVED SIGNALS
The shoot-derived signals that modulate NO; and NH: uptake activity in the root are probably transmitted in the phloem and must be capable of conveying quantitative information about the demand for N in the shoot. Most of the relevant work has been carried out on NO; uptake, and evidence for three different kinds of metabolic signals (amino acids, carboxylates and carbohydrates) is discussed below. It is also possible that hormonal or other nonmetabolic signals could be involved: Lucas has recently proposed the existence of a class of phloem-mobile polypeptides that act as signals carrying information to the root about the nutrient or water status of the shoot (Lucas, 1997). 1. Amino Acids in the Phloem It has been proposed that the flow of amino-N from the leaves to the root, via the phloem, may modulate the activity of the NO; uptake system (Imsande and Touraine, 1994; Touraine et al., 1994). The phloem invariably contains the amino acids that have been identified as putative repressors of NO, transport activity, and it has been shown that there is cycling of amino-N between roots and leaves (Layzell et al., 1981; Lambers et al., 1982; Cooper and Clarkson, 1989; Larsson et al., 1991). The idea would be sustainable if it could be demonstrated that: (1) phloem sap contains amino compounds capable of repressing activity/expressionof the NO, transport systems; (2) the amino acid composition/concentration of phloem sap changes with the N status of shoots; (3) the amino acids delivered in the phloem sap influence the amino compound concentrations in the cytoplasm of cells responsible for NO; absorption; and (4)direct intervention to change the amino acid composition/concentration where the phloem is loaded results in a response of transport systems in the root. There is some evidence on each of the above points.
1. As discussed in Section III.C.3, it is clearly established that amino acids that are normally present in phloem sap inhibit the absorption of NO; if they are supplied to the external medium. 2. There is rather limited information on the time-course of changes in amino acid concentration in phloem sap relative to changes in the kinetic
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parameters of NO; uptake. There are predictable reports that the amino acid concentration in phloem varies with the intensity of N nutrition, but the time-dependency of changes is not clear (e.g. Peuke et al., 1994). If phloem delivery of amino acids to roots is truly a means of signalling the N status of the shoot, then it must change in advance of modulation of NO; influx. Some novel investigations have made use of the pollutant gas NOz, which is readily absorbed and assimilated by leaves of Norway spruce (Picea abies) and beech (Fagus sylvatica) (Schneider et al., 1996). It was estimated that amino-N transport in the phloem of Norway spruce increased three-fold in plants fumigated with NOz. In some, but not all treatments, this was associated with a marked reduction in the rate of NO; uptake by the roots (Muller et al., 1996). These and other consequences of air pollution on shoot-root relationships are concisely reviewed elsewhere (Rennenberg et al., 1996). 3. The relationship between the rate of amino acid delivery in the phloem and the cytosolic concentrations of putative regulatory amino compounds in roots has not been established. Tracer studies, using "N, show that a substantial fraction of the amino-N arriving in the roots of barley (Cooper and Clarkson, 1989) or wheat (Larsson et al., 1991) can be recycled and enters the xylem without mixing with the N of the root as a whole, but this observation does not preclude the possibility that the translocated aminoN mixes with the amino-N in the symplast before being transferred to the xylem. In the work on soybean it was shown that the inhibition of NO; uptake followed the peak of arginine concentration in the phloem sap which originated from exogenous arginine fed to half cotyledons, but the authors did not measure the movement of arginine into the root tissue (Muller and Touraine, 1992). Such measurements present technical difficulties, not least in distinguishing between possible vacuolar and cytoplasmic pools of amino acids. One approach might be to use the technique of secondary ion mass spectrometry (SIMS) which can produce images of the distribution of I5N on freeze-substituted tissue sections (Grignon et al., 1992; Gojon et al., 1996). Loading of "N-labelled amino acids into the phloem could then be correlated with their unloading into root tissues. 4. Indirect loading of the phloem can be achieved if cotyledons of castor bean (Schobert and Komor, 1989) and soybean (Muller and Touraine, 1992) are cut and the cut surfaces bathed in relatively concentrated solutions of amino acids. In soybean, some of the 14 amino acids tested strongly inhibited uptake of NO; by the roots some hours after loading of the cotyledons began. The lag phase was compatible with uptake by the cotyledon tissue, loading into the sieve tubes and downward transport to the root; as mentioned above, a peak of arginine concentration was seen 5 h after the treatment started and was followed 2 h later by a rapid decline in NO; influx. The inhibition seen with different amino acids was
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unrelated to their loading into the phloem sap. Thus, serine, which was without effect, reached greater concentrations than arginine, the most inhibitory. There is a need to develop an alternative method for confirming results of this kind. Because of their large cotyledons, soybean seedlings are well supplied with endogenous N, and rates of NO; uptake are quite low, so that expression of the HATS may well be substantially repressed compared to older plants (Delhon et al., 1995a, b). Experimentally, progress in this area is limited by there being so few species from which phloem sap can reliably be sampled for amino acid analysis. A sampling technique which offers improved reliability, and which is being applied with increasing frequency, employs aphids or other insects that feed on the phloem sap (Wang and Nobel, 1995). This technique involves cutting off the stylet of a feeding aphid (using a laser microsurgical device or by radiofrequency microcautery) and collecting the small amount of sap that exudes from the cut end. In one study on lettuce (van Helden et al., 1994), this technique was compared to two alternative methods in which fluid was collected from petioles treated with EDTA (which prevents blockage of the sieve plates) or the honeydew that collects on the abdomena of feeding aphids was sampled. The results showed marked differences between the composition of the respective samples, with the clear implication that the latter two techniques were unreliable. Doubts about the EDTA method were also raised in an earlier paper (Weibull et al., 1990). Although aphids may favour certain plant species, their range can be increased by restricting choice. For example, the pea aphid, Acyrthosiphon pisum, can be encouraged to feed on the sap of a variety of other legumes that are not its normal food source (Sandstrom and Pettersson, 1994; Girousse et al., 1996). Therefore, there seem to be good prospects for increasing our understanding of quantitative aspects of amino-N movement in the phloem if plant physiologists can join forces with their entomologist colleagues. 2. Carboxylates Carboxylic anions are synthesized in leaf cells in response to imbalances of electrical charges on inorganic ions and to perturbation of cytoplasmic pH by metabolic activities such as NR (Marschner, 1995). Total carboxylate production in tomato leaves, for example, is five-fold greater in plants under NO;, rather than NH:, nutrition. In common with many other crop species, the leaves of tomato are the dominant sites for NO; reduction. A well-known theory was advanced by Ben Zioni et al. (1971) to explain the linkage of NO; reduction in leaves to the rate of NO; uptake in roots (see Imsande and Touraine, 1994, for a more recent interpretation of the model). In essence it is proposed that HCO;/OH-, released from roots via the decarboxylation of phloem-delivered malate, is exchanged for external NO;
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at the root surface. Charge balance for malate in the phloem and NO; in the xylem is provided by K + which cycles in the system. Ever since its inception the model has provoked controversy. The current position appears to be that there are circumstances, for instance in soybean (Touraine et al., 1988, 1992), where the relationships between malate decarboxylation in roots, the release of HCO;, the uptake of NO; and the re-circulation of K + can be quantitatively reconciled according to the model. Internal circulation of malate, fed to a donor root on a soybean plant and then delivered to a receiver root by phloem transport, stimulated NO; uptake by the receiver after a lag of approximately 1.5h (Touraine et al., 1992). There are, nevertheless, numerous examples where carboxylate cycling and malate decarboxylation are not quantitatively related to NO, uptake. Most obviously these come from species where NO; is reduced primarily in roots and no circulation of carboxylate and K + need be envisaged. In a study which sought to explain a marked diurnal variation in the rates of NO; influx and net uptake in soybean (Delhon et al., 1995a), it was found that during the night period, stimulation of leaf NR activity and the provision of malate to either shoots or roots had no positive effect on the low rates of NO; uptake observed at night (Delhon et al., 1996a). Although it is difficult to distinguish experimentally between a H+/NO; symport and an OH-/NO; antiport transport system, there is now a clear balance of opinion in favour of the former. If this is correct, the final stage in coupling to NO; uptake cannot be as the Ben-Zioni model suggests. The attractive feature of the Ben-Zioni model is that it provides a conceptual basis for linking N demand (broadly equivalent to the rate at which NO; can be reduced) and the root transport processes. Controversy over its validity is likely to continue but its Achilles heel would seem to lie in the molecular details of what goes on at the ion-binding sites of the NO; transporters. 3. Carbohydrate Supply It has been proposed that carbon availability to the root may partially control the uptake of N (Jackson et al., 1976). To the extent that NO; uptake depends on the maintenance of the H + electrochemical potential gradient, this is understandable in terms of the ATP available for hydrolysis by the H + ATPase in root cells. In several species a distinct diurnal rhythm has been observed in NO; uptake. In pepper (Capsicum annuum) a maximum rate of NO; uptake occurred 5-7h into the photoperiod and coincided with a channelling of a high proportion of photosynthetically fixed I4C into amino acids and a peak in shoot NR activity measured in vitro (Pearson and Steer, 1977). NR activity declined later in the photoperiod, as did NO; uptake, reaching a minimum in the dark period. The daily pattern of NO; uptake most closely coincided with that of photosynthate translocation from the leaves, and yet analysis revealed no changes in soluble carbohydrate or malate in the roots.
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In soybean, strong circumstantial evidence linked the diurnal variation in NO; uptake with photosynthesis (Delhon et al., 1996b). Shading leaves reduced starch accumulation and amplified the decline in uptake during the dark, whereas the provision of exogenous glucose to roots stimulated NO; uptake to a much greater extent during the night than during the day. Furthermore, removing C 0 2 from the atmosphere in the light prevented the daytime rise in NO, uptake. Stitt and colleagues have used a number of mutant and transgenic tobacco lines with very low NR activities to obtain evidence that accumulation of the NO, ion in the shoot can indirectly modify root growth, possibly through an effect on carbohydrate metabolism (Scheible et al., 1997a, b). The physiological effects that correlated with the accumulation of NO; in the leaves of these plants were various, but included a greatly decreased root:shoot ratio. Splitroot experiments indicated that it was the accumulation of NO, in the shoot that was responsible for inhibiting root growth (Scheible et al., 1997b). The inhibition of root growth was correlated with an inhibition of starch synthesis and breakdown in the leaf and a consequently diminished supply of sucrose to the roots (Scheible et al., 1997a, b). Although the authors did not measure NO, uptake rates in their experiments, the same processes could link NO; accumulation in the shoot with a decreased rate of NO, uptake in the root. These findings appear to point to NO; uptake (and other root activities) being regulated by the availability of photosynthates. This, however, runs counter to the general view (and to the observations on pepper mentioned above) that physiological processes in roots are rarely limited in a direct way by substrate translocation (Lambers et al., 1996). The alternative explanation is that the sugars arriving in the phloem have a signalling role that is distinct from their metabolic role (Jang and Sheen, 1997; Smeekens and Rook, 1997).
X. CONCLUDING REMARKS: LOOKING BACK AND LOOKING FORWARD As we near the end of the millennium it seems appropriate to take a backward glance to see just how far we have come in our understanding of plant nitrogen nutrition. The beneficial effects of saltpetre (KN03) on soil fertility were known at least since the mid-sixteenth century, but it was Mayow (1681) who was amongst the first experimentalists to conclude that plants absorbed ‘nitre’ (NO;) from the soil. Then in the mid-1700s LeBlanc found that several salts of ammonia strongly promoted plant growth (von Meyer, 1906). There still followed many years of confusion and controversy, not resolved until the latter part of the nineteenth century, first over the question of whether plants got their N from the air or the soil, and then whether they used only ammonium or only nitrate (Russell, 1937). Some of the earliest recorded thoughts about uptake mechanisms were published by Jung (1678), who surmised that roots
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had tiny holes capable of selecting from the soil what plants needed for their food. A more mechanistic hypothesis was propounded 50 years later by Jethro Tull, the English inventor of agricultural machinery. He proposed that the pressure caused by the swelling of the growing roots forced minute particles of soil into the ‘lacteal mouths of the roots’ where they entered the circulatory system (Tull, 1731). It has taken nearly three centuries of advances in all branches of the sciences to reach the point we are at today. We still have a long way to go, but from the foregoing discussion it is evident that we are now beginning to get a detailed picture of those ‘lacteal mouths’, their modes of action, their molecular structures and how they are regulated. The recently accelerated rate of progress in this field looks set to continue. It is likely that this progress will be led, in large measure, by advances in molecular genetics and molecular physiology as it is these approaches that allow the dissection of the transport systems and the transport processes in a way that was previously impossible. The cloning of genes and cDNAs for both high- and low-affinity NO; and NH; transporters has already revealed much about the structure of the respective transport systems and their regulation at the mRNA level. The lessons learnt from the complexity of the regulatory mechanisms operating on NR (Huber et al., 1996), illustrate how important it will be to look for possible regulation of the transporters at the ,posttranslational level. There is still much work to be done at the basic level of isolating and characterizing transporter genes. Even in a single-celled organism like S. cerevisiae, the same substrate is often transported by several systems with different kinetic and regulatory properties (Horak, 1997). This makes it less surprising to find that Arabidopsis has at least seven genes controlling NO, and NH; uptake (see Table I and Fig. 8), and that barley may have many more (Trueman et al., 1996a). In addition to the influx systems, there are also likely to be further NO; transport systems responsible for such important processes as efflux across the PM, vacuolar storage and efflux, and xylem loading and unloading. The fact that these systems may not have direct homologues in lower eukaryotes, together with possible problems with developing heterologous expression systems for studying them, could make the identification and cloning of these carriers and channels even more challenging than has been the case for the influx systems. The cloned transporter sequences will certainly be used as tools to manipulate expression of the corresponding genes in transgenic plants. The physiological analysis of these transgenic lines should tell us much about the role of individual transporters in nutrient uptake and the extent to which they control flux through the N assimilatory pathways. Furthermore, there is the exciting prospect of using the same technology to modify the expression of the transporters, or their properties, in such a way as to improve the efficiency of N capture from the soil. Simply placing the relevant transporter under the control of a strong constitutive promoter may not be sufficient to produce the desired
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result; under conditions of adequate N supply the rate of uptake is likely to be determined by the demand for N rather than the capacity of the uptake systems (see Section IX). One way around this limitation might be to create an alternative sink for the absorbed NO;, perhaps by increasing the expression of vegetative storage proteins (Berger et al., 1995). Otherwise a beneficial effect of overexpressing a (high-affinity) NO; or NH; transporter would be most likely under conditions where the growth rate is limited by the supply of N, i.e. when the external [NO,] or [NH4f]is low. One outcome of these experiments may be to highlight our need for a clearer understanding of how N uptake is regulated by the demand for N, and how plant growth is regulated by the availability of
N.
ACKNOWLEDGEMENTS We are very grateful to our colleagues, particularly Tony Miller (IACRRothamsted), Emilio Fernandez (University of Cordoba), Michael Blatt (Wye College, University of London), Tony Glass (University of British Columbia, Vancouver) and Nigel Crawford (University of California, San Diego), for helpful discussions and access to unpublished results. Work in B. G. Forde’s laboratory is partly supported by a grant from the European Union (contract No. BI04-CT97-223 1) in the BIOTECH programme of Framework IV. IACR receives grant-aided support from the BBSRC.
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Vale, F. R., Jackson, W. A. and Volk, R. J. (1987). Potassium influx into maize root systems: influence of root potassium concentration and ambient ammonium. Plant Physiology 84, 14161420. Vale, F. R., Volk, R. J. and Jackson, W. A. (1988a). Simultaneous influx of ammonium and potassium into maize roots: kinetics and interactions. Planta 173, 424-431. Vale, F. R., Jackson, W. A. and Volk, R. J. (1988b). Nitrogen stimulated potassium influx into maize roots: differential response of components resistant and sensitive to ambient ammonium. Plant Cell and Environment 11, 493-500. van der Leij, M., Smith, S. J. and Miller, A. J. (1998). Remobilisation of vacuolar stored nitrate in barley roots. Planta 205, 64-72. van Helden, M., Tjallingii, W. F., Teris, A. and van Beek, T. A. (1994). Phloem sap collection from lettuce (Lactuca sativa L.): chemical comparison among collection methods. Journal of Chemical Ecology 20, 3 191-3206. von Meyer, E. (1906). “A History of Chemistry” (translated by G. McGowan). Macmillan, New York. von Wiren, N., Gazzarrini, S. and Frommer, W. B. (1997). Regulation of mineral nitrogen uptake in plants. Plant and Soil 196, 191-199. Walker, D. J., Smith, S. J. and Miller, A. J. (1995). Simultaneous measurement of intracellular pH and K + or NO; in barley root-cells using triple-barreled, ionselective microelectrodes. Plant Physiology 108, 743-75 1. Wallsgrove, R. M., Hasegawa, H., Kendall, A. C. and Turner, J. C . (1989). The genetics of nitrate uptake in higher plants. In “Molecular and Genetic Aspects of Nitrate Assimilation” (J. L. Wray and J. R. Kinghorn, eds) pp. 15-24. Oxford Science Publications, Oxford. Wang, M. Y., Siddiqi, M. Y . , Ruth, T. J. and Glass, A. D. M. (1993a). Ammonium uptake by rice roots. 11. Kinetics of I3NH4+influx across the plasmalemma. Plant Physiology 103, 1259-1267. Wang, M. Y., Siddiqi, M. Y., Ruth, T. J. and Glass, A. D. M. (1993b). Ammonium uptake by rice roots. I. Fluxes and subcellular distribution of 13NH:. Plant Physiology 103, 1249-1258. Wang, M. Y., Glass, A. D. M., Shaff, J. E. and Kochian, L. V. (1994). Ammonium uptake by rice roots. 111. Electrophysiology. Plant Physiology 104, 899-906. Wang, M. Y., Siddiqi, M. Y. and Glass, A. D. M. (1996). Interactions between K + and NH:: effects on ion uptake by rice roots. Plant Cell and Environment 19, 10371046. Wang, N. and Nobel, P. S. (1995). Phloem exudate collected via scale insect stylets for the CAM species Opuntia ficus Indica under current and doubled C 0 2 concentrations. Annals of Botany 75, 525-532. Wang, R. and Crawford, N. M. (1996). Genetic identification of a gene involved in constitutive, high-affinity nitrate transport in higher plants. Proceedings of the National Academy of Sciences of the United States of America 93, 9297-930 1. Ward, M. R., Tischner, R. and Huffaker, R. C . (1988). Inhibition of nitrate transport by anti-nitrate reductase IgG fragments and the identification of plasma-membrane associated nitrate reductase in roots of barley seedlings. Plant Physiology 88, 1141-1 145. Warner, R. L. and Huffaker, R. C. (1989). Nitrate transport is independent of NADH and NAD(P)H nitrate reductases in barley seedlings. Plant Physiology 91, 947953. Wegner, L. H. and Raschke, K. (1994). Ion channels in the xylem parenchyma of barley roots: a procedure to isolate protoplasts from this tissue and a patch-clamp exploration of salt passageways into xylem vessels. Plant Physiology 105, 799813.
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Wegner, L. H., Deboer, A. H. and Raschke, K. (1994). Properties of the K + inward rectifier in the lasma-membrane of xylem parenchyma cells from barley roots: effects of TEA , Ca2+, Ba2+ and La3+.Journal of Membrane Biology 142, 363379. Weibull, J., Ronquist, F. and Brishammar, S. (1990). Free amino acid composition of leaf exudates and phloem sap: a comparative-study in oats and barley. Plant Physiology 92, 222-226. White, P. J. (1996). The permeation of ammonium through a voltage-independent K + channel in the plasma membrane of rye roots. Journal of Membrane Biology 152, 89-99. Wieneke, J. (1995). Altered influx-efflux relations of nitrate in roots due to nutrient stress. I. effect of phosphorus and zinc deprivation. Journal of Plant Nutrition 18, 1547-1561. Wiesler, F. and Horst, W. J. (1994). Root-growth and nitrate utilization of maize cultivars under field conditions. Plant and Soil 163, 267-277. Wild, A. and Breeze, V. G. (1982). Nutrient uptake in relation to growth. In “Physiological Processes Limiting Crop Production” (C. B. Johnson, ed.) pp. 331-344. Butterworths, London. Wild, A., Jones, L. H. P. and Macduff, J. H. (1987). Uptake of mineral nutrients and crop growth: the use of flowing nutrient solutions. Advances in Agronomy 41, I7 1-2 19. Wray, J. L. (1993). Molecular biology, genetics and regulation of nitrite reduction in higher plants. Physiologia Plantarum 89, 607-612. Wurgler-Murphy, S. M. and Saito, H. (1997). Two-component signal transducers and MAPK cascades. Trends in Biochemical Sciences 22, 172-176. Yompakdee, C., Ogawa, N., Harashima, S. and Oshima, Y. (1996). A putative membrane protein, Pho88p, involved in inorganic phosphate transport in Saccharomyces cerevisiae. Molecular and General Genetics 251, 58&590. Zhang, H. and Forde, B. G. (1998). An Arabidopsis MADS box gene that controls nutrient-induced changes in root architecture. Science 279, 407409. Zhen, R. G., Koyro, H. W., Leigh, R. A., Tomos, A. D. and Miller, A. J. (1991). Compartmental nitrate concentrations in barley root cells measured with nitrateselective microelectrodes and by single cell sap sampling. PIanta 185, 356-361. Zhou, J.-J., Theodoulou, F. L., Muldin, I., Ingemarsson, B. and Miller, A. J. (1998). Cloning and functional characterisation of a Brassica napus transporter which is able to transport nitrate and histidine. Journal of Biological Chemistry 273, 12017-1 2023.
P
Secondary Metabolites in Plant-Insect Interactions: Dynamic Systems of Induced and Adaptive Responses
J. A. PICKETT, D. W. M. SMILEY and C. M. WOODCOCK
IACR-Rothamsted, Harpenden, Hertfordshire ALS 2JQ, UK
I. Introduction ............................................................................................... 11. Feeding and Other Contact Interactions Involving Involatile Metabolites
111.
IV. V.
VI.
A. Plant Defence by Toxic Mechanisms ................................................. B. Disruption of Insect Feeding by Non-toxic Mechanisms .................. C. Sequestration of Toxicants by Insects ................................................ Insect Interactions With Plants Using Volatile Signals ............................. A. Location of Host Plants .............................. ............................. B. Avoidance of Unsuitable Plants .................. Plant Interactions With Predatory and Parasitic Insects ........................... Interactions Between Secondary Metabolites and Insect Pheromone Systems....................................................................................................... Future Prospects ........................................................................................ Acknowledgements..................................................................................... References ..................................................................................................
92 93 93 97 99 100 100 102 103 105 105 107 107
It has long been accepted that plant-insect interactions involve the evolution of defensive secondary metabolic pathways in plants to which insects can adapt. New theories are being proposed to account for the wide diversity of such interactions, particularly where production of plant secondary metabolites is inducible. This review briefly describes the more widely known mechanisms by which secondary metabolites act, such as direct toxicity, antifeedancy and sequestration. Key areas covered in greater depth involve the volatile secondary metabolites produced by plants, both inherently and in response to the stress of insect feeding and colonization. Interactions described include the location of host plants by insects and the corresponding avoidance of unsuitable host plants. The roles of volatile plant compounds in the attraction of predators and parasitoids to plants already hosting insect populations, and in the synergism of insect pheromone activity, are discussed. The potential for exploitation of plant secondary metabolites in novel crop protection strategies, as alternatives to broad spectrum eradicant pesticides, is presented. Advances in Botanical Research Vol. 30 incorporating Advances in Plant Pathology
ISBN 0-12-005930-4
Copyright 0 1999 Academic Press AU rights of reproduction in any form reserved
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I. INTRODUCTION After wide acceptance of the theory that plant-insect interactions resulted from the evolution of defensive secondary metabolism in plants and the adaptation to this by insects, we are now seeing a blossoming of new ideas on the evolutionary origins of these interactions (Berenbaum, 1995a, b), with stressinduced metabolism playing a major role (Stowe et al., 1995; Takabayashi and Dicke, 1996; Karban and Baldwin, 1997). This area is one of the most rapidly developing within the subject of chemical ecology. To some extent, the renewed interest in evolutionary aspects of plant-insect interactions has derived from an enhanced concern over issues relating to species diversity. Demonstrations of the dynamism of such interactions come partially from advances in the study of insect electrophysiology and the dramatic new developments in plant biochemistry and molecular genetics. Jones and Firn (1991) have turned earlier theories around by suggesting that plants which contain a high absolute chemical diversity in secondary metabolism, regardless of activity, have a better chance of being well defended, as the retention of inactive compounds increases the probability of producing new active compounds, with minimal cost to the plant (Firn and Jones, 1996). Indeed, Haslam has suggested that it is the processes of secondary metabolism and not the products (secondary metabolites) themselves which are important, but that this does not in any way preclude the possibility that secondary metabolites have acquired a functional role (Haslam, 1995). Theories developed in connection with marine chemical ecology (Hay and Fenical, 1996; Stachowicz and Hay, 1996), where it has been postulated that herbivores or the smaller carnivores may be protected from predation by choosing hosts that are themselves toxic, have been adapted to explain terrestrial plant-insect relationships (Rothschild, 1993). Hartmann (1996) has referred to ‘the high degree of chemical freedom’ of secondary metabolism which, in contrast to primary metabolism, allows structural modifications with almost no restrictions. This gives the potential for an extremely diverse series of products and modifications to the main biosynthetic pathways. Nonetheless, recent work has emphasized that there can be a high cost for secondary metabolism, particularly true of isoprenoid accumulation (Gershenzon, 1994). It has long been known that toxicants can be released on damage to plant tissues. However, it is now evident that a whole range of biosynthetic pathways associated with secondary metabolism can be induced during damage by insect feeding. These involve endogenous compounds and include systemic signals that initiate defence metabolism in other parts of the plant. Exogenous metabolites can also be induced, for example volatile components which are released into the air and may be used as signals by herbivorous insects and their predators and parasites, and indeed by other plants (Karban and Baldwin, 1997). Such chemicals are termed ‘semiochemicals’, derived from the Greek ‘semeion’ ( ~ ~ E L o vmeaning ), sign or signal, and represent a major aspect of
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the chemical ecology of plant-insect interactions. In the case of insects, they act on the sensory system without direct physiological effects. The term ‘infochemical’ should be avoided as it is flawed, both etymologically and scientifically. In terms of structural characteristics, toxicants are often compounds which are non-volatile or have only low volatility, and with higher molecular weights or hydrophilicity than would allow transfer into the vapour phase. They include a wide range of structural types, from phenolics and alkaloids through to highly oxidized higher isoprenoids (Nahrstedt, 1989; Smith, 1989; Harborne, 1990). Compounds that interfere with insect feeding, but by sensory mechanisms rather than toxic modes of action, are termed ‘antifeedants’ (Griffiths et al., 1989; Klocke and Kubo, 1991). The smaller molecular weight compounds that can be mobile in aqueous systems and, more importantly, are volatile in air, can have many roles as semiochemicals mediating the behaviour of insects, such as in attracting herbivorous insects to plants and in repelling would-be herbivores from inappropriate hosts (Pickett and Woodcock, 1992).
11. FEEDING AND OTHER CONTACT INTERACTIONS INVOLVING INVOLATILE METABOLITES A. PLANT DEFENCE BY TOXIC MECHANISMS
There is an enormous literature, including a number of excellent reviews (D’Mello et al., 1991; Rosenthal and Berenbaum, 1991), covering plant secondary metabolites that are toxic to insects by a wide range of mechanisms. Many of the compounds are biocidal, and the broad activity of these materials precludes them from direct use as bioinsecticides. Some continue to be viewed as good leads for novel insecticides, but the development of the highly successful pyrethroid insecticides from natural insect toxicants, the pyrethrins, has yet to be approached in terms of impact on agriculture or in other areas of insect control (Elliott, 1996; Chamberlain et al., 1998). Perhaps the most exciting developments relate to the induction of toxicant production by a variety of newly identified signals. During attack by insects or pathogens, there is a so-called oxidative burst (Lamb and Dixon, 1997) involving, for example, the polyphenol oxidases. These are copper-containing enzymes that use molecular oxygen to catalyse the oxidation of phenolic and 1,2-diphenolic compounds acting directly as, or as precursors for, defence systems against herbivores, including insects (Bi and Felton, 1995; Constabel et al., 1996). Production of these enzymes can be induced in plants overexpressing prosystemin, i.e. yielding systemin which is a polypeptide signal translocating systemically within the plant (Pearce et al., 1991; Bergey et al., 1996). Such
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induction also occurs with the application of methyl jasmonate (in nature as the epi-isomer, Fig. 1) (Ward and Beale, 1993) relating to the lipoxygenase (octadecanoid) pathway which is activated on wounding. Crop protection is now benefiting directly from the practical development of a signal causing induction of plant defence. Thus, from an understanding of the signals involved in systemically acquired resistance, particularly the benzoic acid derivatives such as salicylic acid (Uknes et al., 1996), commercially useful compounds, for example the benzothiadiazole Bion, have been developed that can be sprayed onto crops to induce defence against pathogens in the recipient plant (Friedrich et al., 1996; Gorlach et al., 1996; Lawton et al., 1996). This approach will eventually be available for pest insect control (Pettersson et al., 1994). A major group of plant toxicants having roles in plant-insect interactions are the alkaloids, and there are a number of studies using jasmonic acid and related compounds to induce a variety of alkaloid biosynthetic pathways (Gundlach et al., 1992; Blechert et al., 1995). Such approaches may be valuable in producing pharmaceuticals in suspension cell cultures, for example paclitaxel from Taxus species (Taxacaceae) (Yukimune et al., 1996). Hydroxamic acids, particularly 2,4-dihydroxy-7-methoxy1,4-benzoxazin-3-0ne (DIMBOA, Fig. l), confer resistance to lepidopterous stem-boring larvae such as the European corn borer, Ostrinia nubilalis (Pyralidae), and also to some homopterous pests such as aphids (Aphididae). There is evidence that this pathway is induced by herbivore feeding (Gianoli and Niemeyer, 1997), and the recent elucidation of the biosynthetic pathway by Gierl’s group and preliminary success in associated molecular biology of the enzymes involved (Frey et al., 1997) would allow exploitation in crop plant protection by recombinant DNA techniques. Low molecular weight biocidal compounds are often employed in plant defence by initial storage as glycosides. For example, the isothiocyanates, which are stored as glucosinolates (Louda and Mole, 1991) (Fig. 1) and are typically produced by plants in the Brassicaceae, have a range of activities against insects and pathogens, although many specialist organisms have adapted positively to these compounds (Blight et al., 1995; Giamoustaris and Mithen, 1995). In molecular biological studies, salicylic acid has been shown to induce genes in oil-seed rape, Brassica napus (Brassicaceae), which may be associated with glucosinolate biosynthesis (Chu and Cho, 1996). Furthermore, methyl jasmonate, which is related to jasmonic acid and can be used as an external signal, when applied to air above B. napus plants causes specific induction of the indolylglucosinolates (Doughty et al., 1995). Again, recent developments in molecular genetics using Arabidopsis thaliana (Brassicaceae) and B. napus (Mithen, unpublished) are providing the technology by which glucosinolate expression can be genetically manipulated for crop protection purposes, at least within this family.
SECONDARY METABOLITES IN PLANT-INSECT INTERACTIONS
R= R' =
u,
R' = H methyl jasmonate
w,
Bion
R = H methyl epijasmonate
4-pentenylglucosinolate (Glucobrassicanapin)
OH DIMBOA
azadirachtin
d &cHo polygodial 'OAc ajugarin-I
Fig. 1 . Structures of plant secondary metabolites.
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J. A. PICKETT, D . W. M. SMILEY and C. M. WOODCOCK
lupulone colupulone
adlupulone
acylphloroglucinols( p-acids)
a::’”?””
-
,$‘
N=C=S
4-pentenyl isothiocyanate
senecionine
linalool
1,8-cineole
(lR,SS)-myrtenal
Fig. 1. continued.
(lS,SR)-pulegol
methyl salicylate
SECONDARY METABOLITES IN PLANT-INSECT INTERACTIONS
4,g-dimethyl-1,3,7-nonatriene
4,8,12-trimethyl-1,3,7,11-tridecatetraene
Q-P-famesene
Q-0-ocimene
H
97
L O H
0
0
4-methyl-3,5-heptanedione volicitin
Fig. 1. continued.
Hydrogen cyanide generation from cyanogenic glycosides is another widely distributed aspect of plant secondary metabolism involved in herbivore interactions (Seigler, 1991). The molecular biology relating to this pathway is even more advanced, with the cloning and functional expression of the gene for the cytochrome P450 responsible for converting amino acid to aldoxime, a key stage in the biosynthesis of these metabolites (Halkier and Maller, 1991; Halkier et al., 1995). The biosynthetic pathway to the cyanogenic glucoside dhurrin has been fully elucidated by Halkier, Maller and co-workers, who have isolated all the enzymes responsible for the biosynthesis (Kahn et al., 1997) (Fig. 2). B. DISRUPTION OF INSECT FEEDING BY NON-TOXIC MECHANISMS
By interfering with feeding behaviour, an essential aspect of insect development particularly at the larval or nymphal stages (Griffiths et al., 1989; Klocke and Kubo, 1991), the antifeedants have great appeal as pest control agents with non-toxic modes of action (Jermy, 1990; Escoubas et al., 1994). Considerable attention is still directed towards the tetranortriterpenoids (limonoids) found in members of the Meliaceae, particularly azadirachtin (Fig. 1) from the Indian Neem tree, Azadirachta indica. This has invited elegant studies in chemical
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HooH P-450 enzyme
HO
NH2
\
L-tyrosine
dhunin
(CYP79)
HO
Q-p-hydroxyphenylacetaldoxime
p-hydroxymandelonitrile
Fig. 2. Key stages in the biosynthesis of the cyanogenic glycoside, dhurrin (Kahn et al., 1997).
synthesis, although a full synthetic route is still not published. Indeed, the absolute configuration of this compound was reported only recently (Ley et al., 1992). Azadirachtin is a multifunctional compound and perhaps should be correctly described as a toxicant, as it possesses direct toxic activity with an insect growth regulant mode of action (Mordue and Blackwell, 1993; Lowery and Isman, 1994). The compound alone is unstable, but can exist long enough for insect control effects to be manifest in the field, provided it is protected by a relatively large biomass, for example that arising from the original plant material. The multifunctionality of the compound may cause difficulties in registration as a pure compound or in development as a lead for new products. Other limonoids can be found in the related families of the Rutaceae, Cneoraceae and Simaroubaceae, and the biological activities, including antifeedancy, of key compounds from these taxa have been reviewed (Champagne et al., 1992). Continued interest remains following the early identification of antifeedants with the diterpenoid clerodane structure, including ajugarin-I (Kubo et al., 1976) (Fig. l), which are active against a wide range of targets (Pickett et al., 1987; Griffths et al., 1988), and the activities of a number of clerodane diterpenoids, obtained from diverse plant sources, have been reviewed by Camps and Coll (1993). The dialdehydic drimanes in the sesquiterpene group, for example polygodial (Fig. l), have
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been investigated in terms of structure-activity relationships with various pest insects, including the Colorado potato beetle, Leptinotarsa decemlineata (Chrysomelidae) (Gols et al., 1996), and the large cabbage white butterfly, Pieris brassicae (Pieridae) (Messchendorp et al., 1996, 1998). Earlier observations showing high activity of polygodial against aphid feeding and transmission of plant virus diseases, even non-persistently transmitted viruses, have been taken further by in-depth behavioural studies, which also investigated comparatively the isoprenolated acylphloroglucinols (Fig. 1) from the oleo resin of hops, Humulus lupulus (Cannabaceae) (Powell et al., 1997). Some alkaloids can show antifeedant activity separate from toxicity, and studies continue on peramine, produced by the endophyte Neotyphodium lolii (Clavicipitaceae) infecting the perennial ryegrass, Lolium perenne (Poaceae). This could be developed for protection of grasses against insects, provided that the presence of the peramine can be established away from undesirable alkaloids that cause neurological problems with livestock (Ball et al., 1997). It is unlikely that a truly broad-spectrum antifeedant will either be found naturally or be developed from natural leads, as many aspects of plant-insect interactions are extremely specific. However, provided that use of such materials can be managed in regimes employing other crop protection strategies, the likely availability in the future of antifeedants through biotechnological means could lead to a greater emphasis in this direction (Pickett et al., 1997). C. SEQUESTRATION OF TOXICANTS BY INSECTS
Sequestration of secondary metabolites by insects, although now being questioned for some small molecular weight compounds (Seybold et al., 1995; Oldham et al., 1996), is a well-proven phenomenon, particularly in the case of toxicants (Bowers, 1991; Harborne, 1991; Malcolm, 1991). Toxicant sequestration is commonly associated with aposematic coloration which can cause learnt avoidance behaviour by, for example, vertebrate predators. Perhaps the most comprehensively studied system is that of the acquisition of the pyrrolizidine alkaloids such as senecionine (Fig. l), either as the free base or as the more hydrophilic N-oxide, by a range of insects as a means of defence (Lindigkeit et al., 1997). Larvae of the European cinnabar moth, Tyria jacobaeae (Arctiidae), store pyrrolizidine alkaloids from their host plant ragwort, Senecio jacobaea (Asteraceae), and retain them during all stages of development (Aplin et al., 1968; Aplin and Rothschild, 1972) typified by bright coloration. In another arctiid moth, Utetheisa ornatrix, which also sequesters pyrrolizidine alkaloids from the larval host plant, both parents provide alkaloids for egg protection (Dussourd et al., 1988) and the behaviour of the female is stimulated by an alkaloid-derived courtship pheromone from the male (Dussourd et al., 1991). Similar biparental contributions to egg defence
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are known for a number of other Lepidoptera (Brown, 1984a, b; Dussourd et al., 1989; Nickisch-Rosenegk et al., 1990). As well as lepidopterous species, the aposematic grasshopper Zonocerus variegatus (Pyrgomorphidae) (Bernays et al., 1977; Biller et al., 1994) and leaf beetles from the genus Oreina (Chrysomelidae) (Pasteels et al., 1988; Hartmann et al., 1997) sequester pyrrolizidine alkaloids from their host plants, the latter accumulating them in their exocrineous defensive secretions at levels well exceeding a 0.5 M concentration. Such adaptation to highly toxic host plants can confer advantages as discussed above (Rothschild, 1993) and can provide precursors for the development of pheromones in herbivorous insects (Bogner et al., 1992), as exemplified previously (Dussourd et al., 1991). There are also examples of sequestration of toxicants giving rise to further specific interactions at higher trophic levels, particularly in the case of parasitism (Pickett et al., 1991).
111. INSECT INTERACTIONS WITH PLANTS USING VOLATILE SIGNALS A. LOCATION OF HOST PLANTS
It is well known that the location of host plants by insects is based on detection not only of general plant volatiles, but also of compounds that are specific to families, genera or even species. The view that non-specific sensory mechanisms underpin the detection of ubiquitous plant compounds has been widely accepted but is now challenged (Pickett et al., 1992, 1997, 1998). However, it was not unexpected to find that, for specific compounds involved in host location such as the organic isothiocyanates released mainly by brassicaceous plants, there are specific mechanisms based on the responses of individual sensory neurons tuned to these compounds. Nonetheless, it was initially surprising to discover that there are specific neurons for particular isothiocyanates or particular structural types. For example, olfactory cells have been identified that respond more sensitively to 3-butenyl and 4-pentenyl isothiocyanate, i.e. the catabolite from 4-pentenylglucosinolate, glucobrassicanapin (Fig. l), than to the analogous ally1 isothiocyanate from sinigrin. Furthermore, there are cells responding only to aromatic isothiocyanates such as the 2-phenylethyl homologue. This system may be general for species which colonize brassicaceous plants but has been demonstrated for three, the cabbage seed weevil, Ceutorhynchus assimilis (Curculionidae) (Blight et al., 1995), the cabbage aphid, Brevicoryne brassicae, and the turnip or mustard aphid, Lipaphis erysimi (Pickett et al., 1992). For many insects, it has now been shown that there are olfactory cells, as specific as those for the isothiocyanates described above, tuned to the detection of ubiquitous plant compounds such as the C-6 or green leaf volatiles. Thus, the widely polyphagous peach-potato aphid, Myzus persicae, has antenna1 cells
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responding strongly to (E)-Zhexenal but showing little or no response to the closely related compounds (9-3-hexen-1-01, (9-3-hexen-1 -yl acetate, hexanal or 2-ethylhexan-1-01 (Pickett et al., 1998). The pea and bean weevil, Sitona lineatus (Curculionidae), has an equally specific olfactory cell for (2)-3-hexen1-01 and an increase in stimulus concentration of at least two orders of magnitude of (2)-3-hexen-l-yl acetate is required for a significant response. Isomers of linalool (Fig. l), another common plant component from a different pathway but often grouped with those previously mentioned, show virtually no stimulation with this particular cell type (Pickett et al., 1997). For the Egyptian cotton leafworm, Spodoptera littoralis (Noctuidae), which is by no means monophagous, there is an olfactory cell tuned specifically to the sesquiterpene humulene, a major component of the cotton plant, Gossypium spp. (Malvaceae) (Hansson, 1995). Such specificity in olfactory acuity to quite general compounds could form the basis of the differential oviposition on plants that has been investigated particularly for lepidopterous species (Honda, 1995). However, other mechanisms must be present where there are insufficient differences in the range of general compounds, or no specific compounds, to allow insects to identify narrow taxonomic groups of plants. The discovery in coleopterous species that olfactory cells, specific for ubiquitous plant volatiles, can be morphologically paired on the antenna may indicate a mechanism by which the ratios of such compounds, as released from plants, can be used as a discriminating feature. Thus, C. assimilis has a series of specific cells which occur in pairs, detecting two distinct compound types, as follows: methyl salicylate and 2-phenylethyl isothiocyanate; (2)-3-hexen- 1-01 and (9-3-hexenI-yl acetate; 2-phenylethanol and the isoprenoid 1,8-cineole (Fig. 1) (Blight et al., 1995). Similar pairings have now been observed in a predatory insect, the seven-spot ladybird, Coccinella septempunctata (Coccinellidae) (Woodcock, unpublished). Further elucidation of these mechanisms is necessary, but this may be the first step towards explaining how it is that insects can be mono- or oligophagous within a narrow taxonomic range of plants, where no compounds aiding such specific colonization can be discerned. Some insects, particularly bees, can effect plant pollination during feedingrelated behaviour. New studies on the honeybee, Apis mellifera (Apidae), have shown that, in a proboscis extension bioassay with bees trained to respond to an extract of oil-seed rape floral volatiles, only a small proportion of the components of the extract are used by the bee in the recognition process (Blight et al., 1997). Even when a simple mixture of electrophysiologically detectable compounds is used in the training process, only some of the components are employed. Furthermore, there is a neurophysiological sensitization during the training process which may take as little as 10min (Pham-Delkgue et al., 1997).
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B. AVOIDANCE OF UNSUITABLE PLANTS
It had been assumed that, for many insects, avoidance of unsuitable plants is based on initial attempts at feeding or colonization which are terminated by lack of appropriate physiology, nutritional aspects or by the detection of potentially toxic secondary metabolites. New work demonstrates that an earlier stage of non-host avoidance exists in which insects can detect, at a distance, volatile metabolites as a cue indicating acceptability or unsuitability of the plant (Pickett et al., 1995, 1997). Although plants produce a wide range of volatiles that will deter herbivores, there are usually insects that will adapt, even to the most generally repellent materials (Eisner et al., 1990), for example the novel but simple monoterpene (lS,SR)-pulegol (Fig. 1) found in Dicerandra frutescens (Lamiaceae). Bark beetles, in their colonization of trees, use a number of isoprenoid host compounds as cues. However, the pine shoot beetle, Tomicus ( = Blastophagus) destruens (Scolytidae), was shown to be repelled by the non-host semiochemical benzyl alcohol (Guerrero et al., 1997). A number of volatiles produced by the onion family, Allium spp. (Liliaceae), repel aphids, including the polyphagous M . persicae (Hori, 1996), whereas the essential oil of rosemary, Rosmarinus officinalis (Lamiaceae), is in turn repellent to the onion aphid, Neotoxoptera formosana (Hori and Komatsu, 1997). The main development in this area, however, is the realization that the specificity of sensory interactions involved in host plant selection is also employed in nonhost avoidance (Pickett et al., 1995, 1998). Thus, although it was not surprising that aphids such as B. brassicae and L. erysimi, which colonize plants in the Brassicaceae, have specific cells for the isothiocyanates, similar cells were found on the antenna of the black bean aphid, Aphis fabae, which does not normally feed on such plants. Indeed, the isothiocyanates have been shown to be repellent to A . fabae and to mask the attractancy of its host plant odour (Nottingham et al., 1991; Hardie et al., 1994). At this point, it was realized that the so-called redundant cells encountered in a number of laboratories studying insect electrophysiology, i.e. olfactory cells which could not be stimulated by pheromones or other positively acting semiochemical cues, may in fact have a role in detection of non-host semiochemicals. Further studies with A . fabae showed olfactory cells that were specifically tuned to (1R,SS)-myrtenal (Fig. I), a compound typically found in many members of the Lamiaceae and as a product of oxidation of monoterpenoids from gymnosperms; this compound again showed repellency for A . fabae and interfered with the attractancy of the host plant volatiles (Hardie et al., 1994). Thus, a mechanism is demonstrated whereby an insect feeding mainly on Fabaceae could avoid a wide range of non-host plants and ecosystems such as conifer forests where host plants are unlikely to be present. The bird-cherry-oat aphid, Rhopalosiphum padi, colonizes as its primary (winter) host the bird-cherry, Prunus padus (Rosaceae), and one of the key distinctive volatile metabolites from this tree is methyl salicylate (Fig. 1). It was
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postulated by Pettersson that primary host components would be repellent to the spring form of the aphid, which migrates from the P . padus tree and colonizes cereal crops. A specific olfactory cell for methyl salicylate was found on the R . padi antenna and in the field, slow release formulations of this compound did indeed reduce colonization of the summer cereal hosts (Pettersson et al., 1994; Pickett et al., 1997). Interestingly, A . fabae was also shown to possess cells specific for methyl salicylate and to be repelled by the compound. Methyl salicylate has now been the subject of an investigation covering over 30 species of insects from four orders, and all species tested showed some electrophysiological response (Woodcock, unpublished). This compound may be a general indicator of an induced defence pathway based on the phenylalanine ammonia-lyase system, with the methyl salicylate being a volatile analogue related to salicylate signalling within the plant. Thus, it appears that, as well as detecting taxonomically inappropriate hosts, insects are able to detect induced defence in plants through their own signalling processes. Furthermore, it was suggested that volatile insect semiochemicals associated with plant stress could act directly on other plants (Pettersson et al., 1994; Pickett et al., 1997). This has recently been demonstrated (Shulaev et al., 1997) for methyl salicylate, which can act as an aerial signal inducing defence against pathogens (Pickett et al., 1999). Insects may therefore provide a means of identifying phytopheromones generally. Although some insects, particularly bark beetles and aphids, actively seek plants that are under stress, giving such signals a positive role in colonization, many insects avoid stressed or damaged plants because of the accompanying induced defence. The molasses grass, Melinis minutiji’ora (Poaceae), releases a number of compounds, notably the homomonoterpene 4,8-dimethyl-1,3,7nonatriene (Fig. l), which are more often associated with plant damage, and is thereby repellent to lepidopterous pests. These include the maize stalk borer, Busseola fusca (Noctuidae), which colonizes a wide range of grasses and cereals (Poaceae) including sorghum, Sorghum bicolor, and maize, Zea mays (Khan et al., 1997). Such activity is being exploited in intercropping systems to protect subsistence cereal crops in Africa (Khan, unpublished).
IV. PLANT INTERACTIONS WITH PREDATORY AND PARASITIC INSECTS When plants are attacked by insects or mites (Arachnida: Acari), compounds can be released from secondary metabolism pathways that cause attraction and increased foraging of parasitic insects (known as ‘parasitoids’ where the host is consequently killed) and predators, including predatory mites (Stowe et al., 1995; Takabayashi and Dicke, 1996). Semiochemicals of this type are termed ‘synomones’ because they are adaptively beneficial to both the emitting and receiving organisms, and a wide range of compounds, including the C-6 or
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green leaf volatiles, may be implicated. Also fulfilling this role are isoprenoids such as the monoterpenoids (e.g. linalool isomers), sesquiterpene hydrocarbons (e.g. farnesenes) and the homoterpenoids (Fig. l), including the homomonoterpene discussed above (Turlings et al., 1990, 1995) and the homosesquiterpene 4,8,12-trimethyl-1,3,7,1 l-tridecatetraene (Fig. I), with the oxidative biosynthesis from higher isoprenoids elegantly elucidated by Boland's group (Donath and Boland, 1995; Boland et al., 1998a). Induction of compounds can involve de novo synthesis, for example with isomers of the farnesenes, ocimenes, linalool, homoterpenes (Fig. 1) and the amino acid metabolite indole, whereas others, including cyclic terpenes and the green leaf volatiles, are released from storage or synthesized from stored intermediates (Park and Tumlinson, 1997a, b). These effects are often systemic, so herbivores feeding on one leaf can stimulate release of such semiochemicals from other parts of the plant (Dicke et al., 1993; Takabayashi et al., 1994). Glucosidases have been implicated as part of the systemically transmitted mechanism for induction of plant volatiles (Mattiacci et al., 1995). However, a recent exciting discovery is that volicitin, N-(l7-hydroxylinolenoyl)-~-glutamine (Fig. I), a compound released from the mouthparts of the beet armyworm, Spodoptera exigua (Noctuidae), is an elicitor inducing the release of a series of volatiles in the host plant, Z . mays, during feeding by the caterpillar. These in turn attract foraging parasitoids (Turlings et al., 1995; Alborn et al., 1997). Volicitin is biosynthetically related to jasmonic acid and jasmonates, which are generated as a consequence of plant damage, including feeding by insects, and can induce biosynthesis of volatile semiochemicals (Boland et al., 1998b). It has been demonstrated for some lepidopterous insects (De Moraes et al., 1998) and aphids (Du et al., 1998) that the composition of the synomones released from a plant relates to the taxonomy of the herbivore feeding on it, even within closely related groupings. This allows specialist parasitoids to distinguish between different species colonizing a common host plant. Thus, Aphidius ervi, a braconid parasitoid specializing on the pea aphid, Acyrthosiphon pisum, is faced with a specific range of compounds relating to A . pisum feeding on bean, Vicia faba (Fabaceae), which is different from those released by either the vetch aphid, Megoura viciae, or A . fabae feeding on the same host plant. The molasses grass, M . minutiflora, referred to earlier as causing repellency in herbivorous insects, at the same time increases foraging of parasitoids, particularly by release of the homomonoterpene. This has been found in intercropping systems to improve control of stem borer pests in the main crop, Z . mays, through increased parasitism of B. fusca by the braconid Cotesia sesamiae (Khan et al., 1997).
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V. INTERACTIONS BETWEEN SECONDARY METABOLITES AND INSECT PHEROMONE SYSTEMS In 1984, the first demonstration that plant compounds could influence behaviour mediated by an insect pheromone was published. Chemicals from the host plant synergistically increased the activity of the aggregation (Fig. l), of the pea and bean weevil, pheromone, 4-methyl-3,5-heptanedione S . lineatus (Blight et al., 1984, 1991). It was then reported that an aphid alarm pheromone component, (a-p-farnesene (Fig. I), was more active in dispersing the turnip aphid, L. erysimi, when compounds from the host plant, specifically the isothiocyanates, were incorporated (Dawson et al., 1987). Such phenomena are predictable within the current understanding of ecology, as it would be disadvantageous in evolutionary terms for an organism to aggregate or mate if inappropriate or insufficient food supplies were available, and host plant cues and pheromonal stimuli could therefore act in concert. Thereafter, other examples were reported of attractant pheromones which are synergized by green leaf volatiles, i.e. for the boll weevil, Anthonomus grandis (Curculionidae), the smaller European elm bark beetle, Scolytus multistriatus (Scolytidae), and the Mediterranean fruit fly, Ceratitis capitata (Trypetidae) (Dickens et al., 1990). It was also shown that females of the corn eanvorm, Helicoverpa (= Heliothis) zea (Noctuidae), delay their reproductive behaviour until a suitable plant host is found, and that host chemicals trigger the production of the sex pheromone (Raina et al., 1992). The sex pheromone of the related tobacco budworm, H . virescens (Noctuidae), was subsequently shown to be synergized by the green leaf volatiles (a-3-hexen-l-yl acetate and (EJ-Zhexen-l-yl acetate (Dickens et al., 1993). The issue of host plant influence on the activity of insect sex pheromones has been reviewed, with the generality of these effects now confirmed (Landolt and Phillips, 1997).
VI. FUTURE PROSPECTS Prospects for extending the science of plant-insect interactions have been mentioned in the foregoing sections. These include an increased understanding of the dynamics of the processes involved, particularly in interactions at higher trophic levels, not only with parasitoids and predators but also with insects parasitizing these organisms, for example hyperparasitoids. Deeper insights into how host plants and unsuitable plants are identified by phytophagous insects will ensue from the discovery of the specificity of the underlying sensory processes and the apparent ability of insects to use morphologically associated olfactory cells to discern minor differences in relative proportions of volatile components (Blight et al., 1995; Baker, 1998). However, a major driving force will be the necessity to use semiochemicals associated with plant-insect interactions as alternatives to broad-spectrum eradicant pesticides in pest
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control, which in turn will involve the exploitation of molecular biology. There is no doubt that semiochemicals cannot be used alone as direct replacements for pesticides. Semiochemicals, and the strategies for their use, must be integrated with other forms of pest control, involving pathogens, predators and parasitoids, and, of course, aspects of resistance conferred by plant breeding and particularly by techniques based on recombinant DNA. The appropriate integration of semiochemicals with other methods of control is now widely considered to involve ‘push-pull’ or stimulo-deterrent diversionary strategies (Miller and Cowles, 1990; Pickett et al., 1997). Thus, deployment of semiochemicals, or plants producing such agents (Khan et al., 1997; Khan, unpublished), will protect the crop by deflecting pests from the vicinity and, at the same time, attract them into traps or trap crops where pathogens, highly selective insecticides or increased predation can be exploited. As new techniques of molecular biology have emerged, tremendous efforts have been made to find ways of using these techniques to improve crop resistance to pests, and a great deal has been achieved in engineering genes which give rise to insecticidal proteinaceous materials. However, the possibility of modifying pathways leading to semiochemical production was also predicted. Although whole pathways were not envisaged initially as being the targets, it was expected that, by an opportunistic transformation, existing pathways to secondary metabolites could be modified to produce more useful metabolites that could act as semiochemicals (Pickett, 1985). Some specific pathways have been chosen (Hallahan et al., 1992) but without, as yet, practical realization of their potential. Nonetheless, the approach to modifying secondary metabolism pathways to give improved products has been demonstrated formally by the successful transfer and expression of a stilbene synthase gene which uses endogenous substrates to create the phytoalexin resveratrol, not normally present in the target plant (Hain et al., 1993). Studies on other pathways leading to secondary metabolite toxicants which could be exploited have been reported recently (Frey et al., 1997), as discussed above. With this knowledge of success and the acknowledgement of limitations, further refinement in targets can be considered (Carozzi and Koziel, 1997; Hick et al., 1997). It is also proving possible to exploit, to a greater extent, production of semiochemicals by plants as a bridging point in the biotechnological production of semiochemicals by molecular biological techniques. A great many studies are being undertaken and an expansion of the subject of physiologically active compounds from plant secondary metabolism is taking place (Copping, 1996; Hedin et al., 1997), with a significance that goes beyond insect activity into pharmaceutical interests (Caporale, 1995). However, even here, behaviourally active compounds should be viewed more positively in the future as chemicals that influence insect behaviour, particularly where the insects interact directly with higher animals (Pickett and Woodcock, 1996) including human beings, are related to, and often identical to, those employed by the higher animals themselves.
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ACKNOWLEDGEMENTS IACR-Rothamsted receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom. Part of this work was supported by the UK Ministry of Agriculture, Fisheries and Food (MAFF). The authors thank R. Mithen and Z.R. Khan for permission to discuss their unpublished results.
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Biosynthesis and Metabolism of Caffeine and Related Purine Alkaloids in Plants
HIROSHI ASHIHARA' and ALAN CROZIER2
'Department of Biology. Faculty of Science. Ochanomizu University. Otsuka. Bunkyo.ku. Tokyo 112-8410. Japan 2Plant Products and Human Nutrition Group. Division of Biochemistry and Molecular Biology. Institute of Biomedical and Life Sciences. University of Glasgow. Glasgow GI2 8QQ. UK
I . Introduction ................................................................ I1. Occurrence of Purine Alkaloids in Plants ............................................ A . Camellia .............................................................................................. B. Coffea ......................................................... C . Ilex ................................................................................................ D . Theobroma and Herrania ......... ................................................ ............................... E . Paullinia ................................................... I11. Purine Nucleotide Metabolism in Higher Plants ....................................... A . Nucleotide Pool in Plant Cells and Tissues........................................ B. Biosynthesis of Purine Ribonucleotides ............................................. C . Catabolism of Purine Nucleotides ...................................................... D . Metabolism of Purine Bases and Nucleosides in Plants .................... E. Other Plant-specific Purine Pathways ................................................ IV . Biosynthesis of Purine Alkaloids ................. ...................................... A . Methylation of the Purine Ring ...... ........................................... B. Enzymes Involved in Methylation S ...................................... C . Caffeine Biosynthesis From Purine Nucleotides ........ D. Purine Alkaloid Biosynthesis in Theobromine-accum E. Physiological Studies on Caffeine Biosynthesis.................................. F. Other Routes .......... ....................................................................... V . Metabolism of Purine Alkaloids in Plants ................................................. A . Catabolism of Caffeine and Related Compounds .............................. B. Diversity of Caffeine Metabolism ........................... .... C . Metabolism of Purine Alkaloids as Xenobiotics in Alkaloid-forming Plants ........................................... ............... Advances in Botanical Research Vol . 30 incorporating Advances in Plant Pathology ISBN 0-12-005930-4
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Copyright Q 1999 Academic Press All rights of reproduction in any form reserved
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VI. Biotechnology of Purine A. Caffeine Production and Tissue Cultures B. Decaffeinated Beverages ..................................................................... VII. Summary .................................................................................................... Acknowledgements. ................................................. ......................... References ..................................................................................................
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Caffeine and other purine alkaloids, including theobromine and theophylline, have played a major role in the long-standing popularity of non-alcoholic beverages and foods such as coffee, tea, cocoa, matP, chocolate and a wide range of soft drinks. Nearly 100 plant species have been identified as containing these purine alkaloids; the more common are from the genera Camellia, Coffea, Cola, Ilex; Paullinia and Theobroma.
H
A
3 O
c
~
N
5
~
~
Y
CH3 CH3 CH3 Caffeine Theobromine Theophylline ( I ,3,7-Trirnethylxanthine) (3,7-Dimethylxanthine) ( 1,3-Dimethylxanthine)
This review begins by summarizing those aspects of general purine metabolism in plants that are related to purine alkaloid metabolism, and then provides an up-to-date account of the biosynthesis of caffeine and theobromine in a variety of plant species. Recent information on the properties and isolation of key enzymes, such as the caffeine synthase, are presented. Physiological studies on caffeine biosynthesis in tea and coffee plants including the authors' own work are also introduced. Catabolism of caffeine via demethylation to xanthine and degradation via the purine catabolism pathway in higher plants is then reviewed. The diversity of caffeine catabolism between species and between tissues of different age is considered. In young tea leaves, theophylline, a catabolite of caffeine, is reutilized for caffeine synthesis, but in aged Coffea arabica leaves 7methylxanthine accumulates. Some Coffea species convert caffeine to methyluric acids. Finally, biotechnology of purine alkaloids including caffeine production in tissue and cell cultures and the possibilities of producing decaffeinated beverages through the use of transgenic coffee and tea plants are described.
I. INTRODUCTION Methylxanthines and methyluric acids are known as purine alkaloids. Caffeine (1,3,7-trimethylxanthine)and theobromine (3,7-dimethylxanthine) are the best known purine alkaloids, being found in chocolate products and various nonalcoholic beverages and soft drinks. The pharmacological effects of purine
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alkaloids in animals, such as stimulation of the central nervous system, have been investigated extensively (see Kihlman, 1977; Scheline, 1991). Caffeine is utilized in cytological studies to induce the formation of binucleate cells and is, therefore, frequently used to measure the duration of the mitotic cycle (Kihlman, 1977). In some higher plants, such as Coffea arabica (coffee) and Camellia sinensis (tea), large amounts of purine alkaloids are synthesized from purine nucleotides. Although caffeine itself was discovered in these species in the early 1820s, the biosynthetic and catabolic pathways were not fully established until the 1990s. The physiological role of endogenous purine alkaloids and related compounds in higher plants remains undetermined, although it is known that that the production of 1-methyladenine in starfish induces oocyte formation (Kanatani, 1969; Kanatani et al., 1969; Mita et al., 1996). Degradation of caffeine is relatively slow even in aged leaves of most species, and it appears not to act as a nitrogen reserve as considerable amounts remain in detached leaves after abscision (Suzuki and Waller, 1987b). There are two hypotheses concerning the role of caffeine in plants. The ‘chemical defence theory’ proposes that the high concentrations of caffeine in young leaves, fruits and flower buds of Cof. arabica and Cam. sinensis act as a chemical defence to protect soft tissues from predators, such as insect larvae (see Nathanson, 1984; Harborne, 1993). The ‘allellopathic or autotoxic function theory’ proposes that caffeine in seed coats is released into the soil and inhibits germination of other seeds (see Rizvi et al., 1981; Rice, 1984; Suzuki and Waller, 1987a; Waller, 1989). While these hypotheses are interesting, there is little evidence to indicate that such phenomena actually operate in nature. The most recent comprehensive review on purine alkaloid metabolism was published 7 years ago (Suzuki et al., 1992). Although many studies on caffeine metabolism were considered, the article was very concise and written for specialists in the field. A review article on caffeine was published in German in 1996 which focused primarily on the author’s intriguing specialized interests (Baumann, 1996). More recently, two chapters in a book on caffeine contained brief descriptions of purine alkaloid metabolism (Tarka and Hurst, 1998; Balentine et al., 1998). Current studies were not discussed, and the caffeine biosynthetic pathway in tea introduced by Balentine et al. (1998) was incorrect. In this review we will introduce recent research and provide relevant background information to enable the subject to be appreciated more widely. For this reason, purine metabolism in plants, a topic that is all but neglected in recent plant biochemistry textbooks (Dennis et al., 1997; Dey and Harborne, 1997; Heldt, 1997), is included and its relationship with purine alkaloids discussed.
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11. OCCURRENCE OF PURINE ALKALOIDS IN PLANTS Compared with other alkaloids, such as nicotine, morphine and strychnine, purine alkaloids are distributed widely in the plant kingdom. The presence of caffeine has been reported in 13 orders of plants (Fig. 1). Most caffeinecontaining plants are members of the Dicotyledoneae, although Scilla maritima belongs to the Monocotyledoneae. In some species theobromine or methyluric acids accumulate rather than caffeine. The presence of purine alkaloids has currently been reported in at least 80 plant species. However, a number of the early studies made use of what would now be regarded as unreliable analytical methods which, in some intances, may well have produced inaccurate data. Among the purine alkaloid-containing plants, by far the majority of studies have been with species belonging to the genera Camellia, Coffea, Cola, Paullinia, Ilex and Theobroma. A number of species in these genera are used to produce non-alcoholic beverages, and it has become the custom of food chemists and plant breeders to express the purine alkaloid content of plants as a percentage of either the dry weight (d.w.) or fresh weight (f.w.). Although this unit is not in common scientific use, for the sake of convenience and tradition it will be used at some points in this review. Those unfamiliar with this method of quantification should note that 1YOcaffeine equates with 0.51 mmol g-' while 1% of a dimethylxanthine, such as theobromine, theophylline or paraxanthine, is equivalent to 0.56mmol g-'. A. CAMELLIA
The genus Camellia includes more than 82 species which are classified into 12 subgeneric sections (Sealy, 1958). Nagata and Sakai (1984) examined the distribution of caffeine in 23 species belong to the genus Camellia. Caffeine was found in young leaves of first flush shoots of Cam. sinensis var. sinensis (2.8% d.w.), Cam. sinensis var. assamica (2.4%), Cam. taliensis (2.5%) and Cam. kissi (<0.02%), but was not detected in 20 other species of Camellia including Cam. japonica and Cam. sasangua. Theobromine was the predominant purine alkaloid in young leaves of Cam. irrawadiensis (<0.8%) (Nagata and Sakai, 1985) and Cam. ptilophylla (5.&6.8%) (Ye et al., 1997). The hybrids, such as Cam. sinensis x Cam. japonica and Cam. sasanqua x Cam. sinensis, contain caffeine, indicating that the ability to synthesize purine alkaloids is a dominant characteristic. The stamens and petals of several species of Camellia plants also contain caffeine and/or theobromine (Fujimori and Ashihara, 1990), and the species to species pattern of purine alkaloid distribution is very similar to that found in young leaves (Ashihara and Kubota, 1987) Cam. sinensis generally consists of two variants, Cam. sinensis var. assamica and Cam. sinensis var. sinensis, with the latter divided into Chinese and Japanese subgroups (Sealy, 1958). Using ca 1500 cultivars, Takeda (1994)
Fig. 1. Phylogenetic relationship between caffeine-synthesizing plant orders (bold). The phylogenetic tree for angiosperms by Takhtajan (1959) was used. (Reproduced courtesty of Dr Satoshi Yamaguchi, Ehime University, Japan.)
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investigated the variation in the caffeine content of young leaves of first flush shoots of Assam (var. assamica), China (var. sinensis) and Japan (var. sinensis) tea. The caffeine content of the Assam cultivars ranged from 2.7 to 5.5% and the mean value was 4.1 YO,while levels in China tea averaged 3.1YOand varied from 1.6 to 4.6%, the equivalent figures for Japan cultivars were a 2.9% mean value and a range of 1.9-3.9%. Distribution of purine alkaloids in seeds and seedlings of Cum. sinensis has been examined by Ashihara and Kubota (1986). In seeds, most of the caffeine was located in the seed coat, and only a trace was detected in the embryo. More than 99% of caffeine in 4-month-old seedlings was in the leaves with very minor pools in stems, roots and cotyledons. A small amount of theobromine was found in young leaves, but theophylline and paraxanthine did not accumulate in detectable quantities in any part of the seedling. Tea leaves contain small amounts of 1,3,7,9-tetramethyluric acid (threacrine) (Johnson, 1937) which has also been detected in Camellia assamicacv. Kucha (C. Ye, personal communications). B. COFFEA
The caffeine content of seeds of different Coffea species varied from 0.4 to 2.4% (Mazzafera and Carvalho, 1992). Most cultivars of Cof. arabica contained 1.&l. 1?LOcaffeine, although less (0.6%) was present in cv. Laurina. Some CoSfea species, including Cof. canephora cv. Robusta (1.7%) and cv. Guarini (2.4%), Cof. dewevrei (1.2%) and Cof. liberica (1.4%), contained higher concentrations of caffeine than Cof. arabica. In contrast, the caffeine content of the seeds of other species, such as Cof. eugenioides (0.4%), Cof. salvatrix (0.7%) and Cof. racemosa (0.8%), was lower than that of Cof. arabica. During development of Cof. arabica fruits, the caffeine content of the pericarp fell from 2 to 0.2% d.w., while it remained constant at 1.3% in the seeds (Keller et al., 1972). Young expanding leaves of Cof. arabica plants also contain theobromine, albeit in lower levels than caffeine (Frischknecht et al., 1986; Fujimori and Ashihara, 1994) Mature leaves of Coffea liberica, Coffea dewevrei and Coffea abeokutae, contain the methyuric acids, theacrine (1,3,7,9-tetramethyluric acid), liberine (0(2), 1,9-trimethyluric acid) and methylliberine (0(2), 1,7,9tetramethyluric acid (Baumann et al., 1976; Petermann and Baumann, 1983). C . ILEX
Purine alkaloids are also present in the leaves of mat6 (Zlex paraguariensis). Mat6 is used to produce a herbal tea, a drink that was originally restricted to rural areas of South America, such as the Brazilian Panthanal and the Pampas in Argentina. Its consumption is now becoming more widespread, perhaps
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aided by articles in the popular press claiming that it has Viagra-like qualities (Veash, 1998). Young mat& leaves contain 0 . 8 4 9 % caffeine, 0.084.16% theobromine and < 0.02% theophylline. Much lower levels are found in mature leaves and trace amounts of caffeine and theobromine are also present in fruits, bark and wood of mat6 (Mazzafera, 1994). D. THEOBROMA AND HERRANIA
Theobromine is the dominant purine alkaloid in seeds of cocoa (Theobroma cacao). Changes in the levels of theobromine and caffeine during the growth of cocoa beans were monitored by Senanayake and Wijesekera (1971). The cotyledons of mature beans contained 2.2-2.7% theobromine and O.M.8% caffeine, while lower levels (0.64.7% theobromine and 0.5-0.6% caffeine) were found in the shell of the seed. Cupu (Theobroma grandiflorum) contains 0.25% liberine in cotyledons and 0.08% in nut shells (Baumann and Wanner, 1980). Recent studies by Hammerstone et al. (1994) found that the seeds of 11 Theobroma species contained tetramethyluric acid as the principal purine alkaloid. The major alkaloid in the immature seeds of Theobroma bicolor was theobromine, but in mature seeds, theobromine was replaced by tetramethyluric acid. Herrania is similar morphologically to Theobroma and tetramethyluric acid was also found in seeds of nine Herrania species, including H . purpurea and H . cuatrecasana) although the leaves of these plants were devoid of purine alkaloids. E. PAULLINIA
Baumann et al. (1995) reported on the distribution of purine alkaloids in the various parts of the fruit and seed of guarana (Paullinia cupana). In seeds, caffeine was the major methylxanthine with the cotyledons containing 4.3 YO and the testa 1.6%. Both tissues also contained traces of theobromine (< 0.2%) and theophylline (< 0.01YO).The pericarp contained lower levels of purine alkaloids but theobromine (0.2%) was the main component together with trace amounts of caffeine (0.02%) and theophylline (0.001%).
111. PURINE NUCLEOTIDE METABOLISM IN HIGHER PLANTS Background information on purine nucleotide metabolism in plants is relevant to this review because purine alkaloids are secondary metabolites derived from purine nucleotides. Compared with other organisms, most notably microorganisms and mammals, there has been limited research on purine nucleotides in higher plants. There are, nonetheless, reviews on nucleotide biosynthesis (Ross, 1981; Wasternack, 1982) and nucleotide metabolism in cultured plant
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H. ASHIHARA and A. CROZIER
cells (Wagner and Backer, 1992). This article will outline those areas of purine metabolism in higher plants that are related to purine alkaloids. Purine nucleotides are precursors of nucleic acids, universal sources of energy, activated intermediates in many biosynthetic pathways, components of major co-enzymes and they also act as metabolic regulators (Henderson and Peterson, 1973; Kelley and Weiner, 1978; Stryer, 1995). There are extensive studies on purine nucleotide metabolism in bacteria (Munch-Petersen, 1983; Neuhard and Nyggard, 1987) and animals (Henderson and Peterson, 1973). The biosynthesis of purine nucleotides falls into three categories: (i) purine nucleotide biosynthesis de novo: (ii) interconversion of purine nucleotides; and (iii) purine salvage. A. NUCLEOTIDE POOL IN PLANT CELLS AND TISSUES
Measuring profiles of purine nucleotides, nucleosides and bases was problematical until the 1980s. This was because plant extracts contain very small amounts of nucleotides and large quantities of UV-absorbing compounds which make it difficult to analyse nucleotides accurately in plant extracts. Application of high-performance liquid chromatography (HPLC) overcame these difficulties (Meyer and Wagner, 1985a-c; Ashihara et al., 1987, 1990), and the levels of purine nucleotides and related compounds have now been investigated in a number of higher plants including tea and mate (Table I). The profiles of nucleotide pools in tea and mat6 leaves are similar to those of plants that do not produce caffeine. The adenine nucleotide pool is always the largest followed by uridine nucleotides and, in turn, the guanine nucleotide pool which is 1&25% of the adenine nucleotide pool. The cytidine nucleotide pool is the smallest of these pools. The intracellular levels of nucleotides in plant cells are influenced by several environmental factors. For example, inorganic phosphate (Pi) starvation of cultured plant cells results in a marked decrease in the levels of nucleotides, especially nucleoside triphosphates (Ashihara and Ukaji, 1986; Ashihara et al., 1988; see Table I). In contrast, the proportion of adenylates, i.e. the adenylate energy charge ([ATP] + i[ADP])/(ATP + ADP + AMP) proposed by Atkinson (1977), in actively growing plant cells is maintained at an almost constant 0.8-0.9. The energy charge in young tea leaves (0.86) is within this range, but in mate lower than average values are obtained (0.58) because of the higher concentrations of adenosine 5’-diphosphate (ADP) and adenosine 5’monophosphate (AMP). The energy charge value observed in mate is similar to that of Pi-starved Catharanthus roseus cells (Table I). Takino et al. (1972) reported that the major nucleotides in green tea products are AMP, uridine 5’-monophosphate (UMP) and ADP. However, this does not mean that they are necessarily the major nucleotides in fresh tea leaves because, during the manufacture of green tea, ATP is converted to ADP and AMP,
125
CAFFEINE A N D RELATED PURINE ALKALOIDS IN PLANTS
TABLE I Concentration of nucleotides in plant cells and tissues determined by HPLC. Mean values are expressed as nmolg-'fresh weight f standard deviation (C,total nucleotide content; EC, adenylate energy charge; tr. trace) Catharanthus roseusd
Nucleotides ATP ADP AMP CAN EC GTP GDP GMP CGN XMP UTP UDP UMP CUN CTP CDP CMP CCN
Tea"
Mateb
Tobacco'
+ Pi
-Pi
177 f 21 35 f 6 14 f 3 225 0.86 26 f 4
121 f 10 110 f 16 71 1 302 0.58 30 f 5 21 f 1 9 f 3 60
82 f 3 50 9 141 0.76 23 f 1 11 f 1 3 f 1 37
100 f 4 18 f 3 16 f 3 134 0.81 26 f 2 3 f 0 3 f 0 32
22 f 3 16 f 2 12 4 50 0.60 8 f 2 3 f 2 2 f 1 13
-
-
-
-
tr tr 26 29 f 1 88 f 3 46 f 10 55 f 23 189
tr tr 32 f 3 32
*
-
-
-
28.0 f 0 15 f 0 14 f 3 57 8 f 3 4 f 0 tr 12
55.0 f 0 29 f 3 33 f 3 117 7 f O 4 f 0 6 f l 17
*
*
10 0 22 It 1 24 f 6 56 2*0 2*1 3 k 1 7
"Young shoot with two leaves (H. Ashihara, unpublished data). bYoung leaves from flush shoot. Only purine nucleotides were determined (Ashihara, 1993).
Young leaves (the seventh leaf from the apex) (Meyer and Wagner, 1986). dSuspension-cultured cells grown in complete ( + Pi) and phosphate-deficient (-Pi) media for 24 h (Ashihara et al., 1988).
while uridine 5'-triphosphate (UTP) and uridine 5'-diphosphate (UDP) are converted to UMP. There are few reports of the concentrations of inosine 5'phosphate (IMP) and xanthosine 5'-phosphate (XMP) in plant cells. In tea leaves, IMP is not present in detectable quantities, although significant amounts of XMP (3&50nmolg-' f.w.) accumulate (Table I, H. Ashihara, unpublished data). Cellular pools of purine nucleosides and bases are usually low in plants as well as in other organisms. This is probably because of the very high activity of salvage enzymes, such as adenosine kinase and adenine phosphoribosyltransferase. Exceptionally high levels of purine nucleosides and bases do, however,
126
H. ASHIHARA and A. CROZIER
occur in leaves of cereals (Sawert et al., 1987, 1988). Earlier reports of the accumulation of purine nucleosides and bases could be due to extraction artefacts, but sometimes they may be stored in vacuoles which probably do not contain salvage enzymes. B. BIOSYNTHESIS OF PURINE RIBONUCLEOTIDES
I . De novo Pathway The purine nucleotide biosynthetic pathway was elucidated independently by Buchanan and Greenberg in the 1950s (see Buchanan, 1986). The purine ring is assembled from several small molecules. The N-1 atom originates from aspartate, C-4, C-5 and N-7 are from glycine, N-3 and N-9 come from the amide group of the side chain of glutamine, C-2 and C-8 are from activated derivatives of tetrahydrofolate, and C-6 is from COz (Fig. 2) (Henderson and Peterson, 1973). It is interesting that early studies on purine biosynthesis used pigeon liver, as well as Cof. arabica and Cam. sinensis plants (Anderson and Gibbs, 1962; Proiser and Serenkov, 1963). Birds synthesize purines from excess amino nitrogen, the purines are then degraded to urate and excreted. Recent investigations on purine biosynthesis in plants have utilized Nz-fixing root nodules of tropical legumes which actively synthesize ureides, such as allantoin and allantoate. The ureides are then translocated to leaves and rapidly growing parts of the shoots where they are utilized as a nitrogen source (Schubert and Boland, 1990). Precise details of purine biosynthesis in higher plants are still obscure. The available evidence implies that the same basic pathways that operate in animals and microorganisms are also functional in higher plants (Atkins et al., 1982; Lovatt, 1983; Hirose and Ashihara, 1984a; Ashihara and Nygaard, 1989;
Glutamine
Glutamine
Fig. 2. Origins of the N and C atoms in the purine ring.
CAFFEINE AND RELATED PURINE ALKALOIDS IN PLANTS
127
Nazario and Lovatt, 1993a). Figure 3 illustrates the proposed de novo biosynthetic pathway of purine nucleotides in plants. The de novo pathway is usually defined as the pathway that is responsible for the synthesis of IMP from 5-phosphoribosylamine (PRA) (Henderson and Paterson, 1973). PRA is formed from 5-phosphoribosyl- 1-pyrophosphate (PRPP) which is a common phosphoribosyl donor for de novo and salvage pathways of purine and pyrimidine nucleotide synthesis, and in the biosynthesis of histidine and tryptophan. PRPP is synthesized from ribose-5-phosphate, an intermediate of the pentose phosphate pathway (Hirose and Ashihara, 1984a). PRPP synthetase and the other enzymes of the de novo biosynthetic pathway that have been characterized in higher plants are summarized in Table 11, while information on the genes encoding these enzymes are presented in Table 111. TABLE I1 Enzymes of purine nucleotide biosynthesis de novo and the interconversion demonstrated in plants Enzyme (EC number)
Plant source
Reference
5-Phosphoribosyl- 1-pyrophosphate synthetase (EC 2.7.6.1)
Black gram hypocotyls Spinach leaves Catharanthus cells
Ashihara and Komamine (1974) Ashihara (1977a, b) Ukaji and Ashihara (1987) Ashihara (1990b)
Bry opsis Tea leaves Tea flowers
5-Phosphori bosylamine synthetase (EC 2.4.2.14) AICAR transformylase (EC 2.1.2.3) Adenylosuccinate synthase (EC 6.3.4.4) Adenylosuccinate lyase (EC 4.3.2.2) IMP dehydrogenase (EC 1.1. 1.205)
Hevea brasiliensis soybean nodules
Fujimori et al. (1991) Fujimori and Ashihara (1993) Gallois et al. (1997) Reynolds et al. (1984)
Pea seeds
Iwai et al. (1972)
Wheat germ
Hatch (1966)
Maize Wheat germ
Walters et al. (1997) Hatch (1966)
Pea seeds
Turner and King (1961)
Cowpea nodule
Shelp and Atkins (1983); Atkins et al. (1985) Nishimura and Ashihara (1993)
Tea leaves
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H. ASHIHARA and A. CROZIER
TABLE 111 Genes encoding enzymes of purine nucleotide biosynthesis de novo demonstrated in higher plants Enzyme name (gene designations)
Plant source
Reference
5-Phosphoribosyl- 1-pyrophosphate synthetase (prs )
Spinach leaves
Krath and Hove-Jensen (1999)
5-Phosphoribosylamine synthetase b u r 1) GAR synthetase (pur 2) AIR synthetase (pur 5 )
Soybean, mothbean
Kim et al. (1995)
Arabidopsis thaliana Arabidopsis thaliana
AIR carboxylase (pur 6) SAICAR synthetase (pur 7 )
Mothbean Arabidopsis thaliana
Schnorr et al. (1996) Senecoff and Meagher (1993) Chapman et al. (1994) Senecoff et al. (1996)
The PRPP content of cultured Catharantus roseus cells varies from 0.4 to 2.2nmolg-' f.w. during the growth cycle, but turnover of PRPP is very fast (Hirose and Ashihara, 1983a). Recently, four cDNAs encoding PRPP synthetase were isolated from a spinach cDNA library. In vitro the four gene products produced PRPP from ribose-5-phosphate and ATP. Two of the enzymes (isozymes 1 and 2) required Pi for activity, whereas others (isozymes 3 and 4)were Pi independent. Several pieces of evidence indicate that isozymes 2 and 3 may be transported to chloroplasts and mitochondria, respectively, while isozyme 4 may be located in cytosol (Krath and Hove-Jensen, 1999).
Fig. 3. De novo biosynthetic pathway of purine nucleotides in plants. Metabolites: PRPP, 5-phosphoribosyl-1-pyrophosphate;PRA, 5-phosphoribosyl amine; GAR, glycineamide ribonucleotide; FGAR, formylglycineamide ribonucleotide; FGAM, formyl glycine amidine ribonucleotide; AIR, 5-aminoimidazole ribonucleotide; CAIR, 5-aminoimidazole 4-carboxylate ribonucleotide; SACAIR, 5-aminoimidazole-4-Nsuccinocarboxyamide ribonucleotide; ZMP, 5-aminoimidazole-4-carboxyamideribonucleotide; FAICAR, 5-formamidoimidazole-4-carboxyamideribonucleotide. Enzymes: (1) amido phosphoribosyltransferase (EC 2.4.2.14); (2) GAR synthetase (EC 6.3.4.13); (3) GAR formyl transferase (EC 2.1.2.2); (4) FGAM synthetase (EC 6.3.5.3); (5) AIR synthetase (EC 6.3.3.1); (6)AIR carboxylase (EC 4.1.1.21); (7) SACAIR synthetase (EC 6.3.2.6); (8) adenylosuccinate lyase (EC 4.3.2.2); (9) ZMP formyltransferase (EC 2.1.2.3); (10) IMP cyclohydrolase (EC 3.5.4.10). N.B. The abbreviation AICAR is often used for both 5-aminoimidazole-4-carboxyamideribonucleotide and 5-aminoimidazole4-carboxyamide ribonucleoside. In this review ZMP is used for the ribonucleotide and AICAR for the ribonucleoside. The names of the intermediates and enzymes used in this review are not based on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, because the more traditional terminology is still well established in this field.
129
CAFFEINE AND RELATED PURINE ALKALOIDS IN PLANTS
PRA
PRPP
GAR
0
Fumarate
L HO
OH ATP
CAIR
ADP+P~
HO
OH
SAICAR
HO
OH
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H. ASHIHARA and A. CROZIER
Accumulation of 5-aminoimidazole carboxyamide (CAIR) in folate-deficient pea seedlings has been reported (Iwai et al., 1972), but there is no information on the size of the intermediate pools in higher plants. Significant accumulation of the intermediates of purine nucleotides de novo has not been observed in mammals. This suggests that purine biosynthesis de novo is an active process with a rapid turnover of intermediates. It has been reported that four multifunctional proteins participate in purine biosynthesis de novo in mammalian cells (Christopherson and Szabados, 1997). They are: (i) a trifunctional protein which shows glycineamide ribonucleotide (GAR) synthetase, GAR transformylase and 5-aminoimidazole ribonucleotide (AIR) synthetase activities (Fig. 3, steps 1, 2 and 4); (ii) a bifunctional protein with AIR carboxylase and 5-aminoimidazole-4-N-succinocarboxyamide ribonucleotide (SACAIR) synthetase activities (steps 5 and 6); (iii) a bifunctional enzyme (now called IMP synthase) which possesses 5-aminoimidazole-4-carboxyamide ribonucleotide (ZMP) transformylase and IMP cyclohydrolase activities (steps 8 and 9); and (iv) a trifunctional enzyme, C1-tetrahydrofolate (THF) synthase, which contains 5,lO-methylene-THF dehydrogenase, 5,lO-methenyl-THF cyclohydrolase, and 10-formyl-THF synthetase. 10-Formyl-THF produced by a sequence of reactions is a substrate for GAR transformylase (step 3) and ZMP transformylase (Fig. 3, step 9). An association of the enzymes of the de novo pathway of purine nucleotide biosynthesis with folate enzymes is believed to lead to the formation of a multienzyme complex called a ‘metabolon’ in animals (Christopherson and Szabados, 1997). A similar complex has not been demonstrated in plants, and the limited information on the genes encoding enzymes (Table 111) in the purine nucleotide pathway provides no evidence for the involvement of multifunctional proteins. The cDNA clones encoding amidophosphoribosyltransferase, the first enzyme of the de novo pathway, have been isolated from cDNA libraries obtained from root nodules of soybean (Glycine max) and Vigna aconifolia (Kim et al., 1995). The amino acid sequence deduced from the soybean clone showed > 85% homology with the sequence of the Vigna enzyme, and 3347% homology to those of bacteria, yeast, chicken, rat and humans. From a cDNA library of Arabidopsis thaliana, cDNAs encoding GAR synthase, GAR formyltransferase and AIR synthase, the second, third and fifth enzymes of the de novo purine biosynthesis pathway, have been cloned by functional complementation of corresponding Escherichia coli mutants (Shnorr et al., 1994). Each of the cDNAs encoded peptides comprised the complete enzymatic domain of either GAR synthase, GAR transformylase or AIR synthase. Comparisons of the three Arabidopsis purine biosynthetic enzymes with corresponding polypeptide fragments from procaryotic and eucaryotic sources indicate a high degree of conserved homology at the amino acid level, especially with the procaryotic enzymes. Sequence analysis, as well as Northern blot analysis, indicate that Arabidopsis has a single monofunctional enzyme.
CAFFEINE AND RELATED PURINE ALKALOIDS IN PLANTS
131
Chapman et al. (1994) have isolated cDNA clones encoding AIR carboxylase and SAICAR synthase (Fig. 3, enzymes 5 and 6) from moth bean. Sequencing these clones revealed that the two enzymes are distinct proteins in this plant, unlike animals where both activities are associated with a single bifunctional protein. Genes encoding AIR synthase and SACAIR synthase, (Fig. 3, enzymes 5 and 7), have also been isolated from Arabidopsis thaliana (Senecoff and Meagher, 1993; Senecoff et al., 1996). These results suggest that, unlike other eucaryotes, higher plants have no multifunctional enzymes in the de novo purine biosynthetic pathway, but resemble the one geneone enzyme relationship found in procaryotes (Schnorr et al., 1994). 2. Interconversion of Purine Nucleotides IMP produced by de novo purine biosynthesis is converted to AMP and guanosine-5‘-monophosphate(GMP) (Fig. 4). Aspartate and IMP are converted to adenylosuccinate in a reaction that utilizes guanosine-5’-triphosphate (GTP) but not ATP (step 1). Adenylosuccinate is cleaved yielding AMP and fumarate (step 2). GMP is synthesized from IMP via XMP. IMP is converted to XMP by oxidation at C-2 in a reaction that is catalysed by NAD+dependent IMP dehydrogenase (step 3). ATP-dependent GMP synthetase is then responsible for the substitution of an amino group at the C-2 position of XMP, resulting in the formation of GMP (step 4).The two purine nucleotides are further phosphorylated to nucleoside diphosphates and finally to the nucleoside triphosphates, ATP and GTP, some of which are utilized as building blocks for RNA synthesis (Ross, 1981; Wagner and Backer, 1992). Purine deoxyribonucleotides are formed from ADP and guanosine-5‘diphosphate (GDP) (Ross, 1981; Wagner and Backer, 1992). These are obviously important compounds for DNA synthesis, but the contribution of these deoxyribonucleotides to purine alkaloid synthesis may be extremely limited because deoxynucleotide pools in plant tissues are much smaller than those of the ribonucleotides (Nygaard, 1972). AMP is deaminated to IMP by AMP deaminase (step 5 ) (see also Section III.C.l) and IMP is the precursor for both adenine and guanine nucleotides (Fig. 4).Therefore, conversion of adenine nucleotides to guanine nucleotides is often observed. In contrast, the conversion of GMP to AMP (step 6) occurs less readily (Le Floc’h et al., 1982; Ashihara et al., 1991) probably because in higher plants there is little or no GMP reductase activity to catalyse the conversion of GMP to IMP. The enzyme is active in certain bacteria but only weakly so in animals (Mager and Magasanik, 1960; see also Ross, 1981).
3. Salvage Pathways The synthesis of purine nucleotides from purine bases and nucleosides is referred to as ‘purine salvage’ (reutilization of purines). Purine bases and nucleosides are not intermediates of purine nucleotide biosynthesis de n o w , but are formed as catabolites of purine nucleotides and nucleic acids. In plants
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H. ASHIHARA and A. CROZIER
AMP
NADP' + NH3
kbose-P
NADPH
Fig. 4. Interconversion of purine nucleotides in higher plants. Metabolites: IMP, inosine-9-monophosphate; SAMP, adenylosuccinate; AMP, adenosine-5/-monophosphate; XMP, xanthosine-5~-monophosphate;GMP, guan0sine-5~-rnonophosphate. Enzymes: (1) SAMP synthetase (EC 6.3.4.4); (2) adenylosuccinase (EC 4.3.2.2); (3) IMP dehydrogenase (EC 1.2.1.14); (4) GMP synthetase (EC 6.3.4.1); (5) AMP deaminase (EC 3.5.4.6);(6) GMP reductase (EC 1.6.6.8).GMP reductase activity has not yet been detected in any plant extracts. The bar over reaction 6 indicates that GMP reductase is probably not present in plants and, as a result, conversion of guanine nucleotide to adenine nucleotides is unlikely.
CAFFEINE AND RELATED PURINE ALKALOIDS IN PLANTS
133
purine salvage may have an important role in certain physiological processes. For example, during germination, ribonucleic acids in storage organs are degraded and the purine bases and nucleosides produced may be reutilized for the formation of nucleotides (Nobusawa and Ashihara, 1983; Ashihara, 1993). There is evidence that in germinating castor bean seeds adenosine, guanosine and adenine are transported from the endosperm to the cotyledons (Kombrink and Beevers, 1983). Similarly, free purine bases and nucleosides that accumulate during the senescence of leaves and other organs may be translocated to younger tissues and used as a substrate for nucleotide synthesis. There are at least four purine salvage enzymes in plants: adenine phosphoribosyltransferase, adenosine kinase, hypoxanthine-guanine phosphoribosyltransfease and inosine-guanosine kinase (Table IV; Fig. 5, reactions 1 , 5, 3 and 7, respectively). Conversion of purine bases to nucleosides is catalysed by adenosine phosphorylase and inosine-guanosine phosphorylase (reactions 2 and 6) which were discovered in wheat germ during the course of a study on
Ribose-I-P
ATP
pi
PRPP
A?p
PPi
Fig. 5. Salvage reactions of purine bases and nucleosides in higher plants. Enzymes: (1) adenine phosphoribosyltransferase (EC 2.4.2.7); (2) adenosine phosphorylase; (3) adenosine kinase (EC 2.7.1.20); (4) nucleoside phosphotransferase (EC 2.7.1.77); (5) hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8); (6) inosine-guanosine phosphorylase (EC 2.4.2.1); (7) inosine-guanosine kinase (EC 2.7.1.73). Solid arrows: major reactions; dashed arrows: minor reactions. PRPP, 5-phosoribosyl-1-pyrophosphate; PPi, pyrophosphate.
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H. ASHIHARA and A. CROZIER
TABLE IV Purine salvage enzymes demonstrated in higher plants Enzyme (EC number)
Plant source
Reference
Adenine phosphoribosyltransferase (EC 2.4.2.7)
Catharanthus roseus
Hirose and Ashihara (1982, 1983a, b); Ukaji et al. (1986) Le Floc'h and Lafleuriel (1978) Ashihara and Ukaji (1985) Guranowski and Barankiewicz (1979) Lee and Moffatt (1993) Gallois et al. (1996) Le Floc'h and Lafleuriel(l98 1b)
Jerusalem artichoke Spinach leaves Lupin seeds
H ypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) Adenosine kinase (EC 2.7.1.20)
Arabidopsis thaliana Hevea brasiliensis Jerusalem artichoke
Lupin seeds
Guranowski (1979a)
Spinach leaves
Ashihara and Ukaji (1985) Faye and Le Floc'ch ( 1997) Combes et al. (1989)
Peach Inosine-guanosine kinase (EC 2.7.1.73) Adenosine phosphorylase
Jerusalem artichoke
Inosine-guanosine phosphorylase (EC 2.4.2.1) Nucleoside phosphotransferase
Wheat germ
Wheat germ
Lupin cotyledons Barley seedlings
Chen and Petschow (1978) Chen and Petschow (1978) Guranowski (1979b) Prasher et al. (1982) . .
cytokinin metabolism (Chen and Petschow, 1978). However, these enzyme activities were not detected in tobacco leaf protoplasts (Barankiewicz and Paszkowski, 1980) and cultured Cutharanthus roseus cells (Hirose and Ashihara, 1983a). In addition to the nucleoside kinases, phosphorylation of purine nucleosides is also catalysed by nucleoside phosphotransferase (reaction 4) which has been purified from lupin cotyledons (Guranowski, 1979a, b) and barley seedlings (Prasher et al., 1982). However, in Catharanthus roseus cells, the activity of nucleoside phosphotransferase activity measured with [8-I4C]adenosine and AMP was much lower (< 6%) than that of adenosine kinase assayed with [8-'4C]adenosine and ATP (Hirose and Ashihara, 1984b).
CAFFEINE AND RELATED PURINE ALKALOIDS IN PLANTS
135
So, direct conversion of purine bases and nucleosides to nucleotides with PRPP and ATP by phosphoribosyltransferases and kinases, respectively, is the major salvage reactions in higher plants, although the two step-conversion (purine bases + purine nucleoside + purine nucleotide) pathway may also operate. The gene encoding adenine phosphoribosyltransferase has been isolated from Arabidopsis thaliana (Moffat et al., 1994). The gene contained five introns. Adenine phosphoribosyl transferase genes from animals have fewer introns. The promoter region of this gene lacks conventional promoter elements, such as TATA, CCAAT or G + C-rich sequence elements. A mutant of Arabidopsis, aprt, deficient in the purine salvage enzyme, adenine phosphoribosyl transferase, is male sterile due to abortion of pollen development soon after the pollen mother cells have completed meiosis (Moffatt and Somerville, 1988; Regan and Moffatt, 1990). This enzyme may have a function in cytokinin metabolism as, unlike the wild type, the aprt mutant is unable to metabolize the synthetic cytokinin base benzyladenine to its nucleotide form (Moffatt et al., 1991). C. CATABOLISM OF PURINE NUCLEOTIDES
In most higher plants, purine nucleotides are degraded via ureides, allantoin and allantoate to NH3 and C 0 2 via the pathways illustrated in Fig. 6 . However, ureides rather than C02, are the end products of this pathway in tissues of ureide-accumulating plants, such as tropical legumes. The enzymes associated with these pathways are summarized in Table V.
I . Deamination In the catabolism of purine nucleotides, there is diversity in the deamination reactions in different species. These reactions occur at the nucleotide, nucleoside or nucleobase level. In higher plants, AMP deaminase and guanosine deaminase are the predominant deamination enzymes for adenine and guanine nucleotides, respectively. Adenosine deaminase, which is widely distributed in animals, and adenine deaminase, which is found in various microorganisms, are not usually present in plant cells (Wagner and Backer, 1992). While guanine deaminase has been detected in plants, its activity is generally lower than that of guanosine deaminase (Negishi et al., 1994; Ashihara et al., 1997a). GMP reductase activity, which catalyses the conversion of GMP to IMP, has not been detected in higher plants. 2. Dephosphorylation and Glycosidic Bond Cleavage Dephosphorylation of purine nucleotides in plant extracts is catalysed by various enzymes, including 5’-nucleotidase and phosphatase. However, the identity of the enzyme(s) that dephosphorylate adenine and guanine nucleotides in vivo have not yet been determined. AMP, GMP, IMP and XMP are
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H. ASHIHARA and A. CROZIER
hydrolysed easily to their respective purine nucleosides, i.e. adenosine, guanosine, inosine and xanthosine. Glycosidic bond cleavage, which releases the ribose from ribonucleosides, is accomplished either hydrolytically or phosphorolytically. In higher plants, hydrolytic cleavage is common, although in many biological systems phosphorolytic cleavage of nucleosides is the major reaction. Two distinct purine nucleosidase enzymes, adenosine nucleosidase and ionosineguanosine nucleosidase, appear to participate in glycosidic bond cleavage of purine nucleosides in plant cells. The former is specific for adenosine, while the latter enzyme hydrolyses guanosine, inosine and xanthosine (Le Floc’h and Lafleuriel, 198la, b; Guranowski, 1982). These nucleosidases were often detected in plant extracts (Hirose and Ashihara, 1984b; Burch and Stuchbury, 1986). 3. Formation of Hypoxanthine and Xanthine from AMP and GMP The initial step of adenine nucleotide catabolism is deamination of AMP which is catalysed by AMP deaminase, and the product, IMP, is dephosphorylated to inosine which, in turn, is hydrolysed to hypoxanthine (Fig. 6). These conversions are catalysed by 5’-nucleotidase (and/or phosphatase) and purine nucleosidase (Atkins et al., 1989). ATP is essential for higher plant AMP deaminase activity, thus, catabolism of adenylates is dependent on the cellular ATP level (Yabuki and Ashihara, 1991, 1992). It has been proposed that in root nodules IMP is initially converted to XMP by IMP dehydrogenase and then dephosphorylated. Therefore, two possible routes may operate: (i) IMP 4 inosine 4 hypoxanthine; and (ii) IMP + XMP 4 xanthosine + xanthine (Fig. 6). The major pathway for the catabolism of guanine nucleotides begins with a dephosphorylation reaction which yields guanosine. Guanosine deaminase then catalyses the conversion of guanosine to xanthosine which is metabolized to xanthine by purine nucleosidase (Fig. 6).
Fig. 6. Catabolism of purine nucleotides in higher plants. Enzymes: (1) AMP deaminase (EC 3.5.4.6); (2) IMP dehydrogenase (EC 1.2.1.14); (3) 5’-nucleotidase (EC 3.1.3.5); (4) inosine-guanosine nucleosidase (EC 3.2.2.2); (5) guanosine deaminase (EC 3.5.4.15); (6) guanine deaminase (EC 3.5.4.3); (7) xanthine dehydrogenase (EC 1.1.1.204); (8) uricase (urate oxidase, EC 1.7.3.3); (9) allantoinase (EC 3.5.2.5); (10) allantoate amidinohydrolase (allantoicase, EC 3.5.3.4); (11) ureidoglycolate lyase (EC 4.3.2.3); (12) urease (urea aminohydrolase, EC 3.5.1.5); (13) allantoate amidohydrolase (decarboxylating) (allantoin deiminase, EC 3.5.3.9); (14) ureidoglycine amidohydrolase (no EC number given); (15) ureidoglycolate hydrolase (ureidoglycolate amidohydrolase (decarboxylating), EC 3.5.3.19). There is only indirect evidence for ureidoglycine amidohydrolase (Winkler et al., 1988).
137
CAFFEINE AND RELATED PURINE ALKALOIDS IN PLANTS
Inosine
Xantiosine
Guar;osine
Xan~ine
-
OH Allantoin hydrate
.
COOH
COOH
OH
OH
OH Hydroxyacetylenediureine-carboxylicacid
5-Ureide-2-imidarole-4,5dol-4-carboxylicacid
coo(9)
Allantoin
coo'
::
H2N-C-i-E-NH3' I
+ N H 3 + coz
Ureidoglycine
o:c,,NH2 H2N-C
It
-W-E-OH
+NH3
+
NH2 Urea
:
H2N-C-
:
I
1-H
cooC-OH
Ureidoglycolate
Ureidoglycolate ZNH3
2NH3+C02
+
COOH I CHO Glyoxylate
+
C02
COOH I CHO Glyoxylate
HzN,
,C=O
+
H2N
Urea
1
2NH3 +
(12)
coz
138
H. ASHIHARA and A. CROZIER
TABLE V Enzymes of the purine nucleotide catabolic pathway demonstrated in higher plants Enzyme (EC number)
Plant source
Reference
AMP deaminase (EC 3.5.4.6)
Spinach
Yoshino and Murakami (1980) Le Floc’h and Lafleuriel (1983)
Jerusalem artichoke
Pea Tea leaves
Yabuki and Ashihara (1991, 1992) Dancer et al. (1997) Negishi et al. (1994)
Tea leaves
Negishi et al. (1994)
Wheat Maize Peanut
Polya and Ashton (1973) Carter and Tipton (1986) Mittal et al. (1988)
Jerusalem artichoke
Le Floc’h and Lafleuriel (1981a) Guranowski (1982)
Catharanthus roseus
Guanosine deaminase (EC 3.5.4.15) Guanine deaminase (EC 3.5.4.3) 5’-Nucleotidase (EC 3.1.3.5) 5‘-AMP nucleotidase (EC 3.1.3.5) Adenosine nucleosidase Inosine-guanosine nucleosidase (EC 3.2.2.2) Xanthine dehydrogenase (EC 1.1.1.204)
Lupin seeds Pea Phaseolus vulgaris (nodules) Soybean (nodules)
Uricase (EC 1.7.3.3)
Allantoinase (EC 3.5.2.5)
Allantoicase (allantoin amidinohydrolase, EC 3.5.3.4) Alantoate deiminase (EC 3.5.3.9) Urease (EC 3.5.1.5)
Soybean Soybean Cowpea (nodules) Soybean Cajanus cajan (nodules) Peanut
Nguyen and Feierabend (1978) Boland (198 1) Triplett et al. (1982); Boland et al. (1983) Tajima and Yamamoto (1975) Bergmann et af. (1983) Rainbird and Atkins (1981) Thomas et al. (1983) Amarjit and Singh (1985) Singh (1968)
Soybean
Winkler et al. (1985)
Soybean
Kerr et al. (1983)
CAFFEINE A N D RELATED PURINE ALKALOIDS IN PLANTS
139
4. Oxidation of Hypoxanthine and Xanthine Hypoxanthine is converted to uricate via xanthine by xanthine oxidoreductase (Fig. 6). There are two forms of xanthine oxidoreductase. One form has a requirement for molecular oxygen and is called xanthine oxidase (EC 1.2.3.2). This form is particularly abundant in bovine milk. The other form, xanthine dehydrogenase (EC 1.2.1.37), is an NAD-dependent enzyme. In higher plants, oxidation of hypoxanthine and xanthine seems to be catalysed by xanthine dehydrogenase, although the two forms of xanthine oxidoreductase may be interconverted (see Nguyen, 1986). Allopurinol (4-hydroxypyrazolo-3,4-~pyrimidine) is a specific inhibitor of xanthine oxidoreductase (Weir and Fisher, 1970). Addition of 2pM allopurinol to pea leaf extracts results in almost complete inhibition of xanthine dehydrogenase activity (Nguyen and Feirebend, 1978). Allopurinol is often used as an inhibitor of purine catabolism in studies with higher plants (Fujiwara and Yamaguchi, 1978; Ashihara, 1983; Hammer et al., 1985; Ashihara et al., 1996b, 1997a).
5 . Breakdown of Uricate Uricate is catabolized rapidly by higher plants, although it is the end product of human purine metabolism (Henderson and Paterson, 1973). Uricase catalyses the formation of allantoin from uricate. Several intermediates in the uricase catalysed degradation of uricate are either known or their existence has been postulated (Fig. 6). Allantoin amidohydrolyase (allantoinase) catalyses the hydrolysis of the internal amide bond of allantoin resulting in its conversion to allantoate. This enzyme is found in many plant species (Schubert and Boland, 1990). Different routes for allantoate degradation have been proposed in higher plants (Winkler et al., 1988; Schubert and Boland, 1990). The classic pathway is the ‘allantoate amidohydrolase (allantoicase)’ pathway in which allantoate is hydrolysed to urea and ureidoglycolate. Ureidoglycolate is further degraded to glyoxylate and urea. Urea formed via this pathway may be hydrolysed to ammonia and C 0 2 by urease (Fig. 6, steps 10-12). However, recent studies suggest the existence of an alternative route, the ‘allantoate deiminase (allantoate amidohydrolase)’ pathway. Allantoate is hydrolysed to C02, NH3 and ureidoglycine. Ureidoglycine is unstable and can be deaminated, either spontaneously or enzymatically, to ureidoglycolate which is cleaved by ureidoglycolate amidohydrolase to produce NH3, COz and glyoxylate (steps 13-15). In the earlier studies, urea was often found as a degradation product of allantoin, however, Winkler et al. (1988) pointed out that non-enzymatic degradation of allantoate to urea can occur. Some plant species accumulate allantoin and allantoate which then play an important role in the storage and translocation of nitrogen. The ureides (allantoin plus allantoate) increase in the spring in maple and comfrey and move from root to shoot in the xylem, arriving in the leaves where they are utilized as substrates for protein synthesis (Reinbothe and Mothes, 1962). At peak concentrations during this period ureide nitrogen in bleeding sap of maple
140
H. ASHIHARA and A. CROZIER
accounts for as much as 7&100% of the total soluble nitrogen. In the early stages of seed germination and development of black gram (Phaseolus mungo) seeds, purine derivatives in the form of storage RNA in the cotyledons are utilized for purine nucleotide and nucleic acid synthesis. However, in the later stages of germination they are degraded to allantoin and allantoate, and reutilized as a nitrogen source for the growth of shoot and root axes (Ashihara, 1983). D. METABOLISM OF PURINE BASES AND NUCLEOSIDES IN PLANTS
Radiolabelled purine bases and nucleosides are useful substrates for studies on the biosynthesis of purine alkaloids in higher plants. In contrast to other common precursors, such as sugars and amino acids, special care must be taken when using purine precursors. For this reason, information on the uptake and metabolism of exogeneously supplied labelled purine precursors obtained from non-caffeine-forming plants (Ashihara and Nobusawa, 1981; Shimazaki and Ashihara, 1982; Hirose and Ashihara, 1983~;Ashihara, 1983, 1990a; Le Floc'h and Fyte, 1995; Ashihara el al., 1997a) is summarized. Studies on the biosynthesis of purine alkaloids in plants have made use of ''C-labelled adenine, adenosine, guanine, guanosine, inosine, hypoxanthine, xanthosine and xanthine as precursors. Upon uptake by plant cells these compounds are immediately either metabolized to nucleotides or degraded to COz. The major pathways involved in the metabolism of purine nucleosides and bases are illustrated in Figs 7 and 8. Labelled AMP and GMP are not used as substrates for purine alkaloid biosynthesis because they do not readily enter plant cells. However, the equivalent nucleosides and nucleobases are easily taken up. Thus, when investigating the metabolic fate of adenine nucleotides in plants, they are pre-labelled with [14C]adenineand ['4C]adenosine (see Yabuki and Ashihara, 1991). 1 . Metabolism of Adenine and Adenosine [14C]Adenine and ['4C]adenosine are taken up rapidly by plant tissues and, in the absence of adenine deaminase and adenosine deaminase, they are phosphorylated by adenine phosphoribosyltransferase and adenosine kinase to AMP, and enter the adenylate pool which consists of ATP, ADP and AMP (Fig. 7). Significant amounts of radioactivity are then incorporated into RNA and DNA. Turnover of adenine nucleotides in plant cells is rapid (Yabuki and Ashihara, 1991), so after longer incubation periods small amounts of radioactivity are incorporated into degradation products of purine catabolism, such as allantoin, allantoate and C02. In most instances, relatively little radioactivity remains as adenine and adenosine probably because salvage of these compounds by plants is extremely high.
141
CAFFEINE AND RELATED PURINE ALKALOIDS IN PLANTS
NA Hypoxanthine
Hypoxanthine
1
Guanine
Fig. 7. Major salvage pathways of exogenously supplied I4C-labelled purine nucleosides and bases by higher plant cells. Dotted arrows show very minor pathways. AIC, 5-aminoimidazole-4-carboxamide;AICAR, 5-aminoimidazole-4-carboxamide riboside. ZMP, 5-aminoimidazole-4-carboxamide ribotide. FAICAR, S-formamidoimidazole-4-carboxyamide ribonucleotide.
2. Metabolism of Guanine and Guanosine The uptake of ['4C]guanosine by the intact suspension cultured cells of Catharanthus roseus is more than 20 times higher than that of [I4C]guanine (Ashihara et al., 1998). The difference between the rates of uptake suggests that some specific mechanism(s) for the transport of guanosine might operate in the cell membranes. In contrast to adenine and adenosine, guanine and guanosine are not converted primarily to their nucleotides. Instead guanine and guanosine are substrates for purine salvage enzymes for GMP synthesis, as well as for guanine deaminase and guanosine deaminase. Therefore, a significant portion of [14C]guanine and [ ''C]guanosine taken up by the cells is catabolized. Nevertheless, rates of incorporation of radioactivity from [ 14C]guanineand ['4C]guanosine into nucleic acids are sometimes higher than from [I4C]adenine and [I4C]adenosine. This may simply be a consequence of the smaller size of the guanine nucleotide pool.
142
H. ASHIHARA and A. CROZIER
Inosine
Inosine
Xanthine
e
Guanine Xanthosine Guanosine
-
IMp
-
uriate -Allantoin
Guanine Xanthosine
Allanoate
Guanosine
Fig. 8. Major degradation pathways of ''C-labelled purine nucleosides and bases by higher plant cells. Dotted arrows show very minor pathways. Abbreviations: see the caption to Fig. 7.
3. Metabolism of Inosine and Hypoxanthine Inosine and hypoxanthine are not only good precursors for purine catabolism, but are also salvaged by specific guanosine-inosine kinase and/or non-specific nucleoside phosphotransferase and hypoxanthine-guanine phosphoribosyltransferase, and converted to IMP. Therefore, only a small proportion of these compounds is converted to nucleotides and nucleic acids, and significant amounts of radioactivity from ['4C]hypoxanthine and [14C]inosineare recovered as 14C02. In the presence of allopurinol the release of 14C02 is almost completely repressed. 4. Metabolic Fate of Xanthosine and Xanthine Little work has been carried out on the metabolic fate of xanthosine and xanthine in higher plants, except in caffeine-forming tea and coffee plants. These compounds are not utilized for nucleotide and nucleic acid biosynthesis, and most of the radioactivity from [14C]xanthine and [ ''C]xanthosine is released as 14C02. The biosynthesis of purine alkaloids from xanthosine is discussed in Section IV. There are no reports that indicate the presence of salvage enzymes for xanthine and xanthosine, although specific xanthine phosphoribosyltransferases have been demonstrated in bacteria (see Henderson and Paterson, 1973).
CAFFEINE A N D RELATED PURINE ALKALOIDS IN PLANTS
143
5. Metabolism of 5-Aminoimidazole-4-carboxamide and S-Aminoimidazole-4-
carboxamideriboside 5-Aminoimidazole-4-carboxamide (AIC) and 5-aminoimidazole-4-carboxamideriboside (AICAR) are converted to 5-aminoimidazole-4-carboxamideribotide (ZMP), an intermediate of purine biosynthesis de novo. AICAR appears to be a much better precursor for purine nucleotide biosynthesis than AIC, because mutant cells of Datura innoxia, which lack the enzymes involved in the initial stages of the de novo pathway, grow well in the presence of AICAR but not AIC (Ashihara et al., 1991). [2-14C]AICAR is converted to AMP and GMP, and utilized for the nucleic acid synthesis. AICAR is phosphorylated by adenosine kinase and the product, ZMP, is converted to adenine and guanine nucleotides via IMP (H. Ashihara, unpublished data). There is only one paper in the literature on AIC metabolism in plants: Shuster (1963) reported that incorporation of [2-14C]AIC into guanine was approximately five times greater than incorporation into adenine in germinating wheat embryos. E. OTHER PLANT-SPECIFIC PURINE PATHWAYS
In addition to purine alkaloids, higher plants possess the metabolic pathways for the production of some specialized purines. Among the most important are the pathways that lead to production of cytokinins, such as zeatin, which are of widespread occurrence in higher plants. The biosynthesis and metabolism of cytokinins are reviewed in detail by Chen (1997) and Crozier et al. (1999).
IV. BIOSYNTHESIS OF PURINE ALKALOIDS A. METHYLATION OF THE PURINE RING
1. Methyl Acceptors What is the initial methyl acceptor for purine alkaloid biosynthesis? There are two possible origins for the purine ring of caffeine: (i) the methylated nucleotides in nucleic acids (Ogutuga and Northcote, 1970a); and (ii) methylated purines derived from the nucleotide pool (Looser et al., 1974; Suzuki and Takahashi, 1976a). Ogutuga and Northcote (1970a, b) proposed two caffeine biosynthetic pathways, although they considered the nucleic acid methylation pathway the more likely route. This pathway is as follows: methylated nucleic acids 4 7methylguanylic acid 4 7-methylguanosine + 7-methylxanthosine 4 (7methylxanthine) --+ theobromine + caffeine (Fig. 9, pathway I). However, several pieces of evidence now seriously question the relevance of this route. Suzuki and Takahashi (1976a) demonstrated the presence of tRNA methyltransferase in cell-free extracts from leaves, cotyledons and roots of tea seedlings. By far the highest tRNA methyltransferase activity was observed in
144
H. ASHIHARA and A. CROZIER
Pathway I
4
7-Methyl GMP Pi
7-Methylpanosine
Purine Pool
I
I
Pathway 11
-the
[M~I 3-Methylxanthine
i.--
$.
7-Methylxanthosine
Theobromine
[Me1 Fig. 9. The classic routes of caffeine biosynthesis suggested by Ogutuga and Northcote (1970a). ‘Pathway I’ that includes the methylation of tRNA seems to be not operative while ‘Pathway 11’ is now considered to be a minor pathway of caffeine biosynthesis (see Fig. 11). [Redrawn from Ogutuga and Northcote (1970a).]
preparations from roots which are not a site of caffeine biosynthesis. Furthermore, 1-methyladenosine, rather than 7-methylguanosine, was the sole methylated RNA base detected in the cell-free preparations. When [methyl-’4C]methioninewas applied to tea shoot tips, label was incorporated into 7-methylxanthine, theobromine and caffeine as well as 1-methyladenylic residues of tRNA (Suzuki and Takahashi, 1976b) Looser et al. (1974) showed that incorporation of the radioactivity from [methyl-14C]methionine into caffeine in leaf discs of Cof. arabica was stimulated by the presence of theobromine, 7-methylxanthine and 7-methylxanthosine, but not 7-methylguanosine. These observations do not support the view that caffeine is synthesized from tRNA-derived 1-methyladenine. Pathway I1 proposed by Ogutuga and Northcote (1970b) is now considered to be one of the minor pathways of caffeine biosynthesis (see Section 1V.C). There is now a general consensus that the purine skeletons of purine alkaloids are derived almost exclusively from purine nucleotides. As purine
145
CAFFEINE AND RELATED PURINE ALKALOIDS IN PLANTS
alkaloids are xanthine derivatives, methylation may be performed at the nucleotide (XMP), nucleoside (xanthosine) or nucleobase (xanthine) levels. Recent experiments with cell-free preparations from tea and coffee leaves indicate that N-methyltransferases involved in caffeine biosynthesis can use xanthosine and mono- and dimethylxanthines as methyl acceptors (Kato et al., 1996, 1999). The available evidence indicates that xanthosine, 7-methylxanthine and theobromine are the in vivo methyl acceptors. There is, however, one report in which it is proposed that XMP, rather than xanthosine, is a methyl acceptor in coffee leaves (Schulhess et al., 1996). Xanthine is a weak methyl acceptor yielding only trace quantities of 3-methylxanthine (Negishi et al., 1985b; Kato et al., 1996). 2. Methyl Donors Anderson and Gibbs (1962) and Inoue and Adachi (1962) demonstrated that the methyl group of methionine and other C-1 compounds could serve as methyl group donors for caffeine biosynthesis. [Methyl-14C]methioninehas been used as a methyl donor for caffeine biosynthesis in the shoot tips of Cam. sinensis (Suzuki and Takahashi, 1976a), and leaf disks (Looser et al., 1974; Schulthess et al., 1996) and fruits of Cof. arabica (Roberts and Waller, 1979; Suzuki and Waller, 1984a). Incorporation of label from [methyl-14C]yglutamylmethylamide (theanine) and [I4C]methylamine into the N-methyl groups of caffeine has also been reported (Konishi et al., 1972; Suzuki, 1973). However, the in situ methyl donor for caffeine biosynthesis is S-adenosylmethionine (SAM). Suzuki (1972) was the first to demonstrate the involvement of SAM in caffeine biosynthesis in tea leaves. Subsequently, SAM has been used widely as the in vitro methyl acceptor for N-methyltransferases in studies on caffeine biosynthesis (Suzuki and Takahashi, 1975a; Roberts and Waller, 1979; Baumann et al., 1983; Negishi et al., 1985b; Fujimori et al., 1991; Mazzafera et al., 1994b; Ashihara et al., 1995, 1998; Kato et al., 1996, 1999; TABLE VI Time-course study with young leaves of Camellia sinensis on the incorporation of the radioactivity from 9 p M [methyl-’4C/SAM into theobromine and caffeine. The rates of incorporation are expressed as kBq f standard deviation (n = 3 ) and as a percentage of total radioactivity taken up by the leaves (parentheses). Segments of tea leaves (200mg fresh weight) were incubatedfor 2 , 6 and 18 h with radioactive SAM and 10 mM sucrose in 2.0 ml of 30 mM potassium phosphate buffer (pH 5.6)
2h Theobromine Caffeine
0.9 f 0.0 (6.9) 1.5 f 0.1 (12.0)
Incubation period 6h
18h
2.2 f 0.2 (1 1.4) 4.6 f 0.5 (23.5)
3.1 f 0.1 (6.8) 16.3 f 0.9 (36.4)
From S. Yama and H. Ashihara, unpublished data.
146
H. ASHIHARA and A. CROZIER
TABLE VII Incorporation of the radioactivity from 9 p M [ m e t h ~ l - ' ~ C ] S A M into theobromine and cafieine in young, mature and aged leaves of Camellia sinensis. The rates of incorporation are expressed as kBq f standard deviation (n = 3 ) and as a percentage of total radioactivity taken up by the leaves (parentheses). Segments of tea leaves (200 mg fresh weight) were incubated for 6 h with radioactive SAM and I0 mM sucrose in 2.0 mi of 30 mM potassium phosphate buffer (pH 5.6)
Theobromine Caffeine
Young leaves
Mature leaves
Aged leaves
2.2 f 0.2 (11.4) 4.6 f 0.5 (23.5)
1.7 f 0.2 (6.1) 3.3 f 0.4 (11.9)
0.7 f 0.1 (3.6) 1.2 f 0.2 (6.3)
Young leaves (newly emerged, small expanding leaves, 1 10mg leaf'), mature leaves (young fully expanded leaves, 450 mg leaf') and aged leaves (1-year-old leaves, 480 mg leaf') were used. From S. Yama and H. Ashihara (unpublished data).
Schulthess et al., 1996; Mosli Waldhauser et al., 1997a,b). Exogenous SAM is utilized efficiently for caffeine biosynthesis with more than 40% of [methyl-'4C]SAM taken up by the young tea leaf disks being incorporated into theobromine and caffeine after 18 h (Table VI). Utilization of SAM for caffeine biosynthesis in tea is highest in young leaves and declines as the leaves age (Table VII) (S. Yama and H. Ashihara, unpublished data). The synthesis and turnover of SAM, the so-called 'activated methyl' cycle (Edwards, 1996) are summarized in Fig. 10. SAM synthetase (EC 2.5.1.6) catalyses the synthesis of SAM from 1-methionine and ATP (Chou and Talalay, 1972). On donation of the S-methyl group to the acceptor molecules, xanthosine, 7-methylxanthine and theobromine, SAM is converted to Sadenosyl-L-homocysteine(SAH). This reaction is catalysed by SAM-dependent N-methyltransferases which are described later in Section IV.B.l. SAH is a very strong inhibitor for many SAM-dependent N-methyltransferases. SAH is hydrolysed to L-homocysteine and adenosine by SAH hydrolase (EC 3.3.1.1). This is reaction is reversible but in vivo SAH hydrolysis is favoured because of the removal of adenosine. Although several enzymes could be responsible for the metabolism of adenosine, in higher plants adenosine kinase is probably involved. [8-14C]Adenosine supplied to the young tea leaves is converted mainly to SAM, adenine nucleotides, theobromine, caffeine and nucleic acids (Table VIII) (S. Yama and H. Ashihara, unpublished data). Part of the adenosine pool is therefore converted to ATP and reutilized for the synthesis of SAM, while the other portion is metabolized to xanthosine via IMP and used for purine alkaloid synthesis (Fig. 10). The homocysteine derived from SAH is converted to 1-methionine, thereby completing the 'activated methyl' cycle.
CAFFEINE AND RELATED PURINE ALKALOIDS IN PLANTS
MTA I
& Adenine
147
Decarboxyl-SAM
A
(lo) (8)
5-methyl-THF
THF xanthosine' 7-Methy lxanthine Theobromine 7-Methylxanthosine Theobromine Caffeine
Fig. 10. S-adenosyl-L-methionine (SAM) cycle in plants. Metabolites: SAM, Sadenosyl-L-methionine; SAH, S-adenosyl-L-homocysteine;MTA, 5'-methylthioadenosine; MTR, 5'-methylthioribose; MTOB, 4-methylthio-2-oxobutyrate; decarboxylSAM, decarboxylated SAM (adenosylmethylthiopropylamine). Enzymes: (1) SAM synthetase (EC 2.5.1.6); (2) SAM-dependent N-methyltransferases; (3) S-adenosylhomocysteine (SAH) hydrolase (EC 3.3.1.1); (4) methionine synthase (EC 2.1.1.13); (5) adenosine kinase (EC 2.7.1.20); (6) adenosine nucleosidase (EC 3.2.2.7); (7)adenine phosphoribosyltransferase (EC 2.4.2.7); (8) SAM decarboxylase (EC 4.1.1.50); (9) aminopropyl transferases (e.g. spermidine synthase, EC 2.5.1.16); (10) 5'-methylthioadenosine (MTA) nucleosidase (EC 3.2.2.9). Adenosine and adenine are salvaged to adenine nucleotides and utilized as ATP (*) in the synthesis of SAM and as xanthosine (9 in the synthesis of caffiene.
3. Sequence of Methylation There are many reports indicating that theobromine is the immediate precursor of caffeine (Suzuki et al., 1992; Fujimori and Ashihara, 1993; Ashihara, 1993; Ashihara et al., 1996a, 1997a). Negishi et al. (1985a) demonstrated that [2-I4C]xanthosine and [2-'4C]7-methylxanthosine were both converted to caffeine via 7-methylxanthine in tea leaves. Pulse-chase experiments with [8-'4C]xanthosine and young Cof. arabica leaves have shown that 7-methylxanthine and theobromine were both labelled during a 6-h pulse, but that radioactivity became associated almost exclusively with caffeine after an 18-h chase (Ashihara et al., 1996a). These studies strongly suggested that the major route from xanthosine to caffeine is a xanthosine + 7-methylxanthosine + 7methyxanthine + theobromine + caffeine pathway (Fig. 11). In addition to
148
H. ASHIHARA and A. CROZIER
TABLE VIII Metabolism of 8 p M [8-14C]adenosine in young tea leaves. The rates of incorporation are expressed as kBq f SD (n = 3 ) and as a percentage of total radioactivity taken up by the leaves (parentheses).Segments of tea leaves (200mg fresh weight) were incubatedfor 2 , 6 and 18 h with radioactive adenosine and 10 mM sucrose in 2.0 ml of 30 mM potassium phosphate buffer ( p H 5.6) (nd, not detected) Incubation period 2h
SAM ATP ADP AMP Xanthine Adenosine Theobromine Caffeine COZ
Nucleic acids Others Total uptake
nd 0.8 0.3 0.1 0.0 0.4 0.7
f 0.1 (15.7)
f 0.1 (5.9) f 0.0 (2.0) f 0.0 (0.0) f 0.0 (7.8) f 0.1 (13.7)
nd 0.1 f 0.1 (2.0) 2.2 f 0.3 (43.1) 0.3 f 0.0 (5.9) 5.1 f 0.6 (100)
6h 1.8 7.3 2.9 1.6 0.8 4.3 28.1 5.6 0.9 10.0 1.5 64.9
f 0.2 (2.8)
1.8 (11.2) f 0.5 (4.5) f 0.3 (2.5) f 0.1 (1.2) f 0.9 (6.6) f 2.4 (43.3) f 0.2 (8.6) f 0.6 (1.4) f 0.5 (15.4) f 0.3 (2.3) f 2.2 (100) f
18h 1.8 2.3 1.1 1.0 1.0 3.5 25.7 28.7 15.4 14.6 2.1 97.3
f 0.5 (1.8) f 1.4 (2.4) f 0.6 (1.1)
0.2 (1.0) f 0.3 (1.0) f 0.3 (3.6) f 6.6 (26.4) f 6.8 (29.5) f 5.4 (15.8) f 2.4 (15.0) f 0.3 (2.2) f 12.6 (100) f
From S. Yama and H. Ashihara (unpublished data).
the main pathway, some minor routes, such as 7-methyxanthine paraxanthine + caffeine, and xanthine -+ 3-methylxanthine + theobromine -+ caffeine, may also function (Kato et al., 1996). These minor pathways may be a consequence of the broad methyl acceptor specificity of the N methyltransferase(s) that is discussed in Section IV.B.2.
-+
4 . Concentration of Intermediates of Caffeine Biosynthesis Intracellular levels of xanthosine and methylxanthines in young, mature and aged tea leaves, as determined by HPLC, are presented in Table IX. As concentrations of these compounds are 200-2000 times lower than that of caffeine, they frequently go undetected. It is noteworthy that the combined 7methylxanthosine and 7-methylxanthine pools total 160nmol g-' f.w. which is very similar to the size of the 177 nmol g-' ATP pool in young tea leaves (see Table I). This suggests that pool sizes of the intermediates involved in caffeine biosynthesis are comparable to those of intermediates in other metabolic pathways, such as glycolysis where metabolites accumulate in concentrations ranging from 10 to 300nmolg-' f.w. (Kubota and Ashihara, 1990).
Xanthine
I
1.1
FMethyl-XMP
t
.*
,*'
(N-3)
Paraxanthine
kbose-P
t
AMP
Fig. 11. Proposed pathways for the biosynthesis of purine alkaloids in tea and coffee plants. Solid arrows indicate main pathways and dashed arrows are minor or uncertain reactions. N-I, N-3 and N-7 in parentheses indicate the position of nitrogen atom in purine ring that is methylated. Only N-7 of xanthosine and XMP is methylated.
150
H. ASHIHARA and A. CROZIER
TABLE IX The sizes of endogenous X M P , xanthosine, xanthine, 7-methyxanthosine and methylxanthines pools in young, mature and aged leaves of Camellia sinensis. Data expressed as nmolg-'fresh weight f standard deviation (n = 3 ) Metabolites
Young
XMP Xanthosine Xanthine 7-Methylxanthosine 7-Methylxanthine 3-Methylxanthine 1-Methylxanthine Theobromine Caffeine
29 f 1 22 f 4 6 f 1 179 f 31 61 f 6 5*3 2 h O 1170 f 100 36400 f 2390
Mature 43 15 4 23 16 38 14 228 20600
* 13 f3
f l f 14 f 7 f
5
f7 f 78 f 4600
Aged 38 f 1 13 f 1 4 f 1 22 f 7 16 f 6 2 f 1 8 f 4 174 48 13 700 f 430
*
For a definition of young, mature and aged leaves see footnote to Table VII. From H. Ashihara and A. Kato (unpublished data).
5. Subcellular distribution of caffeine and related purine alkaloids
Compartmentation of caffeine has not yet been demonstrated directly. However, it is plausible in keeping with many other secondary metabolites, that caffeine and some purine alkaloids are stored in vacuoles (Wink, 1997a). Mechanisms for uptake and sequestration of caffeine in vacuoles remain to be investigated. Active/passive transport by channels and/or transporters, membrane/vesicle fusion may be involved in these processes. Binding of caffeine to polyphenols such as catechins (tea) and chlorogenic acids (coffee) may be functional as a mechanism for the accumulation of these compounds in vacuoles against a concentration gradient. Baumann and his co-workers suggested that vacuolar compartmentation of purine alkaloids depends exclusively on the formation of complexes with chlorogenic acids (Bauman and Rohring, 1989; Molsi Waldhauser and Bauman, 1996). B. ENZYMES INVOLVED IN METHYLATION STEPS
1. N-Methyltransferases The main caffeine biosynthetic pathway from xanthosine, xanthosine 4 7methylxanthosine 4 7-methylxanthine -+ theobromine --+ caffeine, consists of three methylations and the cleavage of a glycosidic bond (Fig. 11). The methylation steps are catalysed by SAM-dependent N-methyltransferases. Activities of 7-methylxanthine N-methyltransferase and theobromine Nmethyltransferase, which catalyse the second and the third methylation steps
CAFFEINE A N D RELATED PURINE ALKALOIDS IN PLANTS
151
in the main pathway, were first demonstrated in crude extracts from tea leaves by Suzuki and Takahashi (1975a) who showed that the two enzymes have identical pH optima and are affected similarly by metal ions and inhibitors. Since then caffeine biosynthesis N-methyltransferase activities have been detected in cell-free extracts prepared from immature fruits (Roberts and Waller, 1979) and cell-suspension cultures (Baumann et al., 1983) of Cof. arabica. The first methylation enzyme, xanthosine N-methyltransferase, which catalyses the formation of 7-methylxanthosine from xanthosine, was demonstrated in tea leaf extracts by Negishi et al. (1985b). Fujimori et al. (1991) confirmed the presence of the activities of the three N-methyltransferases in tea leaf extracts and found that they were highest in very young expanding leaves but absent in fully developed leaves. The purification of N-methyltransferase(s) has been attempted by several investigators. Mazzafera et al. (1994b) reported the purification of a Nmethyltransferase from fruits and leaves of Cof. arabica which possessed 7methylxanthine and theobromine N-methyltransferase activity. However, the activity of the cell-free preparations was extremely labile and the specific activity of the enzyme diminished with each step in a sequential purification procedure. The specific activity of the final preparation was less than 1 fkat mg-' protein. Gillies et al. (1995) purified N-methyltransferase from liquid endosperm of Cof. arabica using Q-Sepharose in the presence of 20% glycerol. The final specific activity was 420 fkat nig-' protein. Kato et al. (1996) partially purified N-methyltransferase from tea leaves by ion-exchange and gelfiltration chromatography. Mosli Waldhauser et al. (1997b) purified Nmethyltransferases from coffee leaves up to 39-fold using ion-exchange chromatography and chromatofocusing, and showed that XMP N-methyltransferase was a different protein to the 7-methylxanthine and theobromine N-methyltransferases. Recently, a N-methyltransferase from young tea leaves, which catalyses the SAM-dependent methylation of methylxanthines, was purified 520-fold to apparent homogeneity by ammonium sulphate fractionation and hydroxyapatite, Shodex IEC QA anion-exchange, adenosine-agarose and Superdex 200 gel-filtration chromatography (Kato et al., 1999). The final specific activity was 5.7 nkat mg-'. The protein exhibited broad substrate specificity and catalysed the second and third methylations in the caffeine biosynthetic pathway, i.e. the conversion of 7-methylxanthine to caffeine via theobromine (Fig. 11). The single N-methyltransferase obtained was, therefore, referred to as caffeine synthase. The properties of this enzyme will be discussed in Section IV.A.3. 2. N-Methyltransferase for the Initial Methylation Step Negishi et al. (1985b, c) demonstrated N-methyltransferase activity in extracts from young tea and coffee leaves which catalysed 7-methylation of xanthosine. The presence of this enzyme was subsequently confirmed by other investigators (Fujimori et al., 1991; Kato et al., 1996). However, Baumann and co-workers
152
H. ASHIHARA and A. CROZIER
suspected that the 7-methylxanthosine was not an intermediate of caffeine biosynthesis, because it was not detected in extracts from cultured coffee cells even when caffeine biosynthesis was stimulated by adenine and ethephon (Schulthess and Baumann, 1995a). Using cell-free extracts containing a nucleotidase inhibitor, Na2M04, they also demonstrated that both xanthosine and XMP were utilized as a methyl acceptor of SAM (Schulthess et al., 1996). On the basis of these observations, they proposed a new XMP -t 7-methylXMP -, 7-methylxanthosine -t 7-methylxanthine pathway (see Fig. 11). However, it is noteworthy that even in the Cof. arabica enzyme preparations used by Schulthess et al. (1996), xanthosine was a better methyl acceptor than XMP. In crude cell-free preparations from young tea leaves, XMP Nmethyltransferase activity is only about 12% of xanthosine N-methyltransferase activity (Negishi et al., 1985b). These investigators believed that the XMP N-methyltransferase activity was an artifact resulting from 7-methylation of xanthosine which was produced from XMP because of the presence of nucleotidase in the cell-free extracts (Negishi et al., 1985b). To investigate whether XMP N-methyltransferase activity is due to nucleotidases and/or phosphatases in crude extracts, partially purified tea N-methyltransferase was prepared by ion-exchange chromatography after ammonium sulphate fractionation as shown in Fig. 12 (M. Kato and H. Ashihara, unpublished data).
3
c
I
r
I
'
30
40
30
f 0 0
10
20 Fraction (ml)
Fig. 12. Profile of N-methyltransferase (NMT) and phosphatase activities eluted from a Shodex IEC QA-824 anion-exchange column. A desalted tea leaf protein extract was applied to the column which was washed with equilibration buffer containing 20mM KCl for 10min followed by a 50-min linear gradient of 20-750mM KCl in equilibration buffer. The flow rate was 0.8 ml min-I. NMT activity was assayed with 0.2mM xanthosine or 0.2 mM XMP in the presence of 4mM [methyl-I4C]SAM, and that of phosphatase were with 2mM IMP. The same buffer (100mM Tris-HCI, pH 8.4) was used. No NMT activity was found with XMP. (Drawn with permission from the unpublished data of M. Kato.)
CAFFEINE AND RELATED PURINE ALKALOIDS IN PLANTS
153
The major peak of xanthosine N-methyltransferase activity had almost no nucleotide-phosphorylating activity, and did not 7-methylate XMP. Therefore, XMP is unlikely to be the methyl acceptor in tea as suggested by Negishi et al. (1985b). Thus, while the possibility that XMP is the initial methyl acceptor in the caffeine biosynthetic pathway in coffee cannot be precluded, the available evidence suggests that XMP is not the methyl acceptor in tea leaves. 3. Properties of Caffeine Synthase from Tea Leaves The native caffeine synthase obtained by Kato et al. (1999) was monomeric with an apparent relative molecular mass of 61 000, as estimated by gelfiltration chromatography, and 41 000 when analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme displayed a sharp pH optimum of pH 8.5. The final preparation exhibited 3and 1-N-methyltransferase activity with a broad substrate specificity, showing high activity toward paraxanthine, 7-methylxanthine and theobromine, and low activity with 3-methylxanthine and 1-methylxanthine. However, the enzyme had no 7-N-methyltransferase activity toward xanthosine and XMP. The K , values of caffeine synthase for paraxanthine, theobromine, 7methylxanthine and SAM were 24, 186, 344 and 21 pM, respectively. The methyl acceptor specificity of the N-methyltransferases in crude, partially purified and highly purified preparations from tea, cocoa tea (Cam. ptilophylla) and coffee are summarized in Table X. The broad substrate specificity of the purified caffeine synthase obtained by Kato et al. (1999) is very similar to that of crude tea leaf extracts reported by Suzuki and Takahashi (1975a). Although paraxanthine is the best methyl acceptor for both tea and coffee N-methyltransferases, the substrate specificity of tea caffeine synthase is different to that of coffee and cocoa tea. Theobromine is a better methyl acceptor than 7-methylxanthine in coffee (Looser et al., 1974; Mazzafera et al., 1994b) but this is not the case with the tea enzyme (Table X). The properties of the N-methyltransferase from cocoa tea, a theobromine-accumulating plant, are different from those of coffee and tea as the cocoa tea enzyme can use 7methylxanthine as a methyl acceptor, but not theobromine or paraxanthine (Ashihara et al., 1998). The data summarized in Table X, therefore, indicate distinct variations in the properties of the N-methyltransferases which may be the cause of the different spectrum of purine alkaloids that accumulate in the three species. The purified tea caffeine synthase did not methylate either xanthosine or XMP and therefore does not catalyse the first methylation step in the caffeine biosynthetic pathway. It is likely that xanthosine N-methyltransferase protein is different to caffeine synthase, and that two different enzymes catalyse the three methylation steps in the caffeine biosynthetic pathway. The K , value for paraxanthine is the lowest and the V,, for this substrate is the largest, hence paraxanthine is the best substrate for caffeine synthase.
TABLE X Comparison of the substrate specijkity of SAM-dependent N-methyltransferases from Camellia sinensis, Camellia ptilophylla and Coffea arabica [7-mX, 7-methylxanthine; 3-mX. 3-methylxanthine; I-mX, 1-methylxanthine; Tb, theobromine; Tp, theophylline; Px, paraxanthine; X,xanthine; X R , xanthosine: XMP, xanthosine 5’-phosphate; nd, not detected; tr, trace; (-), not determined] Substrate Methylation position Tea (Camellia sinensis) Leaves (purified) Leaves (partially purified) Leaves (crude) Leaves (crude) Cocoa tea (Camellia ptilophylla) Leaves (crude) Coffee (Coffea arabica) Endosperm (partially purified) Fruits (crude) Leaves (crude)
7-mX
3-mX
1-mX
Tb
Tp
Px
X
XR
XMP
N-3
N-3
N-3
N-3
N-7
N-3
N-3
N-7
N-7
100 100 100 100
17.6 14.0 tr
4.2 20.0 4.5
nd nd 2.5
210 206 250
0.2
-
-
-
nd 16
nd 10.3 nd 56
nd
-
26.8 21.4 25 20
nd nd
100
-
-
nd
-
nd
-
-
-
Ashihara et al. (1998)
100 100 100
-
-
-
-
-
-
5.7
185 127
-
-
4.6
175
nd
nd
-
-
-
-
-
-
-
-
104
Mazzafera et al. (1994b) Roberts and Waller (1979) Schulthess et al. (1996)
-
-
Reference
Kato et al. (1999) Kato et al. (1996) Suzuki and Takahashi (1975) Negishi et al. (1985b)
CAFFEINE AND RELATED PURINE ALKALOIDS IN PLANTS
155
However, there is limited synthesis of paraxanthine from 7-methylxanthine so it is not an important methyl acceptor in vivo (Kato et al., 1996). The K , value for theobromine is high, >0.3mM, and this low affinity may explain the transient accumulation of theobromine in young tea leaves (Ashihara and Kubota, 1986). The effects of the concentration of SAM and several methyl acceptors on the activity of caffeine synthase show typical Michaelis-Mententype kinetics, and there is no feedback inhibition by caffeine. It is, therefore, unlikely that allosteric control of the caffeine synthase activity is operating in tea leaves. The K, of tea caffeine synthase for SAM (21 pM) in the presence of paraxanthine was similar to the values for 7-methylxanthine and theobromine (25pM) obtained with crude tea enzyme preparations by Suzuki and Takahashi (1975a). These figures are lower than the K, values of coffee leaf N-methyltransferase, 203 pM with 7-methylxanthine and 67 pM with theobromine, reported by Mosli Waldhauser et al. (1997a) who suggested that the presence, as a contaminant, of SAH, which is a competitive inhibitor of SAM, could be one of the reasons for the low K, values for SAM observed by other investigators. This is unlikely to be the reason for the lower K, value obtained for tea caffeine synthase as the addition of adenosine deaminase to remove SAH did not effect the K, values that were obtained by Kato et al. (1999). The discrepancy between the K , values may be due to the use of different species and/or differences in sample purity. One of the major factors affecting the activity of caffeine synthase in vitro appears to be inhibition by SAH. As shown in photo-affinity labelling studies with SAM, caffeine synthase is completely inhibited by SAH. SAH binds to most methyltransferases with higher affinity than SAM (Poulton, 1981). Therefore, control of the intracellular SAM/SAH ratio is one possible mechanism for regulating the activity of many methyltransferases, including caffeine synthase. Nothing is known about such ratios in tea or coffee but the SAM and SAH content of the leaves of 6-day-old pea seedlings is 14.6 and 0.7 nmol g-' f.w., respectively (Edwards, 1995). Maximum caffeine synthase activity is obtained at pH 8.5 (Kato et al., 1999) and similar alkaline pH optima have been reported for several chloroplast stroma enzymes (Foyer, 1984). Caffeine synthase is probably a stroma enzyme, as a study with tea has shown that paraxanthine N-methyltransferase activity is located in chloroplasts (Kato et al., 1998). It is noteworthy that there is a marked decline in caffeine synthase activity between pH 8.0 and 7.0. Upon illumination, stromal pH increases from ca 7.0 to ca 8.0 (Edwards and Waller, 1983), so it is feasible that the activity of caffeine synthase is stimulated by light. Several stromal enzymes, including ribulose-l,5-bisphosphatecarboxyhave lase, fructose- 1,6-bisphosphatase and sedoheptulose-177-bisphosphatase, alkaline pH optima and the regulation of their activities by light is an important mechanism in the control of the Calvin-Benson cycle (Foyer, 1984). However, our current results suggest that net caffeine biosynthesis in tea leaves is not influenced by illumination (See IV.E.2).
156
H. ASHIHARA and A. CROZIER
The 20 amino acid N-terminal sequence obtained for tea caffeine synthase by Kato et al. (1999) does not show similarities with the N-methyltransferase sequence from coffee endosperm reported by Mazzafera et al. (1994). However, a comparison of the amino acid sequences of Acuna et al. (1999) and Mazzafera et al. (1994) using an NIH-NCBI's BLAST search, indicates that the Mazzafera sequence of 19 amino acids shares ca 80% homology with an 11 S storage globulin from endosperm of Cog. arabica seed (personal communication, Dr Takeshi Yasuda, Kobe University). This suggests that the purified endosperm preparations of Mazzafera et al. (1994) were contaminated with the storage protein. Many N-terminal sequences have been reported for plant 0-methyltransferases but there are few relating to plant Nmethyltransferases (Joshi and Chiang 1998). Two genes encoding plant Nmethyltransferases, putrescine N-methyltransferase (Hibi et al., 1994; Walton et al., 1994; Hashimoto et al., 1998) and ribulose-1,5 bisphosphate carboxylase/ oxygenase large subunit N-methyltransferase (Klein and Houtz 1995, Ying et al. 1996), have been cloned, but the amino acid sequences of these enzymes show little homology with the N-terminal sequence of tea caffeine synthase. As has been shown with many secondary metabolism pathways (Poulton, 1981), the biosynthesis of caffeine is closely related to the stage of development and caffeine synthase activity can be detected only in young, expanding tea leaves. Detailed studies on the control of caffeine biosynthesis will be possible when caffeine synthase antibodies and the cDNA-encoding caffeine synthase protein become available. 4. N-Methyl Nucleosidase N-Methyl nucleosidase, from tea leaves, which catalyses the hydrolysis of 7methylxanthosine to 7-methylxanthine, has been purified 30-fold to a specific activity of 0.5 nkat mg-' protein by ammonium sulphate fractionation and DEAE-cellulose, hydroxyapatite and Sephadex G- 100 gel-filtration chromatography (Negishi et al., 1988). The substrate specificity of this partially purified enzyme was broad and high activity was found with several monomethyl purine nucleosides (Table XI). However, it is doubtful that the monomethyl purine nucleosides are endogenous components in tea leaves. The estimated molecular weight was 55 000 and the optimum pH 8.0-8.5. The pH optimum of the N-methyl nucleosidase is similar to that of tea caffeine synthase (Section IV.B.3), but different to the pH 4.5 optimum of adenosine nucleosidase (EC 3.2.2.7). There were several ribonucleosidase activities in the crude extract from tea leaves. Among them, adenosine nucleosidase activity, measured at pH 4.5, was ca 60 times higher than 7-methylxanthosine nucleosidase activity at pH 8.5. N-Methyl nucleosidase was readily separated from adenosine nucleotidase by DEAE-cellulose chromatography. Ribonucleosidase activity with guanosine and inosine was almost negligible, while xanthosine nucleosidase activity at pH 4.5 was approximately 30% of that of 7-methylxanthosine nucleosidase activity at pH 8.5.
157
CAFFEINE AND RELATED PURINE ALKALOIDS IN PLANTS
TABLE XI Substrate specificity of N-methyl nucleosidase and adenosine nucleosidasefrom tea leaves. N-Methyl nucleosidase and adenosine nucleosidase activities were measured at pH 7.5 and 5.5, respectively. Data presented as a per cent of the value obtained with 7methylxanthosine for N-methyl nucleosidase and adenosine for adenosine nucleosidase (nd, no activity detected) Substrate
N-methyl nucleosidase
Adenosine nucleosidase
100 198 12 8 13
nd nd nd nd 515
178
I
22 6
1
7-Methylxanthosine 3-Methylxanthosine 1-Methylxanthosine Xanthosine 7-Methyladenosine 3-Methyladenosine 1-Methyladenosine Adenosine 7-Methylguanosine 3-Methylguanosine 1-Methylguanosine Guanosine 7-Methylinosine 3-Methy linosine 1-Methylinosine
Inosine
189 32
100 nd nd
nd nd
nd
121 128 9
nd nd nd nd
5
nd
Modified from Negishi et al. (1988).
C. CAFFEINE BIOSYNTHESIS FROM PURINE NUCLEOTIDES
1. Origin of Xanthosine for Purine Alkaloid Biosynthesis
As described in Section IV.B.2, the initial methyl acceptor for caffeine biosynthesis in tea and coffee plants is xanthosine and/or XMP. It has been proposed that xanthosine and XMP can originate from: (i) adenine nucleotides; (ii) guanine nucleotides; and (iii) IMP produced via purine nucleotide biosynthesis de n o w . 2. Caffeine Biosynthesis From Adenine Nucleotides (the A M P Pathway) Higher plants lack adenine and adenosine deaminases which results in deamination of adenine-based structures at the nucleotide level (see Section 1II.C). Plant cells, however, do possess very active purine salvage enzymes which readily convert exogenously supplied purine bases and nucleosides to nucleotides. As a consequence, most recent investigations into the biosynthesis of caffeine have made use of labelled purine bases and nucleosides, such as
158
H. ASHIHARA and A. CROZIER
[8-14C]adenine and [8-'4C]adenosine, which are converted to adenylates and then used for the caffeine biosynthesis. Adenosine and adenine are produced as catabolites of SAM in plants. SAM serves as the methyl group donor in various methylation reactions, including caffeine biosynthesis, and the reaction product, SAH, is hydrolysed to adenosine and homocysteine (see Fig. 10). In addition to SAH, 5-methylthioadenosine is another important product of SAM metabolism which is involved in the synthesis of both polyamines and ethylene (see Crozier et al., 1999). In plants 5-methylthioadenosine is hydrolysed to adenine and 5'-methylthioribose by a specific 5-methylthioadenosine nucleosidase (Guranowski et al., 1981). Thus, adenosine and adenine salvage pathways, catalysed by adenosine kinase and adenine phosphoribosyltransferase, may operate in vivo. [8-'4C]Adenine and [8-'4C]adenosine are much better precursors for caffeine synthesis than other l4C-labe1led purine compounds such as hypoxanthine, guanine, xanthine, inosine, guanosine and xanthosine (Suzuki and Takahashi, 1976b; Fujimori and Ashihara, 1993;
I /
NA
co2 .............................. 0
8
1
............................
1.
16
24
Duration of incubation (h) Fig. 13. Purine alkaloid biosynthesis from [8-I4C]adenine (specific activity 2.0 GBq mmol-') in leaves of Camellia sinensis. Fourteen leaf discs obtained from young leaves harvested in May were pulsed [8-14C]adenine for 4 h and then the radioactivity was chased for a further 20 h. Incorporation of radioactivity is expressed as a percentage of total radioactivity taken up by the leaf disks (55.0 & 3.1 kBqg-' fresh weight). Nuc, adenine nucleotides; NA, nucleic acids; Cf, caffeine; Tb, theobromine; COz, carbon dioxide. The dotted line indicates unmetabolized adenine. [Redrawn from the data of Fujimori et al. (1991).]
CAFFEINE AND RELATED PURINE ALKALOIDS IN PLANTS
159
Ashihara et al., 1996a). This may be due to: (i) limited direct degradation of adenine and adenosine; and/or (ii) the efficiency of the adenine and adenosine salvage pathways. Fujimori et al. (1991) showed conversion of AMP to caffeine via theobromine in pulse-chase experiments with young tea leaves using [8-14C]adenine. The most heavily labelled compounds at 4 h were adenine nucleotides but when the radioactivity was chased, the label moved to theobromine and caffeine, and also became increasingly associated with nucleic acids and COz. More than 30% of total radioactivity taken up by the leaf segments were recovered as caffeine after a 20 h chase (Fig. 13). Similar kinetics of [8-14C]adeninemetabolism have been reported in excised tea shoots (Negishi et al., 1992). Theoretically, there are five possible pathways for xanthosine synthesis from AMP: 1. 2. 3. 4. 5.
AMP -+ IMP -+ XMP + xanthosine AMP + adenosine -+ inosine -+ xanthosine AMP -+ IMP -+ inosine --+ hypoxanthine -+ xanthine -+ xanthosine AMP -+ adenosine -+ inosine --+ hypoxanthine -+ xanthine -+ xanthosine AMP -+ adenosine -+ adenine -+ hypoxanthine -+ xanthine -+ xanthosine.
The most likely route is pathway 1, because inosine deaminase, which catalyses the conversion of inosine to xanthosine (pathway 2), adenosine deaminase which catalyses the conversion of adenosine to inosine (pathway 4), adenine deaminase which converts adenine to hypoxanthine (pathway 5 ) are usually not present in plant cells. Furthermore, higher plants contain little or no purine nucleoside phosphorylase activity, which catalyses the reversible conversion of xanthine -+ xanthosine (pathways 3-5) (Wagner and Backer, 1992; Negishi et al., 1992). Therefore, the major route from AMP to caffeine seems to be an AMP -+ IMP -+ XMP --+ xanthosine -+ 7-methylxanthosine -+ 7-methylxanthine -+ theobromine --+ caffeine pathway. This is referred to as the AMP pathway for caffeine biosynthesis. 3. Enzymes Involved in the AMP Pathway for Caffeine Biosynthesis The enzymes involved in the three-step conversion of AMP -+ IMP -+ XMP -+ xanthosine are AMP deaminase, IMP dehydrogenase and 5’-nucleotidase. IMP dehydrogenase and 5’-nucleotidase are also involved in both the GMP and de novo pathways for caffeine biosynthesis (see Sections IV.B.4 and IV.B.6). AMP deaminase. AMP deaminase (EC 3.5.4.6) has been purified from several plant sources (Table V) and its properties characterized. AMP deaminase from cultured Catharanthus roseus cells requires ATP for activity. ATP affects both the K , and VmaXof the enzyme and GTP is a potent inhibitor (Yabuki and Ashihara, 1992). In plants which do not synthesize purine alkaloids, such as Catharanthus roseus, AMP deaminase is a regulatory enzyme in adenylate catabolism, and its activity is controlled by the cellular level by
160
H. ASHIHARA and A. CROZIER
ATP (Yabuki and Ashihara, 1991). AMP deaminase also participates in the conversion of adenylates to guanylates by catalysing the first step in an AMP -+ IMP -+ XMP -+ GMP -+ GDP + GTP pathway. Inhibition of AMP deaminase activity by GMP may be useful in maintaining the proper balance between adenylates and guanylates (Yabuki and Ashihara, 1992). There are no published reports on the properties of AMP deaminase from caffeinecontaining plants although AMP deaminase activity, with a specific activity of 40 pkat g-' f.w., has been detected in desalted extracts from young tea leaves (E. Ito and H. Ashihara, unpublished data). It remains to be determined whether or not the properties of the Cum.sinensis enzyme are similar to those of AMP deaminase from Catharunthus roseus.
IMP dehydrogenuse. The catalytic activity of IMP dehydrogenase can be determined by monitoring the IMP-dependent reduction of NAD in the presence of allopurinol and NaF which inhibit xanthine dehydrogenase and phosphatase, respectively. IMP dehydrogenase activity in crude extracts from young tea leaves was 321 pkat g-' f.w. and fell to 14 pkat g-' in extracts from old leaves (Nishimura and Ashihara, 1993). IMP dehydrogenase is a very labile enzyme so purification is difficult. However, by processing extracts in the presence of glycerol, Nishimura and Ashihara (1993) were able to partially purify IMP dehydrogenase activity from young tea leaves. The K , values of tea leaf IMP dehydrogenase was 28pM, and activity was inhibited by purine nucleotides, with GMP being more effective than XMP and AMP being the least potent of the three purine nucleotides. The Ki value of the enzyme for GMP was 170 pM. Feedback inhibition of IMP dehydrogenase by GMP may have a role in maintaining the size of guanine nucleotide pools. Caffeine and theobromine had little effect on IMP dehydrogenase activity. The enzyme has a broad alkaline pH optimum (8.8-9.8) which falls off rapidly with a 60% loss of activity at pH 7.2. The properties of tea IMP dehydrogenase are similar to those of IMP dehydrogenase from Cutharanthus roseus (Nishimura and Ashihara, 1993) and, therefore, appear not be be affected by whether or not the parent plant produces caffeine. The relatively high activity of IMP dehydrogenase in young tea leaves tends to support the hypothesis that IMP dehydrogenase catalyses a key reaction in the conversion of purine nucleotides to caffeine. +
5'-Nucleotiduse. XMP is easily hydrolysed to xanthosine and Pi in tea extracts. However, vacuoles of higher plant cells contain very active nonspecific phosphatase(s) which in extracts may dephosphorylate a variety of compounds. It is thought that such phosphatases are unlikely to participate in XMP hydrolysis in tea leaves where the conversion of XMP to xanthosine is probably catalysed by a cytoplasmic 5'-nucleotidase. To date, this enzyme has not been characterized in caffeine-forming plants.
CAFFEINE AND RELATED PURINE ALKALOIDS IN PLANTS
161
4. Caffeine Biosynthesis from Guanine Nucleotides (the G M P Pathway) Although the guanine nucleotide pool is always smaller than the adenine nucleotide pool, caffeine is also produced from guanine nucleotides. There are several reports which demonstrate the biosynthesis of caffeine from [8-'4C]guanine and [8-'4C]guanosine (Ashihara et a/., 1996a, 1997a). Metabolism of [8-'4C]guanine to GMP is catalysed by hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8). Xanthosine, the initial methyl acceptor of the caffeine biosynthesis, appears be formed from GMP with guanosine acting as an intermediate and the sequential reactions are catalysed by 5'-nucleotidase and guanosine deaminase. Inosine-guanosine kinase converts limited amounts of [8-'4C]guanosine to GMP which enters the caffeine biosynthetic pathway. However, most [8-14C]guanosine enters the GMP pathway of the caffeine synthesis as an intermediate in the GMP 4 guanosine xanthosine pathway. ---f
5.
Enzymes Involved in the G M P Pathway of Caffeine Biosynthesis
5'-Nucleotidase. As explained in the subsection on '5'-Nucleotidase' in Section IV.C.3, no specific nucleotidases have yet been identified. However, tea leaf extracts do readily hydrolyse GMP to guanosine (H. Ashihara, unpublished data). Guanosine Deaminase. Negishi et al. (1992) demonstrated the presence of guanosine deaminase, which catalyses the conversion of guanosine to xanthosine in cell-free extracts from young tea leaves. The enzyme was partially purified with DEAE-cellulose and Sephadex G- 100 chromatography (Negishi et al., 1994). The guanosine deaminase activity was very labile as after precipitation with ammonium sulphate and dialysis only 6% of the original activity remained. The specific activity of final preparation was 22 pkat mg-I protein and the molecular weight estimated by gel-filtration was 18000. The optimum pH was 7.5 and the K, value for guanosine was 9.5 pM. Guanosine deaminase also catalyses the deamination of 2'-deoxyguanosine and cytidine at 54 and 30%, respectively, of the rate observed with guanosine. Tea leaf preparations contain guanine deaminase as well as guanosine deaminase, but the two enzymes are readily resolved by gel-filtration chromatography. While guanosine deaminase appears to participate in caffeine biosynthesis in tea leaves, guanine deaminase is more likely to be associated with the catabolism of guanine nucleotides as it catalyses the conversion of guanine to xanthine which enters the purine catabolism pathway and is not an effective precursor of caffeine. 6. De Novo Biosynthesis of Caffeine from Small Molecules Anderson and Gibbs (1962) and Proiser and Serenkov (1963) first demonstrated that the carbon atoms of purine skeletons of caffeine, purine nucleotides and nucleic acids are derived from the same precursors.
S A M y Xanthosine SAH
4
AICAR
(14) GAR
H H z O q '
Ribose 7
;
PRA
(Purine Alkaloid Biosynthesis)
F
Ribose
+
PRPP (1)
Ribose-SP
H 7-Methy lxanthine
SAM
SAH
CH~
SAM SAH
3,7-Dimethylxanthine (Theobromine)
CH~
1,3,7-Trimethylxanthine (Caffeine)
A
Pentose Phosphate Pathway
CAFFEINE AND RELATED PURINE ALKALOIDS IN PLANTS
163
Subsequently, there have been a number of reports on the incorporation of radioactivity into purine alkaloids from the ''C-precursors of purine biosynthesis de novo (see Suzuki et al., 1992). However, there is still little information on whether caffeine and other purine alkaloids are formed directly from an intermediate of the de n o w pathway of purine biosynthesis. Ito and Ashihara (1999) reported that young tea leaves incorporated the "N atom from ["Nlglycine into theobromine and caffeine, and that incorporation was reduced markedly by azaserine and aminopterin, inhibitors of purine biosynthesis de novo. Furthermore, the radioactivity from [2-14C]AICAR, a precursor of ZMP, was also incorporated into theobromine and caffeine. Pulse-chase experiments with ["Nlglycine and [2-14C]AICAR suggested that theobromine is the immediate precursor of caffeine. Figure 14 outlines the possible pathways of caffeine biosynthesis in tea leaves. ["IVIGlycine appears' to enter the purine biosynthetic pathway at step 3 and is then utilized for the synthesis of caffeine and theobromine. Azaserine is thought to block steps 2 and 5, and aminopterin steps 4 and 9, of the purine nucleotide biosynthesis de now. Both inhibitors reduced markedly the incorporation of ['SN]glycine into theobromine and caffeine by tea leaves. Most of the exogenously supplied [2-14C]AICAR was metabolized to its nucleotide, ZMP, seemingly via the action of adenosine kinase, a conversion that has been shown to occur in mammals (Sabina et al., 1982; Jimenez et al., 1990). Adenosine kinase has been detected in tea leaves (Negishi et al., 1992). Nucleic acid, primarily RNA, synthesis from AICAR was also detected (Fig. 1 9 , indicating that AICAR can also be converted to purine nucleotides such as ATP and GTP. In order to determine whether purine alkaloids are produced directly via the de novo pathway (steps 1-17) or from the preformed adenine nucleotide pool (Fig. 14, step 20 and steps 12-17), the effect of coformycin on the biosynthesis of purine alkaloids from [2-14C]AICAR was investigated. Coformycin is typically used as an inhibitor of adenosine deaminase in mammalian test
Fig. 14. Biosynthetic pathways of purine alkaloid operating in tea leaves. Solid arrows represent main pathways, the solid arrows broken with a vertical bar represent blocked conversions by inhibitors and dotted arrows indicate a minor route. Wide arrows indicate conversions from exogenously supplied precursors. Abbreviations of metabolites are shown in the caption to Fig. 3. Inhibitors: AMZ, aminopterin; AZS, azaserine; COF, coformycin. Enzymes: (1) PRPP synthetase; (2) amido phosphoribosyltransferase; (3) GAR synthetase; (4) GAR formyl transferase; (5) FGAM synthetase; (6) AIR synthetase; (7) AIR carboxylase; (8) SACAIR synthetase; (9) adenylosuccinate lyase; (10) ZMP formyltransferase; (11) IMP cyclohydrolase; (12) IMP dehydrogenase; (13) GMP synthetase; (14) xanthosine N-methyltransferase; (15) 7-methylxanthosine nucleosidase; (16) 7-methylxanthine N-methyltransferase; (17) theobromine N-methyltransferase; (18) adenylosuccinate synthetase; (19) adenylosuccinate lyase; (20) AMP deaminase; (21) adenosine kinase; (22) nucleoside phosphotransferase; (23) adenine phosphoribosyltransferase.
164
H. ASHIHARA and A. CROZIER
60
t
40
20
0 0
4
8
12
16
20
24
Duration of incubation (hr) Fig. 15. Distribution of radioactivity in metabolites from [2-l4C]A1CAR after a pulse-chase experiment with young tea leaves. Leaf segments were incubated with 21 p M AICAR (specific activity 0.89 GBq mmol-') for 4h, after which the radioactivity was chased for a further 20 h. Incorporation of radioactivity is ex ressed as a percentage of total radioactivity taken up by the leaves (3.8 f 0.3 kBq g-' fresh weight). AICA, 5aminoimidazole-4-carboxyamide;AICAR, 5-amino imidazole-4-carboxyamide ribonucleoside; Cf, caffeine; Tb, theobromine; NA, nucleic acids; COz, carbon dioxide. (Redrawn from the data of Ito and Ashihara (1999).]
systems (Nakamura et al., 1974; Cha et al., 1975). Adenosine deaminase is not present in most plant cells (see Section 111) where coformycin acts by inhibiting AMP deaminase (Yabuki and Ashihara, 1992). Inhibition of the conversion of AMP to IMP by coformycin did not influence significantly the rate of purine alkaloid synthesis from AICAR. It is therefore unlikely that AICAR is converted to theobromine and caffeine via the adenylate pool. Coformycin did, however, reduce slightly both the uptake and degradation of [2-14C]AICAR by tea leaf segments. Caffeine biosynthesis from IMP was confirmed in experiments with [8-'4C]inosine and [8-'4C]adenine (Fig. 16). As the presence of nucleoside phosphotransferase (Negishi et al., 1992) and IMP dehydrogenase (Nishimura and Ashihara, 1993) has been demonstrated in tea leaves, it is probable that inosine is metabolized initially to IMP, then converted to XMP and, thereafter, utilized for caffeine synthesis (Fig. 14, step 22, steps 12-17). Inosine is unlikely to be converted directly to xanthosine, as cell-free extracts from young tea leaves do not contain detectable inosine dehydrogenase activity (Negishi et al., 1992). Adenine, on the other hand, is converted to AMP by the
165
CAFFEINE AND RELATED PURINE ALKALOIDS IN PLANTS (A) Theobromine
(B) Nucleotides r T
........*........
750
500
p
,.." 250
0 0
10
20
30
40
0
10
20
30
40
Time of incubation (min)
Fig. 16. Incorporation of radioactivity from [8-14C]inosine and [8-I4C]adenine into theobromine (A) and nucleotides (B) in young tea leaves. Leaf se ents were incubated with 8.9pM [8-I4C] inosine (specific activity 1.9GBqmmol- ) or 9.6pM [S-'4C] adenine (specific activity 2.1 GBqmmol-I). (Redrawn from the data of Ito and Ashihara (1999).]
gn
action of adenine phosphoribosyltransferase (step 23) and IMP derived from AMP (step 20) then enters the purine alkaloid biosynthetic pathway (steps 1217). The data from short-term experiments shown in Fig. 16 suggest that newly synthesized IMP is utilized directly for theobromine biosynthesis in young tea leaves. These results indicate that caffeine is synthesized via theobromine from purine nucleotides synthesized de novo. Incorporation of [2-'4C]AICAR into the intermediates could not be detected so the pool sizes of the compounds in the pathway between ZMP and theobromine (steps 10-17) must be very small and, because of the relatively large amounts of theobromine and caffeine that accumulate, probably subject to a rapid rate of turnover. Several enzymes related to the de novo purine pathway and caffeine biosynthesis are located in chloroplasts. These are 5-phosphoribosyl-~-pyrophosphate synthetase (step 1: Ashihara, 1990b), 5-phosphoribosyl-Lpyrophosphate amidotransferase (step 2: Doremus and Jagendorf, 1984), 5'nucleotidase (step 13: Eastwell and Stumpf, 1982), and 3-N-methyltransferase (step 16: Kato et al., 1998). Although based on fragmentary data, these observations imply that chloroplasts are the subcellular site for caffeine biosynthesis from the de novo pathway. There are other examples of the localization of enzymes related to alkaloid biosynthesis in chloroplasts (Wink
166
H. ASHIHARA and A. CROZIER
et al., 1980; Roberts, 1981; Wink and Hartmann, 1982; Wink, 1997b). Although it is difficult to rule out the participation of caffeine biosynthetic pathways from preformed purines, the de novo pathway is probably the main route to caffeine, especially in young tea leaves, in which a very rapid net accumulation of purine alkaloids is observed. D. PURINE ALKALOID BIOSYNTHESIS IN
THEOBROMINE-ACCUMULATINGPLANTS
Theobromine, rather than caffeine, is the predominant purine alkaloid in Zlex paraguariensis, Theobroma cacao, Camellia irrawadiences and Cam. ptilophylla (Section 11) but reports on theobromine biosynthesis are restricted to the two Camellia species. I . Camellia irrawadiensis Camellia irrawadiensis tissues contain theobromine and little or no caffeine, and young leaves convert both [8-14C]adenine and [8-14C]hypoxanthine into theobromine, without detectable incorporation of label into caffeine (Ashihara and Kubota, 1987). A similar conversion of [8-14C]adenine also occurs in stamens and petals isolated from Cam. irrawadiensis flower buds where ca 40% of the radioactivity taken up by these organs was recovered as theobromine (Fujimori and Ashihara, 1990). The obvious explanation for the lack of caffeine in Cam. irrawadiensis is limiting theobromine N-methyltransferase activity. However, attempts to obtain experimental data on this point have been thwarted because, for presumably technical reasons, preparations from the thick-walled young leaves of Cam. irrawadiensis do not contain any detectable N-methyltransferase activity.
TABLE XI1 Levels of endogenous purine alkaloidr in young, mature and aged leaves of cocoa tea (Camellia ptilophylla). Data expressed ar pmolg-' dry weight f standard deviation ( n = 6 ) (nd, not detected) Purine alkaloid
Young leaves
Theobromine
145.4 f 1.2 9.4 f 0.4
Theophylline Paraxanthine Caffeine
nd nd
Data from Ashihara et al. (1998).
Mature leaves
Aged leaves
55.5 f 3.3 1.1 f 0.1
48.8 f 2.2 3.9 f 0.2 nd nd
nd nd
CAFFEINE AND RELATED PURINE ALKALOIDS IN PLANTS
167
2. Camellia ptilophylla Caffeine is not present in detectable quantities in leaves of Cam. ptilophylla (cocoa tea) which contain theobromine, the highest concentrations of which, together with trace amounts of theophylline, are present in young expanding leaves (Table XII) (Ashihara et al., 1998). Incubation of young Cam. ptilophylla leaves with [8-14C]adenine resulted in 10% of the absorbed radioactivity being recovered as theobromine, while there was a 10-fold reduction in theobromine synthesis in 1-year-old leaves. Despite containing endogenous theophylline, the young Cam. ptilophylla leaves did not convert [8-14C]adenine into radiolabelled theophylline in detectable quantities (Ashihara et al., 1998). The substrate specificity of Cam. ptilophylla N-methyltransferase activity was examined using gel-filtrated extracts from young leaves. Activity was detected with 7-methylxanthine as a methyl acceptor, but not with dimethylxanthines, i.e. theobromine, theophylline and paraxanthine (see Table X). This indicates that the major caffeine biosynthesis pathway via theobromine is blocked, and this was confirmed when it was shown that leaf segments of Cam. ptilophylla could not convert [2-I4C]theobromine to caffeine. The substrate specificity of the Cam. ptilophylla N-methyltransferase activity is such that as well as the main biosynthesis pathway, the alternative minor routes to caffeine, via substrates such as paraxanthine (see Fig ll), are also likely to be blocked. Interestingly, leaves of Cam. ptilophylia possess enzymes which convert caffeine to theobromine (see Section V.B.4), so even if a small amount of caffeine were synthesized, it would be immediately converted to theobromine. E. PHYSIOLOGICAL STUDIES ON CAFFEINE BIOSYNTHESIS
1. Effect of Age of Tissues on Caffeine Biosynthesis It has been observed that active synthesis of caffeine and related purine alkaloids is found only in young tissues of Camellia, Coffea and Ilex. Although only limited data are available, this appears to be due primarily to the presence of high levels of the N-methyltransferase activities involved in purine alkaloid biosynthesis. Camellia sinensis seedlings. Ashihara and Kubota (1986) examined the concentration of endogenous caffeine and related purine alkaloids, and estimated the capacity for caffeine biosynthesis from the incorporation of radioactivity from [8-14C]adenine into purine alkaloids in leaves, stems, cotyledons and roots of 4-month-old Cam. sinensis seedlings with nine leaves. More than 99% of the [14C]caffeinethat was synthesized accumulated in the leaves. Incorporation of label into theobromine was found only in the upper four leaves, while I4C-labelled theophylline and paraxanthine were either undetectable or present in only trace amounts. Prior to germination the
168
H. ASHIHARA and A. CROZIER
caffeine content of tea seeds was 2 pmol per seed with highest levels in the seed coat. This compares with 24pmol caffeine per seedling observed in 4-monthold plants. The young tea seedlings are therefore clearly synthesizing endogenous caffeine. The biosynthetic capacity for caffeine estimated with [8-'4C]adenine was six-eight times higher in the youngest leaves than in older more basal leaves at positions 5 and 8 below the apex. The caffeine content per leaf, as opposed to the concentration of caffeine g-' of leaf, is higher in older than in young expanding leaves. Caffeine is synthesized rapidly and accumulates in young leaves and, thereafter, as the leaves mature, the rate of caffeine biosynthesis declines but seemingly does not cease completely. Hence, as caffeine is metabolized extremely slowly in the young seedlings, the amount of caffeine in the leaf continues to increase, albeit very gradually, as the leaf matures. Seasonal variations in leaves of Camellia sinensis. Month-to-month variations in the caffeine content of tea leaves have been reported by Suzuki and Waller (1986), while seasonal variations in the capacity of leaves to synthesize purine alkaloids have been investigated by Fujimori et al. (1991) using material obtained from cultivated tea bushes grown under field conditions. In Japan, new shoots begin to emerge from winter buds in late March and early April, and continue to elongate until October. Levels of caffeine in these flush shoots increased to a maximum value of 34.5 f 2.1 pmolleaf' in July and then decreased. The concentration of caffeine in tea leaves was highest in the spring in May (52.9 f 4.9pmolg-') and lowest (22.1 f 8.9pmolg-I) in February. Figure 17 shows seasonal variations in the capacity of tea leaves to convert [8-I4C]adenine into purine alkaloids (caffeine plus theobromine), nucleic acids (RNA plus DNA) and COz. The rates of incorporation of [8-I4C]adenine into purine alkaloids were highest in April and May, but declined markedly in June and no incorporation was observed thereafter. This finding confirms that the biosynthesis of caffeine occurs primarily young tea leaves. Incorporation of [8-14C]adenine into nucleic acids was also high in April and May, declined in June, was reduced further in July and then continued at a similar level for the duration of the experiment. The release of I4CO2 from [8-I4C]adenine, which is an index of the extent of the catabolism of adenine nucleotides, was highest in the young leaves harvested in April. It is noteworthy that the rate of release of 14C02was severely reduced in leaves harvested in May in which the rates of incorporation into purine alkaloids and nucleic acids were still high. It is unclear whether the catabolism of adenine nucleotides was very active in the leaves in April. However, this ambiguity may be a reflection of the rapid turnover of nucleic acids and adenine nucleotides in the leaves. These data demonstrate that the young leaves have a high capacity for caffeine biosynthesis when they emerge in the spring which diminishes after 3 months as the leaves mature. The regulation of the rate of caffeine production, therefore, appears to be achieved through the control of the amount and/or
CAFFEINE AND RELATED PURINE ALKALOIDS IN PLANTS
APR
MAY
JUN
JLJL
AUG
SEP
(XT NOV
DEC
(B)
+ hthosioe
JAN
F'EB
MAR
169
APR
7-Methyinanthine
* Thcobromine
G AUG
+
$
SEP
OCT
;
;
e
NOV
DFC
+ JAN
; FEB
; MAR
APR
Time of year (months)
Fig. 17. Seasonal variations in biosynthetic capacity for the s nthesis of caffeine in tea leaves. The rate of incorporation of radioactivity from [S- 74C ] adenine (specific activity 2.0 GBq mmol-') into purine alkaloids (theobromine plus caffeine), nucleic acids and carbon dioxide in tea leaf disks (A) and the levels of N-methyltransferase activity with xanthosine, 7-methylxanthine and theobromine in the extracts from leaves (B) harvested on the first day of every month. [Redrawn with permission from the data of Fujimori et al. (1991).]
activity of the N-methyltransferases associated with purine alkaloid biosynthesis. The induction and repression of these enzymes, arguably, provides the primary control mechanism with a secondary control being imposed by the availability of substrates, such as xanthosine and SAM. Flowers of Camellia sinensis. The flowers of Cam, sinensis contain purine alkaloids with caffeine present both in the stamens and in the petals, and theobromine accumulating in detectable quantities in the stamens (Suzuki,
170
H. ASHIHARA and A. CROZIER
1985, Fujimori and Ashihara, 1990). In order to examine whether the biosynthesis of caffeine occurred within the flowers themselves or was transported to the flowers from leaves, the metabolism of [8-'4C]adenine in stamens and petals excised from flower buds was investigated by Fujimori and Ashihara (1990). During a 48-h incubation, there was a 30% incorporation of label into caffeine and traces of theobromine by stamens and 20% by petals. Incorporation decreased to 19 and lo%, respectively, when the stamens and petals were obtained from flowers that had opened. Caffeine is, therefore, synthesized in stamens and petals, and the capacity is higher prior to the opening of the flower bud. Fruit of Camellia sinensis. Flowering of tea plants is usually observed in November in Japan. After abscission of petals and stamens, the fresh weight of fruits does not change, initially, but in the following spring increases rapidly until the fruits are fully ripe and desiccated. The seeds are shed in November and the dried pericarp, including its pedicel, is lost by abscission in December (Suzuki and Waller, 1985). In parallel with the growth of the fruit, the levels of theobromine and caffeine increase markedly during the growing season (Suzuki and Waller, 1985). The capacity for caffeine biosynthesis in tea fruits was also examined using segments of fruits harvested at 1- or 2-month intervals during their development (Terrasaki et al., 1994). Figure 18 shows seasonal
--O- Pericarp Seedcoat
APR MAY JUN
JLY AUG SEP OCT
Fig. 18. Seasonal variations in biosynthetic capacity for the s nthesis of caffeine in tea fruits. The rate of incorporation of radioactivity from [8- y4C] adenine (specific activity 2.0 GBq mmol-') into purine alkaloids (theobromine plus caffeine) are shown. In April and May, whole fruits were used because they consisted only of pericarp and very small embryos. The data are plotted as figures for the pericarp. Similarly, in June the value for whole seeds is shown as the value for the seed coat. [Redrawn from the data of Terrasaki et al. (1994).]
CAFFEINE AND RELATED PURINE ALKALOIDS IN PLANTS
171
variations in the rate of incorporation of radioactivity from [8-14C]adenine into purine alkaloids (theobromine plus caffeine). The highest rate of incorporation of radioactivity from [8-I4C]adenine to purine alkaloids was observed in fruits harvested in May with > 56% of the total radioactivity taken up by the fruits being converted to purine alkaloids. A significant rate of purine alkaloids biosynthesis was also observed in fruits harvested in April, although the rate was lower than that in May. From July onwards, the rate of synthesis of purine alkaloids in the pericarp declined gradually and was at its lowest in October. Purine alkaloids biosynthesis was also observed in seed coats and cotyledons. From these results, it is evident that caffeine biosynthesis occurs in young tissues of all parts of tea fruits, with the pericarp exhibiting a particularly high biosynthetic capacity. Leaves of Coffea arabica and Ilex paraguariensis. Frischknecht et al. (1986) examined purine alkaloid formation in buds and developing young leaves of Cof. arabica. [Frischknecht et aL(l986) used the term 'leaflet' for the very young small-size leaf in all their papers. However, the botanical term 'leaflet' means one of the small leaf-like division of a compound leaf (see Sugden, 1984). Therefore, the term young leaf will be used in this review.] Once the young leaf begins to emerge from the bud, there is an acceleration in purine alkaloid formation but as the leaf matures the rate of caffeine biosynthesis decreases exponentially. Fujimori and Ashihara (1994) compared the metabolism of [8-I4C]adenine in young (small) and mature (fully developed) leaves from 1-year-old Cof. arabica plants and detected incorporation of label into theobromine and caffeine only in young leaves. Similar experiments were carried out with young, mature and aged leaves from older Cof. arabica plants using not only [8-I4C]adenine, but also [8-I4C]guanine, [8-'4C]xanthosine and [2-14C]theobromine as precursors for caffeine biosynthesis (Ashihara et al., 1996a). Active biosynthesis of caffeine from [8-I4C]adenine, [8-I4C]guanine and [8-'4C]xanthosine occurred only with young leaves. In contrast, the rate of conversion of [2-I4C]theobromine to caffeine was not influenced by leaf age to the same extent, with incorporation of label into caffeine being reduced 34% in mature leaves and 67% in aged leaves. This implies that the capacity of leaves to carry out 1-N-methylation of theobromine does not decline with age to the same extent as other sections of the purine alkaloid biosynthesis pathway. Studies with matC have been more limited but have demonstrated that only young leaves can convert [8-14C]adenine to theobromine and caffeine (Ashihara, 1993). Fruit of Coffea arabica. Suzuki and Waller (1984a) demonstrated that caffeine biosynthesis from [methyl-'4C]methionine is higher in immature green coffee fruits than in red mature fruits. Radiolabelled caffeine applied to the pericarp of Cof. arabica fruit is translocated to the seed, implying that caffeine is synthesized in pericarp and transported to the seeds (Baumann and Wanner, 1972; Keller et aZ., 1972). However, it is has also been reported that 1-N- and 3-
172
H. ASHIHARA and A. CROZIER
N-methyltransferase activities for caffeine biosynthesis are present in the liquid endosperm of immature fruits of CoJ arabica (Mazzafera et al., 1994a, b; Gillies et al., 1995). 2. Effect of Light on Caffeine Biosynthesis Light is not essential for the biosynthesis of caffeine because net caffeine synthesis has been observed in seedlings of coffee and tea after germination in the dark (Baumann and Gabriel, 1984; Suzuki and Waller, 1985b). In both instances, the caffeine concentration was slightly higher in dark-grown seedlings. A study in which segments of young leaves from light-grown Cam. sinensis plants were incubated with [8-'4C]adenine in light and darkness indicated that light has little effect on the rate of caffeine biosynthesis. Incorporation of label into theobromine and caffeine, respectively, was 6.9 f 1.0% and 45.4 f 0.8% in the dark, and 4.8 f 0.3 and 45.8 k 1.6% in the light (E. Ito and H. Ashihara, unpublished data).
TABLE XI11 Overall metabolism of 9.1 p M [8-I4C]adenine in naturally grown and shaded young tea leaves. Duration of incubation was 2 and 18 h. The rates of incorporation of radioactivity into individual metabolites are expressed as kBqg-' fresh weight f standard deviation and percentage of total radioactivity taken up by the leaf segments (parentheses) (nd, not detected)
Metabolite ATP + ADP + SAM AMP Theobromine Caffeine Allantoin Allantoate Adenine Nucleic acid COZ Total uptake
Naturally grown leaves 2h 18h 8.5f0.6 (30.4)
6.7f1.3 (5.1)
2h
Shaded leaves 18h
14.0f 1.5 (47.1) 24.1 h0.7 (15.4)
0.8ItO.l (2.9) 0.9f0.04 (0.7) 6.6h1.0 (24.1) 13.7f2.2 (10.3)
0.5f0.02 (1.7) 1.5f0.3 (5.1)
1.7zk0.8 (6.2) 35.1zk1.4 (26.4) nd 0.2 f 0.1 (0.1) 0.8 f 0.08 (2.9) 0.6 f 0.02 (0.3) 0.7f0.02 (2.6) 0.8zkO.l (0.5) 6.9It1.4 (25.1) 32.4313.4 (24.4)
1.2h0.5 nd 0.9 It0.2 0.5zk0.1 7.9f0.7
1.3zk0.1 (0.8) 8.8It0.1 (5.6)
(4.0)
24.0h3.2 (15.3) 0.5 f 0.1 (0.3) (3.0) 0.7 f 0.02 (0.4) (1.7) 0.7hO.l (0.4) (26.6) 61.7h6.5 (39.3)
0.5f0.1 (1.8) 41.7f1.4 (31.4) 1.8f0.5 (6.1) 34.61t3.0 (22.1) 133.0zk 1.0 29.7 zk 2.4 156.9zk 1.2 27.4 f 1.O
From A. Kato and H. Ashihara (unpublished data).
CAFFEINE A N D RELATED PURINE ALKALOIDS IN PLANTS
173
Caffeine synthase is located in the chloroplasts of young leaves of Cam. sinensis (Kato et al., 1998). The activity of this enzyme has been measured in preparations from naturally grown young green leaves and partially etiolated leaves obtained by shading plants with layers of black cheesecloth for 13 days. Caffeine synthase activity in the etiolated leaf extracts was < 20% of the activity obtained from light-grown green leaves. The capacity for caffeine biosynthesis determined by the incorporation of [8-I4C]adenine into caffeine was also reduced significantly in etiolated leaves (Table XIII). This could be a consequence of loss of chloroplast function in the shaded leaves. F. OTHER ROUTES
Nazario and Lovatt (1993b,c) presented a novel hypothesis that in leaves of Cof. arabica separate de novo and salvage purine pools are involved in the biosynthesis of theobromine but not caffeine, which they proposed was derived from xanthine rather than theobromine. They used fully expanded leaves, equivalent to mature leaves defined by Ashihara et al. (1996a), which were incubated with 2-5 mM doses of l4C-labe1ledprecursors. This is an exceedingly high substrate level, ca 10&1000-fold higher than is used typically in such studies and well in excess of endogenous pool sizes. Furthermore, no kinetic data were presented and the overall metabolism of added precursors was not investigated. There are also a number of instances where both the experimental design and interpretation of data by Nazario and Lovatt (1993b) were somewhat simplistic. For example, in studies with [14C]formate,a precursor for de novo purine biosynthesis, incorporation into theobromine was reduced by adenosine and adenine, but not by XMP, inosine, hypoxanthine and xanthine, while incorporation of [8-14C]adenine and [8-14C]adenosine into theobromine was inhibited by XMP, inosine, hypoxanthine and xanthine. On the basis of these observations it was concluded that theobromine was generated from two separate pools. If the unlabelled purines added together with the radiolabelled substrate were stable and not metabolized by the Cof. arabica leaves, this hypothesis might be more plausible. However, as described in Section 111, this is clearly not the case. Adenosine and adenine, for instance, are converted rapidly to adenine nucleotides and, at the higher adenine concentrations, AMP accumulates (Figs 5 and 7). AMP is a feedback inhibitor of amidophosphoribosyltransferase, the first enzyme of de no vo purine biosynthesis (Fig. 3), and Hirose and Ashihara (1984~)have reported that 0.1-1.0mM adenine reduced the incorporation of ['4C]formate into total purines by > 70% in cultured Cutharanthus roseus cells. The inhibition of the incorporation of [14C]formateinto both theobromine and caffeine by adenine and adenosine is, therefore, easily explained. Other compounds used by Nazario and Lovatt (199313) are also likely to have been metabolized extensively with significant quantities of XMP, xanthosine, inosine, hypoxanthine and xanthine eventually
174
H. ASHIHARA and A. CROZIER
entering the purine catabolism pathway and being degraded to C 0 2 and NH3. Utilizing PRPP and ATP, hypoxanthine and inosine will also be salvaged by hypoxanthine/guanine phosphoribosyltransferase and inosine/guanosine kinase and converted to IMP. As a consequence, there is likely to be reduced salvage of adenine and adenosine due to depletion of PRPP and ATP (Fig. 5, steps 1 and 3). In view of these complications, it is impossible to come to any realistic conclusions about the incorporation of the 14C precursors into theobromine and caffeine Nazario and Lovatt (1993b) argued that theobromine is not the immediate precursor of caffeine but an end product derived from separate de n o w and salvage purine pools. The theobromine concentration in mature Cof. arabica leaves is very low, ca 0.13mgg-' f.w., so if theobromine were an end product the two pathways involved in its biosynthesis would be extremely unimportant in quantitative terms. However, contrary to the proposals of Nazario and Lovatt (1993b), there is a wealth of data, including pulse-chase experiments with [8-'4C]adenine in young coffee leaves (Ashihara et al., 1996a) and with [15N]glycine,[2-I4C]AICAR and [8-14C]adenine in tea leaves (Fujimori ef al., 1991; Ito and Ashihara, 1999), establishing unequivocally that theobromine is the direct precursor of caffeine biosynthesis from both de n o w and salvage purine synthesis.
V. METABOLISM OF PURINE ALKALOIDS IN PLANTS Caffeine is degraded at a variety of rates in different species of higher plants via a series of demethylation steps that are in a different sequence from the methylation steps of the biosynthesis pathway from xanthosine. There is evidence of diversity in the caffeine catabolism pathways operating in different plant species and, in some species of Coffea, caffeine undergoes further methylation and oxidation rather than being demethylated. A . CATABOLISM OF CAFFEINE AND RELATED COMPOUNDS
In studies using radiolabelled caffeine and paper chromatography, Kalberer (1965) demonstrated catabolism of caffeine in ageing leaves of Cof. arabica. Suzuki and Waller (1984a, b) identified theophylline and theobromine as the first degradation products of caffeine in immature and mature Cof. arabica fruits with the main pathway proceeding via theophylline. Subsequently, more sophisticated analysis with HPLC-radiocounting and gas chromatographymass spectrometry demonstrated that degradation via theophylline is the major route for caffeine catabolism in leaves of Cof. arabica (Ashihara et al., 1996b). This study also demonstrated that theobromine is not a direct catabolite of caffeine and that catabolism proceeds via the caffeine -+ theophylline 4 3methylxanthine -, xanthine pathway (Fig. 19). Several demethylases may
CAFFEINE AND RELATED PURINE ALKALOIDS IN PLANTS
175
participate in the sequential demethylation reactions, but as yet no enzyme has been demonstrated in plant species. In the bacterium Pseudomonas putida, caffeine degradation occurs via a different route, with caffeine being converted to theobromine in the following reaction which is catalysed by a caffeine demethylating mono-oxygenase (Asano et al., 1994): Caffeine + 0
2
+ NADH + Hf
-,Theobromine
+ H20 + NAD' + HCHO.
Leaves of Cam. sinensis, Cam. irrawadiensis and math degrade caffeine in a manner similar to Cof. arabica, but unlike Cof. arabica they also operate a salvage pathway via which theophylline can be reutilized for caffeine biosynthesis (Ashihara et al., 1997b; Ito et al., 1997). The methyluric acids, theacrine, liberine and methylliberine, have been identified in leaves of Cof. dewevrei, Cof. liberica and Cof. abeokutae (Wanner et al., 1975; Petermann et al., 1977; Peterman and Baumann, 1983). In these species, young plants accumulate caffeine, but in later stages of plant development caffeine is gradually replaced by the methyluric acids (Pertermann and Baumann, 1983) which are probably produced by a caffeine -, theacrine + methylliberine -+ liberine pathway (Fig. 19). B. DIVERSITY OF CAFFEINE METABOLISM
The outline provided above indicates that there is significant species-to-species variation in the manner in which caffeine is catabolized. In order to appreciate fully the qualitative and quantitative variations a more detailed picture will now be provided for the relevant species.
I . Coffea arabica Caffeine accumulates in leaves and fruits of Cof. arabica in concentrations of ca 1% d.w. Young leaves and buds have a high capacity for caffeine biosynthesis which, as mentioned earlier, declines with age. Although endogenous caffeine levels decrease as leaves mature, substantial quantities remain in older leaves. The available evidence suggests this is because Cof. arabica leaves have a very limited capacity for caffeine catabolism and, as a consequence, most caffeine that is produced accumulates and is not subjected to active turnover (Suzuki and Waller, 1988; Suzuki et al., 1992; Fujimori and Ashihara, 1994; Mazzafera et al., 1994a; Ashihara et al., 1995). Biodegradation of caffeine to xanthine, which is further catabolized by the purine catabolism pathway via urate to C 0 2 and NH3, was first demonstrated in Cof. arabica leaves by Kalberer (1964, 1965). Subsequently, Suzuki and Waller (1984a, b) showed that theophylline and theobromine were the first degradation products of caffeine in Cof. arabica fruits. Mazzafera et al. (1991) also demonstrated varying degrees of catabolism of caffeine to both
i (9) H3c,N\5cH3 OAN N
cH3
Caffeine
\ -
a
(1)
-
H3c-N5gJ O N N CH3 Theophylline
(7 '-*.,
(5. ,*
0
,CH3
,,"
..
,*'
*
. . :.: ,' '. '.
H
Xanthe
,,*' (8)
'4
H3c 'N
5
a
O N
CH3 Theobromine
#
3-Methylxanthine I
N>
Uric acid
Allantoin
N
H 1 -Methylxanthine
Allantoate
NH,
+ COZ
CAFFEINE AND RELATED PURINE ALKALOIDS IN PLANTS
177
theobromine and theophylline by immature fruits of Cof. arabica and other species of coffee. Ashihara et al. (1996b) reported on detailed investigation of caffeine catabolism in which Cof. arabica leaves were incubated with a number of 14Clabelled purine alkaloids. The release of I4CO2 from [ l-methyl-14C]-, [3methyl-14C]- and [2-'4C]caffeine by mature leaves was 1.0, 0.1 and 0.03% of total radioactivity taken up by the leaves, respectively, when the leaves were incubated for an 18-h pulse followed by a 24-h chase. Caffeine is clearly catabolized very slowly by leaves of Cof. arabica. [2-14C]Theobromine was converted to [14C]caffeineand small quantities of 14C02by young, mature and aged leaves of Cof. arabica. Thus, as anticipated, theobromine acts primarily as the immediate precursor of caffeine. Although relatively small, at 5% of the recovered radioactivity, the amount of 14C02 released from mature leaves incubated with [2-'4C]theobromine was much greater than the 0.03% observed in equivalent incubations with [2-14C]caffeine. This implies that a minor portion of the applied [2-'4C]theobromine was not converted to caffeine but instead was probably subjected to demethylation, yielding xanthine, which was further catabolized to 14C02. In contrast to the very slow metabolism of caffeine, [8-'4C]theophylline is catabolized very rapidly (Table XIV). More than 70% of the [8-'4C]theophylline taken up by young, mature and aged Cof. arabica leaves was recovered as 14C02. Radioactivity was also associated with allantoate, allantoin, xanthine, 3-methylxanthine and 7-methylxanthine. Incorporation of radioactivity into 3methylxanthine was highest in young leaves while the accumulation of xanthine and 7-methylxanthine was greatest in aged leaves. The inclusion of 5mM
Fig. 19. Metabolism of caffeine in Coffea and Cam. sinensis plants. Solid arrows indicate the main pathway (steps 1-3) and dotted arrows minor routes which have been observed in a limited number of species. Caffeine is catabolized to theophylline in Cof. eugenioides but this conversion is the rate-limiting reaction of caffeine degradation in Cof. arabica, Cof. bengalensis and Cam. sinensis. Theophylline is rapidly catabolized to xanthine (steps 2 and 3) by Cof. arabica and Cof. eugenioides, and also by Cof. bengalensis, but at a slower rate. Theophylline as well as caffeine degradation is blocked in Cof. salvatrix. Most xanthine enters the purine catabolic pathway and is degraded to CO1 and N H 3 . In Cof. arabica small amounts of xanthine are remethylated to form 7-methylxanthine (step 6 ) . In Cam. sinensis, theophylline is demethylated to xanthine (steps 2 and 3) and degraded via the purine catabolism pathway, but there is also resynthesis of caffeine via a theophylline + 3-methylxanthine + theobromine + caffeine salvage pathway (steps 4 and 5). Small amounts of xanthine can converted to 3methylxanthine and salvaged for the resynthesis of caffeine via this route. In Cof. dewevrei, Cof. liberica, and Cox abeokutae, caffeine is converted to theacrine, methylliberine and liberine (steps 9-1 1). Conversion of methylxanthines is catalysed by at least two different N-methyltransferases and by demethylase(s). Demethylase and enzymes which participate in the formation of methyluric acid have not yet been demonstrated in any plant species.
TABLE XIV Summary of [8-14C]theophylline metabolism by young, mature and aged leaves of Coffea arabica in the absence or presence of 5 m M allopurinol. Segments of leaves were incubated with 7.0pM [8-14C]theophylline for 18h and then radioactivity was chased for a further 24 h. Incorporation of radioactivity into metabolites is expressed as percentage of total uptake f standard deviation ( n = 3 ) (Tp, theophylline; 3-mX, 3-methylxanthine; 7-mX, 7-methylxanthine; X , xanthine; Aln, allantoin; Ala, allantoic acid; nd, not detected) Leaf type
Treatment
Tp
3-mX
7-mx
X
Aln
Ala
co2
Control Allopurinol
nd nd
2.2 f 0.2 5.0 f 0.2
nd 46.7 f 1.5
0.8 f 0.1 28.3 f 1.0
4.2 f 0.7 nd
6.3 f 0.2 nd
80.0 f 2.5 2.5 f 0.2
Control Allopurinol
nd nd
17.8 f 0.8 52.0 f 2.5
nd 18.8 f 0.8
1.3 f 0.0 45.3 f 2.5
20.5 f 0.7 nd
7.5 f 0.2 nd
88.9 & 4.2 3.8 f 0.2
Control Allpurinol
nd nd
5.8 h 0.2 10.8 f 0.5
9.7 f 0.2 86.4 f 4.2
5.8 f 0.1 10.8 f 0.5
3.3 f 0.1 nd
7.5 f 0.2 nd
90.0 f 4.7 1.7 f 0.1
Young
Mature
Aged
Data from Ashihara et al. (1996b).
CAFFEINE A N D RELATED PURINE ALKALOIDS IN PLANTS
179
allopurinol in the incubation medium had major effects on [8-'4C]theophylline metabolism. The production of 14C02declined dramatically, as a consequence of xanthine degradation being blocked, and there were concomitant increases in the incorporation of label into xanthine, 3-methylxanthine and 7methylxanthine. It is noteworthy that > 70% of the radioactivity recovered from aged leaves treated with allopurinol was incorporated into 7-methylxanthine. While the presence of 3-methylxanthine and xanthine as catabolites of theophylline is to be anticipated, the detection of 7-methylxanthine, especially in such large amounts, was unexpected as its role in Cof. arabica is as a precursor of theobromine in the caffeine biosynthesis pathway (see Fig. 11). [2-'4C]Xanthine underwent extensive metabolism with > 80% of the radioactivity recovered after incubation with young, mature and aged leaves being released as I4CO2.The remainder of the radioactivity was in the form of residual [2-14C]xanthine together with allantoin and allantoate. Inclusion of 5mM allopurinol in the incubation medium resulted in a large reduction in I4CO2 production, a larger pool of unmetabolized [2-14C]xanthine and a marked enhance in the accumulation of radiolabelled 7-methylxanthine. This demonstrates that xanthine is the immediate precursor of 7-methylxanthine (Fig. 19). The results obtained by Ashihara et al. (1996b) demonstrate that catabolism of caffeine in Cof. arabica leaves involves a caffeine + theophylline -, 3methylxanthine -+ xanthine pathway. Xanthine is further degraded by the conventional purine catabolism pathway to C 0 2 and NH, via uric acid, allantoin and allantoate. The very slow degradation of [2-14C]caffeine, compared with that of [8-'4C]theophylline which is accompanied by a high incorporation of label into 14C02, indicates that the conversion of caffeine to theophylline is a major rate-limiting step in the catabolism of caffeine and provides a ready explanation for the high endogenous caffeine content of Cof. arabica leaves. The feeds with [2-14C]theobromine to Cof. arabica leaves indicate that its main role in caffeine biosynthesis is as the immediate precursor rather than a catabolite of caffeine. As well as being degraded to C02 via the purine catabolism pathway, xanthine was converted to 7-methylxanthine (Fig. 19). The function of 7-methylxanthine in purine alkaloid metabolism was previously thought to be limited to that of an intermediate between 7methylxanthosine and theobromine in the caffeine biosynthesis pathway (Fig. 11). There would appear to be some form of subcellular compartmentation in operation as, in the biosynthetic pathway, 7-methylxanthine formed from 7methylxanthosine is converted to theobromine and caffeine whereas 7methylxanthine derived from xanthine is not salvaged for caffeine biosynthesis. 2. Low Caffeine-containing Coffea Plants Catabolism of caffeine has been compared in leaves of Cof. arabica and three low caffeine-containing species of coffee, Cof. salvatrix, Cof. eugenioides and
TABLE X V Summary of [8-14Cjcaffeine metabolism by young and mature leaves of Coffea eugenioides, Coffea salvatrix and Coffea bengalensis. Segments of young leaves were incubated with 8.9 p M [8-I4C]caffeine for 18 h. Incorporation of radioactivity into metabolites is expressed as percentage of total uptake f standard deviation (n = 3 ) . Total uptake of radioactivity is expressed as kBqg-' of leaf (fresh weight) (CJ caffeine; Tp, theophylline; 3-mX 3-methylxanthine; I-mX, I-methylxanthine; X , xanthine; nd, not detected)
Species
Leaves
Coffea salvatrix Young Mature Coffea eugenioides Young Mature Coffea bengalensis Young Mature
Cf
100fO.O 99.7k0.1
TP
3-mx
1-mx
X
Ureides
Urea
nd nd
nd nd
nd nd
nd nd
nd nd
nd nd
1.8h0.2 4.1hO.O
0.6f0.5 1.5k0.1
2.0h0.1 4.5h0.1
nd nd
nd nd
nd nd
23.6*2.5 18.5f0.7
37.6h 1.3 14.3*0.9
100hO.O 96.5h1.0
nd 3.3h1.0
From Ashihara and Crozier (1999).
21.6f0.1 9.1k 0.5 29.7~k1.4 11.6k0.3 nd nd
nd nd
COZ
Total radioactivity recovered (kBq + SD)
nd 0.3hO.l
25.3 f2.9 45.2f0.1
0.8kO.O 13.5h1.8
91.5h3.2 80.957.2
nd 0.2f0.1
49.1 h6.4 42.8h3.2
CAFFEINE A N D RELATED PURINE ALKALOIDS IN PLANTS
181
Cof. bengalensis (Ashihara and Crozier, 1999). Data on the catabolism of [8-'4C]caffeine by young and mature leaves from Cof. salvatrix, Cof. eugenioides and Cof. bengalensis are presented in Table XV. Little or no catabolism occurred in leaves of Cof. salvatrix and Cof. bengalensis. Similar results were obtained in an earlier study with Cof. arabica leaves (Ashihara et al., 1996b), the data from which are included in Table XV for comparative purposes. In contrast, very rapid catabolism of [8-I4C]caffeine was observed in leaf segments from Cof. eugenioides. More than 75% of [8-14C]caffeine taken up by the segments was catabolized in both young and mature Cof. eugenioides leaves, with radioactivity recovered as theophylline, 3-methylxanthine, 1methylxanthine, xanthine, ureides, urea and C02. This indicates that [8-'4C]caffeine undergoes demethylation, probably by the main pathway illustrated in Fig. 19, but also via a minor caffeine -+ theophylline -, 1methyxanthine -+ xanthine route. In order to obtain further information on the pathway utilized for the catabolism of caffeine in Cof. eugenioides, pulse-chase experiments with [8-14C]caffeine were carried out with mature leaves (Table XVI). Caffeine, theophylline and 3-methylxanthine were the most heavily labelled compounds after the 4-h pulse. The radioactivity associated with caffeine declined after the leaves were transferred to the non-radioactive medium. In contrast, I4Clabelled theophylline, 3-methylxanthine, 1-methylxanthine, xanthine, ureides, TABLE XVI Metabolism of [8-'4C]caffeine in a pulse-chase experiment with mature leaves of Coffea eugenioides. Leaf segments (100 mg fresh weight) were incubated with 18 p M [8-'4C]caffeine for 4 h (pulse) and then the incubation medium was replaced by fresh medium without radioactive compounds. The radioactivity was 'chased'for a further 4 and 20 h. Incorporation of radioactivity into each compound is expressed as a percentage of the total radioactivity recovered. Mean values f standard deviation (n = 3) are shown. Total radioactivity taken up by the tissues was 31.2 f 4.0kBqg-' leaf (nd, not detected) Metabolite
4 h (pulse)
8 h (chase)
Residual caffeine Theophylline 3-Meth ylxanthine 1 -Methylxanthine Xanthine Allantoin Allantoate Urea
52.0 f 0.1 21.4 f 0.9 16.0 f 0.1 6.1 f 0.6 nd 0.9 f 0.1 1 . 1 f 0.0 1.2 f 0.1 1.3 f 0.2
4.9 f 0.1 24.2 f 0.6 44.0 f 0.2 6.6 f 0.4 3.9 f 0.4 1.8 k 0.0 2.5 f 0.0 3.5 f 0.1 8.3 f 0.9
co2
Data from Ashihara and Crozier (1999).
24 h (chase) 3.4 f 8.1 f 46.9 f 7.4 f 5.8 f 1.4 f nd 2.9 f 24.2 f
0.1 0.5 1.9 0.0 0.2 1.1 0.1 2.0
TABLE XVII Summary of metabolism of [8-’4C]theophylline by youn and mature leaves of Coffea eugenioides, Coffea salvatrix and Coffea bengalensis. Segments of young leaves were incubated with 9.1 p M [8- 5 Cltheophyllinefor 18 h. Incorporation of radioactivity into metabolites is expressed as percentage of total uptake f standard deviation (n = 3 ) . Total uptake of radioactivity is expressed as kBqg-’ of leaf (fresh weight) (Tp, theophylline; 3-mX. 3-methylxanthine; 1-mX, 1-methylxanthine; X , xanthine; A h , allantoin; Ala, allantoate; nd. not detected)
Species
Leaves
Coffea salvatrix Young Mature Coffea eugenioides Young Mature Coffea bengalensis Young Mature
TP
3-mx
1-mx
X
Aln
Ala
Urea
COz
Total radioactivity recovered (kBq f SD)
nd
nd nd
nd 0.5 f 0.1
nd nd
nd nd
nd 0.5 f 0.0
0.5 f 0.0 1.0 f 0.4
54.8 f 12.2 50.2 f 5.2
1.3 f 0.2
4.8 f 0.7 0.8 f 0.1
1.0 f 0.0 1.3 f 0.0
3.3 f 0.1 3.5 f 0.3
7.9 f 2.9 23.5 + 0.7
86.3 f 14.2 85.7 f 1.5
nd nd
nd nd
1.3 f 0.4 0.9 f 0.1
41.7 f 1.8 34.5 f 3.6
99.5 f 0.0 95.2 f 0.7
2.2
26.6 f 1.0 36.4 f 0.3
53.8 f 1.0 26.2 f 0.7
0.8 f 0.1 0.8 f 0.0
4.8 f 0.3
82.4 f 1.0 78.2 f 0.3
6.9 f 0.0 14.2 f 0.2
2.0 f 0.4 3.1 f 0.2
nd nd
From Ashihara and Crozier (1999).
f
0.2
3.4 3.7
f f
0.2 0.4
CAFFEINE AND RELATED PURINE ALKALOIDS IN PLANTS
183
urea and C 0 2 increased after a 4-h chase, with more than 40% of the radioactivity taken up during the pulse being incorporated into 3-methylxanthine. After a further 20-h chase radioactivity associated with theophylline, ureides and urea declined, while the levels of 14C associated with 3methylxanthine, 1-methylxanthine and xanthine changed little, and l4Co2 evolution increased from 8.3 to 24.2% of the recovered radioactivity (Table XVI). As Cof. arabica leaves catabolize theophylline much more rapidly than caffeine, it was of interest to investigate the fate of theophylline when incubated with young and mature leaves of Cof. salvatrix, Cof. eugenioides and Cof. bengalensis. The data obtained are presented in Table XVII. [8-14C]Theophylline was converted to a range of catabolites, most extensively by Cof. eugenioides, with the evolution of 14C02 from mature leaves being three times greater than from young leaves where more than half of the radioactivity was recovered as 3-methylxanthine. More label was associated with 3-methylxanthine than 1-methylxanthine indicating that the main route for catabolism of theophylline to xanthine is via 3-methylxanthine in Cof. eugenioides. Metabolism of [8-'4C]theophylline was slower in Cof. bengalensis with proportionally more radioactivity being recovered as unmetabolized theophylline and less as I4CO2 and intermediates of purine catabolism. There was relatively little catabolism of [8-14C]theophylline by leaves of Cof. salvatrix with more than 90% of the recovered radioactivity being the unmetabolized substrate and only minimal incorporation of label into purine catabolites. This study revealed that among three low caffeine-containing Coffea species, only Cof. eugenioides possessed a strong capacity for caffeine catabolism with the demethylation of theophylline to xanthine proceeding mainly via 3methylxanthine and to a lesser extent via 1-methylxanthine. In Cof. salvatrix and Cof. bengalensis, catabolism of both caffeine and theophylline is very slow, therefore the low caffeine content of these Coffea species appears to be due primarily to their reduced biosynthetic activity. In Cof. eugenioides, Cof. salvatrix and Cof. bengalensis, [8-14C]theophylline was neither converted to 7methylxanthine nor salvaged for the synthesis of caffeine as it is in Cof. arabica leaves. 3. Camellia sinensis leaves Only a small amount of I4CO2 was released from [8-14C]caffeine incubated with young, mature and aged tea leaves over a 48-h period, and no radiolabelled xanthine derivatives were detected (Ashihara et al., 1997b). These observations indicate that the rate of caffeine catabolism is extremely low and, as a consequence, caffeine accumulates as the major purine alkaloid in tea leaves. Unlike [2-14C]caffeine, [8-'4C]theophylline was metabolized to a variety of compounds (Table XVIII). Metabolism of [8-14C]theophylline was especially rapid in mature and aged leaves where more than 75% of the total radioactivity taken up by the tea leaf segments was catabolized and recovered
TABLE XVIII Summary of the metabolism of [8-'4C]theophylline (36.7kBq) by young, aged and mature leaves of Camellia sinensis in the presence and absence of 5 mM allopurinolfor 24 h. Total radioactivity recoveredpresented as kBqper 50 mg fresh weight (young leaves) andper 80mg fresh weight (mature and aged leaves) f standard deviation ( n = 3). Levels of residual [8-'4C]theophylline ( T p ) and radiolabelled metabolites 3methylxanthine (3-mX), xanthine ( X ) , allantoin ( A h ) , C02, theobromine ( T b ) and caffeine (Cf)are expressed as a percentage of the total radioactivity recovered at the end of the 24-h incubation period (nd, not detected)
Leaves
Treatment
Young
Control Allopurinol Control Allopurinol Control Allopurinol
Mature Aged
TP 47.3 48.1 17.2 22.1 15.7 26.3
3-mx f
+ f f
f f
Data from Ashihara et al. (1997b).
0.5 0.1 0.4 2.2 0.9 0.1
11.6 f 0.4 17.5 f 0.9 8.4 f 0.4 10.9 f 4.5 8.3 f 0.2 12.5 f 0.4
X 3.1 11.4 1.7 52.5 3.7 50.7
Aln 0.1 f 1.1 f 0.5 f 3.3 f 1.4 3.3 f
*
5.0 f 0.9 1.0 f 0.3 1.4 f 0.2 1.6 f 0.1 2.7 f 0.2 1.9 f 0.1
co2 10.8 4.6 67.8 3.4 68.2 6.5
1.5 f 2.6 & 0.8 f 0.3 f 3.1 f 2.6
f
Tb 4.0 f 0.1 5.5 f 0.9 1.9 f 0.1 4.3 f 0.4 0.6 f 0.2 0.9 f 0.1
Total radioactivity recovered (kBq f SE)
Cf 18.2 11.9 1.7 1.3 0.4 0.8
f
f f
f f f
0.3 0.1 0.1 0.5 0.1 0.2
4.00 f 0.02 3.90 f 0.03 6.26 f 0.36 5.48 f 0.27 6.59 f 0.07 5.16 f 0.06
CAFFEINE AND RELATED PURINE ALKALOIDS IN PLANTS
185
as 14C-labelled C02, 3-methylxanthine, xanthine and allantoin, with trace amounts of label also being incorporated, surprisingly, into theobromine and caffeine. These data indicate that theophylline is catabolized, via 3methylxanthine, to xanthine which is degraded to C02 by the conventional purine catabolic pathway (Fig. 19). Young tea leaves catabolized [8-14C]theophylline more slowly than mature and aged leaves, with a marked reduction in 14C02 output (Table XVIII). Approximately 20% of the total radioactivity taken up by the young leaf segments was associated with [14C]caffeineand, to a lesser extent, [14C]theobromine. The incubation of young, mature and aged tea leaves with [2-14C]xanthine resulted in the release of very large amounts of 14C02 with most of the remaining radioactivity being associated with residual [2-14C]xanthine and allantoin. In young leaves treated with allopurinol, the reduced activity of the purine catabolism pathway was also associated with a very large increase in the radioactivity incorporated into 3-methylxanthine, theophylline, theobromine and caffeine, with these compounds representing more than 40% of the recovered radioactivity compared to 1.4% in control leaf segments (Table XVIII). The metabolism data of Ashihara et al. (1997b) demonstrate that there are certain similarities in caffeine catabolism in leaves of Cam. sinensis and Cof. arabica with the purine alkaloid accumulating as a consequence of its conversion to theophylline being very effectively blocked. Both species rapidly demethylate [8-14C]theophylline via 3-methylxanthine to xanthine, which enters the purine catabolism pathway and is broken down to C 0 2 and NH3. In Cof. arabica xanthine can also be metabolized to 7-methylxanthine, a conversion that does not occur in tea leaves. In tea leaves, however, a unique salvage pathway operates in which theophylline and xanthine can be salvaged, via the routes illustrated in Fig. 19, for the synthesis of theobromine which is converted to caffeine. No such salvage pathway operates in the leaves of Cof. arabica, Cof. eugenioides, Cof. salvatrix and Cof. bengalensis. 4.
Camellia ptilophylla (cocoa tea) leaves Theobromine-accumulating leaves of Cam. ptilophylla do not convert [2-14C]theobromine to caffeine. The dimethylxanthine is catabolized slowly and only a small amount of radioactivity enters the purine catabolism pathway and is recovered as I4CO2. However, when leaves are incubated with [8-14C]caffeine there is a 6 8 % incorporation of label into theobromine. The l-N-demethylase that catalyses this conversion is unlikely to play a significant role in the production of endogenous theobromine in Cam. ptilophylla as the leaves do not contain detectable quantities of caffeine (Ashihara et al., 1998).
186
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C. METABOLISM OF PURINE ALKALOIDS AS XENOBIOTICS IN NON-PURINE ALKALOID-FORMING PLANTS
Although purine alkaloids have been used for a variety of purposes in plant cell research (Kihlman, 1949, 1977; Kihlman and Leven, 1949; Mineyuki et al., 1989; Amino and Nagata, 1996; Manandhar et al., 1996), to the best of our knowledge there is only one report on theophylline metabolism in plants which do not contain purine alkaloids (It0 er al., 1997). In contrast, the metabolism of caffeine and theophylline has been studied in detail in humans because of their therapeutic value as diuretics, relaxants and vasodilators (Scheline, 1991). Compared to the species which synthesize purine alkaloids, the uptake and metabolism of [8-'4C]theophylline by leaf segments of Avena sativa, roots of Vigna mungo and suspension cultures of Catharanthus roseus cells, all of which are devoid of purine alkaloids, is extremely slow. Relatively small amounts of radioactivity were associated with 3-methylxanthine, xanthine and COz. There was no detectable incorporation of label into theobromine and caffeine nor into 1-methyluric acid and 1,3-dimethyluric acid, which in mammals are the main metabolites of theophylline (Scheline, 1991). The effects of allopurinol treatment were minimal indicating that relatively minor amounts of the theophylline were being catabolized to xanthine and entering the purine catabolism pathway. When given to mammals as a xenobiotic drug, theophylline is converted to 3-methylxanthine, 1-methyluric acid, 1,3dimethyluric acid and caffeine (Scheline, 1991). Studies with premature infants have shown that theophylline is metabolized to caffeine by a direct N-7 methylation step which contrasts with the indirect theophylline --+ 3methylxanthine 4 theobromine 4 caffeine salvage pathway that operates in tea and Camellia irrawadiensis. Unlike mammals, higher plants, irrespective of whether or not they contain purine alkaloids, do not convert theophylline to either 1-methyluric acid or 1,3-dimethyIuric acid. A further key difference is that in humans, 3-methylxanthine is an end product of theophylline metabolism (Scheline, 1991), whereas in higher plants it is further converted to xanthine and/or theobromine.
VI . BIOTECHNOLOGY OF PURINE ALKALOIDS A. CAFFEINE PRODUCTION A N D DEGRADATION IN CELL A N D TISSUE CULTURES
There is demand for caffeine for medicines, aspirin tablets containing caffeine are being sold on an increasing basis, and large amounts of caffeine are used to supplement soft drinks. The caffeine required for these products is obtained by either large-scale chemical synthesis, which is relatively straightforward, or as a by-product of procedures used to decaffeinate tea and coffee (Baumann and Frischknecht, 1988; Baumann and Neuenschwander, 1990). Caffeine produc-
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tion by tissue and cell cultures of coffee and tea has had no economic impact, although such systems have the potential to be very useful in studies on the mechanisms involved in purine alkaloid biosynthesis. Formation of purine alkaloids in tissue cultures of Coffea arabica was initially demonstrated by Keller et al. (1972). Since then, caffeine and theobromine synthesis by cultured coffee cells has been reported many times (Frischknecht et al., 1977; Baumann and Frischknecht, 1988). Waller et al. (1983) established high caffeine-producing callus cultures from apical portions of orthotropic Cof. arabica branches using Murashige-Skoog medium supplemented with kinetin (0.1 mg 1-') and 2,4-dichlorophenoxyacetic acid (0.1 mgl-'). The callus tissue produced 5&100 times more caffeine than the original explants. In many Cof. arabica culture systems the rate of caffeine production is far higher than in the intact plant tissues, and significant amounts of purine alkaloids are released into the culture medium (Waller et al., 1983). N-Methyltransferase and N-methylxanthosine nucleosidase activities have been extracted from the cultured cells (Baumann et al., 1983; Waller et al., 1983). Theophylline and paraxanthine, which are not accumulated in the parent plant, are often found in cell cultures of coffee (Baumann and Frischknecht, 1982; Suzuki and Waller, 1982). This is probably due to a cultured cell's high capacity for purine alkaloid metabolism (Baumann and Frischknecht, 1982). Purine alkaloid formation in tea callus cultures was first reported by Ogutuga and Northcote (1970a, b). Since then, purine alkaloid-forming callus and suspension cultures have been established, but the amount of caffeine produced in cultured tea cells is lower than found in Cof. arabica in vitro (Tsushida and Doi , 1984; Furuya et al., 1989, 1990; Shervington et al., 1998). Purine alkaloid-producing callus and suspension cultures of Theobroma cacao have also been established (Gurney et al., 1992). Biotransformation of theobromine and caffeine in suspension or immobilized C . arabica cells has been reported (see Baumann and Frischknecht, 1988). Theobromine is readily converted to caffeine while caffeine is transformed to theobromine by Cof. arabica and Cof. humilis, and to theobromine and paraxanthine by Cof. liberica and Cof. eugenioides. High transformation activity was found in suspension cultures of Cof. humilis, Cof. liberica and Cof. eugenioides (Baumann and Frischknecht, 1982). The yield of secondary products can be manipulated empirically in vitro by the carbohydrate and nitrogen sources, phosphate level, phytohormones and elicitor molecules, light regime, temperature and osmotic stress (Stafford, 1991). There are many reports on the effects of these factors on the purine alkaloid production. Exposing in vitro cultures of Coffea to stress with high light intensity and elevated NaCl levels increased the production of purine alkaloids (Frischknecht and Baumann, 1985; Kurata et al., 1997). Immobilization of suspension-cultured Cof. arabica cells by entrapment in calcium
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alginate gels increased caffeine and theobromine production up to 13-fold (Haldimann and Brodelius, 1987). B. DECAFFEINATED BEVERAGES
1. Coffee In economic terms, coffee is one of the most valuable agricultural products exported by developing countries in Central and Southern America and Africa. Cof. arabica (Arabica coffee) is cultivated extensively and represents ca 70% of the market. The remaining 30% consists mainly of Cof. canephora (Robusta coffee). Beans of Arabica and Robusta coffee contain ca 1 and 2% caffeine, respectively. Small-scale cultivation of Cof. liberica, Cof. racemosa and Cof. dewevrei occurs in some African countries but most of the beans are sold locally rather than exported. Since the early 1970s demand for decaffeinated coffee has increased rapidly and it currently accounts for more than 20% of coffee sales. This is because of a growing belief that ingestion of large amounts of caffeine, which is a stimulant, can have adverse effects on health, especially in the elderly. There has been extensive debate in the medical literature on this point without any clear conclusions being drawn. Nevertheless, caffeine is perceived by the general public as being injurious to health. This applies especially to those over 60years of age, and in the United States this group accounts for the consumption of more than 55% of all decaffeinated coffee. Extraction of caffeine from coffee seeds with organic solvents was first used to produce decaffeinated coffee in 1905 by Roselius and Wimmer of Kaffee H.A.G., Germany. Commercial decaffeination is now a sophisticated process and, although it is not completely free of solvent residues, there have been major improvements in the quality of the product in recent years (Katz, 1985). The latest methodology involves the use of supercritical fluid extraction with COz to eliminate the health problems posed by the toxicity of residues from solvents such as dichloromethane. However, this process is likely to be extremely expensive for a commercial-scale operation. In the long term, it seems likely that the increasing demand for decaffeinated coffee could be better met by the use of Coffeea species with beans that contain significantly lower levels of caffeine than either Cof. arabica or Cof. canephora. Although such material is available in the form of species such as Cof. eugenioides, Cof. salvatrix and Cof. bengalensis (Mazaferra et al., 1991), none is suitable for commercial exploitation because of either the poor quality of the resultant beverage and/or the form and low productivity of the trees. The main characteristic of many Coffea species of the section Mascarocoffea is an absence of caffeine in the seed. Commercial exploitation is, however, impaired by poor agronomic characteristics and the bitter taste of the coffee caused by the presence of cafamarine. Attempts to transfer the caffeine-free trait from Mascarocoffea to Cof. arabica and Cof. canephora have been
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unsuccessful because of the infertility of the progeny. As Cof. arabica is polyploid and most other species of Coffea are diploid (n = 22), there are also genetic barriers that prevent effective hybridize between Cof. arabica and Coffea species, such Cof. salvatrix and Cof. bengelensis, that contain much lower levels of caffeine. The few progeny that are produced exhibit a wide range of phenotypes, with great variations in their caffeine content. The use of a breeding programme to obtain low caffeine producers would, therefore, probably take 20 years or more as back crosses would be required to establish and stabilize the desired traits. For instance, C. arabica cv. Icatu, which is tolerant to yellow rust disease and recently began to be used by commercial growers in Brazil, is the product of a 42-year breeding programme. In the circumstances, the use of genetic engineering to produce transgenic caffeine-deficient Cof. arabica may ultimately be a more practical proposition than breeding programmes. For this approach to be successful, information is required on the enzymes and genes controlling key steps in the biosynthesis and/or catabolism of caffeine. As discussed in Section IV.B.3, recent studies have obtained a 20 amino acid N-terminal sequence for caffeine synthase from tea, the N-methyltransferase catalysing the two-step conversion of 7methylxanthine to caffeine (Kato et al., 1999). In the near future this should lead to the cloning of the cDNAs encoding the methyltransferase. This, in turn, will enable antisense techniques to be used to produce transgenic Cof. arabica plants which are deficient in caffeine because of an absence of 7methylxanthine and theobromine N-methyltransferase activity. One complication is that this may well lead to the accumulation of 7-methylxanthine instead of caffeine and this may bring its own problems. The situation is potentially more straightforward once the gene encoding xanthosine N-methyltransferase activity is cloned, as its antisense expression will produce transgenic plants that cannot convert xanthosine to 7-methylxanthosine. Xanthosine, however, is unlikely to accumulate as it will be converted to xanthine and degraded by the purine catabolism pathway (Crozier, 1997) (see Fig. 6). There is an alternative approach whereby genetic engineering has the potential to produce caffeine-deficient Cof. arabica. As discussed in Section V.B.l caffeine accumulates in Cof. arabica because its conversion to theophylline, via cleavage of the 7-N methyl group, is blocked. Theophylline, in contrast, is rapidly degraded to C 0 2 and NH3 via 3-methylxanthine and xanthine. The 7-N-demethylase is active at least in the leaves of CoJ eugenioides which contain relatively low levels of caffeine (Section V.B.2). Thus, the cloning of the demethylase from Cof. eugenioides, and its overexpression in transgenic Cof. arabica, is a further route to the production of natural, caffeine-deficient coffee. Several species of caffeine-degrading bacteria, such as Pseudomonas cepacia, Pseudomonas putida and Serratia marcescens have been isolated through the use of screening procedures that involve culturing bacteria on a medium in which caffeine is used as the sole source of nitrogen and/or carbon. The
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bacterial degradation is different to that operating in higher plants as it appears to involve a caffeine theobromine --+ 7-methylxanthine xanthine --+ NH3 pathway. The catabolic capacity of Psu. cepacia, in particular, is extremely high with 100-ml cultures in the exponential phase of growth being able to catabolize > 50 mg caffeine within 24 h. The expression of the bacterial genes encoding caffeine degradation enzymes, in transgenic Cof. arabica, offer another route by which genetic engineering could be used to produce caffeinedeficient coffee. --+
---f
2. Tea Sales of decaffeinated tea are also rising but not on the scale of increases in the consumption of decaffeinated coffee. Leaves of Cam. sinensis contain ca 5% caffeine d.w., but some cultivars obtained from the Japanese National Research Institute of Vegetable, Ornamental Plants and Tea have been shown to contain < 2% caffeine (Takeda, 1994; Takeda and Saba, 1998). Ashihara et al. (1995) suggested that these low caffeine-containing cultivers have a slower rate of caffeine biosynthesis and a higher rate of caffeine catabolism than cultivars with a high endogenous caffeine content. Decaffeinated tea, however, is currently produced by solvent/supercritical fluid extraction and there is little, if any, interest in developing either low caffeine-containing varieties of tea or transgenic tea deficient in caffeine for the commercial production of decaffeinated plants.
VII. SUMMARY The major route to caffeine in higher plants is a xanthosine -+ 7methylxanthosine -+ 7-methylxanthine --+ theobromine -+ caffeine pathway, while the trimethylxanthine is catabolized via a caffeine 4 theophylline 4 3methylxanthine --+ xanthine pathway. Several minor biosynthesis and catabolism pathways also operate, probably as a consequence of the broad substrate specificity of N-methyltransferase (caffeine synthase) and purine alkaloid demethylase(s). As yet no demethylases related to the caffeine catabolism have not been characterized. Diversity of caffeine metabolism between different purine alkaloid-containing plants appears to be due to variations in the substrate specificity of the demethylases and caffeine synthases in different species. Caffeine is synthesized in young tissues, and in developing leaves caffeine synthase is located in chloroplasts. However, light is not a prerequisite for caffeine biosynthesis as caffeine accumulates in dark-grown Cof. arabica seedlings. The precursors of caffeine are derived from purine nucleotides which originate from de n o w purine biosynthesis, as well as adenine and guanine nucleotide pools that are formed by the salvage pathways of adenosine, adenine and other related compounds. The rate of caffeine biosynthesis
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appears to be regulated primarily by the induction and repression (so-called ‘coarse control’) of caffeine synthase, the activation and inhibition of caffeine synthase activity by the SAM/SAH ratio and the availability of purine precursors (‘fine control’). However, feedback control of caffeine synthase activity by caffeine is not evident. The rate-limiting reaction in caffeine catabolism, and the reason for the accumulation of caffeine in Cof. arabica and Cam. sinensis, is the conversion of caffeine to theophylline. Once formed, theophylline is rapidly degraded to C 0 2 and NH3 via the conventional purine catabolism pathway found ubiquitously in plants. There are currently no reports on molecular biology and genetic engineering of caffeine metabolism. However, as a 20 amino acid N-terminal sequence has been obtained for high purified caffeine synthase it is likely that the gene encoding this key enzyme in the caffeine biosynthesis will be cloned in the near future. Studies on caffeine will then enter a new phase, and the major challange will be to provide an understanding of the molecular mechanisms that regulate purine alkaloid metabolism. Accompanying these developments will be the possibilities of using biotechnology to produced caffeine-deficient transgenic Cof. arabica and Cam. sinensis plants to meet the growing demand for decaffeinated coffee and tea.
ACKNOWLEDGEMENTS We would like to thank Dr Satoshi Yamaguchi (Ehime University), Dr Takeo Suzuki (Kyoto Institute of Technology) , Dr. Masatoshi Mita (Waseda University) and Dr T. W. Baumann (Zurich University) for their helpful advice and for sending reprints. Some original data cited in this article were the unpublished results of research carried out with co-workers, Dr Misako Kato, Emi Ito, Ayako Kato and Sachiko Yama (Ochanomizu University). Part of the work is supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (No. 08454255 and 10640627) to H. Ashihara, and UK-Japan travel grants from the British Council and Royal Society to A. Crozier. We also would like to thank Dr C. T. Wheeler (Glasgow University) for critical reading of the manuscript, Dr Roger Mandel (Glasgow University) for preparing Figure 1 and Professor Takao Yokota (Teikyo University) for preparing Figure 2.
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Suzuki, T. and Takahashi, E. (1975a). Biosynthesis of caffeine by tea-leaf extracts: Enzymic formation of theobromine from 7-methylxanthine and of caffeine from theobromine. Biochemical Journal 146, 87-96. Suzuki, T. and Takahashi, E. (1975b). Metabolism of xanthine and hypoxanthine in the tea plant (Thea sinensis L.). Biochemical Journal 146, 79-85. Suzuki, T. and Takahashi, E. (1976a). Metabolism of methionine and biosynthesis of caffeine in the tea plant (Camellia sinensis L.). Biochemical Journal 160, 171-179. Suzuki, T. and Takahashi, E. (1976b). Caffeine biosynthesis in Camellia sinensis. Phytochemistry 15, 1235-1239. Suzuki, T. and Takahashi, E. (1976~).Further investigation of the biosynthesis of caffeine in tea plants (Camellia sinensis L.): Methylation of transfer ribonucleic acid by tea leaf extracts. Biochemical Journal 160, 181-184. Suzuki, T. and Waller, G. R.(1982). Metabolism of caffeine in sterile callus tissue cultures of Coffea arabica. In “Plant Tissue Culture 1982” (A. Fujiwara, ed) pp. 385-386, Maruzen, Tokyo. Suzuki, T. and Waller, G. R. (1984a). Biosynthesis and biodegradation of caffeine, theobromine, and theophylline in Coffea arabica L. fruits. Journal of Agricultural and Food Chemistry 32, 845-848. Suzuki, T. and Waller, G. R. (1984b). Biodegradation of caffeine: Formation of theophylline and caffeine in mature Coffea arabica fruits. Journal of the Science of Food and Agriculture 35, 6670. Suzuki, T. and Waller, G. R. (1985a). Purine alkaloids of fruits of Camellia sinensis L. and Coffea arabica L. during fruit development. Annals of Botany 56, 537-542. Suzuki, T. and Waller, G. R. (1985b). Effects of light on the production and degradation of caffeine in Camellia sinensis L. seedlings. Plant and Cell Physiology 26, 765-768. Suzuki, T. and Waller, G . R. (1986). Total nitrogen and purine alkaloids in the tea plant throughout the year. Journal of the Science of Food and Agriculture 37, 862-866. Suzuki, T. and Waller, G. R. (1987a). Alleopathy due to purine alkaloids in tea seeds during germination. Plant and Soil 98, 131-136. Suzuki, T. and Waller, G. R. (1987b). Purine alkaloids in tea seeds during germination. In “American Chemical Society Symposium Series No. 330. Allelochemicals: Role in Agriculture and Forestry” (G.R. Waller, ed) pp. 291-294. American Chemical Society. Suzuki, T. and Waller, G. R. (1988). Metabolism and analysis of caffeine and other methylxanthines in coffee, tea, cola, guarana and cacao. In “Modern Methods of Plant Analysis, New Series, Volume 8. Analysis of Nonalcoholic Beverages” (H. F. Linskens and J. F. Jackson, eds) pp. 184-220. Springer, Berlin. Suzuki, T., Ashihara, H. and Waller, G. R. (1992). Purine and purine alkaloid metabolism in Camellia and Coffea plants. Phytochemistry 31, 2575-2584. Tajima, S. and Yamamoto, Y. (1975). Enzymes of purine catabolism in soybean plants. Plant and Cell Physiology 16, 271-282. Takhtajan, A. (1959). “Die Evolution der Angiospermen”. Gustav Fisher, Jena. Takeda, Y. (1994). Differences in caffeine and tannin contents between tea cultivars, and application to tea breeding. Japan Agricultural Research Quarterly 28, 117123. Takeda, Y. and Saba, T. (1998). Breeding of low-caffeine tea (Part 2). Tea Research Journal 87, Suppl., 8. (In Japanese.) Takino, Y., Imagawa, H. and Shishido, K. (1972). Studies on the nucleotides in tea Part I. Nucleotides in green tea. Japanese Journal of Food Science and Technology 19, 2 13-2 18.
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Arabinogrrlactan-proteins In the Multiple Domains of the Plant Cell Surface
MARCEL0 D. SERF%' and EUGENE A. NOTHNAGEL2 'Department of Biology, Boise State University, Boise, ID 83725, USA 'Department of Botany and Plant Sciences, University of California, Riverside, CA 92521, USA
I. Introduction...............................................................................................
............. A. BiochemicalCharacterization ............................................................. B. Expression and Function.................................................................... 111. Cell Wall AGPS (CW-AGPs)..................................................................... A. Biochemicalcharacterization ............................................................. B. Expression and Function.................................................................... Iv. Plasma Membrane AGPs (PM-AGPs) ...................................................... A. Biochemical characterization ............................................................. B. ExptesSion and Function.................................................................... V. Conclusions and Future Prospects ............................................................ A c ~ o ....................................................................................... w ~ References.................................................................................................. 11. Soluble AGPS in the Cell Wall and Exttacellular Spaces
208 211 211 225 238 240 244 256 257 265 270 274 274
This review examines recent information on the structure, expression and function of arabinogalactan-proteins,a class of plant proteoglycans with very broad taxonomic and anatomic djstributions. Plants typicallyproduce at kast several arabinogalactan-proteins that exhibit heterogeneity in both the predominant ( >90%) carbohy&ate portion and in the polypeptide core. Developmentally regulated expression and various other lines of evidence indicate that arabinogalactan-proteinsfaction in p h t development, although precise functions remain to be identiyid. The approach m this review is to discuss the structure and fMction of arabinogalactan-proteins with special consideration to their localization in three domains of the plant cell surface: the aqueousphase of the cell wall and extracelllular spaces; the solid phase of the cell wall; and the stnface of the plarma membrane. Particular emphasis isplaced on d r n experimental approaches, such as use Advaacu in Bot.Il*rl Redeucb VOL 30
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M. D. SERPE and E. A. NOTHNAGEL
of monoclonal antibodies and recombinant DNA technology, and on the recent finding that some arabinogalactan-proteins are synthesized with a glycosyl-phosphatidylinositollipid anchor.
I. INTRODUCTION The plant cell surface plays a fundamental role in the regulation and support of plant life. Processes central to plant development, such as the regulation of cell expansion and differentiation, are intimately related to the structure and metabolism of the cell surface (Roberts et al., 1984; Bacic et al., 1988; Carpita and Gibeaut, 1993; Carpita, 1996). Furthermore, the plant cell surface participates in the perception of environmental signals, in the recognition of pathogen infection and in cell-to-cell communications (Roberts et al., 1984; Bolwell, 1993). The variety of functions played by the plant cell surface is partially attributed to its complexity. The plant cell surface is composed of various domains including the outer surface of the plasma membrane, the plasma membrane-cell wall interface, the solid phase of the cell wall polymers, the aqueous phases between and beyond cell wall polymers, and the middle lamella (Wyatt and Carpita, 1993; Nothnagel, 1997). Each of these domains has distinct physical and chemical properties, and each contains an array of macromolecules such as polysaccharides, glycoproteins and phenolic substances, all of which undergo modifications throughout the life of plant cells. Among the components of the plant cell surface are arabinogalactanproteins (AGPs) (Clarke et al., 1979a; Fincher et al., 1983; Roberts, 1990; Pennell, 1992; Knox, 1995, 1996; Nothnagel, 1997). These proteoglycans comprise a group of macromolecules with certain structural characteristics. A macromolecule is considered an AGP if it has both carbohydrate and polypeptide components, and if the carbohydrate component is rich in galactosyl and arabinosyl residues (Fincher et al., 1983; Nothnagel, 1997). Furthermore, the galactosyl residues in AGPs occur predominantly in (1,3)-,0D-, (1,~)-P-Dand (1,3,6)-P-~-galactopyranosyllinkages. These linkages are characteristic of type I1 arabinogalactans (Aspinall, 1973; Carpita and Gibeaut, 1993), and distinguish AGP glycans from other arabinogalactans found in plants and micro-organisms (Clarke et al., 1979a; Daffe et al., 1993). In type I arabinogalactans of the plant cell wall, for example, galactosyl residues occur predominantly in (1,4)-P-~-galactopyranosyl linkages (Clarke et al., 1979a). While certain structural characteristics are common features of AGPs, considerable variation occurs among these macromolecules in both their polypeptide and carbohydrate components. For many AGPs, the protein proportion typically falls in the range of 1-lo%, although AGPs with higher
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protein proportions have been reported (Bacic et al., 1987; Norman et al., 1990; Baldwin et al., 1993). The amino acid composition and sequence of core polypeptides can also differ significantly between distinct AGPs. For example, analysis of core polypeptides of several AGPs suggests few homologies at the level of amino acid sequence (Mau et al., 1995; Bacic et al., 1996; Du et al., 1996a; Gerster et al., 1996; Li and Showalter, 1996; Qiu et al., 1997). Furthermore, the carbohydrate component of AGPs shows notable complexity. In addition to arabinose and galactose, at least eight other sugars have been found as residues in AGPs (Jermyn and Yeow, 1975; Clarke et al., 1979a; Akiyama and Kato, 1981; Fincher et al., 1983; Tsumuraya et al., 1984, 1988). Variations in the proportion, linkage and sequence of these sugars offer an enormous potential for chemical and structural diversity. Various AGPs are present at different subcellular locations, and in different cell types and tissues. On the cell surface, AGPs are found on the outer surface of the plasma membrane (Knox et al., 1989; Pennell et al., 1989; Norman et al., 1990; Komalavilas et al., 1991; Serpe and Nothnagel, 1996; Smallwood et al., 1996; Kjellbom et al., 1997), bound to the cell wall (Basile and Basile, 1987; Schopfer, 1990; Serpe and Nothnagel, 1995; Kido et al., 1996) or as soluble molecules in the cell wall space and plant secretions (Akiyama et al., 1984; Gleeson et al., 1989; Komalavilas et al., 1991). Each of these domains has a complement of AGPs that is partially distinct and partially overlapping (Komalavilas et al., 1991; Serpe and Nothnagel, 1995, 1996). During plant development, the various tissues and organs of plants express some unique AGPs that can be distinguished from each other on the basis of physical, structural and/or immunological characteristics (van Holst and Clarke, 1986; Knox et al., 1989; Stacey et al., 1990; Pennell et al., 1991). In addition to their wide distribution at the anatomical level, AGPs are also ubiquitously distributed throughout the plant kingdom. Arabinogalactanproteins have been found in bryophytes, seedless vascular plants, and in many gymnosperms and angiosperms (Jermyn and Yeow, 1975; Clarke et al., 1978; Bobalek and Johnson, 1983; Basile and Basile, 1990). Certain monoclonal antibodies that recognize higher plant AGPs also recognize epitopes on macromolecules from the alga Chara fragilis and the cyanobacterium Nostoc, although structural characterizations of these macromolecules are still needed to ascertain if they are AGPs (Snogerup, 1997). The widespread occurrence of AGPs at the taxonomic level indicates that AGPs were conserved through evolution, presumably because they play a vital role in plants (Snogerup, 1997). Although the functions of AGPs are not established, several lines of evidence indicate that AGPs participate in the regulation of plant development. This hypothesis was first inferred from observations that tissue-specific and spatiotemporal variations in AGPs correlate with certain aspects of plant development (Gleeson and Clarke, 1980; Bobalek and Johnson, 1983; Gel1 et al., 1986; van Holst and Clarke, 1986; Tsumuraya et al., 1987, 1988; Knox, 1995; Pennell et al., 1995; Kreuger and van Holst, 1996). Other experimental
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approaches, such as application of exogenous AGPs to cell cultures (Kreuger and van Holst, 1993; Kreuger et al., 1995; Egertsdotter and von Arnold, 1995) or perturbation of endogenous AGPs by application of AGP-binding agents (Serpe and Nothnagel, 1994), yielded results consistent with the notion that AGPs are involved in plant development. Various experimental systems exhibited different responses to perturbation of endogenous AGPs, but the observed alterations in cell division, cell expansion, cell wall assembly and/or cell death (Serpe and Nothnagel, 1994; Jauh and Lord, 1996; Willats and Knox, 1996; Langan and Nothnagel, 1997; Ding and Zhu, 1997; Roy et al., 1998) are all intimately related to plant development. While these and other similar results represent promising leads about AGP function, the exact role(s) of these molecules remains to be ascertained. Successes in isolating cDNAs that encode the core polypeptide of various AGPs have enabled additional approaches for analysing AGP function(s). Regulated expression of AGPs is being examined at the level of mRNA corresponding to the core polypeptide, and production of transgenic plants producing sense and antisense mRNAs for a core polypeptide of an AGP has been reported (Cheung et al., 1996). These and other genetic approaches are likely to shed additional light on the function and biochemistry of these ubiquitous molecules. Recent reviews have covered developments in AGP research with emphasis placed on the role of AGPs in plant development and on the structure of these molecules (Knox, 1995, 1996; Bacic et al., 1996; Du et al., 1996b; Kreuger and van Holst, 1996; Nothnagel, 1997; Sommer-Knudsen et al., 1997, 1998a; Schultz et al., 1998). The approach in this review is to discuss the structure and function of AGPs with special consideration to their localization in the various domains of the plant cell surface. In particular, separate discussions will be presented for AGPs that are soluble in the aqueous phase of the cell wall and extracellular spaces, AGPs that are bound to the cell wall and AGPs that are bound to the plasma membrane. Each of these three domains have quite distinct chemical and biophysical properties. Recent studies have increased our understanding of the structure of AGPs at these three cell surface locations and have provided valuable clues regarding AGP function. Our knowledge in these areas, however, remains incomplete. How many distinct AGPs are present at the plant cell surface? What mechanisms determine the targeting of AGPs to particular cell surface sites? Is the function(s) of AGPs similar throughout the cell surface? We will review information addressing these and similar questions, and highlight those areas where more information is needed.
ARABINOGALACTAN-PROTEINS IN THE PLANT CELL SURFACE
2 11
11. SOLUBLE AGPS IN THE CELL WALL A N D EXTRACELLULAR SPACES Early studies showed that AGPs are secreted in large amounts in special tissues. For example, the bark of Acacia senegal secretes large amounts of gum arabic, a mixture containing AGPs exuded as tears in response to wounding (Smith, 1939a, b; Stephen, 1983; Akiyama et al., 1984; Defaye and Wong, 1986; Qi et al., 1991; Osman et al., 1993; Whistler, 1993). Secretion of AGPs is also common in the gynoecium. As a result, AGPs accumulate on the stigma, in the extracellular matrix of the stylar transmitting tissue and in the ovary (Clarke et al., 1979b; Gleeson and Clarke, 1979; Cheung et al., 1995; Gane et al., 1995a; Jauh and Lord, 1996; Lennon et al., 1998). Likewise, the peripheral cells of the root cap secrete a mucigel that is rich in AGPs (Bacic et al., 1986; Moody et al., 1988). Arabinogalactan-proteins are also secreted in large amounts by cells in suspension cultures (Aspinall et al., 1969; Anderson et al., 1977). These AGPs accumulate in the culture medium, presumably after exiting the cell via cell wall pores. It is not certain, however, that these AGPs are actually targeted beyond the cell wall. An alternative possibility is that culture medium AGPs and other extracellular polysaccharides are targeted to the cell wall but pass to the medium upon failure to assemble in the cell wall under the culture conditions (Shea et al., 1989). A variety of evidence indicates that AGPs are also present in the intercellular and cell wall spaces of organized tissues in whole plants. Some AGPs can be extracted from organized tissues using dilute salt solutions (Jermyn and Yeow, 1975; Clarke et al., 1978), thus suggesting that they are not tightly bound to the cell. Furthermore, Samson et al. (1984) collected AGPs from bean hypocotyl segments by using vacuum infiltration and low speed centrifugation. Because these procedures do not rupture the plasma membrane, the collected material was assumed to come from intercellular and cell wall spaces. On the other hand, while AGPs are present in the intercellular and cell wall spaces of organized tissues, they do not seem to accumulate there to the same extent as they do in cultured cells. Knox (1995) noted that immunocytochemistry generally detects no AGPs in the intercellular spaces of plant tissues. Interpretation of these results can be complicated, however, by uncertainties regarding the extent to which soluble molecules might be washed out of the intercellular and cell wall spaces during fixation and staining procedures. A. BIOCHEMICAL CHARACTERIZATION
Because soluble AGPs can be obtained in relatively large quantities, they have been structurally characterized to a greater detail than cell wall or plasma membrane AGPs (Table I). Nevertheless, no AGP has yet been characterized in full detail. Arabinogalactan-proteins are among the most complex molecules
TABLE I Selected examples of arabinogalactan-proteins (AGPs) localized in identified cell surface domains Molecule
Location
Probes binding
Acacia robusta AGP
Gum exudate
Acacia senegal gum arabic glycoprotein
Gum exudate
(P-D-G~c)~
Nicotiana tabacum TTS-1 and TTS-2 glycoproteins Nicotiana alata NaPRPS Nicotiana alata AGPs Nicotiana tabacum AGP Lolium multijlorum AGP
Intercellular matrix of the transmitting tissue of style
(P-D-GlC)3
Intercellular matrix of the transmitting tissue of style Stigma and intercellular matrix of style Culture medium of suspensioncultured cells Culture medium of suspensioncultured cells
Carrot hyp- deficient AGP
Culture medium of suspensioncultured cells
Rose CM-AGPa and CM-AGPb
Culture medium of suspensioncultured cells
(P-D-Gk)3 (P-D-G~c)~ 5539 antibody MAC207 monoclonal antibody, (P-D-Gk)3 (P-D-G~C)~
Structural information
Reference
Glycosyl linkages", aminoacyl composition Glycosyl and aminoacyl compositions, carbohydrat+ polypeptide linkages CDNA~, giycosyi linkages
Churms and Stephen (1984) Qi et al. (1991)
Cheung et al. (1993); Wu et al. (1995)
cDNA, glycosyl linkages Glycosyl linkages
Lind et al. (1994); Schultz et al. (1997) Gane et al.-(1995b)
Glycosyl linkages, aminoacyl composition Glycosyl linkages, aminoacyl composition and partial sequences Glycosyl linkages, aminoacyl composition
Akiyama and Kato (1981) Bacic et al. (1987); Gleeson et al. (1989)
Glycosyl linkages', aminoacyl composition, N-terminal sequence
Komalavilas et al. (199 1)
Pennell et al. (1989); Baldwin et al. (1993)
Molecule
Location
Pear AGPPcl and AGPPc2 Acacia senegal Hypdeficient AGP Nicotiana alata AGPNa2 glycoprotein Rice srAGPl and srAGP2
Culture medium of suspensioncultured cells Culture medium of suspensioncultured cells Culture medium of suspensioncultured cells Culture medium of suspensioncultured cells
Rose CW-AGPl and GGP Nicotiana alata NaPRP4 glycoprotein
Cell wall of suspensioncultured cells Cell wall of stylar cells
Brassica oleracea AGP
Cell wall of leaves
Nicotiana glutinosa PM-AGP
Plasma membrane of suspensioncultured cells
Rose PM-AGPl and PM-AGP2
Plasma membrane of suspensioncultured cells
Probes binding
Structural information
Reference
(P-D-G~C)~
cDNA, glycosyl linkages
(P-D-GIc)~
Glycosyl linkages', aminoacyl composition cDNA, glycosyl linkages
Chen et al. (1994); Mau et al. (1995) Mollard and Joseleau ( 1994) Mau et al. (1995)
Glycosyl composition
Smallwood et al. (1996)
Glycosyl linkages', aminoacyl composition cDNA, carbohydratepolypeptide linkages, glycosyl linkages Glycosyl and aminoacyl compositions Glycosyl and aminoacyl compositions
Serpe and Nothnagel (1 995) Sommer-Knudsen et af. (1996)
Glycosyl linkages', aminoacyl composition
Serpe and Nothnagel (1996) . ,
LM2, JIM13 monoclonal antibodies, (P-D-GIC)3 (P-D-Gk)3
PN16.4B4 monoclonal antibody (P-D-G~c)~
Kid0 et al. (1996) Norman et al. (1990)
Examples are limited to AGPs that have been at least partially purified and biochemically characterized. The types of data that identify the molecules as AGPs are indicated. Whenever the glycosyl linkage composition was determined, the glycosyl residue composition was also determined. %henever a cDNA was characterized, the aminoacyl composition and sequence were also predicted. 'Analysis of glycosyl linkage composition included determination of predominant anomeric configurations.
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M. D. SERPE and E. A. NOTHNAGEL
known, and consequently characterization of the complete structure of any AGP is a demanding undertaking. Soluble AGPs show considerable variation in molecular size and protein content (Akiyama et al., 1984; Bacic et al., 1987; Komalavilas et al., 1991; Qi et al., 1991; Osman et al., 1993). The molecular size of soluble AGPs ranges between 60 and 300 kDa. The core polypeptide of these AGPs usually accounts for 1-10% of this mass, with carbohydrate accounting for the dominant remaining portion (Clarke et al., 1979a). While most of the information in this section refers to soluble AGPs, some mention is also made of AGPs of yet unknown subcellular localization. Similarly, this section includes a brief discussion of AGP biosynthesis that applies to AGPs throughout the cell.
I . Polypeptide Component The polypeptide portion of soluble AGPs is typically rich in Hyp, Ala, Ser, Thr and Gly residues, although variations have been reported. Three Hyp-deficient AGPs (Hillestad et al., 1977; Baldwin et al., 1993; Mollard and Joseleau, 1994), one His-rich AGP (Kieliszewski et al., 1992) and one Ala-poor AGP (Qi et al., 1991) have been found. Direct determination of amino acid sequences for AGPs has been difficult because AGP preparations are often heterogeneous in both carbohydrate and polypeptide components. Also, AGPs require extensive deglycosylation prior to sequencing. As adequate purification and deglycosylation procedures were developed, partial amino acid sequences from carrot (Jermyn and Guthrie, 1985), ryegrass (Gleeson et al., 1989), rose (Komalavilas et al., 1991) and maize (Kieliszewski et al., 1992) gradually appeared in the literature. Progress in AGP purification and determination of partial amino acid sequences led to the cloning of cDNAs that encode core polypeptides of several confirmed AGPs (Chen et al., 1994; D u e t al., 1994, 1996a; Mau et al., 1995). In these cases, macromolecules were purified and confirmed to be AGPs based on analyses of their glycosyl composition and linkages, and their affinity for (p-DGlc)3 Yariv phenylglycoside. Chemically named as 1,3,5-tri-(p-glucosyloxyphenylazo)-2,4,6-trihydroxybenzene, (P-D-G~c)~ is a synthetic (Yariv et al., 1962; Ganjian and Basile, 1997), chromophoric molecule that specifically binds and precipitates AGPs (for details see Nothnagel, 1997). After chemical deglycosylation, the core polypeptides were analysed to obtain partial amino acid sequences. These sequences were used to generate nucleotide probes for isolating cDNA clones, which were then sequenced. Two of the isolated cDNAs encode core polypeptides of AGPs from the culture medium of pear cells, and were named AGPPc1 and AGPPc2 (Chen et al., 1994; Mau et al., 1995). Two other cDNAs, AGPNa1 and AGPNa3, encode core polypeptides of AGPs isolated from Nicotiana alata styles (Du et al., 1994, 1996a), while AGPNa2 encodes the core polypeptide of an AGP isolated from the culture medium of Nicotiana alata cells (Mau et al., 1995).
ARABINOGALACTAN-PROTEINS IN THE PLANT CELL SURFACE
2 15
In addition to these five cDNAs that encode confirmed AGPs, several other cDNAs have been isolated and predicted to encode polypeptides that might be the cores of AGPs. Most of these other cDNAs were obtained through screening for organ- or tissue-specific gene expression. For example, screening for style-specific gene expression in Nicotiana species led to the isolation of three cDNAs, named NaPRP4, TTS-1 and TTS-2, that encode Pro-rich polypeptides (Chen et al., 1993; Cheung et al., 1993). Glycoproteins containing the polypeptides encoded by these cDNAs were subsequently analysed and found to possess some characteristic features of AGPs. In both the NaPRP4 and TTS glycoproteins, the carbohydrate component contained arabinosyl residues present in terminal linkage and was rich in galactosyl residues present in terminal, (1,3)-, (1,6)- and (1,3,6)-galactopyranosylforms (Wang et al., 1993; Cheung et al., 1995; Wu et al., 1995; Sommer-Knudsen et al., 1996). The NaPRP4 glycoprotein, however, showed only very weak affinity for (P-D-GIc)~ Yariv phenylglycoside, suggesting that this glycoprotein might not be a typical AGP (Sommer-Knudsen et al., 1996). Still another cDNA, NaPRP5, was isolated through use of amino acid sequence information determined from a 120-kDa glycoprotein that is specifically expressed in the style of Nicotiana d a t a (Schultz et al., 1997). This glycoprotein contains the (1,3)-, (1,6)- and (1,3,6)-linked galactopyranosyl residues that are characteristic of AGPs, but it also has (1,2)-linked arabinofuranosyl residues and polypeptide sequences that are characteristic of extensins, the well-studied structural glycoproteins of the cell wall. Screening by specific gene expression or other approaches in a variety of plant materials, including pea nodules, pine xylem, cotton fibers, tomato fruit, Brassica stamens and alfalfa pollen, led to the isolation of cDNAs that may encode the core polypeptides of AGPs (Scheres et al., 1990; John and Keller, 1995; Loopstra and Sederoff, 1995; Pogson and Davies, 1995; Gerster et al., 1996; Li and Showalter, 1996; Qiu et al., 1997). The hypotheses that these cDNAs encode AGPs remain tentative because the corresponding mature translation products have not been characterized. Instead, the identifications have been based on predicted core polypeptide properties such as: an overall amino acid composition similar to that of known AGPs; sequence domains rich in Pro, Ala and Ser; and/or the presence of Ala-Pro repeats which commonly occur in AGPs. Similarly, five genes marked by expressed sequence tags in an Arabidopsis thaliana database have been suggested to encode AGP core polypeptides (Schultz et al., 1998). In all of these cases, where AGPs have been putatively identified through screening for specific gene expression or through searching of sequence databases, the subcellular localizations and other characteristics of the mature translation products are unknown. The availability of cDNAs corresponding to confirmed and putative AGPs has prompted searches for homologies in amino acid sequences among the predicted polypeptides (Mau et al., 1995; Bacic et al., 1996; Du et al., 1996a; Gerster et al., 1996; Li and Showalter, 1996; Qiu et al., 1997). Except for TTS-1
216
M. D. SERPE and E. A. NOTHNAGEL
from Nicotiana tabacum (Cheung et al., 1993), and NaPRP4 and NaPRP5 from Nicotiana alata (Chen et al., 1993; Schultz et al., 1997), the cDNAs of confirmed and putative AGPs show little homology at the level of amino acid sequence. The greatest level of homology among sequences from different genera is 36.8% between AGPPc1 and a putative AGP from the stamen of Brassica napus (Gerster et al., 1996). While little homology exists at the level of particular amino acid sequences, two features that are common among all of the predicted polypeptides are a Nterminal signal sequence for entry into the endoplasmic reticulum and a Prorich domain. In the cases where data from the mature AGP are available, posttranslational hydroxylation of many of these Pro residues to Hyp residues has been evident. Beyond these two common features, other homologies are limited to certain AGPs which have thus been designated as particular types. At the broadest level, AGPs have been designated as either ‘classical’ or ‘non-classical’ (Du, 1995; Mau et al., 1995; Bacic et al., 1996; Du et al., 1996b). The core polypeptides of classical AGPs are predicted to be synthesized with three domains: the N-terminal signal sequence; a central hydrophilic portion rich in Pro, Ala, Thr and Ser; and a C-terminal hydrophobic region (Fig. 1A). The Nterminal signal sequence has been found to be cleaved from the mature AGP in all cases where relevant data are available. Recent results indicate that the Cterminal hydrophobic domain is cleaved from the polypeptide and replaced by a glycosyl-phosphatidylinositol(GPI) lipid anchor (see Section IV.A.2). Thus, only the central hydrophilic domain survives in the mature classical AGP. Non-classical AGPs contain at least three predicted domains: the N-terminal signal sequence; a central Pro-rich domain; and a C-terminal domain rich in either Asn or Cys (Fig. 1B and D). Some non-classical AGPs have a fourth predicted domain which is rich in Asn, and occurs between the signal sequence and the Pro-rich domain (Fig. 1C) (Mau et al., 1995). Non-classical AGPs with a predicted Cys-rich domain at the C-terminal seem to retain this domain in the mature AGP (Du et al., 1996a). While predicted Asn-rich domains seem to be absent from isolated AGPs, the point at which these domains are lost during the biosynthetic/secretory process remains unclear (Mau et al., 1995). A typical feature of structural cell wall proteins is the presence of repetitive peptide motifs such as the Ser-Hyp4 sequence of extensin (Showalter and Varner, 1989; Keller, 1993; Kieliszewski and Lamport, 1994). This classical extensin sequence is not typical of AGPs, although many Ser-ProzP7 motifs occur in the N-terminal domain of the polypeptide encoded by NaPRP5, a cDNA corresponding to a 120-kDa glycoprotein with characteristics of both extensins and AGPs (Schultz et al., 1997). Several authors have noted that some cDNAs predict the occurrence of repetitive peptide motifs and/or runs of at least three consecutive occurrences of an amino acid in core polypeptides of confirmed or putative AGPs (Loopstra and Sederoff, 1995; Li and Showalter, 1996; Nothnagel, 1997; Qiu et al., 1997). Compared to the peptide motifs of
ARABINOGALACTAN-PROTEINS IN THE PLANT CELL SURFACE
2 17
"Classical" AGPs: A. H2NC -OOH "Nonclassical"AGPs: B. H2N
COOH
C. H2N D. H
Key:
COOH 2
m
N
P
C
O
O
H
Secretion signal 0 Hydrophobic Pro-rich Asn-rich Cys-rich
Fig. 1. Domain structures of various AGP core polypeptides. The designation as either 'classical' or 'non-classical' is based on the aminoacyl composition of the predicted polypeptide encoded by the cDNA (Mau et al., 1995; Du et al., 1996b). (A) Domain structure exhibited by AGPPc1 (Chen et al., 1994) and AGPNal (Du el al., 1994). (B) Domain structure exhibited by AGPNa2 (Mau et al., 1995). (C) Domain structure exhibited by AGPPc2 (Mau et ul., 1995). (D) Domain structure exhibited by AGPNa3 (Du et al., 1996b). Several additional cDNAs predicted to encode classical AGPs are discussed in the text. The secretion signal has been shown to be excised from the mature AGP in all classical and non-classical examples that have been carefully examined thus far. The hydrophobic domain has been shown to be excised and replaced by a C-terminal ethanolamine, as is characteristic of glycosyl-phosphatidylinositol anchors (see Section IV.A.2), in mature AGPPcl and AGPNul, the only two AGPs examined so far (You1 et al., 1998). Reproduced with permission from Nothnagel (1997).
extensins and other structural wall proteins, however, these A G P motifs tend to exhibit low repetition frequency and little consensus from one A G P to another. The occurrence of short runs of Pro alternating with Ala or Ser has also been noticed in several AGPs (Showalter and Varner, 1989; Loopstra and Sederoff, 1995; Li and Showalter, 1996). While a more generalized form of this sequence, Pro-X-Pro-Z-Pro (where X and Z are any amino acids), occurs in many confirmed and putative AGPs, even this is not a consensus sequence as it does not occur in the confirmed AGPs AGPNu1 and AGPNu3 (Nothnagel, 1997). Thus, a t the level of amino acid sequence, AGPs d o not appear to exhibit a definitive common characteristic. As additional cDNAs encoding AGPs become available, sequence homologies may become more evident. Because carbohydrate accounts for 90% or more of the mass of most AGPs, the amino acid sequences that encode glycosylation sites are of great importance. Potential N-glycosylation sites occur at Asn in the sequences Asn-X-Ser or Asn-X-Thr where X is not Pro (Montreuil et ul., 1994). Structural analysis of purified AGPs has rarely yielded any evidence of Nglycosylation (Clarke et ul., 1979a), however, and only a few of the known
218
M.D. SERPE and E. A. NOTHNAGEL
cDNAs encoding core polypeptides of confirmed or putative AGPs contain any potential N-glycosylation sites (Chen et al., 1993; Cheung et al., 1993; Loopstra and Sederoff, 1995; Li and Showalter, 1996). Among these potential N-glycosylation sites, only those in the TTS and NaPRP4 glycoproteins of Nicotiana have thus far been shown to be actually glycosylated (Wang et al., 1993; Sommer-Knudsen et al., 1996). The glycan-polypeptide linkages in AGPs are typically present as 0glycosidic linkages. Determining the exact aminoacyl residues involved in these linkages is a major problem as the precise sequence codes for O-glycosylation sites are not yet established. Furthermore, in contrast to the N-glycosidic bond which always occurs with Asn, the O-glycosidic bond can occur with several aminoacyl residues including Hyp, Ser, Thr and hydroxylysine (Montreuil et al., 1994). Carbohydrate-polypeptide O-glycosidic linkages have been chemically identified in only a few AGPs. In an arabinogalactan-peptide from wheat endosperm (Strahm et aE., 1981) and in an AGP fraction purified from gum arabic (Qi et al., 1991), galactosyl-O-Hyp was identified as a carbohydratepolypeptide linkage. Evidence of arabinosyl-O-Hyp linkages was found for AGPs from sycamore (Pope, 1977) and bean (van Holst and Klis, 1981). Unidentified glycosyl residues were also found linked to Hyp residues in tobacco AGPs (Kawasaki, 1987a) and probably in an AGP from Lolium multijlorum (Bacic et al., 1987). Post-translational hydroxylation of Pro residues in the core polypeptide is required for glycosylation of AGPs through O-glycosidic linkages to Hyp. Although the codes that govern hydroxylation of Pro have not been confirmed, some rules have been proposed on the basis of hydroxylations found in extensins. Kieliszewski and Lamport (1994) suggested that the sequences LysPro, Tyr-Pro and Phe-Pro are not hydroxylated, while Pro-Val is always hydroxylated. Matsuoka et al. (1995) suggested that hydroxylation of Pro is common if this residue occurs in a reverse turn region of a polypeptide. Once hydroxylation of Pro to Hyp has occurred, other unidentified codes presumably specify which of the Hyp residues will be glycosylated. The appearance of Hyp in blocks, such as Hyp4, increases the likelihood of attachment of arabinose oligosaccharides in extensins (Kieliszewski et a[., 1995). While such Hyp-arabinose oligosaccharides have been found in an AGP from gum arabic (Qi et al., 1991), it remains to be ascertained whether they occur in other AGPs. In addition to Hyp, the aminoacyl residues Ser and Thr have been found in O-glycosidic linkages in some AGPs. Galactosyl-O-Ser linkage has been reported for leaf AGPs from Cannabis sativa (Hillestad et al., 1977) and radish (Tsumuraya et al., 1984). Unidentified glycosyl residues were linked to Thr in AGPs from radish seeds (Tsumuraya et al., 1987) and grape berries (Saulnier and Brillouet, 1989).
ARABINOGALACTAN-PROTEINS IN THE PLANT CELL SURFACE
2 19
Whether more than one type of aminoacyl residue is involved in O-glycosidic bonds in a single AGP is unknown. Similarly, the total number of carbohydrate-polypeptide linkages per macromolecule has been estimated for only a few AGPs (Kawasaki, 1987a). Such information is useful in inferring the average size of the glycans attached to the polypeptide core, thus improving our understanding of the macromolecular organization of AGPs. 2. Carbohydrate Component Characterization of the structural details of the carbohydrate chains of AGPs may ultimately prove to be a key to understanding AGP function. While function is primarily associated with the polypeptide portion rather than the carbohydrate portion of many glycoproteins, the situation may be reversed for AGPs which typically consist of more than 90% carbohydrate. Analyses of AGPs by nuclear magnetic resonance (NMR) spectrometry have shown that the NMR signals from the (1 + 3)-P-~-galactanbackbone are flattened and broadened relative to the signals from the (1 + 6)-P-~-galactanside chains and their substituents. This flattening and broadening of NMR signals has been attributed to restricted mobility toward the inner region of AGP molecules. Signals from aminoacyl residues in AGPs have not been observed by NMR, apparently because motion at the protein core is so restricted that the NMR signals are flattened into the background (Saulnier et al., 1992; Gane et al., 1995b). These NMR observations suggest that the core polypeptide is locked in position towards the interior of the AGP. Thus, functional interaction with other macromolecules is sterically much more possible for the peripheral glycosyl residues than for the internalized aminoacyl residues of AGPs. As described in some following sections in this review, a large literature involving immunocytochemistry with monoclonal antibodies links expression of AGP epitopes with plant development. These observations likewise point to a possible functional importance of peripheral glycosyl residues in AGPs, as the epitopes of most of the monoclonal antibodies occur among these peripheral residues (Knox, 1997; Nothnagel, 1997). Thus, variations in terminal glycosyl sequences or other subtle structural features of AGPs may be involved in cellto-cell communications, cell identity expression or other processes of plant development. A distinctive characteristic of AGP glycans is the presence of a (1 -+ 3)-P-Dgalactan backbone with (1 --t 6)-P-~-galactanside chains (Aspinall, 1973; Clarke et al., 1979a; Fincher et al., 1983) (Fig. 2). Arabinose and lesser amounts of other sugars such as glucuronic acid and rhamnose are attached to the galactan framework (Clarke et al., 1979a; Bacic et al., 1987). Although most AGPs share these characteristics, considerable differences among AGPs arise through variations in the galactan framework, in the size of the glycan chains attached to polypeptide core, and in the type and proportions of sugars attached to the galactan framework.
I &!=O
I
=-r= =-I-= z-I I
010
I
W
0
t W
n
3
2
\
,
1
POLYPEPTlDE
a
ARABINOGALACTAN-PROTEINS IN THE PLANT CELL SURFACE
22 1
Although most AGPs seem to contain a pure (1 + 3)-/?-~-galactan backbone, exceptions to this characteristic have been reported. In an AGP from the culture medium of Lolium multzjlorum, the (1 + 3)-/3-~-galactan backbone is interrupted by residues of (1,6)-linked galactopyranose or (1,5)linked arabinofuranose (Bacic et al., 1987). Similarly in an AGP secreted from Acacia robusta (Churms and Stephen, 1984), blocks of four residues of 3-linked galactopyranose are separated by single (1,6)-linked galactopyranose residues. The effects of these backbone variations on molecular shape and function remain unknown. The molecular shape of AGPs is likely influenced by the size of the arabinogalactans attached to the polypeptide core. The size of these glycans varies among different AGPs, with estimates falling in the range of 30-150 sugar residues (Tsumuraya et al., 1984; Kawasaki, 1987a; Qi et al., 1991; Gane et al., 1995b). Some AGP molecules have been shown to contain glycans of more than one size. Analysis of an AGP from gum arabic revealed the presence of large glycan chains but also smaller Hyp-arabinose oligosaccharides, similar to those of extensins (Qi et af., 1991). Macromolecules that seem to contain both the large glycans of AGPs and the small arabinose oligosaccharides of extensins have also been found in Zea mays (Kieliszewski et al., 1992) and Nicotiana alata (Lind et al., 1994; Schultz et af., 1997).
Fig. 2. Hypothetical structural model of an arabinogalactan-protein (AGP) carrying a glycosyl-phosphatidylinositol (GPI) lipid anchor. The ellipse represents the 15 x 25 nm size of carrot AGPs, as observed by electron microscopy (Baldwin et al., 1993). The wavy line along the long axis of the ellipse represents the core polypeptide. For a 141-kDa AGP containing 5.6% protein (Nothnagel, 1997), the length of the core polypeptide in the polyproline I1 conformation found in AGPs (van Holst and Fincher, 1984) is calculated to be 24 nm, essentially the same length as the long axis of the ellipse. The GPI anchor is similarly drawn to approximate scale. The presence of ethanolamine at the C-terminus of the polypeptide has been demonstrated for AGPPcI and AGPNal (You1 et al., 1998), and the ceramide lipid consisting of tetracosanoic acid and phytosphingosine is as found in rose AGPs (Svetek et al., 1999). The linker oligosaccharide is drawn to match the consensus GPI linker core ethanolamine -+ PO4 -+ 6Manal --+ 2Mana1 + 6Mancul + 4GlcNal + 6myo-inositoll -+ PO4 -+ lipid found in animals and micro-organisms, but this structure has not yet been verified for higher plants. The site of cleavage by phosphatidylinositol-specific phospholipase C (PIPLC) is indicated. The type I1 arabinogalactan chains in AGPs typically consist of 3& 150 sugar residues (Tsumuraya et al., 1984; Kawasaki, 1987a; Qi et al., 1991; Gane et al., 1995b), and such chains would typically be attached to a number of the amino acid residues in the core polypeptide. The structures shown as side chains on the (1 + ~)-P-Dgalactan backbone are based on oligosaccharides cleaved from various AGPs and structurally characterized as P-~-(4-O-methyl-GlcpA)-(1 + 6)[a-~-Araf(1 + ~)]-P-DGalp-(1 -t 6)-~-Gal(Misawa et al., 1996), a-L-Rhap-(l + 4)-P-~-GlcpA-(l-+ ~)-P-DGalp-(1 -+ (Defaye and Wong, 1986; Pellerin et al., 1995) and a-L-Araf(1 3)-a-~Araf(1 -+ 3)-P-~-Galp-(l + (Defaye and Wong, 1986). The placement of these oligosaccharides as shown, however, is hypothetical. --+
222
M. D. SEFWE and E. A. NOTHNAGEL
Based on the size of the glycan chains attached to the polypeptide core, two models have been proposed for the macromolecular organization of AGPs: the ‘wattle blossom’ model (Fincher et al., 1983) and the ‘twisted hairy rope’ model (Qi et al., 1991). In the ‘wattle blossom’ model, the glycan chains contain about 100 sugar residues and fold into a globular shape. Attachment of several or many such glycans to the polypeptide core results in an AGP of overall spheroidal shape (Fincher et al., 1983). In the ‘twisted hairy rope’ model, the carbohydrate is present as both short linear oligosaccharides and medium-sized (about 30 residues) glycans of extended conformation. According to this model, the AGP has an overall elongated shape with short projections along its length. Some of the data supporting the ‘twisted hairy rope’ model have been criticized (Osman et al., 1993; Islam et al., 1997). Observation of the shape of some AGPs has been accomplished by rotary shadowing/transmission electron microscopy. Images of AGPs from the culture medium of carrot cells were ellipsoidal of size 15 x 25nm (Baldwin et al., 1993), while images of AGPs from the tobacco style were spheroidal with 30nm diameter (Cheung et al., 1995). Both of these images seem consistent with the predictions of the ‘wattle blossom’ model. In contrast, an apparent AGP from gum arabic appeared as a rod-like and flexible structure of about 5nm diameter and 150nm length (Qi et al., 1991), consistent with the ‘twisted hairy rope’ model. How AGPs of any of these shapes and sizes pass through the cell wall is not clearly understood. Depending on the plant material and the method of measurement, diameters of the largest pores in primary cell walls generally fall in the range of 4-1Onm (Carpita er al., 1979; Tepfer and Taylor, 1981; BaronEpel et al., 1988; McCann et al., 1990), except for one report of 18nm (Itoh and Ogawa, 1993). These pores might allow the rod-like molecule from gum arabic to thread its way through the cell wall, but would seem to restrict the movement of the carrot and tobacco AGPs. Nevertheless, the carrot and tobacco AGPs were found outside the cell wall. This apparent discrepancy remains to be resolved. Elucidation of the mechanism by which AGPs pass through the cell wall would be valuable with regard to understanding both the sieving properties of the cell wall and the conformation and flexibility of AGPs. While D-galactosyl and L-arabinosyl residues are characteristic of all AGPs, a wide variation exists regarding the other sugar residues that might be also present. Some AGPs, such as one from Lolium multijlorum (Bacic et al., 1987) and another from Nicotiana d a t a (Gane et al., 1995b), consist almost solely of galactosyl and arabinosyl residues in approximately 2: 1 molar ratio, with only trace amounts of other neutral sugars and no uronic acids present. More often, however, AGPs contain some amounts of other sugars that might include Lrhamnose, D-mannose, D-xylose, D-glucose, L-fucose, D-glucosamine and the uronic acids, D-glucuronic acid and D-galacturonic acid (Jermyn and Yeow, 1975; Clarke et al., 1979a; Akiyama and Kato, 1981; Fincher et al., 1983; Tsumuraya et al., 1984, 1988). Among these sugars, the one most commonly
ARABINOGALACTAN-PROTEINS IN THE PLANT CELL SURFACE
223
found in AGPs has been glucuronic acid, sometimes also present as the 4-0methyl glucuronic acid derivative. While glucuronic acid typically accounts for less than 10mol% of the total sugar content of AGPs, amounts up to 43 mol% have been found (Clarke et al., 1979a). Rhamnose in amounts less than 10mol% has also been commonly found in AGPs (Clarke et al., 1979a). An interesting exception in this regard is radish which has AGPs that contain no rhamnose, but instead contain either xylose or fucose, but not both (Nakamura et al., 1984; Tsumuraya et al., 1984, 1987, 1988). Structural analyses have shown that the sugars other than galactose tend to be attached to the (1 + 6)-P-~-galactanside chains of AGPs. These sugars are usually in terminal positions (Clarke et al., 1979a; Fincher et al., 1983), although rarely has their exact placement been ascertained. Analyses in this area have been largely limited to structural characterization of a few oligosaccharide fragments from gum arabic (Defaye and Wong, 1986), from AGPs present in grape pulp and red wine (Saulnier et al., 1992; Pellerin et al., 1995), and from AGPs purified from radish leaves and roots (Tsumuraya et al., 1984, 1990; Misawa et al., 1996). Some linkages found in these AGPs were a-LAraf(1 + 3)-P-~-Galp-(l+ (Defaye and Wong, 1986; Saulnier et al., 1992), aL-Araf-(1 + 3)-a-~-Araf(l +. 3)-P-~-Gaip-(l (Defaye and Wong, 1986) (Fig. 2), /3-~-(4-O-methyl-GlcpA)-(1 6)-P-~-Galp-(1 +. 6)-P-~-Galp-( 1 + 6)P-D-Galp-( I + 6)-~-Gal(Tsumuraya et al., 1990), P-~-(4-O-methyl-GlcpA)(1 + 6)[a-~-Araf(l +. 3)]-P-~-Galp-(l+ 6)-~-Gal(Misawa et al., 1996) (Fig. 1 + 6)-P-~-Galp-( 1 + (Defaye and Wong, 2), a-L-Rhap-(1 +. 4)-P-~-GlcpA-( 1986; Pellerin et al., 1995) (Fig. 2) and a-~-Fucp-(l+. 2)-a-~-Araf(l + (Tsumuraya et al., 1984). Complex sugar composition, extensive branching and large size combine to make structural analysis of AGP glycans a challenging problem. Contributions to solving this problem are coming from a variety of techniques such as NMR spectrometry (Agrawal, 1992; Saulnier et al., 1992; Gane et al., 1995b), methylation followed by gas chromatography-mass spectrometry (Carpita and Shea, 1989) and fragmentation by specific glycosidases (Gleeson and Clarke, 1979; Tsumuraya et al., 1984, 1990; Brillouet et al., 1991; Saulnier et al., 1992; Pellerin and Brillouet, 1994). Monoclonal antibodies have proven to be sensitive probes for distinguishing AGPs that differ in carbohydrate epitopes (Roberts, 1990; Knox, 1992, 1997; Pennell, 1992; Pennell and Roberts, 1995), and as the epitopes of these antibodies are more precisely identified (Steffan et al., 1995; Yates et al., 1996) the antibodies may become useful tools in the structural characterization of AGPs. Structural characterization and epitope identification studies are both likely to be facilitated by recent advances in chemical synthesis of oligosaccharides related to AGPs (Valdor and Mackie, 1997). Another major challenge in the study of the carbohydrate portion of AGPs is the elucidation of its biosynthesis. Although few of the steps in the biosynthesis of AGPs have been experimentally examined, the evidence +.
+.
224
M. D. SERPE and E. A. NOTHNAGEL
available thus far is consistent with the conventional endomembrane/secretory pathway for the biosynthesis of other glycoproteins and proteoglycans in eukaryotic cells. If this conventional pathway holds for AGP biosynthesis, then co-translational insertion of the AGP core polypeptide into the lumen of the endoplasmic reticulum is directed by the N-terminal signal sequence, which is subsequently excised. Recent results indicate that classical AGPs are synthesized with a glycosyl-phosphatidylinositol (GPI) lipid anchor (see Section IV.A.2). If the pathway of biosynthesis of GPI-linked proteins in plants is assumed to be the same as in other organisms (Kinoshita et al., 1997), then the C-terminal hydrophobic domain anchors the AGP core polypeptide to the endoplasmic reticulum membrane until this domain is excised and replaced by the GPI lipid, which then serves as the membrane anchor through the remainder of the secretory pathway to the plasma membrane. Post-translational hydroxylation of Pro residues occurs while the AGP core polypeptide is still in the endoplasmic reticulum. Glycosylation of the polypeptide possibly begins in the endoplasmic reticulum, but most of the glycosylation occurs after vesicular transport of the polypeptide to the Golgi apparatus (Gibeault and Carpita, 1994). The enzymes principally responsible for the biosynthesis of the glycan chains of glycoconjugates and polysaccharides are glycosyltransferases (Gibeaut and Carpita, 1994). While mRNA serves as a template for the synthesis of a polypeptide chain, no analogous template has been found for the assembly of any complex carbohydrate. Thus, current models hold that the information specifying a particular carbohydrate linkage is contained within the glycosyltransferase that forms that linkage, the implication being that a separate glycosyltransferase is required for each type of linkage. Because of the great structural complexity of AGPs, many glycosyltransferases could be required for the synthesis. None of the glycosyltransferases involved in AGP synthesis have been isolated and characterized, but appropriate activities have been detected in membrane preparations. For example, unfractionated membranes from cultured cells of ryegrass endosperm incorporated radioactive galactosyl residues from uridine 5'-diphosphate (UDP)-['4C]galactose into various macromolecules, including some having the (1 + 6)-P-~-galactanlinkage characteristic of AGPs (Mascara and Fincher, 1982). Subsequent fractionation of these membranes revealed that the synthesis of (1 ---f 6)-P-~-galactanwas localized in the Golgi apparatus (Schibeci et al., 1984). Synthesis of (1 -+ 6)-P-~-galactanwas also detected in a pea microsomal fraction (Hayashi and Maclachlan, 1984). Other glycosyltransferases that participate in AGP synthesis are arabinosyltransferases. Evidence for the involvement of these enzymes in AGP synthesis was reported by Bolwell (1984) and Kawasaki (1987a, b) who detected the incorporation of arabinosyl residues into bean and radish AGPs, respectively. Arabinosyltransferase activity is not unique to AGP synthesis, however, as several other plant polysaccharides and glycoconjugates also contain
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arabinosyl residues. The incorporation of these residues into products such as arabinans and extensins is likely to be mediated by arabinosyltransferases different from those implicated in AGP synthesis. While arabinosyltransferase activity is typically associated with Golgi membranes (Gardiner and Chrispeels, 1975; Kawasaki, 1981; Owens and Northcote, 1981; Rodgers and Bolwell, 1992), this activity has also been reported to occur in the endoplasmic reticulum (Andreae et al., 1988). Among the many other transferases required for AGP synthesis, only fucosyltransferases appear to have been investigated. Misawa et al. (1 996) reported a fucosyltransferase with an activity appropriate for transferring fucosyl residues to radish AGPs. This transferase was able to move a L-fucosyl residue from guanosine 5’-diphosphate (GDP)-fucose onto a trisaccharide acceptor. The resulting tetrasaccharide, CX-L-FUC~-( 1 + 2)-a-~-Araf-(1 + 3)-PD-Galp-(l + 6)-~-Gal,is a terminal oligosaccharide in radish AGPs (Tsumuraya et al., 1984). This fucosyltransferase activity was correlated with the synthesis of fucosylated AGPs and was localized in the Golgi apparatus. The transfer of individual glycosyl units from nucleotide phosphate sugars may not be the only mechanism of AGP assembly. Some observations suggest that AGPs synthesis may also involve block assembly. In some AGPs (Churms and Stephen, 1984; Bacic et al., 1987), the (1 --t 3)-P-~-galactanbackbone exists in blocks interspersed with a different residue. Furthermore, certain tissues contain oligosaccharides with linkages characteristic of AGPs (Kawaguchi et al., 1996). These observations leave open the possibility that (1 -+ ~)-P-Dgalactan blocks or other oligosaccharides are separately synthesized and then incorporated into the growing AGP molecule. The mechanism of assembly might be analogous to the formation of N-linked glycoproteins. In these molecules, oligosaccharides are initially synthesized as dolichol-phosphate glycolipids and then transferred from the lipid to the polypeptide. Although no definitive evidence has been found for the participation of dolichol-phosphate glycolipids in AGP synthesis, Mascara and Fincher (1982) and Hayashi and Maclachlan (1984) observed incorporation of galactosyl residues from radioactive UDP-galactose into glycolipids. An alternative mechanism of AGP assembly might involve the transfer of oligosaccharides to sugar residues already attached to the polypeptide core. Glycosyltransferases capable of splicing oligosaccharides onto sugar residues in macromolecules have been found in plants (Fry, 1995). Little is known, however, about the role of these or similar enzymes in the biosynthesis of cell surface components. B. EXPRESSION AND FUNCTION
The expression of extracellular AGPs has been analysed with various molecular probes and approaches. Probes used in the study of AGPs include Yariv phenylglycosides (Yariv et al., 1967), lectins (Schopfer, 1990), polyclonal
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and monoclonal antibodies (Knox, 1992, 1997; Kikuchi et al., 1993; Yates and Knox, 1994; Smallwood et al., 1996) and, more recently, nucleotide probes corresponding to the mRNAs encoding core polypeptides of putative and confirmed AGPs (Chen et al., 1993; Cheung et al., 1993; Du et al., 1994; Mau et al., 1995). Analytical and preparative biochemical methods have also been used to detect, separate and characterize the AGP complements of various tissues and exudates (Tsumuraya et al., 1984, 1987, 1988, 1990; van Holst and Clarke, 1986; Osman et al., 1995). Results from these analyses have shown that the types and amounts of some extracellular AGPs vary in response to stress, age and/or stage of tissue development. These variations in expression, combined with results from other experimental approaches, have provided an insight into the function of extracellular AGPs (Kreuger and van Holst, 1993, 1995; Cheung et al., 1995; Wu et al., 1995; Jauh and Lord, 1996; Roy et al., 1998). This section will focus on the expression and function of extracellular AGPs in those plant tissues where they are relatively abundant. 1. AGPs in Mucilages and Gums Mucilages and gums are complex mixtures of various polymers including polysaccharides, proteoglycans, proteins, phenolic compounds and/or lipids (Aspinall, 1970; Clarke et al., 1979a; Stephen, 1983; Whistler, 1993). Gums and mucilages accumulate in intercellular spaces, such as gum ducts or mucilage cavities, and in the lumen of dead cells, such as tracheids and vessels (Clarke et al., 1979a; Fahn, 1981). Mucilages and gums are also secreted from some plant tissues. These secretions are of widespread occurrence in the root tip but also occur, either naturally or in response to injury, in aerial parts of certain plants (Clarke et al., 1979a; Fahn, 1981; Whistler, 1993). Although mucigel or slime secretion by the root cap occurs in most plant species, the chemical composition of this mucigel has been studied in detail for only a few species (Moody et al., 1988). The available data suggest that AGPs are an important component of this extracellular secretion. In cowpea, wheat and maize, the root slime contains (1,3)-, (1,6)- and (1,3,6)-linked galactopyranosyl residues and/or Hyp residues (Bacic et al., 1986; Moody et al., 1988), both of these being possible indicators of AGPs. Furthermore, components of these slimes bind to (P-D-GIc)~Yariv phenylglycoside and to the mouse myeloma IgA 5539, a monoclonal antibody that recognizes (1,6)-linked pgalactopyranosyl residues (Glaudemans et al., 1986; Moody et al., 1988). Similarly, a polyclonal antibody directed against (1 + 6)-,&~-galactotetraose oligosaccharide strongly labelled the periphery of root cap cells of radish (Kikuchi et al., 1993). The p-(1 + 6)-galactan linkage is considered indicative of type I1 arabinogalactans, as found in AGPs, because it is not common in other plant polymers (Kikuchi et al., 1993). Taken together, the above results strongly suggest the presence of AGPs in the root mucigel. The root mucigel has several functions that include lubricating the root tip for passage through the soil, protecting the tip against desiccation, facilitating
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absorption of nutrients and mediating interactions with soil microorganisms. Because AGPs have a high water-holding capacity and can form gels, Moody et al. (1988) speculated that AGPs contribute to the protective and lubricative properties of the slime. Acidic sugar residues of AGPs might contribute to absorption of nutrient cations from the soil. Somewhat conversely, AGPs might be degraded and serve as a source of nutrients for soil microflora. Performance of these and similar functions, however, might also involve other mucigel components such as pectins, xyloglucans and glucans (Bacic et al., 1986; Moody et al., 1988). Consequently, the precise contribution of AGPs to mucigel function is still uncertain. Arabinogalactan-proteins are also abundant components of certain gums. The best known and most studied of these gums is gum arabic, secreted upon wounding the bark of Acacia senegal (Clarke et al., 1979a; Stephen, 1983; Whistler, 1993). Gum arabic has been used for various purposes by humans for 4000years. The Ancient Egyptians used gum arabic as an adhesive when wrapping mummies, and in mineral paints when making hieroglyphs (Whistler, 1993). While gum arabic has also been used as an adhesive in modern times, such as in envelope closures, the most important applications of gum arabic have been in the food and pharmaceutical industries. Gum arabic has the unique combination of being an excellent emulsifier but yet having low viscosity even at high concentrations. These properties make gum arabic very useful as a flavor encapsulator, an agent to prevent sucrose crystallization in confections and, especially, as a stabilizer of citrus oil emulsion concentrates in the soft-drink industry. The emulsifying and coating characteristics of gum arabic are also the basis of its applications in the pharmaceutical and cosmetic industries. Its easy wettability, spreadability and stabilization of other chemicals have likewise made gum arabic useful in lithography (Alain and McMullen, 1985; Randall et al., 1989; Whistler, 1993; Ray et al., 1995; Islam et al., 1997). Although gum arabic has been studied for many years, the characteristics of the AGPs present in this gum, or even how many different AGPs are present, have not been clearly established. A complicating factor in analysing gum arabic is the variation in composition that occurs among samples collected from different trees and during different seasons (Clarke et al., 1979a; Yates and Knox, 1994). Nevertheless, several recent investigations have involved use of advanced chromatography or other techniques to fractionate gum arabic and identify the AGPs present in this gum (Randall et al., 1989; Qi et al., 1991; Osman et al., 1993, 1995). The results of these studies indicate that gum arabic contains several AGPs that can be distinguished from each other on the bases of sugar composition, protein content and/or molecular weight. Randall et al. (1989) used hydrophobic interaction chromatography to separate gum arabic into three fractions, all being rich in arabinosyl and galactosyl residues but differing in protein content of 0.4, 12 or 47% (w/w). The fractions with 0.4 and 12% protein accounted for 88.4 and 10.4% of the
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total gum, respectively. The protein-rich fractions appeared to determine the emulsifying properties of gum arabic (Randall et al., 1988; Ray et al., 1995). Somewhat similar results were obtained by Qi et al. (1991), who used gel permeation chromatography to separate gum arabic into a high molecular weight fraction containing 10% protein and a lower molecular weight, heterogenous, fraction containing less than 1YO protein. Fractionation by anion-exchange chromatography was more effective and separated gum arabic into five or six fractions (Osman et al., 1995). These fractions had similar carbohydrate compositions, and ranged between 0.3 and 2.8% in protein content. All of the fractions bound to (P-D-GIc)~Yariv phenylglycoside and to several members of a panel of monoclonal antibodies directed against AGPs. Different fractions bound different subsets of antibodies from the panel, however, indicating that they contained distinct AGP epitopes. These results suggest that gum arabic contains at least five distinct AGP molecules. While Acacia senegal is the most important source of gum arabic for commercial use, other Acacia species produce gums that are also called gum arabic (Stephen, 1983; Whistler, 1993; Islam et al., 1997; Mocak et al., 1998). Acacia seyal and, to a lesser extent, Acacia laetia are the commercially most important of these other species and together account for about 20% of the total gum arabic production (Whistler, 1993; Islam et al., 1997; Mocak et al., 1998). Overall, at least 90 Acacia species produce gums that have been chemically investigated (Stephen, 1983). Fractionation and subsequent structural analyses of many of these gums have revealed that they contain AGPs (Churms and Stephen, 1984; Churms et al., 1986; Gammon et al., 1986; de Pinto et al., 1998). Variations in AGP structure and gum composition occur among Acacia species and have been used to establish chemotaxonomic differences within the genus (Anderson and Dea, 1969; Anderson and Gill, 1975). Investigations into the chemical composition and average molecular mass of Acacia gums also have practical value because these characteristics affect the emulsifying and binding power of the gum, as well as its hydration and solubility properties (Clarke et al., 1979a; Alain and McMullen, 1985). Changes in these attributes affect the quality of the food and pharmaceutical products that contain gum arabic as an ingredient. Gums from some species outside the Acacia genus probably also contain AGPs. Within the Fabaceae family, which includes Acacia, gum exudates from species belonging to various genera have been analysed for carbohydrate and amino acid compositions (Anderson and de Pinto, 1985; Anderson and Weiping, 1990; Anderson et al., 1990). Most of these gums are rich in galactosyl, arabinosyl and glucuronosyl residues, and have a proteinaceous component that, in some gums, has a high proportion of Hyp residues (Churms et al., 1986; Gammon et al., 1986; Anderson et al., 1987). These features suggest that AGPs are components of these gums, although further tests are required to establish this conclusion.
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Species from several families other than the Fabaceae also produce gums that contain type I1 arabinogalactans (Clarke et al., 1979a; Stephen, 1983). Within the angiosperms, type I1 arabinogalactans are found in gums from species in the Combretaceae, Rosaceae, Anacardiaceae, Meliaceae and Cactacea families (Stephen, 1983; de Pinto et af., 1994; Goycoolea et af., 1997). Similarly, within the gymnosperms, gums containing type I1 arabinogalactans are produced by species in the Pinophyta, Cycadophyta and Gnetophyta divisions (Clarke et al., 1979a). Some of these arabinogalactans have no detectable protein component and consequently are not AGPs. Such is the case, for example, with an arabinogalactan from Larix, a gymnosperm that accumulates large amounts of mucilage in the lumen of tracheids (Clarke et af., 1979a; Stephen, 1983). For most gums, however, the available chemical data neither definitively establish nor rule out the presence of AGPs. In these cases, tests for the presence of (P-~-Glc)3-bindingmolecules may provide valuable information. Using this approach, Harris et af. (1992) detected (P-D-GIc)~binding molecules, presumably AGPs, in mucilage ducts of Cofosia escufenta,a monocot plant that is an important food crop in tropical areas. Although many plant species produce gums, little is known about their function. Similarly, the biological function of the AGPs within the gums remains to be established. Various functions have been proposed, as reasoned from knowledge of the factors that stimulate gum secretion or from the observed physico-chemical properties of the gum. Because mechanical injuries promote the production of gums and sometimes the secretion of extracellular AGPs (Clarke et al., 1979a, Whistler, 1993), action of these molecules in wound healing has been proposed (Whistler, 1993), although the exact nature of such action remains uncertain (Fincher et al., 1983). One possibility is that AGPs act as adhesives that facilitate sealing or plugging of wounded tissue, thereby preventing desiccation and/or pathogen invasion. Alternatively, some gums contain oils and secondary metabolites (Clarke et af., 1979a; Anderson et al., 1990) and in these gums AGPs may act as emulsifying agents that contribute to the stability of such compounds within the gum mixture. At present, however, none of these proposed functions is supported by direct evidence. From the perspective of AGP research, gums have been valuable in the production of antibodies directed against AGPs and in the identification of AGP epitopes. In some cases, antibodies have been produced using whole gums as the immunogen (Pazur et af., 1991). This approach yields antibodies directed against unknown epitope(s), especially when the gums are complex mixtures of various macromolecules. This limitation can be avoided by partially fragmenting the gum to yield a mixture of oligosaccharides and then using a particular oligosaccharide from this mixture as a hapten. For example, Kikuchi et al. (1993) purified a (1 + 6)-P-~-galactotetraoseoligosaccharide from a partial hydrolysate of gum ghatti. This oligosaccharide was then coupled to a protein and used as the immunogen for the production of polyclonal antibodies. Upon affinity purification, the resulting antibody
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preparation was highly specific for the (1 -+ 6)-P-~-galactanlinkages in AGPs (Kikuchi et al., 1993). Another important use of gums, especially gum arabic, has been in the identification of antibodies that recognize AGP epitopes. When complex mixtures of plant components such as the conditioned medium of cells in culture, membrane vesicle preparations or whole protoplasts have been used as the immunogen for the production of monoclonal antibodies (Anderson et al., 1984; Villaneuva et af., 1986; Knox et al., 1989; Norman et al., 1990; Pennell et al., 1991; Smallwood et al., 1996), a useful approach for characterizing the resulting antibodies has been to test their reactivity with gum arabic (Pennell, 1992; Yates and Knox, 1994). Gum arabic, however, contains certain sugar residues such as terminal a-L-arabinosyl residues (Akiyama et al., 1984) that are not unique to AGPs. These residues are present in various cell wall polymers including arabinans, rhamnogalacturonan I, glucuronoarabinoxylans and extensins (Darvill et al., 1980; Bacic et al., 1988; Carpita and Gibeaut, 1993). Consequently, these other polymers or appropriate haptens have been used as competitive inhibitors in binding assays to assess the specificity of antibodies for AGPs (Pennell, 1992). Gums have also been valuable as sources of complex oligosaccharides for use in the precise identification of antibody epitopes. A standard approach to characterizing antibody epitopes has been to use oligosaccharides of known chemical structure as competitive inhibitors in binding assays (Pazur et al., 1991; Lind et al., 1994; Steffan et al., 1995). An interesting alternative approach was taken by Yates et al. (1996) who partially hydrolysed gum karaya to produce complex oligosaccharides which were then screened by antibody binding to find an oligosaccharide containing the epitope. Structural overlap between epitopes of different monoclonal antibodies has also been evaluated in epitope-mapping experiments with gums such as gum ghatti, gum karaya and gum tragacanth (Yates and Knox, 1994). Taken together, these various studies involving gums have been an important part of AGP research. It seems likely, however, that much more could be gained here. Progress in biological research has often been greatly facilitated by studies of a model system wherein the process of interest is more prominent than in the norm. Acacia trees and other plants that produce massive quantities of gum exudates seem to await exploitation as model systems for studies of AGP synthesis, expression and function. 2. Extracellular AGPs in the Gynoecium A model system that has been effectively exploited in the study of AGPs is the gynoecium of flowers. Arabinogalactan-proteins are major components of the exudates of the stigma and style (Knox et al., 1976; Gleeson and Clarke, 1979; 1980; Hoggart and Clark, 1984). The abundance of extracellular AGPs in these exudates has suggested roles for these molecules in pollen-stigma recognition and in pollen tube extension (Knox et af., 1976; Clarke et al., 1979b). This
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23 1
notion has been further supported by observations of variations in AGP types and/or abundances during gynoecium development. Using crossed electrophoresis with (P-D-G~c)~ embedded in the second-dimension gel, Gel1 et al. (1986) showed that stigma and style extracts of Nicotiana alata contain different classes of AGPs. The amounts of these AGPs changed during flower development and again after pollination. Although this study did not distinguish the subcellular localization of AGPs, work by Sedgley and Clarke (1986) indicated that some of the observed changes were attributed to extracellular AGPs. Sedgley and Clark (1986) used a monoclonal antibody directed against a-L-arabinosyl residues to locate AGPs in the transmitting tissue of Nicotiana alata. This antibody labelled the Golgi apparatus, multivesicular bodies, cell wall and intercellular matrix. A general increase in intensity of labelling with the antibody was observed during flower development, as was a relative increase in the proportion of labelling on the intercellular matrix of the transmitting tract, the space along which pollen tubes grow towards the ovary. These results indicate an increase in the secretion of extracellular AGPs during gynoecium development. More recently, expression of stylar AGPs during development has been investigated at the level of mRNAs corresponding to the core polypeptides of confirmed and putative AGPs. At least seven examples of expression of mRNAs of AGPs or AGP-like molecules in the style and/or stigma have been reported. Five of these mRNAs correspond to cDNAs from Nicotiana alata: AGPNa1 (Du et af., 1994), AGPNa2 (Mau et af., 1995), AGPNa3 (Du et al., 1996a), NaPRP4 (Chen et al., 1993) and NaPRP5 (Schultz et al., 1997); and the other two correspond to cDNAs from Nicotiana tabacum: TTS-1 and TTS-2 (Cheung et af., 1993). The mRNAs corresponding to AGPNa1 and AGPNa2 were expressed in various other parts of the plant as well as in the style (Du et al., 1994; Mau et af., 1995). In contrast, expression of mRNAs corresponding to AGPNa3 (Du et al., 1996a), NaPRP4 (Sommer-Knudsen et al., 1996), NaPRP5 (Schultz et al., 1997), TTS-1 and TTS-2 (Wang et al., 1993) occurred exclusively or primarily in the style and/or stigma. The levels of expression of these mRNAs increased during gynoecium maturation and, at least in some cases, declined again as maturity was reached (Schultz et af., 1997). For the TTS mRNAs, the increase in expression occurred first near the stigma end of the style and progressed toward the ovary end (Wang et al., 1993). Premature pollination caused a three- to five-fold increase in the abundance of TTS mRNAs. Changes in mRNA expression following normal pollination were also observed with some cDNAs. After pollination, the AGPNa3 mRNA declined (Du et al., 1996a) and the TTS mRNAs became smaller due to poly(A) tail-shortening (Wang et af.,1996). The reduction in size of the TTS mRNAs was associated with pollen tube penetration of the transmitting tissue (Wang et al., 1996). This effect may have been mediated by the plant hormone ethylene. Pollen tube penetration caused increases in the abundance of mRNAs that encode ethylene biosynthetic enzymes. Also,
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application of exogenous ethylene induced TTS poly(A) tail shortening in unpollinated flowers (Wang et al., 1996). In contrast to these cases where pollination influenced expression, the expression of the mRNA corresponding to NaPRPS was unaffected by pollination with either compatible or incompatible pollen. Expression of NaPRP5 was strong with or without pollination, as its mature 120-kDa glycoprotein product accounted for approximately 9% of the total buffer-soluble protein in the transmitting tissue of styles (Schultz et al., 1997). Within the style and stigma, spatial regulation of the expression of mRNAs encoding AGPs has been demonstrated using in situ hybridization. The AGPNa3 mRNA was abundant in the stigma and weakly present in the transmitting tissue of the style (Du et al., 1996a). In contrast, the mRNAs corresponding to NaPRP4, TTS-1 and TTS-2 were abundant in the transmitting tissue (Cheung et al., 1993; Sommer-Knudsen et al., 1996). For some cDNAs, localization of the corresponding mature AGP or AGPlike molecule at cell surface sites within the style has been examined. To detect the TTS glycoproteins in the style of Nicotiana tabacum, Wang et al. (1993) generated a polyclonal antibody directed against bacterially-produced TTS-2 protein. This antibody bound evenly to the intercellular matrix of the transmitting tissue but did not bind appreciably to the walls of the transmitting tissue cells nor to other cells in the style (Wang et al., 1993). In contrast, the glycoprotein corresponding to NaPRP4, whose core polypeptide is 96.9% identical to the polypeptide encoded by TTS-1 (Chen et al., 1993; Cheung et al., 1993), was not found in the intercellular matrix but instead was found ionically bound to the walls of stylar cells in Nicotiana alata (Sommer-Knudsen et al., 1996). These contrasting results have been critically compared (SommerKnudsen et al., 1998b) and seem to suggest that post-translational modifications or differences in glycosylation might be important in determining the final localization of AGPs. While the glycoprotein corresponding to NaPRP4 was bound to the walls of stylar cells (Sommer-Knudsen et al., 1996), the 120-kDa glycoprotein corresponding to NaPRP5 was found in the intercellular matrix of the stylar transmitting tissue of Nicotiana alata, as judged by cytochemistry with a polyclonal antibody raised against the polypeptide backbone (Lind et al., 1994). The polypeptide backbones encoded by NaPRP4 and NaPRP5 share antigenic determinants (Sommer-Knudsen et al., 1996) and, within their Cterminal domains, are 57% identical at the amino acid level and exhibit a conserved pattern of six Cys residues (Schultz et al., 1997). Thus, NaPRP4 and NaPRPS provide another example of closely related AGP-like molecules having different localizations and perhaps different functions at the cell surface. Localization of the NaPRP5 product in the intercellular matrix of the stylar transmitting tissue is probably important with regard to the observation that this molecule seems to be taken up by growing pollen tubes in the style (Lind et al., 1996).
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Jauh and Lord (1996) used antibodies directed to the carbohydrate moiety of AGPs to investigate the localization of AGPs in the gynoecium of lily and found that AGPs in the transmitting tissue are distinct from those in other nearby cells. In the lily the transmitting tissue consists of a layer of secreting epidermal cells that lines the internal cavity of the hollow style. The LM2 monoclonal antibody, which recognizes an epitope containing a P-D-glucuronopyranosyl residue (Smallwood et al., 1996), bound to the surface of every cell in the style except the cells of the transmitting tissue. In contrast, the JIM13 monoclonal antibody, which recognizes an epitope contained within the @-~-GlcpA-( 1 -+ 3)a-D-GalpA-(1 + 2)-~-Rhaoligosaccharide (Yates et al., 1996), bound only to the transmitting tissue cells and their secretions (Jauh and Lord, 1996). The JIM 13 epitope was similarly detected in the secretions of septum epidermal cells in the transmitting tissue of Arabidopsis thaliuna (Lennon et al., 1998). The spatial regulation of AGP epitope expression in the transmitting tissue of the lily suggests that AGPs may play a role in pollen tube elongation. More direct evidence of such a role was obtained by using (O-D-GIC)~ to perturb AGPs in living tissue. Jauh and Lord (1996) injected (P-D-GIc)~into the hollow lily style and observed reduced pollen tube growth and fertilization. It was uncertain, however, that this inhibition of pollen was due to perturbation of stylar AGPs as (P-D-GIC)~ profoundly inhibits lily pollen tube growth in vitro (see Section III.B.2). Also, because (P-D-GIc)~binds to many AGPs, it was not possible to distinguish whether one or more AGPs were involved in the observed phenomenon. Among AGPs or AGP-like molecules present in stylar secretions, the TTS glycoproteins from Nicotiana tabucum and the NaPRP4 glycoprotein from Nicotiana data have been the most studied with regard to function. While the TTS-1 and NaPRP4 cDNAs predict that the core polypeptides of these glycoproteins are 96.9% identical (Chen et al., 1993; Cheung et al., 1993), investigations of function in the two species have produced diverse results. Several lines of evidence showed that TTS glycoproteins can affect the rate and direction of pollen tube elongation in N. tabacum (Cheung et al., 1995). Addition of 2 pg ml-' of TTS glycoproteins to sugar-depleted growth medium caused a three-fold increase in the in vitro growth rates of pollen tubes. In semiin vivo conditions, pollen tubes grew towards regions of higher TTS glycoprotein concentration. Growth of N . alata pollen tubes, on the other hand, was neither stimulated by, nor chemotactic toward the NaPRP4 glycoproteins (Sommer-Knudsen et al., 1998b). At NaPRP4 glycoprotein concentrations above 0.4 mg ml-', which were estimated to be comparable to the actual concentrations in the cell walls of transmitting tract cells, the growth of N . alatu pollen tubes was inhibited. Reported differences in glycosylation between the N . tabacum TTS glycoproteins and the N . alata NaPRP4 glycoprotein may also be relevant to function. The TTS glycoproteins were reported to exhibit a gradient of increasing glycosylation from the stigma to the ovary end of the style in N .
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tabacum (Wu et al., 1995). Such a gradient might help guide the growth of the pollen tubes towards the ovary. Furthermore, under in vitro conditions, N . tabacum pollen tubes were observed to deglycosylate TTS glycoproteins (Wu et al., 1995). This activity was attributed to cell wall- or plasma membrane-bound glycosidases in the pollen tube rather than to secreted glycosidases, as deglycosylation was observed on TTS glycoproteins that were bound to the pollen tube wall but not on TTS glycoproteins that were present in the growing medium (Wu et al., 1995). During the incubation, the pollen tubes appeared to completely deglycosylate TTS glycoproteins because they ultimately reached the same molecular weight as chemically deglycosylated TTS glycoproteins. In contrast, no stigma-to-ovary glycosylation gradient was detected among NaPRP4 glycoproteins in the style of N . alata, nor was deglycosylation of NaPRP4 glycoproteins detected during pollen tube growth (Sommer-Knudsen et al., 1998b). Through experiments involving transgenic plants, Cheung et al. (1995) further demonstrated that TTS glycoproteins are important for pollen tube elongation under in vivo conditions. Plants transformed to express an antisense TTS transgene had reduced levels of TTS mRNAs and glycoproteins. The transgenic plants showed normal vegetative and floral growth, but they had low seed production. Wild-type pollen applied to the low-TTS transgenic plants also showed reduced growth and seed production. In contrast, pollen from the low-TTS transgenic plants produced normal seed yield when applied to the wild-type plants. These results indicated that TTS glycoproteins are needed to maintain normal pollen tube growth in the style. Additional evidence of the effect of TTS glycoproteins on pollen tube growth was obtained by producing transgenic tobacco plants that express TTS glycoprotein in tissues other than the style. In one approach, Cheung et al. (1996) produced such plants by constitutively expressing a TTS transgene. Although the TTS polypeptides accumulated in all vegetative and floral tissues of the transformed plants, the extent of glycosylation of these polypeptides was very low except in the style. This observation suggested that some enzymes involved in glycosylation of TTS glycoproteins are normally expressed only in the style. By constitutively expressing an Agamous transgene, Cheung et al. (1996) were able to overcome this problem. Agarnous is a floral identity gene that expresses a MADS box protein required for normal stamen and gynoecium development. Constitutive expression of the Agarnous gene can cause development of abnormal characteristics in sepals and petals. In tobacco plants transformed to constitutively express this gene, the sepals developed stigma-like tips and contained detectable levels of TTS glycoproteins produced from the endogenous genes. These TTS glycoproteins appeared to be glycosylated to the same extent as TTS glycoproteins in the style of wildtype plants. Pollen grains applied to the exudate-covered, stigma-like tip of the abnormal sepals germinated and grew. In contrast, pollen grains did not
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germinate when applied to sepals of wild-type plants or plants transformed to constitutively express a TTS transgene. While species-to-species differences and other issues continue to limit our understanding of the functions of AGPs and AGP-like molecules in the gynoecium, several hypotheses clearly deserve continued evaluation. One hypothesis holds that these molecules serve as a nutrient source for the growing pollen tube. The reports of stimulation of pollen tube growth by TTS glycoproteins (Cheung et al., 1995), the deglycosylation of TTS glycoproteins by pollen tubes (Wu et al., 1995) and the uptake of NaPRP5 glycoprotein by growing pollen tubes (Lind et al., 1996) all seem to be consistent with the nutrient hypothesis. On the other hand, while TTS, NaPRP5 or other AGP-like molecules may be important for pollen tube nourishment, their effect on pollen tube elongation seems unlikely to be entirely due to a nutritional effect. The TTS glycoproteins still promoted pollen tube elongation even when excess free sugars were present in the growth medium and when TTS deglycosylation was inhibited by addition of galactose (Wu et al., 1995). Thus, another important hypothesis, this one focused on adhesion properties, deserves further evaluation as well. Because TTS glycoproteins adhere to the pollen tube surface, Wu et al. (1995) proposed that this property might be partially responsible for the promotion of pollen tube growth. Close adhesion between pollen tubes and the stylar matrix may facilitate transfer of sugars, minerals and water that the pollen tube needs for elongation. In addition to this passive effect, a more active role might also be played by the stylar matrix. Sanders and Lord (1992) proposed that the growth of pollen tubes may be analogous to the migration exhibited by certain animal cells. In these animal cells, focal adhesion points form between the extracellular matrix and the plasma membrane through substrate adhesion molecules such as vitronectin. Through interactions with contractile microfilaments or perhaps through yetto-be-characterized dynamic properties of their own, the substrate adhesion molecules enable animal cell migration. In the plant gynoecium, AGPs and AGP-like molecules such as TTS glycoproteins are good candidates for mediating pollen tube-stylar matrix interactions, and a crude stylar exudate containing such molecules has been reported to promote in vitro adhesion and growth of pollen tubes on an artificial matrix (Jauh et al., 1997). No clear evidence exists, however, to show that interactions involving AGPs or AGPlike molecules actively propel the pollen tube tip towards the ovary. An alternative hypothesis, more consistent with growth mechanisms of other plant cells, is that TTS glycoproteins or similar molecules affect the yielding properties of the pollen tube wall. For example, Wu et al. (1995) proposed that TTS glycoproteins bind to negatively charged pectins in the pollen tube wall. Such binding might reduce the formation of pectate gels and result in a more extensible pollen tip wall, thereby enabling a faster rate of pollen tube extension.
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3. Culture Medium AGPs (CM-AGPs) Plant cells grown in suspension culture secrete an array of macromolecules including polysaccharides, glycoproteins, proteoglycans, proteins and other molecules that accumulate in the culture medium. The altered medium is called conditioned medium, and the macromolecules it contains are generally considered to be representative of those forming the cell wall (Aspinall et al., 1969). Arabinogalactan-proteins are abundant in the conditioned medium of many plant cell cultures (Aspinall et al., 1969; Anderson et al., 1977: Hori et al., 1980; Akiyama and Kato, 1981; Stevenson et al., 1986; Bacic et al., 1987; Cartier et al., 1987; Gleeson et al., 1989; Knox et al., 1989; Pennell et al., 1989; Shea et al., 1989; Komalavilas et aE., 1991; Baldwin et al., 1993; Kreuger and van Holst, 1993; Smallwood et al., 1996). Estimates of the amount of AGPs that accumulate in conditioned medium during a 7-day culture cycle ranged between 0.5 and lOOpgml-' (Gleeson et al., 1989; Komalavilas et al., 1991; Egertsdotter and von Arnold, 1995; Langan and Nothnagel, 1997). The considerable breadth of this range might be partially due to species-to-species variation. For example, the conditioned medium of Norway spruce cells contained AGPs at 0.5-5 pgml-' while that of carrot and rose cells contained 2&100 pgml-' and 14-57 pgml-', respectively (Komalavilas et al., 1991; Kreuger and van Holst, 1993; Egertsdotter and von Arnold, 1995; Langan and Nothnagel, 1997). Variation within a species also occurs, however, and involves factors such as the age of the cell line with respect to the moment when it was derived from the mother plant tissue. Both carrot (Egertsdotter and von Arnold, 1995) and rose (Langan and Nothnagel, 1997) cell cultures exhibited several-fold increases in the amount of AGPs secreted into the conditioned medium as the cell line aged. These investigations did not consider whether this increase in secretion occurred only with AGPs or with other macromolecules as well. The age of the cell line can also affect the types of AGPs present in the conditioned medium. As the age of a cell line increases, the AGPs secreted into the conditioned medium become different from those present in the mother tissues from which the culture was derived. Using crossed electrophoresis, Kreuger and van Holst (1993) observed that CM-AGPs from a 19-day-old carrot cell line had an electrophoretic pattern similar to that of AGPs from the hypocotyl, which was the explant used to initiate the cell line. As passage number increased over a 2-year period, the electrophoretic mobilities of the CM-AGPs gradually decreased compared to the mobilities of AGPs from the initial explant. Similar results were obtained by Langan and Nothnagel (1997) with rose cell cultures. Culture medium AGPs from an old rose cell line had generally lower electrophoretic mobilities than CM-AGPs from a cell line recently initiated from stems. Several investigations have focused on CM-AGPs in the context of their role in somatic embryogenesis. (Investigations of cell wall AGPs relative to somatic
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embryogenesis will be considered in Section III.B.3.) Kreuger and van Holst (1993) reported that changes in CM-AGPs with increasing age of carrot cell cultures were related to the potential of a cell line to produce somatic embryos. Recently initiated cell lines were embryogenic but lost this potential after approximately 1 year in culture. A preparation of total CM-AGPs from an old, non-embryogenic cell line inhibited embryogenic potential when added to the medium of a recently initiated cell line. Conversely, old cell lines regained embryogenic potential when the medium was supplemented with a preparation of total AGPs from carrot seeds. In subsequent work, Kreuger and van Holst (1995) generated ZUM15 and ZUM18, two monoclonal antibodies that recognize unidentified epitopes on certain AGPs, and used them to fractionate seed AGPs by affinity chromatography. The seed AGPs bound by ZUM15 antibodies inhibited embryogenesis when added to a moderately embryogenic carrot cell line. In contrast, the seed AGPs bound by ZUM18 antibodies markedly increased embryogenesis. This promoting effect on somatic embryogenesis was not limited to carrot cells. The carrot seed AGPs recognized by ZUM 18 also promoted somatic embryogenesis in cell cultures of Cyclamen persicum (Kreuger et al., 1995). Similarly, a fraction of tomato seed AGPs recognized by ZUM 18 antibodies increased embryogenic potential in a carrot cell culture. The carrot and tomato AGPs recognized by ZUM18 differed, however, in their electrophoretic mobilities (Kreuger and van Holst, 1995). These results suggested that the epitope itself, rather than more general structural features of the CM-AGPs, determined the effect of CM-AGPs on embryogenesis. Toonen et al. (1997) subsequently performed experiments similar to those of Kreuger and van Holst (1993, 1995) but obtained results that were in only partial agreement with the earlier results. Like Kreuger and van Holst (1993), Toonen et al. (1997) observed that a total AGP preparation from carrot seeds increased somatic embryogenesis in a carrot cell culture that otherwise produced very few embryos. To observe this enhancement, however, Toonen et al. (1997) found it necessary to partially fractionate the cell culture to retain clusters of cytoplasmic-rich cells while discarding large vacuolated and other cell types. Toonen et al. (1997) also fractioned total carrot seed AGPs by antibody affinity purification. In contrast to Kreuger and van Holst (1995), Toonen et al. (1997) found that AGPs bound by the ZUM18 monoclonal antibody did not enhance embryogenesis in an embryogenic carrot cell line, although they did observe that embryogenesis was inhibited by AGPs bound by the JIM8 monoclonal antibody, which recognizes an unidentified epitope in the carbohydrate portion of AGPs (Pennell et al., 1991). The effects of exogenous AGPs on somatic embryogenesis have also been studied with Norway spruce, a species where propagation by somatic embryogenesis is economically desirable (Egertsdotter and von Arnold, 1995). A major problem in propagating this and other conifers by somatic embryogenesis is that some cell lines produce somatic embryos, but these
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embryos do not mature and thus do not develop into plants (Gupta et al., 1993; Egertsdotter and von Arnold, 1995). Egertsdotter and von Arnold (1995) compared the CM-AGPs from two embryogenic cell lines of Norway spruce that produce distinct somatic embryos, designated group A and group B. Group A embryos have larger embryonic regions than group B embryos, and only the former produce mature somatic embryos upon addition of abscisic acid. Electrophoretic analysis showed that CM-AGPs from group A cultures were different from those in group B cultures. Addition of either CM-AGPs from group A or AGPs from seeds caused group B embryos to advance in their development, although this advance did not fully convert them into group A embryos. Such conversion occurred only when a total aqueous seed extract, containing other unidentified components in addition to AGPs, was added to the group B embryos. These observations suggest that the presence of particular CM-AGPs is important for somatic embryogenesis in Norway spruce, although other seed components are also needed to complete the conversion of group B into group A embryos. Further research into the effects of CM-AGPs on somatic embryogenesis is needed to improve our basic understanding of the phenomenon, as well as to realize its practical economic value. The experimental variables responsible for inconsistencies in results between different research groups should be precisely identified (Kreuger and van Holst 1995, Toonen et al., 1997). Likewise, further work is needed on other conditioned medium components that affect somatic embryogenesis (Hari, 1980; van Engelen and de Vries, 1992). For example, Cordewener et al. (1991) and De Jong et al. (1992) used inhibitors of glycosylation or mutant cell lines of carrot to identify secreted enzymes that are required for completion of somatic embryogenesis. Similarly, Gavish et al. (1 992) identified extracellular glycoproteins, of yet unknown structure, that inhibit somatic embryogenesis in Citrus cell cultures. Thus, various secreted molecules, including AGPs, play a role in somatic embryogenesis. It remains unclear whether these various molecules act by independent mechanisms or in concert through a single mechanism during the induction of somatic embryos.
111. CELL WALL AGPS (CW-AGPS) Cytochemical and immunocytochemical studies have revealed that AGPs are present in the cell walls of bryophytes (Basile and Basile, 1987, 1990) as well as species belonging to dicots (Clarke et al., 1975, 1978; Van Aelst and Van Went, 1992; Kikuchi et al., 1993; Gane et al., 1994) and monocots (Schopfer, 1990; Harris et al., 1992; Schindler et al., 1995). Results from microscopic and biochemical analyses of binding of (P-D-GIc)~Yariv phenylglycoside to cell walls of epidermal cells (Schopfer, 1990; Gane et al., 1995a), xylem elements (Schindler et al., 1995), pollen tubes (Jauh and Lord, 1996, Roy et al., 1998) and undifferentiated cells in culture (Serpe and Nothnagel, 1994, 1995; Langan
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and Nothnagel, 1997) can be interpreted as evidence that AGPs occur as wall components in various cell types, provided that such studies included proper control experiments with ( a - ~ - G a l )or~ other Yariv phenylglycoside that does not bind AGPs (Triplett and Timpa, 1997). Immunocytochemical studies using monoclonal antibodies that recognize specific AGP epitopes showed that certain variations in CW-AGP expression are closely related to developmental events (Pennell et al., 1992; Schindler et al., 1995). Although histochemical and immunocytochemical studies have indicated the presence of AGPs in the cell wall, these studies have usually not distinguished whether the detected AGPs were soluble in the aqueous phase of cell wall space or were tightly bound to other wall polymers, i.e. the solid phase of the cell wall. Uncertainties regarding the persistence of localization of AGPs in the cell wall also arise from pulse-chase experiments with radioactive sugars (Takeuchi and Komamine, 1980; Gibeaut and Carpita, 1991). These experiments showed that cells in suspension cultures secrete AGPs to the cell surface and then rapidly release them to the culture medium. Based on these results, the possibility remains that AGPs are only transiently associated with the cell wall rather than being an integral part of it. Evidence that some AGPs, or at least some type I1 arabinogalactans, are an integral part of the cell wall came from analyses of glycosyl linkage composition of extracted cell wall polymers. Polymers containing (1,3)-, (1,6)-and (1,3,6)-galactopyranosylresidues, which are characteristic of AGPs, were found in the cell walls of dicots (O’Neill and Selvendran, 1985; Iraki et al., 1989a; Shea et al., 1989; Gane et al., 1994) and cereals (Carpita, 1989). The presence of these residues in both dicot and cereal walls is noteworthy because the cell walls from these two classes of plants differ markedly in their structural characteristics, especially in their hemicellulosic components (Carpita and Gibeaut, 1993). The polymers containing the (1,3)-, (1,6)- and (1,3,6)galactopyranosyl residues were tightly bound to the cell wall, as they could not be extracted with high salt but instead required solvents that extract hemicelluloses or pectic polysaccharides (Carpita, 1989; Shea et al., 1989). While these results indicated the presence of type I1 arabinogalactans, they did not distinguish whether these arabinogalactans were linked to polypeptides, as occurs in AGPs, or whether they were linked to other cell wall polysaccharides, such as pectic rhamnogalacturonans (Pellerin et al., 1995) or xylans (Kwan and Morvan, 1995). More definitive evidence that some AGPs are tightly bound to the cell wall was obtained by Serpe and Nothnagel(l994, 1995). Rose cell walls were found to bind (P-D-G~c)~ in amounts of 1-5nmol (P-D-G~c)~ per mg dry cell wall (Serpe and Nothnagel, 1995; Langan and Nothnagel, 1997). For control comparison, less than 3% of this amount of binding was observed with (p-DMan)3, a Yariv phenylglycoside that does not bind AGPs (Langan and Nothnagel, 1997). The (P-~-Glc)~-binding molecules were extracted from the cell walls and were identified as AGPs based on their glycosyl and aminoacyl
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residue compositions, glycosyl linkage compositions and NMR spectra (Serpe and Nothnagel, 1995). A. BIOCHEMICAL CHARACTERIZATION
Knowledge regarding the biochemistry of CW-AGPs (Table I) is relatively limited when compared to knowledge regarding other structural cell wall molecules or extracellular AGPs. Isolation and structural characterization of AGPs from plant cell walls has been conducted for only a few sources, these including suspension-cultured rose cells (Serpe and Nothnagel, 1995), Nicotiana data styles (Sommer-Knudsen et al., 1996) and cabbage leaves (Kido et al., 1996). For both the rose and Nicotiana data materials, the conditions used to solubilize CW-AGPs indicated that these molecules were bound to the cell wall by ionic forces. A crude fungal cellulase preparation was used to release CW-AGPs from cabbage leaf cell walls (Kido et al., 1996). Various procedures were tested for efficacy in solubilizing AGPs from the cell wall of rose cells (Serpe and Nothnagel, 1994, 1995). Solutions of salts and chelators were ineffective at ambient or lower temperatures, but extraction with concentrated salt solutions or chelators became effective at elevated temperatures. The procedure affording the best extraction and recovery of AGPs was incubation with 0.5 M potassium phosphate buffer (PH 4.5) for 2.5 h at 90°C. Fractionation of the extract yielded two major CW-AGPs and at least two minor CW-AGPs. While all of these molecules contained sugars characteristic of AGPs, they differed markedly in the proportions of arabinosyl and glucuronosyl residues. In particular, one major CW-AGP contained more glucuronosyl than arabinosyl residues and consequently was named glucuronogalactan-protein (GGP). All four CW-AGPs contained protein but differed in amounts, ranging between 4.5 and 11.5% (w/w). Similarly, all four CW-AGPs were rich in Hyp and Ala but differed in the proportions of various amino acids, including Hyp, Ala, Ser and Gly. Methylation and NMR analyses showed that the two major AGPs from rose cell walls, cell wall AGPl (CW-AGP1) and GGP, had galactosyl and arabinosyl linkages characteristic of type I1 arabinogalactans (Serpe and Nothnagel, 1995). The two molecules differed in linkage of glucuronosyl residues, which were mostly (1,4)-linked in CW-AGP1 but terminal in GGP. Furthermore, the NMR spectrum of GGP indicated the presence of 0acetylated sugars, which were not detected in CW-AGP1 nor in other rose AGPs. Structural comparison of rose CW-AGPs and CM-AGPs indicated that some of these molecules were probably related, while others were unique to the cell wall (Komalavilas et al., 1991; Serpe and Nothnagel, 1994, 1995). Similarities between CW-AGP1 and the two major CM-AGPs, CM-AGPa and CM-AGPb, included generally comparable electrophoretic mobilities, amino-
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24 1
acyl compositions, glycosyl residue compositions, glycosyl linkage compositions and NMR spectra. Differences were detected in particular details, however, including a Hyp content in CW-AGP1 that was 1.3 and 2 times higher than in CM-AGPa and CM-AGPb, respectively, and a molecular size of CW-AGP1 that was intermediate between the sizes of CM-AGPa and CMAGPb. Branching, as indicated by (1,3,6)-galactopyranosylresidues, was lower in CW-AGP1 than in either CM-AGPa or CM-AGPb. While these details differed, the general resemblance between CW-AGP1 and the CM-AGPs suggested possible precursor-product relationships among these molecules. The possibility of a precursor-product relationship between GGP and CMAGPs seemed much less likely. Compared to CM-AGPs, GGP had much higher native electrophoretic mobility and molecular size, as well as distinctive proportions of galactosyl, arabinosyl, glucuronosyl, Ala and Ser residues. The scarcity of GGP-like molecules in the culture medium indicated that this AGP is efficiently retained in the cell wall. While the mechanism of this retention has not been established, the larger size and ionic charge of GGP may be relevant factors. These characteristics would restrict diffusion and favour ionic interactions, thus favouring the retention of GGP in the cell wall. The GGP also demonstrated an extraordinary ability to bind (P-D-G~c),, the stoichiometry being about six times greater than for other rose AGPs (Serpe and Nothnagel, 1995). Although the biological significance of the affinity of AGPs for (P-D-G~c)~ has not been established (Fincher et al., 1983; Carpita and Gibeaut, 1993), it may be a characteristic that enables GGP to interact with other cell wall molecules. Identification of a cell wall AGP or AGP-like molecule in stylar cells of Nicotiana alata originated from screening for style-specific gene expression (Chen et al., 1993; see also Sections II.A.l and II.B.2). The NaPRP4 cDNA thus isolated predicted a polypeptide with some characteristics similar to a type of non-classical AGP (Fig. 1D) but without prominent Asx and Glx contents. The glycoprotein corresponding to this polypeptide was subsequently isolated from homogenized styles (Sommer-Knudsen et al., 1996). Solubilization of the NaPRP4 glycoprotein from the styles did not occur with low-salt buffers but was achieved by repeated extractions with a high-salt buffer (0.4 M NaCl, 14mM P-mercaptoethanol and 1 mM EDTA). These results, combined with results from localization studies using polyclonal antibodies, indicated that the NaPRP4 glycoprotein was ionically bound to the cell walls of stylar cells (Sommer-Knudsen et al., 1996). Structural characterization of the isolated NaPRP4 glycoprotein revealed a core polypeptide consistent with that predicted by the NaPRP4 cDNA (Sommer-Knudsen et al., 1996). Post-translational processing included elimination of the signal sequence, hydroxylation of half of the Pro residues, and glycosylation with both 0- and N-glycans. Carbohydrate accounted for 75% (w/w) of the NaPRP4 glycoprotein, and included glycosyl residues and linkages characteristic of AGPs. The NaPRP4 glycoprotein was basic,
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however, while most AGPs are acidic due to the presence of glucuronosyl residues. The NaPRP4 glycoprotein also differed from most AGPs in that its capacity to bind (P-D-G~c)~ was very low, the stoichiometry being 30 times less than that of gum arabic. While the requirement for high salt during extraction indicated an ionic interaction between the NaPRP4 glycoprotein and the stylar cell wall (Sommer-Knudsen et al., 1996), the details of this interaction await further elucidation. Investigation of this issue might be facilitated by available comparisons with related molecules that are not bound to the cell wall but instead are present in intercellular secretions. In particular, the polypeptide predicted by the TTS-1 cDNA from Nicotiana tabacum (Cheung et al., 1993) is 96.9% identical to that predicted by the NaPRP4 cDNA, but the TTS-1 glycoprotein was found in intercellular secretions instead of in the cell wall (Wang et al., 1993). Likewise, the polypeptide backbones of the NaPRP4 and NaPRP5 glycoproteins share antigenic determinants (Sommer-Knudsen et al., 1996) and, within their C-terminal domains, are 57% identical at the amino acid level and exhibit a conserved pattern of six Cys residues (Schultz et al., 1997). The NaPRP5 glycoprotein was found in intercellular secretions in the Nicotiana afata style, however, instead of bound to the stylar cell walls like the NaPRP4 glycoprotein. These differences in localization among glycoproteins with similar core polypeptides suggest that structural features of the carbohydrate moieties might be determinants of localization. In this context it seems noteworthy that the NaPRP4 glycoprotein was 75% carbohydrate (Sommer-Knudsen et al., 1996) while the TTS glycoproteins were only 35% carbohydrate (Cheung et af., 1995). Kido et al. (1996) reported the preparation of several CW-AGP fractions from head leaves of Rakuyo cabbage (Brassica oleracea L. var. Capitata L.). The cell wall fraction from homogenized leaf tissue was washed with chloroform and then extracted with 80°C water to remove soluble AGPs and pectins. Prolonged digestion of the cell wall residue with a crude fungal cellulase preparation resulted in solubilization of polymers that were subsequently fractionated by gel permeation and anion-exchange chromatographies. The five fractions resulting from these chromatographies were all suggested to contain AGPs, as judged by the presence of Hyp and large proportions of galactosyl and arabinosyl residues. Extensins and rhamnogalacturonans were also suggested to be present, especially in the most anionic fractions where higher levels of rhamnose and uronic acids were detected. The first fraction eluting from the anion-exchange column, however, was suggested to be nearly pure AGP. This material was approximately 180 kDa in size, and consisted of 15.4% (w/w) protein, 78.8% neutral sugar and 5.8% uronic acid. The protein was 28.3 mol% Hyp, and the neutral sugar was 59.2% arabinose, 36.9% galactose and 3.9% xylose. Additional tests that could distinguish extensins and AGPs, such as binding of (P-D-G~c)~ Yariv phenylglycoside or analysis of glycosyl linkage composition, were not reported.
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While the studies of rose (Serpe and Nothnagel, 1995), Nicotiana d a t a (Sommer-Knudsen et al., 1996) and cabbage (Kido et al., 1996) show that some AGPs are tightly bound to the cell wall, the wall component of this interaction remains unidentified. As the interactive forces appear to be ionic, CW-AGPs might be binding to pectic polymers through calcium or other cations. An AGP-pectin interaction would also be consistent with the presence of (1,3)-, (1,6)- and (1,3,6)-linked galactopyranosyl residues in pectic fractions obtained during sequential extraction of isolated cell walls (O’Neill and Selvendran, 1985; Carpita, 1989; Iraki et al., 1989a; Shea et al., 1989). Similarly, the procedure optimized to solubilize AGPs from isolated rose cell walls also released polymers rich in galacturonosyl and rhamnosyl residues, which are indicative of pectic polymers. No covalent binding between the CW-AGPs and pectic polymers was apparent in this case as the latter separated from the CWAGPs under mild conditions (Serpe and Nothnagel, 1994, 1995). Because coextraction may occur by coincidence, however, these observations do not establish an interaction between AGPs and pectic polymers. More direct evidence that AGPs can bind to pectin was obtained by Baldwin et al. (1993). Using dot blotting, they demonstrated a calcium-dependent binding between an extracellular carrot AGP and a pectin fraction extracted from carrot cell walls. The ability of AGPs to bind certain Yariv phenylglycosides has also been proposed as an attribute that may allow AGPs to interact with cell wall molecules (Fincher et al., 1983; Rohringer et al., 1989; Carpita and Gibeaut, 1993). The exceptionally high (P-~-Glc)~-binding capacity of GGP (Serpe and Nothnagel, 1995) and the exceptionally low (P-~-Glc)~-binding capacity of the NaPRP4 glycoprotein (Sommer-Knudsen et al., 1996) present opposing implications relative to this hypothesis as both of these molecules bind to the cell wall. Because the Yariv phenylglycosides that bind to AGPs generally contain D-sugars in P-anomeric configuration (Yariv et al., 1967; Jermyn and Yeow, 1975; Anderson et al., 1977; Larkin, 1977, 1978; Jermyn, 1978a, b; Nothnagel and Lyon, 1986), AGPs have sometimes been called ‘all P-lectins’ (Jermyn and Yeow, 1975; Larkin, 1977). While this name suggests that AGPs might exhibit lectin-like binding to cell wall polysaccharides containing plinked sugars, the requirements for binding of AGPs to Yariv phenylglycosides are distinctly different from those for binding of conventional lectins to sugars (Jermyn, 1978a; Nothnagel and Lyon, 1986). For example, the interaction between AGPs and (P-D-GIc)~was not competed by monosaccharides or methylglycosides (Larkin, 1978) and was only very weakly inhibited by simple phenylglycosides (Clarke et al., 1975; Jermyn, 1978b; Larkin, 1978; Samsom et al., 1983). Even a fluorescent phenyl-P-D-glucoside (Nothnagel and Lyon, 1986) and various multivalent phenyl-P-D-glucosides did not exhibit any binding to AGPs (Jermyn, 1978a; Nothnagel and Lyon, 1986). The binding of AGPs to Yariv phenylglycosides seems to rely on the ability of the latter to selfassociate in aqueous solutions to form complexes of 10-50 molecules (Woods
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et al., 1978). This self-associated complex of Yariv phenylglycosides apparently presents a higher-order structure that interacts with AGPs (Nothnagel and Lyon, 1986). It remains possible that this higher-order structure mimics a cell wall polymer that is the natural interactive partner with some AGPs. Our knowledge of the biochemistry of CW-AGPs has only begun to develop. Structural characterization of these molecules over a wider spectrum of plant species and cell types is needed. Such characterization may reveal structural differences between CW-AGPs among taxa or in relation to developmental processes. Much remains to be learned about the interactions of CW-AGPs with other wall molecules, and about the biosynthesis and turnover of CWAGPs. Studies in these areas may provide valuable insights into the way that AGP affects cell wall structure and function.
B. EXPRESSION AND FUNCTION
While various probes have been employed to study AGP expression, use of monoclonal antibodies has led to most of the advances in characterizing the expression of CW-AGPs. Currently, at least 15 AGP-binding monoclonal antibodies are available (Knox, 1997; Nothnagel, 1997). Most of these antibodies have been shown to recognize epitopes in the carbohydrate portion of AGPs. In the few cases where additional structural details of the epitopes have been elucidated, the epitopes appear to be part of the (1 + 6 ) - p - ~ galactan side chains and/or their substituents. Labelling experiments with AGP-binding monoclonal antibodies have revealed that the expression of cell surface AGPs is highly regulated (Knox, 1995, 1996; Pennell and Roberts, 1995). Particular AGP-binding monoclonal antibodies label the surface of only certain cell types, and different monoclonal antibodies show distinct labelling patterns. In some cases, the expression of particular cell surface AGP epitopes has been shown to presage future patterns of development. These and other demonstrations of correlations between expression of cell surface AGP epitopes and pattern formation, or other events in the differentiation of vascular tissues, development of reproductive structures or somatic embryogenesis, have led to a consensus hypothesis that AGPs function in, and perhaps even regulate, certain aspects of plant development. Developmental expression of both CW-AGPs and plasma membrane AGPs (PM-AGPs) has been observed through immunolocalization studies. In fact, reliable distinction between labelling in the cell wall and labelling on the plasma membrane cannot usually be done by light microscopy but instead requires immunogold labelling and high-resolution electron microscopy. Thus, while the focus of this section is on CW-AGPs, some discussion of PM-AGPs is inevitable when considering results from experiments that could not clearly distinguish between CW-AGPs and PM-AGPs. Whether involving CW-AGPs
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or PM-AGPs, however, changes in expression will be considered with attention to resulting implications regarding possible AGP functions. 1. C W-AGP Epitopes in Vascular Tissues The expression of AGP epitopes has been analysed during differentiation of vascular tissues in various plants. In Arabidopsis thaliana roots, for example, developmental and spatial variations in CW-AGP epitopes have been detected with JIMl 3, JIM 14 and CCRC-M7 monoclonal antibodies. Xylem development in Arabidopsis roots was associated with the expression of the JIM13 epitope, which is contained within the ,&~-GlcpA-(1 + 3)-a-~-GalpA-( 1 + 2)L-Rha oligosaccharide (Yates et al., 1996). The JIM13 antibody labelled the central metaxylem initial cell located just above the quiescent centre (Dolan et al., 1995). Further up from the meristem, JIM 13 epitopes were also detected on the plasma membrane and the inner portion of the cell wall of cells in the protoxylem, pericycle and endodermis (Dolan et al., 1995). The expression of the JIM 13 epitope gradually decreased as the xylary elements became lignified (Dolan and Roberts, 1995). In contrast to xylary elements, phloem sieve elements were not labelled by the JIM13 antibody, but instead were labelled by the JIM 14 and CCRC-M7 monoclonal antibodies. The JIM14 antibody, which recognizes an unidentified epitope in the carbohydrate portion of AGPs (Knox et al., 1991), labelled the cell wall of mature sieve elements and, much less intensely, the plasma membrane of most root cells (Dolan et al., 1995). Similarly, the CCRC-M7 antibody labelled the cell walls of sieve tubes and also labelled most other root cells on the plasma membrane or in the immediately adjacent region of the cell wall (Freshour et al., 1996). Labelling with CCRCM7 is, however, not necessarily indicative of the presence of AGPs. This antibody binds to arabinosylated (1,6)-O-~-galactanswhich are present in both AGPs and rhamnogalacturonan I, a pectic polysaccharide of the cell wall (Steffan et al., 1995). Expression of AGP epitopes in the root apex has also been described for carrot, pea, radish and onion (Knox et al., 1989, 1991; Casero et al., 1998) with some variability in results. For example, the JIMl 3 epitope was associated with xylem development in carrot, radish and pea but with phloem development in onion (Casero et al., 1998). Thus, each species exhibited its own characteristic pattern of specific epitope occurrence linked to vascular tissue development. Regulated expression of the JIM13 epitope has also been observed during differentiation of tracheids and sclerenchyma cells in maize coleoptiles (Schindler et a[., 1995). In cells destined to become sclerenchyma, the JIM13 antibody labelled the plasma membrane and multivesicular bodies in the vacuole. In future tracheids, the JIM 13 antibody labelled the thickened secondary wall. The AGPs containing the JIM13 epitope did not seem to be tightly bound to the cell wall as molecules containing this epitope were extracted from the coleoptiles with detergent and low salt, while further treatment with high salt extracted little additional JIM 13-reactive material.
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Expression of AGP epitopes in maize coleoptiles was further probed with MAC207 and JIM14 monoclonal antibodies (Schindler et al., 1995). The MAC207 antibody, which recognizes an epitope contained within the p-DGlcpA-(1 + 3)-a-~-GalpA-(l--+ 2)-~-Rhaoligosaccharide (Yates et al., 1996), labelled plasma membranes throughout the coleoptile, although the label was most conspicuous in the outer epidermis and vascular bundles. The JIM14 antibody, on the other hand, labelled the inner layer of the cell wall of sclerenchyma cells whose protoplasts had begun to disintegrate. Like JIM 13, MAC207 reacted with molecules extracted by detergent/low-salt treatment of the coleoptiles. In contrast, JIM 14 reacted with molecules that were extracted only by high-salt treatment. These JIM 14-reactive molecules did not, however, show detectable affinity for (P-D-GIc)~,a probe that binds most AGPs including those extracted from the coleoptiles with detergent and low salt. Thus, the nature of the molecule containing the JIM14 epitope remains uncertain. Regulated expression of the JIM 13 epitope was also observed during differentiation of Zinnia elegans cells into tracheids (Stacey et al., 1995). Under appropriate culture conditions, mesophyll cells isolated from Zinnia leaves can be induced to form tracheids. During this process of differentiation the cell walls change significantly in composition and then undergo secondary thickening. Prior to secondary thickening, the JIM 13 epitope appeared in the primary wall of some cells and in the culture medium. As differentiation progressed, the JIM 13 epitope was detected in the secondary wall thickenings of the mature tracheids. The expression of the JIM13 epitope was not limited to cells in culture. In Zinnia leaves, two cell types with thickened walls, xylary elements and guard cells, also expressed the JIM13 epitope (Stacey et al., 1995). Taken together, these results from several systems show that the JIM13 epitope is commonly expressed in cells destined to become xylem elements. Because these cells die during the normal course of development, Schindler et al. (1995) proposed that AGPs containing this epitope may mark cells committed to programmed cell death. Expression of the JIM13 epitope, however, does not always lead to cell death. This epitope also occurs in some cells that are alive at maturity, such as epidermal cells (Knox et al., 1991) and guard cells (Stacey et al., 1995). Perhaps a more widespread characteristic of cells expressing the JIM13 epitope is that they have thickened walls. Such thickening requires massive cellulose deposition. Whether AGPs play a role in this process is unknown, but various cell types undergoing wall thickening do contain abundant levels of mRNAs that encode putative AGPs (John and Keller, 1995; Loopstra and Sederoff, 1995). For example, screening for xylemspecific gene expression in loblolly pine (Pinus taeda L.) led to the isolation of the PtX3H6 and PtX14A9 cDNAs. These cDNAs encode putative AGPs, and the corresponding transcripts appear to be the most abundant of all xylemspecific mRNAs (Loopstra and Sederoff, 1995). Similarly, cellulose deposition is very active in developing cotton fibres, and screening for fibre-specific
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expression led to the isolation of pCK-H6 (John and Keller, 1995), a putative AGP-encoding cDNA that shares various characteristics with the PtX3H6 cDNA. These observations suggest that certain AGPs are expressed at high levels when massive cellulose deposition is required. It would be interesting to know if the translation products of these cDNAs are expressed on the cell surface and, in particular, if they are post-translationally modified to contain the JIM 13 epitope. 2. CW-AGP Epitopes in the Male Gametophyte Some CW-AGPs of the gynoecium have already been considered in the discussion of extracellular AGPs in Section II.B.2, as necessitated by the divergent results regarding whether these molecules are bound to the cell wall or soluble in the secretions of the transmitting tissue cells (Wang et al., 1993; Sommer-Knudsen et al., 1996, 1998b). The present section will be focused on spatio-temporal variations in CW-AGP epitopes in another reproductive structure, the male gametophyte. Both pollen grains and pollen tubes contain various sets of CW-AGPs that are recognized by monoclonal antibodies. Some of these AGPs are probably specific to pollen, as indicated by the isolation of three putative AGP-encoding cDNAs, one expressed specifically in alfalfa pollen (Qiu et al., 1997) and two expressed specifically in Brassica anthers late in pollen development (Gerster et al., 1996). The presence of CW-AGP epitopes in pollen grains of Arabidopsis thaliana (Van Aelst and Van Went, 1992) and tobacco (Li et al., 1995) has been investigated at the ultrastructural level using the MAC207 and JIM8 monoclonal antibodies in immunogold labelling. In the mature, ungerminated, tricellular pollen grains of Arabidopsis, MAC207 labelled only the intine, the inner layer of the wall of the vegetative cell (Van Aeslt and Van Went, 1992). The JIM8 antibody did not label the intine but instead labelled the thin matrix layer between the plasma membrane of a sperm cell and the inner plasma membrane of the vegetative cell. In pollen grains of tobacco, the MAC207 and JIM8 antibodies produced a somewhat different labelling pattern (Li et al., 1995). In these pollen grains, the MAC207 and JIM8 antibodies both labelled the intine of the vegetative cell and the thin matrix layer between the inner plasma membrane of the vegetative cell and the plasma membrane of the generative cell, which later divided to form the two sperm cells. Differences in antibody labelling between Arabidopsis and tobacco may reflect interspecies variations in AGP expression and/or differences in the stage of pollen grain development. Arabidopsis pollen grains were examined ungerminated (Van Aelst and Van Went, 1992) while tobacco pollen grains were examined after hydration, just at the start of germination (Li et al., 1995). Localization of AGPs at the surface of Brassica campestris sperm was studied at the immunofluorescence level with MAC207, JIM8 and JIM13 monoclonal antibodies (Southworth and Kwiatkowski, 1996). Like Arabidopsis
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sperm cells, Brassica sperm cells did not label with MAC207. Whether in pollen grains, in pollen tubes or isolated from pollen, Brassica sperm cells labelled with both JIM8 and JIM13, the two antibodies yielding similar results. Labelling at the sperm surface was uniform and bright when the sperm cells were in pollen grains or tubes, or when sperm cells were isolated as intact pairs. Sperm cells isolated as individuals, however, showed a patchy, weaker labelling pattern. Southworth and Kwiatkowski (1996) interpreted these results as demonstrating the presence of AGPs on both the sperm plasma membrane and the inner plasma membrane of the vegetative cell, the reasoning being that sperm cells isolated as individuals showed weaker labelling because they had lost the fluorescent inner plasma membrane of the vegetative cell. An alternative interpretation, based on observed immunogold labelling of the thin matrix layer between the sperm plasma membrane and the inner plasma membrane of the vegetative cell in Arabidopsis (Van Aeslt and Van Went, 1992), would be that most of the AGPs were localized in the thin matrix layer but were lost to the medium when the inner plasma membrane of the vegetative cell ruptured during sperm isolation. Single isolated generative cells and sperm cells of Lilium longiflorum likewise showed patchy surface labelling with either JIM8 or JIM13 (Southworth and Kwiatkowslu, 1996). Spatial regulation in the localization of AGP epitopes at the surface of pollen tubes has been investigated in several species with somewhat divergent results. In tobacco, no AGP epitopes of either JIM8 or MAC207 were found at the tube tip, except in a few unusually short tubes (Li et al., 1992, 1995). In lily, on the other hand, AGP epitopes of MAC207, JIM13, LM2 and several other anti-AGP monoclonal antibodies were found in abundance at the tip of pollen tubes grown either in vitro or in vivo (Jauh and Lord, 1996). Both tobacco and lily, however, contained AGP epitopes along the lateral walls of the pollen tubes. As viewed by immunofluorescence microscopy, labelling intensity in intact lateral walls was very low. The labelling intensity could be markedly increased by treatment of the pollen tubes with crude commercial cellulase (Li et al., 1992) or pectinase (Li et al., 1992; Jauh and Lord, 1996) preparations. After these treatments, JIM8 and MAC207 labelled the tobacco pollen tubes in circumferential bands spaced at approximately 6pm intervals along the tube length. A somewhat similar banded pattern was observed in lily pollen tubes following pectinase treatment and subsequent labelling with JIM13, LM2 or MAC207. With the higher resolution of immunogold labelling and electron microscopy, the labelling of AGP epitopes was found in the outer pectic layer and the middle cellulosic layer of the lateral cell walls in the tobacco pollen tubes (Li et al., 1995). The mechanism through which cellulase and pectinase increased detection of AGP epitopes in the lateral walls of tobacco (Li et al., 1992) and lily (Jauh and Lord, 1996) pollen tubes remains to be elucidated. Digestion with these enzymes may have increased the accessibility of the antibodies to the AGPs. Alternatively, minor components in the crude enzyme preparations (Rombouts
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et al., 1988; Tagawa and Kaji, 1988; Pellerin and Brillouet, 1994) may have cleaved the AGPs, thereby generating epitopes. Action of cell wall-degrading enzymes has been shown to generate lectin receptors in this way on the surface of protoplasts (Sun et al., 1992). The significance of the banding pattern generated after enzyme treatment is also unknown, but may be related to the pulsatory growth behaviour sometimes exhibited by pollen tubes (Pierson et al., 1995). While use of monoclonal antibodies has provided evidence regarding the localization of AGPs in pollen tubes, the function of these AGPs has been investigated through use of (P-D-GIc)~Yariv phenylglycoside. Treatment with (P-D-G~c)~ blocked both in vitro and in vivo growth of lily pollen tubes (Jauh and Lord, 1996). Red staining from (P-D-GIc)~was visible only at the tube tip, indicating its action at that site. Furthermore, pollen tube growth was not affected by the P-D-mannosyl Yariv phenylglycoside, ( P - D - M ~ ~which ) ~ , does not bind to AGPs. Taken together, these observations suggest that (P-D-GIc)~ blocked pollen tube growth by perturbing AGPs at the pollen tip. On washing (P-D-G~c),from the medium, the pollen tubes resumed growth by generating a new growing tip along the side of the original tip (Jauh and Lord, 1996). Immunogold localization with JIM 13 and electron microscopy indicated that the AGPs at the tip of lily pollen tubes in control conditions were predominantly on the plasma membrane (Jauh and Lord, 1996). In pollen tubes treated with (P-D-GIc)~,however, an accumulation of matrix material was observed between the plasma membrane and the thin cell wall at the tube tip (Roy et al., 1998). The accumulated material was heterogenous as some regions appeared fibrillar and labelled with antibodies to both esterified and unesterified galacturonans, while other regions appeared electron-translucent but contained osmiophilic inclusions and labelled with both JIM 13 antibody to AGP epitopes and an anti-callose monoclonal antibody. These observations were interpreted in terms of a model in which Golgi-derived vesicles, lined with membrane-bound AGPs and carrying pectic polymers, normally fuse at the tube tip and thereby deliver the new wall material needed for tip growth. In pollen tubes treated with (P-D-G~c)~, vesicle secretion and wall assembly at the tip are disrupted and callose deposition is stimulated (Roy et al., 1998). Further testing of this interesting model is needed, particularly in species such as tobacco which do not seem to have AGPs at the tip of pollen tubes (Li et al., 1992, 1995).
3. C W-AGPs in Cell Cultures Cells in culture have been used to investigate the expression and function of CW-AGPs. Variations in CW-AGP expression have been detected in relation to processes of cell differentiation and to modifications of culture conditions. Certain perturbations of AGPs can be most easily achieved in cell cultures, thereby facilitating study of AGP function in living cells.
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A process of differentiation where CW-AGP epitope expression has been analysed is somatic embryogenesis in carrot cell cultures. Early investigations involved light microscopy and use of the JIM4 monoclonal antibody, which recognizes an epitope contained within the p-~-GlcpA-(1 .+ 3)-a-~-GalpA-( 1 .+ 2)-~-Rha oligosaccharide (Yates et al., 1996). Stacey et af. (1990) found that JIM4 bound to surface layers of cells at the future shoot end of the developing somatic embryos, and then later to cells of the cotyledonary provascular tissue and to hypocotyl cells in two regions marking the two sectors of the future stele of the diarch carrot root. Most subsequent studies of developmental variations in CW-AGP epitopes during somatic embryogenesis in carrot have involved use of the JIM8 monoclonal antibody, which recognizes an unidentified epitope in the carbohydrate portion of AGPs (Pennell et al., 1991). Pennell et al. (1992) found that the JIM8 epitope was not present in carrot cells from nonembryogenic cultures, but it was expressed in the cell wall of some cells in embryogenic cultures. Furthermore, an increase in the frequency of cells carrying the JIM8 cell wall epitope was correlated with an increase in the embryogenic potential of the carrot cell culture. On the basis of these and other observations, Pennell et al. (1992) hypothesized that the JIM8 epitope was present in the cell walls of several types of cells in embryogenic carrot cultures. During development of the culture, cells that retained the cell wall JIM8 epitope would elongate and eventually die. Other cells initially contained the cell wall JIM8 epitope but lost it as they divided and became proembryogenic masses. Thus, cells destined to become somatic embryos only transiently expressed the JIM8 epitope (Pennell et al., 1992). The hypothesis that the JIM8 cell wall epitope marks a transitional state in the formation of embryogenic cells was subsequently tested by Toonen et al. (1996). These investigators sieved carrot cell cultures to obtain a population of single cells. By using fluorescence-labelled JIM8 antibodies and a fluorescence microscope with a computer-controlled scanning stage, Toonen et al. (1996) were able to identify single cells that contained the JIM8 cell wall epitope and then track the development of those cells. Tracking of 43 000 cells revealed that only a few JIM8-labelled cells developed into somatic embryos, and instead most somatic embryos developed from cells lacking the JIM8 cell wall epitope. Thus, Toonen et al. (1996) concluded that expression of the JIM8 cell wall epitope was not closely correlated with embryogenic competency of single cells. The importance of the JIM8 cell wall epitope in carrot somatic embryogenesis was reinforced, however, by the recent report of McCabe et af. (1997). These investigators sieved embryogenic carrot cell cultures to obtain a population of single cells, and then used JIM8 antibodies and paramagnetic beads coated with secondary antibodies to immunomagnetically sort the cells into two populations, JIM8( +) cells that expressed the JIM8 cell wall epitope and JIM8(-) cells that did not. On subsequent culturing in fresh medium, the JIM8( +) population generated somatic embryos, as did a control population
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25 1
of unsorted cells. The JIM8(-) population did not generate somatic embryos when cultured in fresh medium. The JIM8( -) population did generate somatic embryos, however, when cultured in medium previously conditioned by either unsorted cells or JIM8( + ) cells alone. Analysis of conditioned medium from unsorted cells revealed the presence of macromolecular fractions that bound the JIM8 antibody and contained sugars characteristic of AGPs, although it was not shown that these were the conditioned medium components responsible for stimulating JIM8( -) cells to generate somatic embryos. Judging from these results, as well as from observations by immunofluorescence and electron microscopy, McCabe et al. (1997) proposed a revision of the hypothesis of Pennell et al. (1992). In the revised hypothesis, embryogenic capacity originates from certain small, spherical, vacuolate cells that contain the JIM8 cell wall antigen. This type of cell, designated state B, becomes polarized and undergoes an asymmetric division. One daughter cell (state F) is small, spherical, vacuolate and contains the JIM8 cell wall antigen. The other daughter cell (state C) is small, spherical, cytoplasmically dense and lacks the JIM8 cell wall antigen. Only the state C cell is competent to progress to a somatic embryo, but such progression requires a signal, this being a soluble molecule released from state B or possibly other cells in the JIM8(+) population. While labelling of JIM8 epitopes relative to somatic embryogenesis in carrot has generated considerable research interest, the somewhat divergent results indicate that further investigations will be needed to reach a satisfactory understanding of the relationship between JIM8 epitopes and embryogenesis. The generation of testable models (McCabe et af., 1997) is certainly a promising advance. Also needed is further research toward the identification of the JIM8 epitope and the cell wall antigen that carries this epitope. Kikuchi et al. (1996) analysed pectic fractions from the cell walls of embryogenic and nonembryogenic callus cultures of carrot and found that structural differences in the arabinose- and galactose-rich side chains of high-mass pectins correlated with cell cluster size. An investigation of JIM8 binding to these high-mass pectin fractions might prove interesting. A different approach to investigating AGPs relative to somatic embryogenesis was taken by Thompson and Knox (1998) who used (P-D-G~c)~ Yariv phenylglycoside to bind and thereby perturb AGPs at various stages of somatic embryogenesis in carrot cell cultures. When applied to cell cultures under embryo-inducing conditions, (P-D-GIc)~promoted production of roots without shoot structures. If applied later, at the globular or early heart-shaped embryo stage, (P-D-GIc)~stimulated root elongation and shoot radial expansion but blocked progression to the next developmental stage. An overall partial inhibition of growth resulted if (P-D-GIc)~was applied to torpedo embryos or plantlets. Combined with the previously described studies involving application of exogenous CM-AGPs (Section II.B.2), these studies on the expression of CW-
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AGP epitopes and the perturbation of AGPs by (P-D-G~c)~ Yariv phenylglycoside lead to a complex and unfinished picture of AGP involvement in somatic embryogenesis. A major remaining challenge is to identify the precise mechanisms that mediate these various effects involving AGPs. Certainly, an intriguing possibility is that AGPs are involved in intercellular signalling (Kreuger and van Holst, 1996). In this role CM-AGPs could be part of a signal transduction pathway that initiates a cascade of events leading to differentiation. Alternatively, the precise expression of CW-AGPs might be needed to establish proper cell adhesion (Kikuchi et al., 1996) or other structural or physiological characteristics essential for embryogenesis. Cells that become somatic embryos are small and form clusters in which the cells tightly adhere to each other (de Vries et al., 1988). Considering that AGPs may be involved in the control of cell wall expansion (Willats and Knox, 1996; Ding and Zhu, 1997) and/or cell adhesion (Clarke et al., 1979a), it is plausible that the expression of particular AGPs helps to produce the morphology of embryonic cells. Thus far, however, no direct evidence exists to support the above explanations. In addition to somatic embryogenesis, other morphogenic events have been linked to changes in the expression of CW-AGPs. Such a link has been observed, for example, in Gymnocolea injlata, a leafy liverwort whose development can be altered by addition of ammonium to the culture medium (Basile and Basile, 1980; 1993). The altered plants develop a variant or desuppressed leaf pattern, which is characterized by the appearance of leaves at locations where they are not normally expressed. Basile and Basile (1993) extracted AGPs from normal and desupressed plants. Analysis of these AGPs by density-gradient centrifugation revealed that normal plants retained a highbuoyant density AGP fraction and released a low-buoyant density AGP fraction into the medium. Conversely, desupressed plants retained a lowbuoyant density AGP fraction and released a high-buoyant density fraction into the medium. Because abnormal leaf development is partially the result of cell proliferation in places where it is normally inhibited, Basile and Basile (1993) suggested that AGPs may be involved in the regulation of cell division. It is not known, however, whether the changes in AGP expression were the cause of the abnormal pattern of cell proliferation. While changes in CW-AGP expression have been documented in relation to developmental events, it remains unclear whether these changes significantly affect the cell wall itself. A correlation between changes in AGP expression and cell wall properties was described by Zhu et al. (1993), who investigated AGPs in suspension-cultured tobacco cells adapted to NaCl stress. These cells maintained high turgor pressure through osmotic adjustment, but they were smaller than control cells (Iraki et al., 1989b). Furthermore, the minimum internal pressure required to burst the cells was higher in salt-adapted cells than in control cells (Iraki et al., 1989b). These observations suggested that the walls of salt-adapted cells were less extensible than those of control cells (Iraki et al.,
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1989a, b; Zhu et al., 1993). Zhu et al. (1993) measured the levels of PM-AGPs and CM-AGPs, and found that both of these were lower in salt-adapted cells than in control cells. Unfortunately, CW-AGPs were not investigated. The correlation between low extensibility and low AGP levels in salt-adapted cells was taken as an indication that AGPs may participate in cell wall expansion (Zhu et al., 1993). A correlation between low AGP levels and reduced cell expansion has also been observed in whole plants. An Arabidopsis mutant that exhibited short hypocotyls, petioles, stems and roots contained lower levels of AGPs than wild-type plants (Takahashi et al., 1995). The mutation was located in a gene encoding a protein that might be involved in transcriptional regulation. The effect of the mutation was not specific to AGPs, however, as the levels of a 0tubulin mRNA were four-fold lower in the mutant than in the wild type. Thus, it remains uncertain whether reduced elongation was due to low AGP levels or to other factors. Correlations between AGP expression and cell differentiation, proliferation or expansion raise the possibility that AGPs are involved in these processes. Other experimental approaches have yielded results consistent with such hypotheses. In several systems, perturbations of cell surface AGPs with (p-DG ~ c Yariv ) ~ phenylglycoside have resulted in inhibition of cell division (Serpe and Nothnagel, 1994; Langan and Nothnagel, 1997; Thompson and Knox, 1998), inhibition of cell expansion (Jauh and Lord, 1996; Willats and Knox, 1996; Ding and Zhu, 1997; Roy et al., 1998) or cell death (Langan and Nothnagel, 1997). The effect of (P-D-GIC)~on living material was first investigated in suspension-cultured rose cells (Serpe and Nothnagel, 1994). In this system, addition of (P-D-GIc)~to the culture medium caused reversible inhibition of growth in a concentration-dependent manner. Cell cultures exposed to 50 pM (P-D-G~c)~ for 7 days did not grow (Serpe and Nothnagel, 1994). In contrast, cultures exposed to the ( p - ~ - M a n or ) ~ (a-D-galactosyl)3Yariv phenylglycoside [(a-~-Gal)~], which do not bind AGPs, grew to the same extent as control cultures. These results indicated that the effect of (P-D-G~c)~ on rose cells was not attributable to the non-glycosidic part of the molecule but was instead due to the affinity of (P-D-GIC)~ for AGPs. Approximately 95% of the (P-D-GIC)~ bound to the cells was associated with the cell wall, and most of the remaining 5% was probably bound to the plasma membrane. The (P-~-Glc)~-binding molecules were purified from the cell wall (Serpe and Nothnagel, 1994, 1995; Langan and Nothnagel, 1997) and plasma membrane (Komalavilas et al., 1991; Serpe and Nothnagel, 1996), and were identified as AGPs based on several structural characteristics. Taken together, these results indicated that (P-D-G~c)~ inhbited growth by binding to cell surface AGPs. The mechanism of inhibition of cell-culture growth upon binding of (p-DG ~ c to ) ~cell surface AGPs could, in principle, involve cell death, cell expansion and/or cell division. In an old rose cell line, (P-D-GIc)~inhibited growth
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without causing cell death (Serpe and Nothnagel, 1994; Langan and Nothnagel, 1997). When cells were transferred to normal medium after 7 days in (P-D-G~c)~, they resumed growth with a time-course similar to that of control cells. Cell sizes in control and (P-~-Glc),-inhibited cultures were similar, indicating that the mechanism of growth inhbition involved suppression of cell division (Serpe and Nothnagel, 1994). The effect of (P-D-G~c)~ was not, however, the same on all rose cell lines. When added to a new cell line, recently initiated from rose stems, (P-D-G~c)~ not only inhibited growth but also killed most of the cells (Langan and Nothnagel, 1997). As passages of the cells in normal medium continued, however, the cells gradually improved their ability to survive the (P-D-GIc)~ treatment. After about 15months in culture, the new cell line recovered from (P-D-G~c)~ treatment as well as the old cell line. Differences in the response to (P-D-G~c)~ treatment were accompanied by differences in the amounts and types of cell surface AGPs present in each cell line. The CW-AGPs were less abundant in the old than in the new cell line, while the old cell line secreted larger amounts of CM-AGPs. Furthermore, as the new cell line improved its survivability to (P-D-GIc)~treatment, the amount of CM-AGPs it secreted also increased (Langan and Nothnagel, 1997). Studies with other species have confirmed that (P-D-GIc)~is broadly effective in inhibiting the growth of cell cultures, but the mechanism of the effect can vary. When applied in the media of Nicotiana edwardsonii and Arabidopsis thafiana cells for several days, (P-D-GIc)~inhibited culture growth by killing the cells (Langan and Nothnagel, 1997). These two cell lines exhibited sharp, but different, threshold concentrations in this effect. In 15 pM (P-D-G~c)~, Nicotiana cells exhibited viability and growth characteristics equivalent to those of untreated controls, but the cells rapidly died in 25pM (P-D-GIc)~. Arabidopsis cells grew normally in 25 pM (P-D-GIc)~but rapidly died in 50 pM (P-D-G~c)~ (Langan and Nothnagel, 1997). Growth of suspension cultures of carrot cells was stopped by (P-D-GIc)~,but the mechanism of stoppage depended on the cell line. Growth by cell elongation normally occurred when L2 carrot cells were transferred into a medium lacking 2,4-dichlorophenoxyacetic acid, but (P-D-GIc)~blocked this cell expansion (Willats and Knox, 1996). When applied in the medium of the L1 line of carrot cells, however, (PD-GIc)~reversibly inhibited cell proliferation (Thompson and Knox, 1998). Inhibition of cell expansion by (P-D-G~c)~ is not limited to cell cultures but has also been observed in whole plants. Several investigators have studied the effects of applying (P-D-GIc)) in the agar- or gellan gum-solidified medium of Arabidopsis thaliana plants. In this system, (P-D-GIc)~did not affect seed germination but did reduce subsequent root growth to less than one-third of that in seedlings growing in control medium or in medium containing (WDGal)3 or ( p - ~ - M a n(Willats )~ and Knox, 1996; Ding and Zhu, 1997). Growth of (P-~-Glc)~-treated roots increased upon transfer of the seedlings to normal medium, thus demonstrating that the inhibitory effect of (P-D-G~c)~ was
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reversible. As observed by light microscopy, the most noticeable effect of the (P-D-G~c)~ treatment was on the zone of root elongation where the epidermal cells became bulbous rather than elongated (Willats and Knox, 1996). Ding and Zhu (1997) observed that this effect of ( p - ~ - G l con ) ~ expansion of root epidermal cells closely copied the phenotype of the rebl-l (root epidermal cell bulging) mutant of Arabidopsis. Upon electrophoretic analysis of AGPs extracted by homogenization of tissue in a low-salt buffer containing 1% (v/v) Triton X-100 detergent, roots of the rebl-l mutant were found to contain only about 70% as much AGP as wild-type roots. In particular, the rebl-1 mutant was relatively deficient in a root AGP having high electrophoretic mobility. No such deficiency was observed when AGPs were extracted from the shoots of rebl-l and wild-type plants, the rebl-1 mutant actually having a higher overall amount of AGPs in this case. These results support the hypothesis that AGPs function in root growth, particularly as regards expansion of epidermal cells. The precise function of the gene mutated in rebl-l, however, remains to be determined. An attractive hypothesis is that this gene encodes an AGP core polypeptide or other protein involved in the synthesis of an AGP, thus directly causing the AGP deficiency in the mutant. Alternatively, the effect of the reblI mutation might be to alter the low-salt/detergent extractability of an AGP, or even to alter AGP levels or extractability through a pleiotropic effect. The mechanisms through which functions of living cells are disrupted by (p~-Glc)~-mediated perturbation of AGPs likewise remain to be determined. One possibility is that binding of (P-D-GIc)~to cell surface AGPs affects the physical properties of the cell wall or plasma membrane. Perturbations of CW-AGPs might, for example, interfere with wall loosening, thereby directly causing the effects of (P-D-GIC)~ on cell expansion. Alternatively, perturbation of AGPs with (P-D-G~C)~ might interfere with wall assembly required for cell expansion, as thought to occur in pollen tubes treated with (P-D-G~c)~ (see Section III.B.2). Effects of (P-D-G~c)~ on wall loosening or assembly might indirectly inhibit cell proliferation as structural and metabolic characteristics of the cell wall are known to alter the rate of cell division (Meyer and Abel, 1975; van Engelen and de Vries, 1992; Hetherington and Fry, 1993). Alternatively, disruption of cell function by ( P - D - ~ ~ Cmight ), involve perturbation of PM-AGPs. Measurements of lateral diffusion of plasma membrane components in living protoplasts have shown that (P-D-G~C)~ increases the variability of the diffusion coefficients, and decreases the mobile fractions of plasma membrane proteins and glycoconjugates (Serpe and Nothnagel, 1994). This observation clearly raises the possibility that (P-D-GIC)~ might perturb normal molecular interactions at the plasma membrane and thereby interfere with transmission of signals that affect cell development (Schindler et af., 1995; Bacic et af.,1996; Kreuger and van Holst, 1996; Schultz et al., 1998). Taken together, these various experiments involving application of (p-DG ~ cto) ~living materials show that perturbations of cell surface AGPs can have
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dramatic effects. On the one hand, the capacity of (P-D-G~c)~ to bind to most AGPs enhances the broad applicability of this approach to studying AGP function. On the other hand, this same capacity of (P-D-G~c)~ to bind to most AGPs frustrates the usual desire to interpret the results of such experiments in terms of a particular AGP. Use of monoclonal antibodies as perturbing agents, instead of (P-D-G~c)~, is an experimental approach that might enable examination of function for selected AGPs. Butowt et al. (1999) have recently used this approach to investigate the function of AGPs in cell wall regeneration by protoplasts. Earlier studies with Vinca rosea (Takeuchi and Komamine, 1978) and carrot (Shea et al., 1989; Mock et al., 1990) protoplasts had shown that accumulation of cell wall material on protoplasts was paralleled by the accumulation of polymers in the culture medium. These culture medium polymers, which were rich in galactose and arabinose, included AGPs as judged by glycosyl linkage analysis (Takeuchi and Komamine, 1978; Shea et a f . , 1989) or precipitation with Yariv phenylglycoside (Mock et al., 1990). In their investigation of sugar beet protoplasts, Butowt et al. (1999) found that the JIM1 3 monoclonal antibody exhibited negligible binding to the surface of freshly isolated protoplasts. Upon culturing the protoplasts, Butowt et al. (1999) found that some wall material formed around protoplasts within 2 days and apparently complete walls regenerated within 4-6 days. Electron microscopy and immunogold labelling demonstrated that the JIM 13 epitope was present in the partial walls at 2days and abundant in the complete walls at 46days. The JIM13 epitope was also abundant in the cell walls of many, but not all, of the suspension-cultured cells from which the protoplasts were isolated. When applied as a perturbing agent in the culture medium of these suspensioncultured cells, the JIM13 antibody had no detectable effect. When applied in the medium of freshly isolated protoplasts, however, JIM 13 completely inhibited proliferation. When applied to cultures at 7 days after protoplast isolation, JIM 13 reduced subsequent callus colony formation by 50% (Butowt et af., 1999). These results point to the function of certain CW-AGPs in wall assembly, and should stimulate further use of antibodies as perturbing agents.
IV. PLASMA MEMBRANE AGPS (PM-AGPS) The presence of AGPs on the plasma membrane was first suggested by the observation that Yariv phenylglycosides stained the membrane-cell wall interface in tissue sections (Clarke et af., 1975, 1978). Agglutination of protoplasts upon addition of (P-D-GIc)~provided further evidence of the presence of AGPs on the outer surface of the plasma membrane (Larkin, 1977, 1978; Nothnagel and Lyon, 1986). Subsequently, several monoclonal antibodies recognizing AGP epitopes on the plasma membrane were generated and used to investigate how these epitopes varied in expression during
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development and among species (Pennell et al., 1989, 1991; Knox, 1992; Pennell, 1992; Pennell and Roberts, 1995; Smallwood et al., 1996). The results obtained indicated that the plasma membrane contains several AGP epitopes exhibiting regulated expression. The presence of AGPs on the plasma membrane was also investigated at the biochemical level. From a tobacco microsomal fraction, Kawasaki (1987a, b) isolated macromolecules with glycosyl and aminoacyl compositions characteristic of AGPs. These results showed that some AGPs were membrane bound, although the use of a microsomal fraction did not permit a determination of whether these AGPs were on the plasma membrane or in other cell membranes. More definitive biochemical evidence for the presence of AGPs on the plasma membrane was obtained by Norman et al. (1990). Using a monoclonal antibody directed against an antigen on the plasma membrane, Norman et al. (1990) purified macromolecules from Nicotiana glutinosa cells by immunoaffinity chromatography and showed that these molecules contained the Hyp, arabinosyl and galactosyl residues that are characteristic of AGPs. Taking an alternative approach, Komalavilas et al. (1991) used aqueous two-phase partitioning to purify plasma membrane vesicles from the microsomal fraction of suspension-cultured rose cells and then used (/3-D-Glc)3-induced precipitation to purify AGPs from the plasma membrane vesicles. Thus, results from several approaches converged to convincingly demonstrate the occurrence of AGPs on the plasma membrane. The existence of PMAGPs has generated great interest because the plasma membrane is a site of important functions including, for example, synthesis and secretion of cell wall components (Gibeaut and Carpita, 1994; Delmer and Amor, 1995). Molecules at the outer surface of the plasma membrane also probably function as developmental clues or as receptors during cell interactions with other cells or with the environment (Roberts, 1990; Knox, 1995). The PM-AGPs are good candidates to participate in these events. In this section we will examine evidence in support of such potential roles, as well as other information regarding the structure of PM-AGPs and their relation to CW- and/or soluble AGPs. A. BIOCHEMICAL CHARACTERIZATION
The first portion of this section will focus on the purification and general structural characterization of PM-AGPs, with particular emphasis on comparison of PM-AGPs with the CW-AGPs and the soluble AGPs of the cell wall space. The second portion of this section will focus on the recent discovery that classical AGPs are synthesized with a lipid substituent that transiently anchors them to membranes.
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1. Purification and General Structural Features The first step in the biochemical analysis of PM-AGPs is their purification from plant tissues. For this purpose, two general strategies are available. In one strategy, monoclonal antibodies that recognize particular AGP epitopes are utilized to purify PM-AGPs by immunoaffinity chromatography (Norman et al., 1990) or to detect AGPs during electrophoretic analyses of membrane fractions (Smallwood et al., 1996; Stohr et al., 1996; Kjellbom et al., 1997). This approach enables the purification of the particular AGPs that contain the epitope recognized by the antibody. In the second strategy, a fraction enriched in plasma membrane vesicles is prepared and then AGPs are purified from these plasma membrane vesicles by (P-~-Glc)~-induced precipitation and subsequent chromatographic steps (Komalavilas et al., 1991). Because (p-DG ~ c binds ) ~ to most AGPs, use of this probe leads to results that provide a more complete picture of the AGPs present at the plasma membrane. The (P-~-Glc)~-based strategy was used to investigate the PM-AGP complement of suspension-cultured rose cells (Komalavilas et al., 1991; Serpe and Nothnagel, 1996). A fraction highly enriched in plasma membrane vesicles was obtained from these cells by aqueous, two-phase partitioning (Komalavilas et al., 1991). The carb0hydrate:protein ratio of these vesicles was 0.72:l (w/w) (Komalavilas et al., 1991), much higher than the ratio of 0.16:l found in human erythrocyte membranes (Guidotti, 1972). A broader survey of plant and animal cell types is needed to determine if this remarkably high carbohydrate content of the plant plasma membrane is a general distinction between plant and animal cells. About 47% of this abundant carbohydrate in rose plasma membranes consisted of approximately equal amounts of arabinosyl and galactosyl residues, the characteristic sugars of AGPs. When AGPs were quantitated on the basis of interaction with (P-D-GIc)~,rose plasma membranes were found to have an AGPs:total protein ratio of 0.067:l (w/w), and the AGPs accounted for 9% of the total plasma membrane carbohydrate. The remaining 91% of the carbohydrate was present in glycolipids and other unidentified glycoconjugates. Although AGPs are quantitatively significant components of the plasma membrane, obtaining sufficient amounts of PM-AGPs for structural characterization is a challenging task. Approximately 100 g (fresh weight) of suspension-culture rose cells were needed to obtain an amount of highlypurified plasma membrane vesicles containing 1 mg total protein. The PMAGPs were solubilized from these vesicles with 1% (w/w) Triton X-100 and subsequently precipitated with (P-D-GIc)~to yield approximately 0.03 mg AGPs per mg plasma membrane protein (Serpe and Nothnagel, 1996). Thus, for isolations starting with lOOg of cells, about 30 preparations of plasma membrane vesicles were required to obtain l m g of total PM-AGPs. Such a preparation contained several distinct AGPs, however, thus necessitating further fractionation before structural analysis.
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Separation of total rose PM-AGPs by several chromatographic steps yielded two major and two minor fractions (Serpe and Nothnagel, 1996). All of these fractions contained macromolecules with typical AGP characteristics including affinity for (P-D-G~c)~ and carbohydrate compositions rich in galactosyl, arabinosyl and glucuronosyl residues. The two most abundant PM-AGP fractions, PM-AGP1 and PM-AGP2, were prepared in sufficient amounts to enable glycosyl linkage analysis which demonstrated the presence of the terminal a-arabinofuranosyl residues and the (1,3)-/3-, (1,6)-P- and (1,3,6)-,8galactopyranosyl residues that form the framework of type I1 arabinogalactans (Aspinall, 1973). The protein moieties of PM-AGP1 and PM-AGP2 were rich in Hyp, Ala and Ser (Serpe and Nothnagel, 1996), which are abundant amino acids in most AGPs (Anderson et al., 1977; Clarke et al., 1979a; Gleeson et al., 1989; Nothnagel, 1997). While both sharing the general characteristics of AGPs, PM-AGP1 and PMAGP2 differed from each other in the details of their glycosyl and aminoacyl compositions and were separable by anion-exchange chromatography, PMAGP2 binding more tightly to the column than PM-AGP1. Furthermore, PMAGPl and PM-AGP2 had distinct molecular sizes of 140 and 217kDa, respectively, and distinct protein proportions of 3.7 and 15% (w/w), respectively (Serpe and Nothnagel, 1996). The minor PM-AGP fractions eluted later from anion-exchange and reversed-phase chromatographic columns than did either PM-AGP1 or PM-AGP2 (see Section IV.A.2). The functional significance of the presence of several distinct AGPs on the plasma membrane of undifferentiated cells remains uncertain. The availability of structural information regarding rose PM-AGPs, CM-AGPs and CW-AGPs (Komalavilas et al., 1991; Serpe and Nothnagel, 1995, 1996), however, enabled a test of the hypothesis that PM-AGPs are precursors to CM-AGPs and CWAGPs (Nothnagel, 1997). Consistent with this hypothesis was a close structural resemblance between PM-AGP1 (Serpe and Nothnagel, 1996), CM-AGPb (Komalavilas et al., 1991) and CW-AGP1 (Serpe and Nothnagel, 1995). As estimated by gel-permeation chromatography, the molecular masses of PMAGP1, CM-AGPb and CW-AGP1 were 140, 141 and 130 kDa, respectively. These three AGPs also had comparable electrophoretic mobilities in agarose and sodium dodecyl sulphate (SDS)-polyacrylamide gels (Serpe and Nothnagel, 1996), and comparable aminoacyl and glycosyl compositions (Nothnagel, 1997). These structural similarities suggested that PM-AGP1 might be released from the plasma membrane to give rise to CM-AGPb or CW-AGP1, and this possibility motivated a search for lipid anchors on PM-AGPs (see Section IV.A.2). While PM-AGP1 shared very similar structural features with certain CMand CW-AGPs, such was not the case for PM-AGP2, the other abundant rose plasma membrane AGP. The 217-kDa size and 15% protein content were unique features of PM-AGP2, as was its exceptionally high Hyp content at 32.2% (Serpe and Nothnagel, 1996; Nothnagel, 1997). These and other
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distinguishing features indicated that PM-AGP2 was localized at the plasma membrane and was not released as a close derivative into either the cell wall or culture medium. Through affinity chromatography with the monoclonal antibody PN16.4B4, which recognizes an unidentified epitope in the carbohydrate portion of AGPs, Norman et al. (1990) purified a family of AGPs present on the plasma membrane of suspension-cultured Nicotiana glutinosa cells. On average, the carbohydrate portion of these AGPs consisted of 3 1mol% arabinose, 46 mol% galactose, 11 mol% glucose, and lesser amounts of rhamnose, mannose and fucose, but no glucuronic acid. The molecules contained 27-37% protein which consisted of 16mol% Ser, 15mol% Gly, 14mol% Ala, 12mol% (Glu + Gln) and lesser amounts of other amino acids. Hydroxyproline was not resolved in the analysis, but the sum of Asp, Asn and Hyp totalled 8mol%. Thus, compared with rose PM-AGPs (Serpe and Nothnagel, 1996), these Nicotiana PM-AGPs had less Hyp but higher overall protein contents. In both rose and Nicotiana PM-AGPs, however, the carbohydrate portion was much larger than the protein portion. These results do not support the hypothesis that membrane-associated AGPs have a larger protein portion than carbohydrate portion (Chasan, 1994). 2. Glycosyl-phosphatidylinositol ( G P I ) Lipid Anchors It is widely recognized that AGPs are generally very hydrophilic molecules, and thus a question arises as to the mechanism through which these molecules associate with the plasma membrane. The initial experiments relative to this question involved treatments with agents that modify the physical forces that generally support protein-membrane interactions. Norman et al. (1990) observed that high salt washes and sonication failed to release an AGP from microsomal vesicles of Nicotiana glutinosa, thus indicating that AGPs are not held on the membrane through ionic forces. On the other hand, several investigators found that detergents were effective in solubilizing AGPs from membrane vesicles, thus indicating that the mechanism of AGP association with membranes involves hydrophobic interactions. The membrane AGPs were not highly hydrophobic, however, as the detergent treatments required to solubilize AGPs from membranes were relatively mild, involving 1YO(v/v) NP40 detergent with Nicotiana microsomal vesicles (Norman et al., 1990) or 1 % (w/w) Triton X-100 with rose plasma membrane vesicles (Komalavilas et al., 1991; Serpe and Nothnagel, 1996). Likewise, the generally hydrophilic nature of PM-AGPs was demonstrated by application of Triton X-114 detergentaqueous phase partitioning to carrot microsomal vesicles (Pennell et al., 1989; Smallwood et al., 1996), Nicotiana plasma membrane vesicles (Norman et al., 1990) and sugar beet plasma membrane vesicles (Pennell et al., 1991; Stohr et al., 1996; Kjellbom et al., 1997), where in each case the PM-AGPs partitioned exclusively into the aqueous phase rather than into the hydrophobic detergent phase. An exception was found with rice microsomal vesicles, where the AGPs
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partitioned preferentially into the hydrophobic detergent phase (Smallwood et af., 1996). An important advance in understanding the mechanism through which AGPs associate with membranes has come with the recent finding, by three laboratories, working independently through different approaches, that some AGPs are synthesized with a glycosyl-phosphatidylinositol (GPI) lipid anchor (Fig. 2). In addition to providing a structural model for understanding association of PM-AGPs with the plasma membrane, the finding of GPI anchors has numerous implications regarding the biosynthesis, targeting and function of AGPs. One approach leading to the finding of GPI anchors on AGPs focused on the C-terminal aminoacyl sequence of the AGP core polypeptide. When the first two cDNAs encoding classical AGPs, AGPPcl and AGPNal, were sequenced and the predicted hydrophobic C-terminal domain (Fig. 1A) was noticed in hydropathy plots, it was suggested that this domain might form a transmembrane a-helix that anchored the AGP to the plasma membrane (Chen et al., 1994; Du et af., 1994). These first cDNAs were obtained through partial aminoacyl sequencing of soluble AGPs from culture medium or extracellular secretions, however, rather than from PM-AGPs. The presence of the hydrophobic C-terminus in these mature, soluble AGPs was uncertain, as it was in the classical AGPs predicted by other cDNAs subsequently cloned. The important breakthrough in this line of research recently came when Youl et al. (1998) used tandem mass spectrometry in the C-terminal sequencing of the core polypeptides of AGPPc1 and AGPNa1. In their mature, soluble forms, both of these AGPs were found to lack their hydrophobic C-terminal domain. Instead, the truncated C-terminus was linked to ethanolamine. This type of C-terminal processing is precisely characteristic of the GPI anchors known to occur on many plasma membrane proteins in animals and microorganisms (Low, 1989; Schneider and Ferguson, 1995). In all structurally characterized GPI anchors from animals and microorganisms, the structure at the C-terminus of the protein is protein + ethanolamine + PO4 + 6Mana1 -+ 2Manal + 6Manal + 4GlcNa1 + 6myo-inositoll + PO4 + lipid (Low, 1989; Fig. 2). In some cases, the mannosyl residues of this consensus core are decorated with additional mannosyl, galactosyl, glucosyl, N-acetylgalactosaminyl or phosphoethanolaminyl residues, while the inositol sometimes carries a palmitate ester (Schneider and Ferguson, 1995). Thus, the detection by Youl et af. (1998) of small amounts of inositol, glucosamine and mannose in the mature forms of AGPPc1 and AGPNa1 was further evidence in support of the hypothesis that these AGPs were modified by a GPI anchor. Youl et al. (1998) were unable, however, to detect any type of lipid component in either AGPPc1 or AGPNal. A different approach to the finding of GPI anchors on AGPs involved the purification and structural comparison of AGPs from different domains at the surface of suspension-cultured rose cells. As described in Section 1V.A. 1, close
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structural resemblances between PM-AGP1 (Serpe and Nothnagel, 1996), CMAGPb (Komalavilas et al., 1991) and CW-AGP1 (Serpe and Nothnagel, 1995) stimulated the hypothesis that PM-AGP1 might be released from the plasma membrane to give rise to CM-AGPb or CW-AGP1. Furthermore, none of the characterized PM-AGPs from rose cells had aminoacyl compositions that were more hydrophobic than the aminoacyl compositions of CM- or CW-AGPs, thus arguing against a hydrophobic polypeptide domain as a membrane anchor in PM-AGPs (Serpe and Nothnagel, 1996). On the other hand, the 'HNMR spectrum of a PM-AGP was found to contain a small peak centred at 1.33parts per million (ppm). Because the position and intensity of this signal were consistent with the PM-AGP containing about 1.7 hydrocarbon chains the size of palmitate, Serpe and Nothnagel(l996) hypothesized that rose PMAGPs are linked to GPI lipid anchors. The hypothesis that GPI anchors are present in rose PM-AGPs was tested in four lines of experimentation by Svetek et al. (1999). First, treatment with exogenous phosphatidylinositol-specific phospholipase C (PI-PLC; Fig. 2) stimulated the release of AGPs from rose plasma membrane vesicles in vitro. In these experiments, a facile inherent release of AGPs from plasma membrane vesicles was also observed. At least some of this inherent release of PM-AGPs seemed to be due to endogenous phospholipases as it was partially inhibited by known inhibitors of PI-PLCs. Second, reverse-phase chromatography revealed that about 18% of the AGPs in the plasma membrane fraction chromatographed as hydrophobic molecules. Most of these hydrophobic AGPs were sensitive to exogenous PI-PLC, which caused their elution to shift to the markedly more hydrophilic position where the other 82% of the AGPs eluted. Total CM-AGP preparations, which could be prepared in larger amounts, were found to contain a smaller proportion of hydrophobic AGPs which were similarly sensitive to exogenous PI-PLC. Third, inositol, glucosamine and mannose were detected in both PM-AGPs and CM-AGPs at abundances roughly consistent with the presence of a GPI core oligosaccharide (Fig. 2). Fourth, long-chain fatty acids were detected at approximately the anticipated level in the hydrophobic CM- and PM-AGPs that were susceptible to conversion to hydrophilic forms by exogenous PI-PLC. The fatty acids were linked to CM-AGPs through an amide bond, as characteristic of ceramides. The predominant long-chain fatty acid of the ceramide was tetracosanoic acid and the predominant long-chain base was phytosphingosine. These results were interpreted as confirming the hypothesis that GPI lipid anchors are present in rose PM-AGPs (Svetek et al., 1999). Facile conversion of PM-AGPs to hydrophilic forms in the plasma membrane vesicle preparations in vitro was interpreted as arising from cleavage of GPI anchors by an endogenous phospholipase, such as the glycosyl inositol phospholipidspecific phospholipase C partially purified from peanut (Butikofer and Brodbeck, 1993). The presence of a small proportion of hydrophobic AGPs in the rose cell-culture medium was hypothesized to be due to the biophysical
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mechanism of partitioning, which in this case arose from the carbohydrate component of PM-AGPs being large and hydrophilic enough to occasionally pull the ceramide lipid out of the plasma membrane into the aqueous phase (Svetek et al., 1999). The third independent finding of GPI anchors on AGPs occurred in experiments on Arabidopsis (Sherrier et al., 1999). This work demonstrated the existence of multiple GPI-anchored proteins that were susceptible to PI-PLC cleavage. The GPI-anchored proteins were found to be a relatively abundant class of plant plasma membrane proteins, some of these being released into the extracellular matrix. At least one of these proteins was an AGP. This work opens the way to the study of GPI-anchored proteins in the important Arabidopsis model system. Taken together, these three independent findings place AGPs at the forefront of knowledge regarding GPI anchors in higher plants. While many membrane proteins of animal, protozoan, yeast or bacterial origin have been demonstrated to contain GPI lipid anchors (Low, 1989; Jones and VarelaNieto, 1998), previous reports of GPI-anchored proteins in plants have been limited to an alkaline phosphatase in the aquatic plant Spirodela oligorrhiza (Morita et al., 1996; Nakazato et al., 1998), nitrate reductases in the plasma membranes of sugar beet and barley (Kunze et al., 1997), and several unidentified plasma membrane proteins in Nicotiana tabacum protoplasts (Takos et al., 1997). The green alga Chlorella saccharophila has also been reported to contain a GPI-anchored plasma membrane nitrate reductase (Stoehr et al., 1995). In each of these reports, the evidence for GPI anchors involved susceptibility to PI-PLC and/or incorporation of radiolabelled precursors such as ethanolamine. These studies, together with the work by Sherrier et al. (1999) on Arabidopsis, suggest that the abundance and diversity of GPI-anchored proteins in higher plants will eventually be found to rival those in animals and microorganisms. Conjectures regarding the abundance and functions of GPI-modified proteins in higher plants might also be reasonably based on results for the yeast Saccharomyces cerevisiae. Among GPI lipid anchors found in various organisms, the most common type of lipid structure is sn- 1-alkyl-2-acylglycerol-3-phosphate-inositol (alkyl-acyl-PI), although diacyl-PI, lyso-acyl-PI, lyso-alkyl-PI and ceramide-PI have also been found (Schneider and Ferguson, 1995). Similarity between yeast and higher plants is indicated here because the predominant form of GPI lipid has been found to be ceramide in both yeast proteins (Fankhauser et al., 1993) and rose AGPs (Svetek et al., 1999). In both cases, the predominant long-chain base is phytosphingosine. The predominant long-chain fatty acid is hexacosanoic acid in Saccharomyces cerevisiae (Fankhauser et al., 1993) and tetracosanoic acid in rose AGPs (Svetek et al., 1999). Thus, it noteworthy that of the 6218 known open reading frames in the fully sequenced yeast genome, 686 are predicted to encode proteins with a N-
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terminal signal sequence for entry into the endoplasmic reticulum and secretory pathway (Caro et al., 1997). Certain aminoacyl sequences have been identified as characteristic of the cleavage site of the C-terminal hydrophobic domain during synthesis of GPI-linked proteins (Udenfriend and Kodukula, 1995). Judging from the presence of these consensus sequences, Car0 et al. (1997) predicted the occurrence of GPI anchors on 58 yeast proteins, 51 of these being among the 686 predicted secretory-path proteins. Some of these 58 predicted GPI-modified proteins are known to be localized in the plasma membrane, and include aspartyl proteases, lysophospholipases and various proteins involved in cell wall synthesis (Caro et al., 1997). Of the 58 GPI-modified proteins predicted in yeast, however, 15 are known to be localized in the cell wall and another 23 are homologous to known cell wall proteins (Caro et al., 1997). All 38 of these known and putative cell wall proteins contain Ser- and Thr-rich regions and are thought to become heavily 0-glycosylated with short mannose side chains. In many of the 38 the aminoacyl sequences include repeats that are rich in Ser, Thr, and, to a lesser extent, Ala, Val, Pro and Glu. In the few cases investigated thus far, the lipid portion of the GPI anchor has been found to be cleaved from these proteins prior to their incorporation into the yeast cell wall. Most of the GPI linker oligosaccharide remains, however, and in some cases has been shown to be bound to cell wall P-1,6-glucans (Caro et al., 1997). The analogy between these GPI-modified yeast cell wall proteins and GPImodified AGPs in higher plants is striking and should stimulate additional searches for GPI-modification of plant cell wall proteins. Upon finding aminoacyl sequences characteristic of the consensus cleavage site of the Cterminal hydrophobic domain of GPI-linked proteins, Schultz et al. (1998) predicted that all known and putative classical AGPs are modified by attachment of GPI-anchors. Five Arabidopsis genes found in an expressed sequence tag database were also predicted to encode AGPs with GPI anchors (Schultz et al., 1998). The relatively small genome of Arabidopsis should facilitate mutational analysis of AGP functions. Such studies will probably be facilitated by existing knowledge of yeast mutants. The cwh6/gpi3 yeast, for example, has a temperature-sensitive mutation in a gene involved in the first step of GPI synthesis, the addition of N-acetylglucosamine to phosphatidylinositol. At semi-permissive temperature, the phenotype of cwh6/gpi3 yeast includes severely reduced growth rate, proliferation of endoplasmic reticulum structures and accumulation of cell wall protein precursors therein, and inefficient incorporation of proteins into the cell wall (Vossen et al., 1997). While the discovery of GPI anchors on AGPs is certain to stimulate new approaches to investigating AGP biosynthesis, targeting and function, it should be remembered that non-classical AGPs are not encoded with a hydrophobic C-terminal domain (Fig. 1) and thus are unlikely to be modified with a GPI anchor. It is also possible that some PM-AGPs interact with the plasma membrane through a mechanism other than a GPI lipid anchor. In
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animal cells, heparan sulphate proteoglycans have been found to interact with plasma membranes through three mechanisms. Some plasma membrane heparan sulphates contain GPI lipid anchors, other heparan sulphates contain a hydrophobic polypeptide domain embedded in the lipid bilayer, and still other heparan sulphates bind non-covalently to other plasma membrane components (Yanagishita and Hascall, 1992). B. EXPRESSION AND FUNCTION
The expression of PM-AGPs has been principally investigated using various monoclonal antibodies that recognize AGP epitopes. Some of these antibodies appear to recognize a general epitope present on a variety of AGP-like molecules and therefore lack developmental or cell specificity (Glaudemans et al., 1986; Pennell et al., 1989; Kikuchi et al., 1993). Certain anti-AGP monoclonal antibodies show higher specificity, however, and bind to PM-AGP epitopes in only certain cell types or during particular stages of plant development (Knox et al., 1989; Pennell et al., 1991; Dolan et al., 1995). Both the general and specific antibodies have been used in immunocytochemistry to reveal that the plasma membrane contains several AGP-like molecules and to elucidate taxonomic and developmental variations in these plasma membrane AGP epitopes.
I . PM-AGP Epitopes Among Different Taxa The MAC207 monoclonal antibody, which recognizes an epitope contained within the p-~-GlcpA-(1 -+ 3)-a-~-GalpA-( 1 -+ 2)-~-Rhaoligosaccharide (Yates et al., 1996), appears to have been the only monoclonal antibody used to compare the expression of PM-AGP epitopes among various taxa. Using highresolution immunogold electron microscopy to distinguish labelling on the plasma membrane from labelling in the cell wall, Pennell et al. (1989) found that MAC207 bound to the plasma membrane of somatic cells of both dicotyledonous and monocotyledonous angiosperms. Families of dicotyledonous plants tested and found to contain the plasma membrane epitope included Papaveraceae, Chenopodiaceae, Cucurbitaceae, Cruciferae, Leguminoseae, Umbelliferae, Solanaceae and Caprifoliaceae. Families of monocotyledonous plants tested and found to contain MAC207 epitopes on the plasma membrane included Liliaceae and Agavaceae. In contrast, MAC207 did not bind to sections cut from a moss, two pteridophytes and two conifers. These observations were interpreted as evidence that the MAC207 epitope is widely distributed among flowering plants but it is absent from non-flowering plants. It would be interesting to further test this hypothesis and to determine if there are major structural differences between PM-AGPs from different taxa which may reveal evolutionary trends in the structure of these molecules. It is of note
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that while MAC207 did not bind to conifer plasma membranes, for example, conifers do contain AGPs (Bobalek and Johnson, 1983). 2. Immunochemical Evidence Indicating the Presence of Several AGP-like Molecules on the Plasma Membrane Early evidence that the plasma membrane contains several AGP-like molecules was obtained using MAC207 (Pennell et al., 1989). In carrot suspension culture cells, MAC 207 bound predominantly to the outer surface of the plasma membrane. On analysis of a microsomal membrane fraction by twodimensional gel electrophoresis and subsequent immunoblotting, Pennell et al. (1989) observed that 18 distinct spots were labelled by MAC 207. Although it remains uncertain whether all of these spots arose from the plasma membrane and whether all were AGPs, the results suggested that the plasma membrane contains at least several AGP-like molecules. The JIM4 monoclonal antibody, which recognizes an epitope contained within the ,Ll-~-GlcpA-(1 -+ 3)-a-~-GalpA-( 1 -+ 2)-~-Rha oligosaccharide (Yates et al., 1996), and the JIM8 monoclonal antibody, which recognizes an unidentified epitope in the carbohydrate portion of AGPs (Pennell et al., 1991), both recognize several PM-AGPs or AGP-like molecules. When microsomal membranes from suspension-cultured carrot cells were analysed by electrophoresis and immunoblotting, JIM4 and MAC207 both labelled a similar series of discrete bands, indicating that these two monoclonal antibodies recognized the same set of membrane molecules (Knox et al., 1989). Pennell et al. (1991) purified plasma membrane vesicles from microsomes of sugar beet leaves, and analysed the preparation by electrophoresis and immunoblotting. The JIM8 antibody stained three bands, while MAC207 stained the same three bands plus an additional fourth band. Thus, the JIM8 antibody bound to a subset of the molecules recognized by MAC207. Norman et al. (1990) used the PN16.4B4 monoclonal antibody, which recognizes an unidentified epitope in the carbohydrate portion of AGPs, to probe immunoblots obtained in electrophoretic analysis of the plasma membrane fraction from aqueous two-phase partitioning of microsomes from suspension-cultured cells of Nicotiana glutinosa. The antibody labelled a family of bands corresponding to relative molecular masses in the range of 135180 kDa. Although this observation suggested that the plasma membrane of N . glutinosa contained several AGPs or AGP-like molecules, other evidence led Norman et al. (1990) to suggest that these various species might represent various levels of glycosylation of a single core polypeptide of size 50 kDa. Using an improved SDS-agarose gel electrophoresis system (Stohr et al., 1996), Kjellbom et al. (1997) found that the appearance of several bands with AGP epitopes in immunoblots of plasma membrane components from sugar beet leaves was due, in part, to oxidative cross-linking of PM-AGPs. When leaves were excised at 6°C and immediately homogenized at WC, two bands of apparent molecular masses 82 and 97 kDa were detected on blots probed with
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either MAC207 monoclonal antibody or with (P-D-G~c)~ Yariv phenylglycoside. Analysis on two-dimensional gels indicated that the 82- and 97-kDa bands each consisted of two isoforms. When leaves were excised and held at 22°C for 15 min prior to homogenization, however, the one-dimensional blots showed five bands at 82, 97, 120, 170 and 210 kDa, the 170-kDa band appearing particularly intense. Upon addition of H202 and horseradish peroxidase to preparations from immediately homogenized leaves, the 170kDa band appeared with a concomitant weakening of the 82-kDa band. Kjellbom et al. (1997) interpreted these results in terms of oxidative crosslinking of PM-AGPs as a wound response induced by leaf excision. Thus, sugar beet plasma membranes appeared to contain two principal PM-AGPs with other bands arising from cross-linking. It remains to be elucidated whether these two sugar beet PM-AGPs are related to PM-AGP1 and PM-AGP2, the two principal PM-AGPs found in rose plasma membranes (Serpe and Nothnagel, 1996; see Section 1V.A.1). This study of PM-AGP cross-linking also produced some interesting anecdotal data regarding specificities of various anti-AGP monoclonal antibodies (Kjellbom et al., 1997). The MAC207, JIM8, PN16.4B4 and OxFB8 antibodies all bound to both the 82- and 97-kDa sugar beet PM-AGPs. The MAC207 and PN16.4B4 antibodies also bound to all three of the apparent cross-link products at 120, 170 and 210 kDa. The Ox-FB8 antibody bound to only the 120- and 170-kDa bands, however, while JIM8 bound to just the 170kDa band. A simple interpretation of these results might be that cross-linking blocked or destroyed the Ox-FB8 and JIM8 epitopes in some cases. More interesting, however, were the results obtained with JIM14, which recognizes an unidentified epitope in the carbohydrate portion of AGPs (Knox et al., 199l), and JIM 15, which recognizes an epitope involving 0-D-glucuronopyranosyl residues (Yates et al., 1996). Kjellbom et al. (1997) found that neither JIM14 nor JIM15 bound to the 82- and 97-kDa sugar beet PM-AGPs, but both of these antibodies bound to the apparent cross-link products at 120, 170 and 210kDa. Earlier work has shown that JIM14 binds to a cell wall epitope whose expression has been linked with development of vascular tissues in roots (Knox et al., 1991; Dolan et al., 1995) and coleoptiles (Schindler et al., 1995) (see Section 1II.B.l), while JIM1 5 binds to a plasma membrane epitope whose expression has been linked with pattern formation in roots (Knox et al., 1991). Taken together, these studies indicate that cross-linking of PM-AGPs may be important relative to several aspects of plant development. 3. PM-AGP Epitopes During Anther and Ovary Development Although MAC207 binds to many PM-AGPs, the epitope recognized by this antibody is notably absent from particular cell types. In pea, MAC207 bound to vegetative cells throughout the plant, but not to certain cells in developing stamens and carpels (Pennell and Roberts, 1990). The MAC207 epitope disappeared from progenitor cell clusters that would later give rise to the pollen
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sac, tapetum, sporangeous tissue, embryo sac and nucellus. All of these reproductive structures, plus the zygote, the endosperm and the early embryo, lacked the MAC207 epitope. This epitope reappeared when the embryo reached the heart stage. These results clearly showed that expression of some PM-AGPs is developmentally regulated. Furthermore, the disappearance of the MAC207 epitope occurred prior to the formation of the various reproductive structures, so this time sequence leaves open the possibility that PM-AGPs could play a regulatory role in sexual development. The expression of PM-AGPs in reproductive structures has also been investigated with the JIM8 monoclonal antibody and high-resolution immunogold electron microscopy (Pennell et al., 199 1). This antibody produced an staining pattern that directly contrasted with that produced by MAC207. In aerial organs of oil-seed rape, the JIM8 epitope was present in anthers and ovules but was absent from vegetative parts, except for the xylem vessels where it appeared transiently after cell wall thickening. During development of anthers and ovules, the expression of the JIM8 epitope progressed through various cells types. In the anthers, the JIM8 epitope was first expressed in the tapetum, then in microspore tetrads and, finally, on the plasma membrane of sperm cells. A temporal change was also observed in the ovule where the JIM8 epitope was first expressed in the nucellus, and then on the plasma membrane of the egg and synergid cells. As the JIM8 epitope appeared on the sperm, egg and synergid cells, this epitope disappeared from the other cells where it was previously present. The functional significance of these changes in PM-AGP epitope expression during sexual development remains unknown. In several of the cell types that contain the JIM8 epitope, the cell wall either disintegrates, as in the tapetum, or is absent, as in sperm cells. Thus, the JIM8 epitope might be involved with cell wall metabolism. Alternatively, the presence of the JIM8 epitope in sperm and egg cells opens the prospect that certain PM-AGPs might be involved in gamete recognition (Pennell et al., 1991). To complement these data on expression of the particular MAC207 and JIM8 epitopes, it seems useful to consider overall expression of AGPs during sexual development. Gane et al. (1995a) showed that the overall concentration of AGPs in the ovary of Nicotiana alata did not change during development or fertilization. The relative amounts of the several different ovary AGPs detected by electrophoresis did, however, exhibit developmental changes. Spatial variations in the distribution of AGPs were also observed. As detected by histochemical staining with (P-D-G~c)~, AGPs were most abundant at the epidermis of the placenta. This technique could not distinguish, however, whether these AGPs were associated with the plasma membrane or cell wall. Comparable studies of overall AGP expression during anther development are lacking. An alternate experimental approach involving analysis of small molecules has yielded data that seem to be relevant to AGP metabolism during
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microsporogenesis. Kawaguchi et al. (1996) found that rice anthers accumulate a tetrasaccharide that is structurally related to AGPs. The abundance of this tetrasaccharide changes dramatically during the different stages of rice microsporogenesis, the maximum abundance amounting to about 1% of wet anther weight at the middle of microsporogenesis. The structure of this tetrasaccharide, P-L-AraF(1 -+ 3)-a-~-Araf-(1 -+ 3)-P-~-Galp-( 1 -+ 6)-~-Gal, contains linkages characteristics of AGPs and differs from a gum arabic side chain only by the anomeric configuration of the terminal arabinosyl residue (Defaye and Wong, 1986). These features suggest that the rice-anther tetrasaccharide is a precursor or a product of an anther AGP, although further work is needed to establish such a relationship. 4. PM-AGP Epitopes During Differentiation of Vascular Tissues As discussed in Section 1II.B.1, the differentiation of vascular tissues is accompanied by the expression of particular AGP epitopes. In root tips (Knox et al., 1991; Dolan et al., 1995) and coleoptiles (Schindler et al., 1995), the epitope recognized by the JIM1 3 monoclonal antibody has been shown to be expressed in both the plasma membrane and the cell wall. In these systems, the presence of the JIM 13 epitope preceded cell wall thickening and/or cell death, thus suggesting that this AGP epitope may be involved in these processes. The JIM4 monoclonal antibody, which recognizes an epitope contained within the @-~-GlcpA-( 1 -+ 3)-a-~-GalpA-( 1 -+ 2)-~-Rhaoligosaccharide (Yates et al., 1996), has also been used to detect changes in expression of PMAGPs during differentiation of vascular tissues. As observed by highresolution immunogold electron microscopy, the JIM4 antibody labelled the plasma membrane of cells in two regions of the stele in transverse sections cut 50-100 pm from the most apical meristematic cells in carrot root tips (Knox e f al., 1989). The centres of these two regions corresponded to the locations of the future protoxylem poles. Within the labelled areas the staining was most intense in the developing pericycle. Other cells of the stele, including those giving rise to the metaxylem, protophloem and metaphloem, were not labelled by the JIM4 antibody. Similarly, the part of the pericycle neighbouring the protophloem lacked the JIM4 epitope. Thus, JIM4 labelled a particular type of cell in some, but not all, regions of the carrot stele, or JIM4 labelled cells that differentiated into more than one cell type. Knox et al. (1989) interpreted these results as indicating that the JIM4 epitope is not a marker of cell type but rather a marker of cell position, namely the position of the protoxylem poles in carrot root tips. As the xylem develops centripetally from these poles, the diarch structure of the carrot root develops. This intriguing hypothesis that the JIM4 epitope marks the position at which the protoxylem differentiates does not seem to be universally applicable, however, as Casero et al. (1998) found that JIM4 labelled pericycle cells in front of both the xylem and phloem in radish (another diarch root) and did not label any cells in either pea (triarch) or onion (pentarch) roots.
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The investigations summarized in this section provide several examples of situations where the expression of PM-AGP epitopes, as detected by MAC207, JIM8, JIM 13 and/or JIM4 monoclonal antibodies, is very highly regulated in space or time during plant development. These alterations in the expression of PM-AGP epitopes are sometimes followed by changes in the fate of daughter or neighbouring cells, thus suggesting that PM-AGPs participate in the control of cell differentiation. In all cases, however, the precise molecular functions of PM-AGPs in these events remain unknown. In addition to their apparent roles in plant development, PM-AGPs may serve other functions in plant cells. For example, PM-AGPs may help to maintain the integrity of the plasma membrane when it is pressed against the cell wall by turgor pressure. A direct contact between the lipid bilayer of the plasma membrane and the cell wall could result in damage to the plasma membrane, especially when these two distinct structures undergo relative motion, as is likely to occur during cell growth or even during stretches/ contractions associated with turgor pressure changes. If such damage to the plasma membrane occurred, the resulting leakage could kill the cell. Consequently by protecting the plasma membrane, PM-AGPs may perform a function essential to the life of all plant cells.
V. CONCLUSIONS AND FUTURE PROSPECTS The organizational approach of this review has been to examine AGPs from the viewpoint of their localization in the multiple domains of the plant cell surface, these being the aqueous phase of the extracellular and cell wall spaces, the solid phase of the cell wall and the surface of the plasma membrane. This approach was motivated by our opinion that localization and function are often linked and thus different AGPs in different domains may have different functions. The two principle challenges of this approach should be apparent to every reader who has come this far. First, although it has been possible to present various hypotheses regarding AGP functions, no precise function has been firmly established for any AGP. Thus, no firm conclusions can be drawn as to whether different AGPs in different domains have different functions. Second, much of the AGP literature cannot be definitively linked to any of the three domains of the cell surface. Biochemical studies of total AGP extracts have often omitted data regarding subcellular localization. Immunocytochemistry at the light microscope level has often lacked the resolution needed to distinguish between labelling at the plasma membrane and labelling in the cell wall. For many experimental systems where data are available on AGPs definitively associated with one cell surface domain, comparable data on AGPs associated with the other domains have been lacking. On the other hand, the approach of examining AGPs from the viewpoint of their localization in domains is particularly timely given the recent finding that
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some, perhaps many, AGPs are synthesized with GPI lipid anchors. With regard to AGP structure, the GPI anchor provides an immediate model for understanding how inherently hydrophilic AGPs associate with plasma membranes and for understanding apparent product-precursor relationships between secreted or CW-AGPs and PM-AGPs, the latter apparently being shed from the membrane through action of a phospholipase. In the contrapositive, data demonstrating that some AGPs seem to occur uniquely in only one cell surface domain serve to caution investigators that not all AGPs might fit the GPI anchor model. This caution is also obvious from cDNA sequences encoding core polypeptides of non-classical AGPs, as these lack the hydrophobic, a-helical, C-terminal domain expected of polypeptides that become modified with GPI anchors (Fig. 1). A consequence of organizing this review to focus on AGP localization in cell surface domains has been to emphasize the importance of monoclonal antibodies in AGP research to date. Currently, the dominant hypothesis regarding AGP function is that these molecules play roles in, and perhaps sometimes regulate, plant development. While this hypothesis originated from biochemical and electrophoretic data and is now being reinforced by gene expression data, it is clear that the hypothesis has been most strongly advanced by the many studies documenting developmentally regulated expression of AGP epitopes. At the same time, however, the widespread use of monoclonal antibodies has generated many questions, some of these involving the antibodies themselves. What are the precise structures of the epitopes recognized by these antibodies? Progress has been made in this difficult area (Pennell et al., 1991; Lind et al., 1994; Steffan et al., 1995; Yates et al., 1996). Much remains to be learnt, however, as illustrated by continuing paradoxes, such as the observation that MAC207, JIM4 and JIM13 all bind to the same P-~-GlcpA-(l-+ 3 ) - a - ~ GalpA-(1 -, 2)-~-Rhaoligosaccharide (Yates et al., 1996) yet show strikingly distinct tissue localization patterns (Knox et al., 1991; Casero et al., 1998). Other questions regarding AGP epitopes are relevant to AGP function. Are different AGP epitopes uniquely linked to different AGP core polypeptides, or might changes in the activities of glycosyltransferases (Gibeaut and Carpita, 1994) or glycosidases (Dopico et al., 1989 a, b; Hirano et al., 1994) cause one core polypeptide to carry different epitopes at different times or locations in the plant? This consideration leads to an even more fundamental question: On what basis should two AGPs be considered to be different molecules? Most investigators would probably agree that if two AGPs have core polypeptides that differ by more than the one or few aminoacyl substitutions that are characteristic of isoforms, then the two AGPs are different molecules. This view may be too restrictive, however, as the core polypeptide typically accounts for less than 10% of the total AGP mass. Thus, two molecules of vastly different sizes due to glycosylation differences might have the same core polypeptide and thus be considered to be the same AGP. In fact, data already
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available show that significant differences in glycosylation of a single AGP core polypeptide can occur within a single organ (Wu et al., 1995). Thus, part of understanding the meaning of regulated expression of AGP epitopes will involve the isolation and structural comparison of AGPs that carry and do not carry the epitopes displaying regulated expression. A fuller understanding of AGP epitope expression, as well as the more general issue of whether two AGPs are different molecules, will require consideration of whether the two molecules perform different functions, a point which is currently inaccessible due to a lack of satisfactory information regarding AGP functions. The identification of AGP functions is clearly the area of AGP research that is in most need of progress. The hypothesis that AGPs play roles in, and perhaps sometimes regulate, plant development has been useful but is too broad to guide further significant progress. Attention must shift to the action of AGPs in the fundamental processes that underlie development. As described in this review, some progress in this direction has come from experiments in which living material has been treated with (P-D-G~c)~ Yariv phenylglycoside. Perturbation of AGPs by complex formation with (P-D-G~c)~ has been shown to disrupt fundamental cellular processes which, depending on the experimental system, include cell proliferation, cell expansion, cell wall assembly and/ or cell death. Further progress through this approach may come if the analyses can be extended to determine the pathway through which perturbation of AGPs is transduced to disrupt these fundamental cellular processes. For example, does inhibition of cell division by (P-D-GIc)~occur through inhibition of a cyclin-dependent protein kinase and, if so, what transduction pathway brings the signal from the AGP to the cyclin-dependent protein kinase? Schultz et al. (1998) have recently emphasized that the presence of GPI anchors on AGPs has implications relative to at least two mechanisms of signalling in plants. One such mechanism would involve the phosphoinositide pathway as phospholipase cleavage of GPI anchors on AGPs might generate some of the intracellular messengers of this pathway. The other mechanism would involve interaction of GPI-anchored proteins with other proteins that span the plasma membrane and activate signal transduction pathways, the contactin system of neuronal tissues of rats being the example described by Schultz et al. (1998). One or both of these mechanisms could provide a focus for research relative to the hypothesis that the functions of AGPs in plant development involve position marking, cell marking or other forms of signalling (Pennell et al., 1995; Knox, 1996; Du et al., 1996b; Kreuger and van Holst, 1996). Another useful focal point for future research into AGP functions might be the plasma membrane-cell wall interface. Functions of molecules at the interface between the plasma membrane and the extracellular matrix have been extensively studied in animal cells (Gallagher, 1989; David, 1993; Edelman et al., 1995). Molecules at this interface are responsible for adhesion of the plasma membrane to the extracellular matrix, and are involved in various cellular
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processes including the regulation of cell shape, cell migration, cell differentiation and programmed cell death (Edelman, 1986; Lin and Bissell, 1993; Fox, 1995; Boudreau et al., 1995a, b). Because some AGPs contain a GPI lipid that anchors to the plasma membrane (see Section IV.A.2) and some AGPs tightly associate with the cell wall (see Section 111), adequate reason exists to hypothesize that a GPI-anchored PM-AGP could serve as a connection bridge between the plant plasma membrane and cell wall. Localized adhesion of the plasma membrane to the cell wall has been observed in plasmolysed plant cells and has generated considerable interest (Wyatt and Carpita, 1993; Pont-Lezica et al., 1993; Reuzeau and Pont-Lezica, 1995), including still inconclusive searches for plant analogues of animal adhesion molecules such as vitronectin and fibronectin (reviewed in Nothnagel, 1997). Apart from their role in plant development, AGPs may perform other functions. For example as components of certain gums and mucilages, AGPs may function in plant defence, plant-symbiont interactions, wound healing and/or moisture retention (Clarke et al., 1979a; Fincher et al., 1983; Whistler, 1993). These are all early and still viable hypotheses of AGP function, but they have not yet been adequately tested. The availability of cDNA clones for AGP core polypeptides has clearly enabled new approaches in the investigation of AGP expression and function, and will continue to do so in the future. Promoter sequence analysis and related approaches in studying the regulation of transcription of genes encoding AGP core polypeptides are now accessible and await exploitation. Production of transgenic plants with altered expression of AGPs has been used to investigate the function of AGPs in stylar secretions (Cheung et al., 1996). The future will surely bring an abundance of additional studies utilizing this approach as methods are devised to solve the associated complications. These complications include the need to control the expression of both the AGP core polypeptide and the glycosyltransferases that accomplish the extensive posttranslational modifications (Cheung et al., 1996), and the need to overcome apparent functional plasticity or redundancy among several AGPs expressed in a single tissue (Du, 1995; Cheung et al., 1996). Use of the Arabidopsis model system with its small genome will probably be beneficial in this regard, and thus the recent identification of five cDNA clones encoding putative AGPs in Arabidopsis (Schultz et al., 1998) is a particularly encouraging development. Two Arabidopsis mutants with altered expression of AGPs have been described (Takahashi et al., 1995; Ding and Zhu, 1997). While neither of these mutations has been shown to occur in a gene encoding an AGP core polypeptide, such an Arabidopsis mutation has been found (Schultz et al., 1998). The future will surely bring intense investigations of these and other AGP mutants yet to be generated.
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ACKNOWLEDGEMENTS Research support from the US Department of Agriculture National Research Initiative Competitive Grants Program (Award No. 97-3531 1-5157 to M. D. Serpe and Award No. 95-37304-2292 to E. A. Nothnagel) is gratefully acknowledged.
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Plant Disease Resistance: Progress in Basic Understanding and Practical Application
N . T. KEEN Department of Plant Pathology. University of California. Riverside. CA 92521. USA
I . Introduction ....................................... ................................................... I1. Pathogenicity and Virulence Mechanisms ................................................. A . Factors Facilitating Pathogen Entry Into and Movement Through the Host .............................................................................................. B . Preformed Resistance Mechanisms .................................................... C . Toxins and Other Virulence Factors .................................................. D . Enzymes and Extracellular Polysaccharides ....................................... E . Newly Discovered Virulence Factors .................................... I11. Active Disease Resistance .......................................................................... A . The Hypersensitive Response and Disease Resistance Genes ............ B . Avirulence Genes and Elicitors .................................................... C . Elicitor Presentation ........................................................................... D . Active Oxygen Species. ................................................................ E . Signal Transduction ..... ................................................................ F. Systemic Acquired Resi ce ............................................................ G . Similarities of the HR and SAR With Defence in Vertebrates and Insects ................................................................................................. H . Defence Response Genes .................................................................... I . Defensins and Related Factors ......... .......................................... Disease Control ............... IV . Approaches to the Use of New Knowled A . Transgenic Plants Expressing Foreign Disease Resistance Genes ..... B . Transgenic Plants That Inactivate Toxins .......................................... C . Transgenic Plants Expressing Regulatory or Defence Response Genes .................................................................................. D . Expression of Pathogen Genes in Plants ............................................ E. Other Rationales .............................. V. Conclusions and Future Directions ........................................................... References ..................................................................................................
Advances in Botanical Research Vol . 30 incorporating Advances in Plant Pathology
ISBN 0-12-005930-4
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Copyright 0 1999 Academic Press All rights of reproduction in any form reserved
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Major advances have occurred over the past 15 years in understanding the molecular basis of factors determining plant resistance to pathogens. Several preformed structural and chemical factors have been proven to be important resistance factors unless pathogens overcome them. Progress has also occurred in understanding active disease defence in plants, collectively called the hypersensitive response ( H R ). An important milestone was the cloning and characterization of avirulence genes from fungal, bacterial and viral pathogens that direct production of specijk elicitors. These elicitors initiate the activation of plant defence response genes only in cultivars carrying the matching or complementary disease resistance genes. Several of these plant resistance genes have been cloned and most contain leucine-rich-repeat ( L R R ) domains that are required for their specificity. Recent data establish that the LRR domains convey specificity for elicitor recognition, but it is possible that other plant proteins function as primary receptors for pathogen elicitors. The occurrence of such receptors has been demonstrated in elicitor-binding studies, but few of them have been characterized. Nonetheless, the available data support the elicitor-receptor hypothesis stating that plants carrying a particular resistance gene have high-affinity receptors specific for the cognate elicitor. Despite these advances in our basic understanding of disease resistance in plants and the emergence of promising rationales for improved disease controI, relatively little use has yet occurred in practical agriculture. It is likely, however, that several strategies now under development will have widespread signijkance on plant disease control in the next century.
I. INTRODUCTION The success of plant disease resistance mechanisms to disease-causing organisms, as with the resistance of medieval castles to attack, depends on the resistance mechanism and on whether pathogens can overcome it. Accordingly, a discussion of plant disease resistance must take into account the sometimes insidious offensive weaponry of pathogens designed to overcome such mechanisms. Fortunately, the last several years have seen dramatic advances in our understanding of the basic mechanisms that permit pathogens to grow in and damage plants, and in the systems employed by plants for resistance against pathogen ingress. Much of this progress resulted from the eruption of molecular cloning, the ability to construct transgenic organisms, and technical progress in the production of defined mutants in plants and pathogens. Unlike earlier eras, where elegant biochemical work frequently resulted in equivocal and/or incomplete conclusions, these genetic tools now allow definitive evaluation of whether particular biochemical functions are causally involved in physiological processes such as disease resistance in plants or virulence in pathogens. This chapter will review some of the progress in understanding why plants do or do not get sick from a particular disease. As the field has been heavily reviewed in recent years, this paper will focus only on recent work, and makes no attempt to be comprehensive. I will try to relate recent research to the quest for new approaches to disease control in the field. Some useful recent reviews germane
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to this article were written by Hahn (1996), Ryals et al. (1996), Alfano and Collmer (1997), Baker et al. (1997), Delaney (1997), DeWit (1997), Glazebrook et al. (1997a), Vivian and Gibbon (1997), Wilson et al. (1997), Yang et al. (1997), Dempsey et al. (1998), Ji et al. (1998a), and Somssich and Hahlbrock (1998).
11. PATHOGENICITY AND VIRULENCE MECHANISMS Plants and pathogens have, for the most part, co-existed for millions of years. The consequence of this selection process has been that relatively few potential pathogens successfully parasitize a particular plant species and most, but not all, pathogens have limited host ranges. The few successful pathogens existing today most certainly have accumulated mechanisms required for entry into and movement within the plant, nutrient supply, escape from plant defence reactions, and, finally, egress of reproductive structures from the host and their dissemination. While our understanding is rudimentary, we are beginning to catalogue mechanisms operative in some of these categories and to appreciate their complexity.
A. FACTORS FACILITATING PATHOGEN ENTRY INTO A N D MOVEMENT THROUGH THE HOST
Classical work has defined important parameters involved in pathogen penetration and infection. For instance, early in this century Brown and Harvey (1927) established that mechanical penetration is an important aspect of plant infection by certain fungi such as Botrytis spp. Recent work with Magnaporthe grisea has given a more detailed view of infection (see Talbot et al., 1996, and references therein). This fungus forms appressoria on the surface of the host that adhere by a remarkably efficient ‘glue’, permitting mechanical penetration of an infection peg through the plant cell cuticle and cell wall. The development of appressoria and their role in pathogenesis have recently been reviewed by Dean (1997). Other pathogens enter through natural openings such as stomata or wounds on plant surfaces. Hoch et al. (1987) made the important discovery that Urornyces phaseoli germ tubes have the impressive ability to gauge the height of guard cell lips on the host plant, bean, in order to direct appressoria formation over open stomata. The finding raised the prediction that disease resistance might result from altering guard cell topology, but this has not yet come to fruition. Once a pathogen enters a plant, it faces the problem of movement through the host. In the case of fungi and nematodes, that can physically move through
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intercellular spaces, this would seem a trivial question, but such is not the case when the poorly understood area of nutrient procurement is considered. For bacteria and viruses, the situation would appear even more complex. Bacteria commonly induce water-soaking conditions at their infection sites, a plausible scheme for facilitating progressive movement of bacteria through the intercellular spaces of plant tissue and their dissemination back out of the plant (see Yang et al., 1996). Viruses, on the other hand, are intracellular pathogens, and possess clever mechanisms for modifying host plasmodesmata in order to facilitate their cell to cell movement (e.g. Lartey et al., 1997; Oparka et al., 1997). B. PREFORMED RESISTANCE MECHANISMS
These ‘static’ defences may be either structural or chemical in nature, and the former may encompass intrinsic features of plant cells such as the ubiquitous cell wall. If a pathogen is successful in attacking a particular plant it must have ways of handling the cell wall and other structural barriers, as well as preformed chemical inhibitors. Unlike animals, plants seem to have evolved the strategy of storing large quantities of chemicals that are toxic per se or toxic after cleavage with ubiquitous plant enzymes such as glycosidases. Although the occurrence of such preformed toxicants has been known for years, evidence firmly linking them with disease resistance has only been provided recently through studies of fungal pathogens that can metabolize the plant chemicals. It has traditionally been difficult to convincingly test the role of preformed antibiotic compounds in pathogen resistance (see Prusky and Keen, 1993). Plant mutants or transgenic plants over- or underexpressing preformed compounds would be a desired tool. In this regard, Maher et al. (1994) and Bi et al. (1997) evaluated transgenic tobacco plants with reduced levels of preformed phenolic compounds, including chlorogenic acid, a major preformed compound in tobacco. The role of chlorogenic acid in disease resistance has been debated for years, but the work of Maher et al. showed that reduced concentrations of chlorogenic acid and related phenolics resulted in greater susceptibility of tobacco to a fungal pathogen, Cercospora nicotianae. This approach opens the door for a more incisive look at the physiological role of preformed compounds in disease resistance. Bowyer et al. (1995) provided strong support for the earlier idea that oat plants are resistant to Gaeumannomyces graminis var. tritici because their roots (but not those of wheat) contain the saponin, avenacin A-1. G. graminis var. avenae, unlike var. tritici, attacks oat plants in addition to wheat and can enzymatically detoxify avenacin. However, mutation of the var avenae gene encoding the detoxifying enzyme converted the oat pathogen to a form that was indistinguishable from the wheat pathogen - that is, the mutant could still attack wheat but not oat plants. The work therefore provides strong evidence
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that avenacin A-1 is an effective preformed resistance factor in oat roots unless an invading fungus has the ability to detoxify or otherwise deal with it. Tomatine is a preformed steroid glycoalkaloid occurring in tomato, and possesses potent antifungal activity. Some fungi, however, are relatively insensitive to tomatine or actively degrade the compound to an inactive form (e.g. Sandrock and VanEtten, 1998). Osbourn et al. (1995) showed that a fungal pathogen of tomato leaves became less virulent when a gene encoding a tomatine-degrading enzyme (tomatinase) was mutated. Furthermore, expression of a cloned tomatinase gene in Cladosporium fulvum (which does not normally degrade tomatine but is a pathogen on tomato) increased virulence on both genetically resistant and susceptible tomato cultivars (Melton et al., 1998). These results make a strong case for the role of tomatinase in fungal virulence, and establish that tomatine is a bonafide resistance factor in tomato unless pathogens can deal with it. Cuticle has been viewed as a preformed structural barrier to pathogen ingress, much as the epidermis of vertebrates. Cutinases produced by certain pathogens have also been proposed as agents that aid the penetration of pathogens into plant tissues. Genetic experiments in which cutinase genes have been mutated in the pathogen (e.g. Rogers et al., 1994) provided evidence for the importance of cutinases. Further evidence was obtained when a cloned cutinase gene was transformed into a pathogen deficient in cutinase activity (Dickman et al., 1989). Such experiments support the role of cutinases in penetration, and establish that the cuticle is an effective resistance mechanism unless pathogens can mechanically or enzymatically breach it. C. TOXINS AND OTHER VIRULENCE FACTORS
Extracellular, generally low molecular weight toxins have long been thought to be involved with the pathogenicity and virulence of plant pathogens. Fungal host-selective toxins have been extensively investigated, and conclusive proof has been obtained for their involvement as virulence factors on particular host genotypes (e.g. Scheffer et al., 1967; Ahn and Walton, 1997). A well-studied case of plant resistance to a fungal toxin, and the consequence of mutations that render the plant unable to detoxify the fungal metabolite, is illustrated by HC toxin. A gene that confers resistance in corn to Cochliobolus carbonum race 1 was cloned and shown to encode a reductase enzyme that chemically inactivates HC toxin produced by the fungus (Meeley et al., 1992; Briggs and Johal, 1994). As HC toxin is essential for high virulence of the fungus, production of the reductase by resistant corn plants represents a defence strategy that may be more widely employed by plants than now realized. The HC toxin paradigm has been successfully utilized in searches for other toxin detoxification or tolerance mechanisms that might be utilized in transgenic plants for disease control. For example, an ornithine carbamoyl
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transferase gene was cloned that encodes an enzyme resistant to phaseolotoxin, produced by Pseudomonas syringae pv. phaseolicola. When transformed into bean plants that are normally affected by phaseolotoxin, the transgenic plants exhibited high resistance to the toxin, as well as to the bacterial disease (Herrera-Estrella and Simpson, 1995). In other work, a tabtoxin degradation gene inserted into transgenic tobacco plants led to reduced disease symptoms by the tabtoxin-producing pathogen, Pseudomonas syringae pv. tabaci (Anzai et al., 1989). Several other toxin-degrading genes have also been cloned from microorganisms (e.g. Zhang and Birch, 1997) and show promise for generating disease-resistant transgenic plants. A particularly appealing aspect of this strategy is that, unlike disease resistance genes, it should be much more difficult for the pathogen to overcome a plant resistance strategy based on degradation or tolerance of a toxin required for high virulence. Studies with bacterial pathogens have identified genes that behave as hostspecific virulence factors (e.g. Waney et al., 1991; Arlat et al., 1994; Yang et al., 1996; Valinsky et al., 1998). Gabriel and co-workers showed that a gene in Xanthomonas citri, called pthA, behaves as a specific virulence/pathogenicity factor on citrus plants. This gene is a member of the avrBs3 family, to be discussed later. Recent work by the Gabriel group demonstrated that transient expression of the pthA gene in citrus cells causes erumpent canker symptoms that are indistinguishable from bacterial infections (personal communication). Thus, while other bacterial virulence factors may be required for a successful infection, pthA appears to be the major factor responsible for canker symptoms. The pthA gene is of further interest because it has also been shown to behave as an avirulence gene in other non-host plants. The results withpthA are accordingly consistent with other work, described later, indicating that plants have co-opted pathogen virulence factors as elicitors of active defence mechanisms. D. ENZYMES AND EXTRACELLULAR POLYSACCHARIDES
Extracellular cell-wall enzymes released by pathogens have been associated with plant diseases since the last century (DeBary, 1886), particularly those involving maceration of plant tissue. The fact that pathogens often produce these enzymes establishes the plant cell wall as an important resistance mechanism against potential pathogens that must overcome this barrier for success. Genetic experiments have, in some cases, established a cause-effect relationship between particular pectic enzymes and virulence (e.g. Ried and Collmer, 1988). However, certain enzymes suspected to be causally involved in disease have not been demonstrated as important when wild-type and gene knockout strains were compared (e.g. Ape1 et al., 1993; Keen et al., 1996). I have argued (Keen, 1995) that such results need be interpreted cautiously because virulence tests are difficult to perform in order to reconstruct
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conditions occurring in natural ecosystems, and differences too small to reliably detect in laboratory contexts may be important in nature. Another complication is that pathogens appear to have redundant, overlapping virulence genes. One of the best studied cases is Erwinia chrysanthemi, which produces at least eight different pectate lyase enzymes attacking pectic polymers in the plant cell wall, all of which are under complex regulation (see Hugouvieux-Cotte-Pattat et al., 1996). Some pectate lyase genes are induced by the substrate, polypectate, but others are not responsive to substrate and instead are induced by as yet undefined plant substances (Kelemu and Coilmer, 1993). Given this complexity, it is not difficult to understand why an early experiment, in which four different pectate lyase genes were mutated in E. chrysanthemi strain EC16, greatly reduced but did not totally eliminate pathogenicity (Ried and Collmer, 1988). Recent work has revealed more complexity in the attack on plant cell walls by Erwinia spp. Shevchik et al. (1997) recently identified a new gene in E. chrysanthemi that encoded a pectin acetyl esterase. Pectin esterase has long been regarded as a ‘de-decoration’ enzyme that converts cell-wall pectic material into a form more susceptible to degradation by the pectate lyases. The new esterase was also shown to be important for full virulence via construction of a gene knockout, and appears to function by removing acetyl groups from native pectin, again presumably to promote greater activity by pectic enzymes. The esterase accordingly illustrates the sophistication of pathogen virulence mechanisms. Ralstonia (formerly Pseudornonas) solanacearum requires the production of extracellular polysaccharide (EPS) for high virulence (see Denny, 1995). The bacterium has at least two important life-cycle stages, free living in soil and pathogenic, and EPS is probably a negatively selected trait during the freeliving state. It is therefore reasonable to suppose that EPS synthesis is under strict regulation. Indeed, transcription of a gene operon involved in EPS synthesis has been shown to be under complex regulation (Denny, 1995). For instance, recent work (Huang et al., 1998) showed that regulation of the EPS promoter requires at least three sensory regulators, VsrD, XpsR and VsrC, that function collectively. Thus, as with E. chrysanthemi discussed above, regulation of EPS production is complex in R . solanacearum. E. NEWLY DISCOVERED VIRULENCE FACTORS
Gene knockouts of pathogen avirulence genes disclosed, somewhat surprisingly, that some of them also have important virulence roles (Kearney and Staskawicz, 1990; Lorang et al., 1994; Ritter and Dangl, 1995). Furthermore, naturally occurring mutant avirulence gene alleles involving point mutations, insertions or deletions, have been discovered that avoid plant surveillance but may retain virulence functions (Kearney et al., 1988; Yucel and Keen, 1994;
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Stevens et al., 1998). Some of the bacterial avirulence gene products are also thought to be delivered to plant cell protoplasts by the hrp gene systems of bacterial pathogens (Alfano and Collmer, 1997; Lindgren, 1997). This raises the probability that, in addition to extracellularly produced toxins and enzymes, plant pathogens also deliver virulence proteins directly to plant protoplasts. Unfortunately, the biochemical functions of pathogen avirulence genes are not known. Of interest, homology was observed between the Xanthomonas campestris avirulence gene, avrRxv (Whalen et al., 1993), and the yopJ virulence gene of Yersinia pseudotuberculosis (Monack et al., 1997). This latter gene has recently been suggested to inhibit the production of tumour necrosis factor-a in vertebrate hosts, and thereby lead to apoptotic cell death (Palmer et al., 1998). As tumour necrosis factor is believed to be important in animal cell resistance to the bacteria, YopJ can be viewed as interferring with host defence. It will be interesting, therefore, to see if AvrRxv exhibits a similar effect in plant cells, particularly in view of its role in eliciting the hypersensitive response (HR) in host plants carrying the cognate disease resistance gene. Two avirulence phenotypes require the function of two genes. These are avrE and avrPphF.RI in Pseudomonas syringae (see Vivian and Gibbon, 1997). avrE from Pseudomonas syringae pv. tomato was cloned as an avirulence gene but also appears to play an important role in bacterial virulence (Lorang et al., 1994). The avrE avirulence phenotype was shown to require two different transcriptional units, and transposon insertions in either unit led to loss of the avirulence phenotype and reduced virulence. Significantly, the avrE locus was also found to be tightly linked to the right terminus of the formal hrp gene cluster in P . s. pv. tomato (Lorang and Keen, 1995). Recently, Bogdanove et al. (1998a) sequenced the P . s. pv. tomato avrE locus and showed that it contained two genes, avrE and avrF. avrE encoded a 195 kDa protein, while avrF encoded a much smaller, 14kDa protein. Significantly, these genes exhibited similarity to two genes required for high virulence in Erwinia amylovora, called dspE and dspF (Gaudriault et al., 1997; Bogdanove et al., 1998a). The genes occurred in tandem in a single transcriptional unit, unlike avrE/avrF which are present in two opposed transcriptional units (Lorang and Keen, 1995). The large proteins AvrE/DspE are suspected to be virulence factors and have been shown to be hrp secreted (Gaudriault et al., 1997; Bogdanove et al., 1998b), while the smaller AvrF and DspF proteins are suspected to be specific chaperones for AvrE/DspE. DspE/F were also shown to be functionally homologous to avrE/ F as the cloned avrE gene locus from P . syringae restored virulence to a dspE mutation in E. amylovora. The dspE/F genes also conferred an avirulence function in P . syringae pv. glycinea race 4 to elicit a hypersensitive reaction in soybean. Thus, dspE/F are the first avirulence genes against a non-host plant identified in E. amylovora, but have a virulence function in normal hosts. The results with avrE/F are illustrative of other data indicating that genes occurring outside, but closely linked to, the formal hrp gene clusters of pathogenic bacteria may be important for virulence/avirulence functions (Arlat
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et al., 1994; Mansfield et al., 1994; Alfano et al., 1997). Kim et al. (1998) recently reported that three possible virulence genes are interspersed among the hrp regulons of Erwinia chrysanthemi. Two were a haemolysin gene homologue as well as a putative activator gene, and the third gene encoded a phospholipase that had been cloned earlier (Keen et al., 1992). All of these examples support the idea that ‘pathogenicity islands’ of functionally related genes occur in plant pathogenic bacteria, as is also the case in animal pathogens (Groisman and Ochman, 1996). Harpin proteins have been identified as products of genes in the formal hrp gene clusters (Alfano and Collmer, 1997). Harpin proteins are suspected to be involved in recognition of bacteria as foreign by non-hosts and facilitate virulence in host plants, but their biological roles are as yet unclear. Recently, E. amylovora and P . syringae were also shown to harbour genes, called hrp W , at the right-hand end of their hrp clusters that encode proteins which have Nterminal domains homologous with known harpins and C-terminal domains homologous with Class I11 pectate lyases (Charkowski et al., 1998; Kim and Beer, 1998). The products of the hrp W genes do not appear to exhibit pectate lyase activity but do cause the HR, similar to harpins. Because they bind pectic material, the HrpW proteins are suspected to function in the plant cell wall.
111. ACTIVE DISEASE RESISTANCE Plants employ several active defence strategies against pathogens and herbivores. Most higher plants are stationary, certainly a penalty regarding pathogen attack, and some plants may have lifespans of hundreds of years. All of these considerations can be expected to influence the disease defence strategies employed by plants, as can the fact that plants are concerned with the survival of populations rather than individuals. Thus, individuals in native populations generally vary in the complement of disease resistance genes in order to maximize variability. A. THE HYPERSENSITIVE RESPONSE AND DISEASE RESISTANCE GENES
The hypersensitive response (HR) will be used here to collectively describe active local defence mechanisms in plants. There are two applications of the HR, one general resistance against non-pathogens of a particular plant species, another specijk resistance in cultivars of a plant species carrying particular disease resistance genes. Because variation often occurs in the avirulence genes of pathogen biotypes, gene-for-gene complementarity is observed. Since cloning of the first HR-modulating disease resistance gene, Pto (Martin et al., 1993), several additional resistance genes have been cloned and characterized (Bent, 1986; Baker et al., 1997; Jones and Jones, 1997).
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Surprisingly, they are all different from Pto (which is a serine/threonine protein kinase), namely members of the leucine-rich-repeat (LRR) class. Several LRR protein genes have recently been cloned by utilizing conserved sequences from LRR resistance gene proteins (e.g. Feuillet et al., 1997; Aarts, M. G. M. et al., 1998; Ohmori et al., 1998; Speulman et al., 1998) and these are often closely linked to known resistance gene loci. Indeed, Botella et al. (1997) estimate that a substantial portion of the Arabidopsis genome, perhaps 5%, is concerned with disease resistance. There have been recent new additions to the LRR list of disease resistance genes. For example, Tornero et al. (1996) reported a LRR protein in tomato plants that was up-regulated during viroid infection and also processed to a smaller size. Yoshimura et al. (1998) cloned the Xal disease resistance gene against Xunthomonus oryzae from rice plants, and reported that its expression is also up-regulated following bacterial inoculation. Li and Chory (1997) identified an Arabidopsis gene required for brassinosteroid signal transduction that, upon sequencing, was revealed to exhibit homology with the LRR disease resistance genes Xa2l and Cp.It is therefore interesting that disease resistance and hormone perception in plants may have co-evolved. It is noteworthy that two different cloned resistance genes have dual specificities. Thus, the Rpml resistance gene from Arabidopsis recognizes bacteria expressing either of two unrelated avirulence genes, avrRpm or avrB (Bisgrove et al., 1994; Grant et al., 1995). More recently, Rossi et al. (1998) showed that the Mi gene in tomato confers resistance to root knot nematodes and to the potato aphid. Thus, a single gene is effective against animal pests in two different phyla. The nature of the pathogen elicitor for Mi is not known, but the primary or processed uvrRpm protein product may play this role with RPMl (J. Dangl, personal communication). Disease resistance genes often occur in large clusters (e.g. Ellis et al., 1997; Hulbert, 1997; Simons et al., 1998; Wang et al., 1998). The most variable regions in these tandem arrays are the LRR domains, and these also correlate with specificity for recognition of pathogens expressing cognate avirulence genes (Parniske et ul., 1997; Thomas et al., 1997). It is not yet known whether the LRR domains are associated with elicitor binding, however, a topic that will be discussed later. B. AVIRULENCE GENES AND ELICITORS
Several avirulence genes have been identified in viral, bacterial and fungal pathogens (Leach and White, 1996). They have not yet been cloned from nematode or insect pests, but avirulence genes were recently demonstrated genetically in Heterodera sp. (Dong and Opperman, 1997) and almost certainly occur in insects based on the specificity of certain plant-insect systems
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(Dweikat et al., 1997). Of the more than 40 avirulence genes sequenced, none show high homology to known genes. Genetic evidence cited earlier has indicated that several bacterial avirulence genes are important for pathogen virulence. This is of interest as avirulence genes also appear to direct the production by the pathogen of specific elicitors. These are factors that elicit the HR in plant cultivars carrying the cognate plant disease resistance genes, but may also play a role in virulence. Relatively few specific elicitors have been identified. In fungal pathogens, several peptides have been shown to function as specific elicitors (see Knogge, 1996; DeWit, 1997), and some of them are, indeed, also virulence factors. Lauge et al. (1998) recently utilized a clever strategy to identify tomato lines carrying a resistance gene complementing a previously identified peptide virulence factor, ECP2, from Cladosporium fulvum. The gene encoding ECP2 was expressed in a potato virus X expression vector, which permitted rapid screening of a large number of tomato lines for their ability to produce a HR rather than normal susceptible virus symptoms. Four tomato lines were identified that exhibited the HR and were also resistant to inoculation with the fungus. As all known isolates of C .fulvum harbour ECP2, and ECP2 mutants are greatly reduced in virulence, it is anticipated that the newly identified tomato resistance gene should exhibit a degree of permanence. In bacterial pathogens, avrD functions by directing production by bacteria of acyl glycoside elicitors called syringolides (Ji et al., 1997). This is the only known example of a bacterial avirulence gene that functions catalytically to generate a low molecular weight specific elicitor. Several bacterial avirulence gene proteins appear to function directly as elicitors and to require delivery to the plant cytoplasm by the hrp gene secretion pathway (Gopalan et al., 1996; Leister et al., 1996; Pirhonen et al., 1996; Van den Ackerveken et al., 1996; Roine et al., 1997; De Feyter et al., 1998). Furthermore, the avrBs3 family of avirulence genes from Xanthornonas contains functional nuclear localization signals (Yang and Gabriel, 1995), as well as sequences at the carboxyl terminus that function as eucaryote transcription activation domains (Zhu et al., 1998). As discussed earlier, the Gabriel group has recently shown that one of the avrBs3 family members behaves as a virulence factor causing overgrowths on citrus leaves. It will accordingly be of considerable interest to deduce not only the nature of plant receptors for the avirulence functions of these proteins but also plant systems that are affected by their virulence functions. The bacterial avrPto protein product also appears to be delivered to plant cells by the bacterial hrp gene proteins (Ham et al., 1998). This protein has been shown to interact with the tomato resistance gene protein, Pto, in the yeast two-hybrid system (Scofield et al., 1996; Tang et al., 1996). Resistance governed by Pto, however, also requires a LRR protein, Prf (Salmeron et al., 1996), that resembles other LRR disease resistance gene proteins. Interestingly, Prf is also required for the function of another ser/thr kinase gene, fen. This kinase confers sensitivity of plants to the insecticide fenthion, but not avrPto,
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despite the fact these two proteins are 80% identical in amino acid sequence. Frederick et al. (1998) recently showed that altering one amino acid offen in the kinase activation loop confers the ability to bind Pto, as well as give Ptomediated defence responses. Similar to previous findings with several bacterial pathogens (see Vivian and Gibbon, 1997), Kamoun et al. (1998) reported that an elicitin peptide produced by Phytophthora infestans mediates non-pathogen resistance in tobacco to the fungus. These workers showed that antisense suppression of elicitin production permitted the transgenic fungus to significantly colonize tobacco leaves, thus implicating genes encoding the elicitin as avirulence genes modulating non-host specificity. Several general elicitors do not appear to result from avirulence gene activity, but instead are either components of the pathogen cell wall or other elements (for reviews see Hahn, 1996; Boller and Keen, 1999) or, alternatively, are plant metabolites liberated by pathogen activity. For example, while disease resistance genes targeted against pathogen pectic enzymes are not known, pectate oligomers liberated from the plant cell wall do elicit plant defence reactions (see Hahn, 1996). Curiously, cutinases, discussed earlier as virulence factors in pathogenic fungi, also lead to products that have elicitor activity (Schweizer et al., 1996) and elicit active defence (Parker and Koller, 1998). C. ELICITOR PRESENTATION
An area of emerging interest is how elicitors are recognized and presented to disease-resistant plant cells. Certain bacterial avirulence proteins appear to be delivered to plant protoplasts by the hrp type I11 secretion system, as discussed earlier. These proteins do not function if applied to the surface of plant cells, but several other elicitors are effective if applied exogenously. These findings invoke the question of elicitor presentation and processing to active forms in plant cells, an area that is much better understood in vertebrate systems. Mammals contain Class I major histocompatibility complex (MHC) genes, the protein products of which specifically recognize and present processed peptide fragments of antigens to the cognate T cells (Maffei et al., 1997). There are, however, uncertainties in antigen recognition. For example, Shen et al. (1998) made the finding that a Listeria epitope-specific antigen was recognized by mouse CD8 T cells only if the bacterium secreted the antigen. This was in contrast to the general case with Listeria cells, in which antigens could be recognized by T cells regardless of their localization in the bacterial cell. This work and other results from vertebrate MHC research bear on the question of elicitor perception by plant cells. For example, it has recently been shown for the first time that certain Class I MHC genes recognize glycolipids as antigens (see Brenner and Porcelli, 1997), a finding of interest in the case of elicitors that
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function in plants, several of which are oligosaccharides, lipids or their conjugates. Several groups have presented evidence for the occurrence of high-affinity plant-binding sites for pathogen-produced general elicitors, but few of them have been well characterized (e.g. Basse et al., 1993; Hahn, 1996; Mithofer et al., 1996; Ito et al., 1997; Nennstiel et al., 1998). Umemoto et al. (1997) identified a putative protein receptor for the heptaglucan elicitor from soybean, and cloned the gene encoding it. Using the yeast two-hybrid system, the Pto protein kinase and the avrPto protein appeared to directly interact (Scofield et al., 1996; Tang et al., 1996), but this has not been biochemically confirmed either in vitro or in planta. The results, however, suggest that an avirulence gene protein can function as an elicitor per se, and initiate active defence by physical interaction with its cognate resistance gene protein. Significantly, none of the elicitor-binding proteins show similarity with LRR resistance gene proteins. As discussed earlier, experiments with LRR resistance gene proteins have established that they convey the specificity of elicitor recognition. However, to date, there is no proof that these proteins function as primary receptors for elicitors. Indeed, the perception of avirulence gene-specific elicitors by resistant plants may be more complex. In two systems, labelled specific elicitors have been shown to bind with similar affinity to plant factors, regardless of the resistance genotype. In the case of the avr9 peptide elicitor from Cladosporium fulvum, Kooman-Gersmann et al. (1996) observed saturable, ligand displaceable binding to plasma-membrane preparations from either C p or c p tomato genotypes. Furthermore, expression of C p in Arabidopsis plants led neither to avr9 peptide sensitivity nor to the binding of avr9 peptide by plasma-membrane preparations from the transgenic Arabidopsis plants (DeWit, 1997). On the other hand, Hammond-Kosack et al. (1998) reported that transgenic C p tobacco and potato plants recognized the avr9 peptide to elicit a HR. This proves that C p is involved in the specificity of a v p peptide recognition, but does not establish that the C P protein is a primary receptor. Unfortunately, avr9 peptide-binding data were not shown for the transgenic and untransformed plants. The experiments therefore do not prove a role for C p as a primary binding site for the peptide elicitor, and instead raise the possibility that a common primary receptor occurs in both c p and C f l tomato plants, as well as tobacco and potato. Labelled syringolides, produced by bacteria expressing the avirulence gene, avrll), also bound specifically to a high-affinity site in the soluble fraction of soybean leaves (Ji et al., 1997). Again, however, no significant differences were observed in binding to extracts from soybean cultivars containing or lacking the cognate disease resistance gene, Rpg4. Affinity chromatography permitted the isolation of a 34 kDa high-affinity binding protein (Ji et al., 1998a), which turned out to be identical to P34, the gene of which was cloned earlier from soybean (Kalinski et al., 1990, 1992). The P34 protein is produced from a 46 kDa precursor protein, and occurs in substantial quantities in seeds but is
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present in very low amounts in leaves and other vegetative plant parts. P34 also has considerable homology with papain and other thiol proteases, but lacks a cysteine residue required for their activity. When expressed in the baculovirus system, P34 but not the 46 kDa precursor protein showed specific syringolidebinding activity (Ji et al., 1998b). As with the avr9 peptide, discussed above, results with the syringolides raise the possibility that these elicitors are not initially recognized by the Rpg4 disease resistance gene product. Such a model has been proposed by Ji et al. (1998a) and follows the emerging pattern of signal transduction patterns in animals, wherein complexes of several proteins are required to initiate signalling (e.g. Pawson and Scott, 1997). This model is also consistent with the results discussed above for the AvrPto and Pto interaction. Pto activity requires the presence of a second gene, Prf (Salmeron et al., 1996), a member of the LRR resistance gene family, but Prf appears to occur in all tomato cultivars and there is no indication that Prf binds AvrPto. Thus, as with Cf9, Prf does not appear to be directly involved in elicitor binding, but is required for resistance to occur. Finally, Warren et al. (1998) recently reported that mutation of the Arabidopsis disease resistance gene, RPSS, suppresses the effects of several other resistance genes against bacteria and fungi. Although the authors interpret otherwise, the finding is consistent with the idea that the Rps5 protein may be required for the formation of complexes with other resistance gene proteins and elicitor-binding proteins. D. ACTIVE OXYGEN SPECIES
It has been known for some time that active oxygen species (AOS) are generated early in plant-pathogen interactions resulting in a HR (see Baker and Orlandi, 1997; Lamb and Dixon, 1997). Active oxygen species have been proposed to act directly in an antibiotic fashion against an invading pathogen or to stimulate cell-wall strengthening events that restrict pathogen movement. However, these speculations neglect the observation that pathogen growth generally does not stop when AOS are generated, but is restricted much later and correlates with the production of phytoalexins and other defence response proteins. Furthermore, Mittler et al. (1996) showed that low oxygen pressure inhibited cell death in the tobacco HR but defence responses occurred normally. These observations question a role for AOS in the restriction of pathogen growth, and have recently been extended by Yu et al. (1998) who isolated the dndl mutant in Arabidopsis that exhibited normal resistance against a range of pathogens, but exhibited essentially no hypersensitive cell death. This paper may force re-evaluation of the role of AOS and cell death in disease resistance. Active oxygen species have been considered as signalling agents causing hypersensitive cell death and active resistance (Levine et al., 1994; Jabs et al.,
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1997; Alvarez et al., 1998). This idea is supported by the work of Kazan et al. (1998), in which transgenic tobacco plants expressed a glucose oxidase gene driven by a pathogen- and wound-inducible promoter. When grown under high light conditions, expected to increases AOS levels, these plants exhibited defence gene activation and visible necrosis. Infiltration of exogenous glucose into tissues, however, induced massive cell death and defence response gene induction. Finally, Kazan et al. observed that plant cell death produced by expression of the glucose oxidase gene was potentiated by salicylic acid (SA). Another experiment supporting a signalling role for AOS in disease resistance was published by Rance et al. (1998). These workers showed that antisense tobacco plant for lipoxygenase exhibited lower levels of this enzyme and also showed reduced resistance to a normally incompatible race of Phytophthora parasitica var. nicotianae. This work therefore implicates lipoxygenase as a mediator of active resistance. The role of lipoxygenase is most probably in the generation of peroxides and their adducts, including jasmonic acid, which may in turn function as activators of defence response genes. In work related to that discussed above, Chamnongpol et al. (1998) recently investigated the role of hydrogen peroxide in transgenic tobacco plants deficient in catalase activity. Elevated levels of hydrogen peroxide could be generated in these plants by increasing the light intensity. The results showed that exposure of leaves to prolonged high levels of hydrogen peroxide resulted in necrosis. Shorter exposure to high hydrogen peroxide levels did not induce necrosis but did systemically cause elevation of defence response proteins and pathogen tolerance. E. SIGNAL TRANSDUCTION
This rapidly growing area will be hurriedly covered, and readers are directed to reviews by Yang et al. (1997) and Boller and Keen (1999). A unifying theme of active resistance is that, once activated, disease resistance involves one or more signal transduction pathways, many involving phosphorylation or dephosphorylation of particular proteins. It has also become clear that, even in the same plant, two or more different transduction pathways may operate (see Somssich and Hahlbrock, 1998). Two experimental approaches have been utilized to study signal transduction pathways associated with active defence in plants. These are: (i) biochemical searches for proteins known to be involved in other systems or for proteins that are specifically phosphorylated/dephosphorylated after induction of defence; and (ii) searches for mutants that are defective in the expression of resistance. Both approaches have uncovered several putative transduction pathway members. Significantly, mutational searches have identified neither putative signal pathway proteins that are differentially phosphorylated nor transcriptional activators. This may be because multiple genes commonly encode these factors.
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Certain mutants in Arabidopsis, however, eliminate the phenotypes of several different disease resistance genes. Examples are the eds genes (Parker et al., 1996; Rogers and Ausubel, 1997) and NDRl (Century et al., 1997). It is accordingly assumed that these genes encode signal transduction members. Aarts, N. et al. (1998) showed that the E D 9 1 gene affected phenotypic expression of the disease resistance genes RPP2, RPPI, RPPS, RPP2l and RPS4. These resistance genes were not significantly affected by the NDRI mutation. On the other hand, phenotypes of RPS2, R P M l and RPSS operate independently of EDS-1, but were greatly reduced in the presence of the NDRl mutation. Although the results to not illuminate the question of function of the two signalling proteins, they do strongly suggest that there are at least two partially independent signalling pathways in Arabidopsis for resistance genemediated signal transduction. Biochemical approaches have, unlike the mutant hunts, identified several proteins that are specifically phosphorylated or dephosphorylated during defence induction (Dietrich et al., 1990; Grosskopf et al., 1990; Felix et al., 1991, 1994; MacIntosh et al., 1994; Xing et al., 1997; Rojo et al., 1998), but the roles for most of them have not been defined. Zhou et al. (1997) used the yeast two-hybrid system to identify three different proteins that interact with the Pto disease resistance gene protein. Although not confirmed either biochemically or in vivo, the new proteins are suspected to regulate the expression of defence response genes. If correct, signalling in the Pto pathway is quite short. In contrast, Droge-Loser et al. (1997) identified a soybean DNA-binding protein, G-HBF- 1, suspected to activate defence response gene expression. A novel serine protein kinase was shown to be activated in response to elicitors and, in turn, converted G-HBF-1 to the active form during defence response gene induction. Accordingly, the soybean system conforms to expectations seen in most other signalling pathways. Yin et al. (1997) studied expression of the defence response promoter ahead of a phytoalexin synthesis gene in tobacco, and observed that several general agents reported to stimulate defence responses had little or no effect on this promoter. These included hydrogen peroxide, salicylic acid and methyl jasmonate. However, treatment with elicitors or incompatible pathogens led to high-level expression, as well as to the particular spatial patterns associated with naturally occurring resistance. This work raises the probability that many putative defence effectors may in fact be stimulating very low-level activity relative to bona fide elicitors. Zhang and Klessig (1998) reported that the tobacco mitogen-activated protein (MAP) kinase, WIPK, is specifically activated following tobacco mosaic virus (TMV) infection, but only in cells carrying the N disease resistance gene. WIPK was induced at the transcriptional, protein and activity levels, and accordingly may be a required element in signal transduction involving the N disease resistance gene. In similar work, Ligterink et al. (1997) showed that the parsley ERMK gene may encode a 45 kDa protein kinase
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specifically activated by a peptide elicitor from Phytophthora sojae. These studies represent progress toward a goal of understanding the complete signal transduction pathway from elicitor recognition to defence response gene activation. F. SYSTEMIC ACQUIRED RESISTANCE
Systemic acquired resistance (SAR) is induced following pathogen challenge of a plant, preferably one that elicits a defence reaction. Such plants typically do not show visible symptoms outside of the original inoculation site, but are commonly found to exhibit varying degrees of resistance to subsequent inoculation of other plant parts with the original or a completely different pathogen (see Ryals et al., 1996; Delaney, 1997). There has been great interest in understanding the basic mechanisms underlying SAR because of its broad effects on a range of pathogens and the memory effect. These considerations have led to attempts to harness SAR in practical disease control. Indeed, chemicals such as isonicotinic acid (INA) were found to be potent inducers of SAR and, more recently, commercial applied chemicals have found their way into the disease control arena. A major advance was the finding that salicylic acid (SA) accumulation correlates with SAR in several plant species. Work by Ryals and associates established the essential role of salicylic acid in tobacco and Arabidopsis via the use of transgenic plants that degrade SA (Gaffney et al., 1993). The plants were transformed with the nahG gene from Pseudomonas putida, which encodes a protein that converts SA to an ineffective metabolite. These experiments established that SA was an essential component of SAR, but also argued that SA is not the as yet unidentified systemic primary signal molecule translocated from initially inoculated tissue. Surprisingly, nahG plants were also compromised in their ability to mount a local, hypersensitive response (Delaney et al., 1994). Thus, SA appeared to be critically required for the local HR as well as SAR. This work accordingly showed that the HR and SAR are closely related processes. Mutant studies with Arabidopsis have provided insight into the mechanism of SAR (see Delaney, 1997). Mutants have been isolated that exhibit constitutive SAR phenotypes or that are deficient in SAR. Of the latter, the NPRl mutant is non-responsive to SAR induction. Upon sequencing (Cao et al., 1997; Ryals et al., 1997) NPRl was found to exhibit strong homology to the IKB family of mammalian signal transduction factors, to be discussed below. Cao et al. (1998) recently showed that Arabidopsis plants constitutively expressing NPRl express disease resistance, a promising approach to disease control. Dong and colleagues have also isolated several constitutive SARexpressing Arabidopsis mutants. These cpr mutants generally require that plants produce SA for their effect. Of interest, cpr6-1 was recently shown to constitutively express three different response gene proteins, and these are still
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expressed in a cpr6-1, nprl double mutant (Clarke et al., 1998). However, resistance to P . syringae pv. maculicola was reduced in the double mutant, indicating that the three defence response proteins must not be involved in resistance to the bacterium. G . SIMILARITIES OF THE HR AND SAR WITH DEFENCE IN VERTEBRATES AND INSECTS
Recent work has shown similarities between active defence in plants and animals (see Wilson et al., 1997). The two major active defence systems of vertebrates are the acquired immunity system (involving B and T lymphocytes specificities that are determined somatically, and are not inherited by successive generations) and the innate immunity system, utilizing germline encoded proteins to identify microbial substances. There are now firm indications that the HR and SAR in plants share features of the innate system, as well as with the somatic humoral defence system in Drosophila (Belvin and Anderson, 1996; Lemaitre et al., 1996, 1997). When the first plant disease resistance genes were cloned, the N gene from tobacco (Whitham et al., 1994) and L6 from flax (Lawrence et al., 1995) showed homology with the Toll gene of Drosophila. Toll is involved, along with several other proteins, in two functions - first, the establishment of dorsal/ventral polarization during development; and, secondly, as a component of the humoral defence system. This system, once triggered, leads to activation of genes encoding antimicrobial proteins, called defensins. The Drosophila Toll gene and several LRR plant disease resistance genes have also recently been shown to have homology with several recently discovered human genes, called TLRs (Medzhitov et al., 1997; Rock et al., 1998). In Drosophila, Toll activates the signal transduction protein, Dorsal, which resembles NF-KB, a well-known participant in innate defence in vertebrates. In mammals, NF-KB activation leads to a number of factors involved in inflammation and immune responses that are required for resistance to viral and bacterial pathogens (Baeuerle and Baltimore, 1996). NF-KB is negatively regulated in vertebrates by I-KB as part of a protein complex signalling cascade (Pawson and Scott, 1997). As discussed in the last section, NPRl, an Arabidopsis protein required for systemic acquired resistance, exhibited considerable homology to I-KB in vertebrates. I-KB behaves as an inhibitor, while NPRl appears to behave as an activator of SAR. Nonetheless, NPRl exemplified the clear similarities between the HR and innate defence in animals that are emerging. Of more importance, the Toll system offers clues on proceeding with studies of active defence in plants. Szyperski et al. (1998) noted structural similarities between a pathogenesisrelated protein in humans (GliPR) and tomato P14a. GliPR, involved in primary brain tumours, is also induced in macrophages, and accordingly may be involved in defence. Comparison of several related sequences in plants, animals, fungi and insects led to the conclusion that the family is ancient, and
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perhaps has been modified for defensive and other purposes as a function of evolution. H. DEFENCE RESPONSE GENES
Defence response genes are a diverse collection of genes unified by their inducible expression during the HR. While it is generally assumed that all defence response genes of a particular plant are expressed following a wide range of elicitors, there is gathering evidence that certain elicitors may control only a subset of defence response genes. In this section I will briefly discuss recent work with several defence response genes, ranging from those that encode defensins similar to the vertebrate and insect innate defence proteins, to gene products that contribute to the synthesis of antibiotic phytoalexins. Several defence response genes encode proteins in phytoalexin synthesis. Phytoalexins have been thought to be important components of active defence in plants for many years, despite the absence of genetic proof via plant mutants. Glazebrook et al. (1997a) have recently identified several phytoalexindeficient (PAD) mutants of Arabidopsis that make reduced levels of camalexin in response to pathogen challenge. While none of the PAD genes have been cloned, PAD4 may play a regulatory role, perhaps as part of a signal transduction cascade. The PAD4 mutant exhibits greater susceptibility to Pseudomonas syringae strains, as well as to incompatible Peronospora parasitica isolates, but shows neither reduced camalexin production nor susceptibility to Cochliobolus carbonum (Glazebrook et al., 1997b). Recently, PAD4 was shown to control other defence responses, indicating that it is not specific to camalexin synthesis, and of little value in assessing the role of camalexin in resistance (Zhou et al., 1998). Some, but not all, of the remaining PAD mutants cause greater susceptibility to compatible P. syringae strains, but do not markedly affect incompatible resistant responses to the same bacteria. While these results may implicate camalexin as a general mechanism restraining bacterial multiplication in Arabidopsis, there is no evidence that it functions in resistance gene-mediated responses. The data do suggest that camalexin may be a factor in Peronospora resistance, but it is possible that other of the identified PAD genes may exhibit pleiotropic effects, as already shown for PAD4. The most impressive plant genetic proof of a physiological role for phytoalexins comes from studies in which genes for phytoalexin biosynthesis were transformed into heterologous plant species. Hain et al. (1993) observed that a gene for stilbene synthase from grapevine plants conferred disease resistance in transgenic tobacco plants. More recently, Thomzik et al. (1997) showed that transgenic tomato plants carrying the stilbene synthase gene produced the grapevine phytoalexin and exhibited enhanced resistance
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against Phytophthora infestans. Because of the functional specificity of the transgene, these observations make a strong case for the stilbene phytoalexins as important defence components. Dixon et al. (1996) explored how these experiments, as well as more sophisticated manipulations of phenylpropanoid biosynthesis, might be used for improved plant disease resistance. Additional proof for a physiological role of phytoalexins in plant defence comes from fungal genetics. In a series of landmark papers, VanEtten and coworkers showed that Nectria haematococca isolates of reduced virulence on pea were, relative to highly virulent strains, frequently deficient in pisatin demethylases which attack the major pea phytoalexin, pisatin (e.g. Wasmann and VanEtten, 1996). Recently, it was shown that transformation of a single cloned pisatin demethylase gene into a low virulence fungus isolate substantially increased its ability to degrade pisatin, as well as virulence, on pea plants (Ciufetti and VanEtten, 1996). This experiment makes a strong case for the importance of pisatin demethylase as a virulence determinant in N . haematococca, and also argues that pisatin is an important component of active defence in pea plants unless a pathogen is equipped to handle it. The findings with Nectria have been followed by similar results indicating that phytoalexin detoxification is an important virulence component on chickpea (Enkerli et al., 1998) and Phaseolus (Li et al., 1995). While ethylene production is frequently observed during the HR, administering ethylene yields conflicting effects on disease resistance. In order to examine the role of ethylene in the HR more critically, Knoester et al. (1998) recently transformed the Arabidopsis etrl-1 ethylene insensitivity gene into tobacco plants. This mutant allele behaves as a dominant character and is believed to function as an ethylene receptor. The etr 1-1 transgenic plants indeed failed to exhibit several indicators of ethylene activity and, notably, these plants also became susceptible to normally non-pathogenic soil-borne fungi such as Rhizopus and Chalara, as well as three Pythium sp. Concomitant with increased susceptibility, the etrl-1 transgenic plants failed to accumulate several defence response gene proteins suspected to be part of active defence. The same transgenic plants, however, exhibited a normal local lesion response against tobacco mosaic virus. The results of Knoester et al. (1998) accordingly make a case for an ethylene role in active resistance, but only against certain pathogens or potential pathogens. Vijayan et al. (1998) made the important discovery that Arabidopsis mutants unable to accumulate jasmonic acid were susceptible to the soil fungus Pythium mastophorum. In concert with other reports, this result indicates that plants have evolved several signalling pathways for defence response gene actiation, and that each of them may function with only certain elicitors.
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I. DEFENSINS A N D RELATED FACTORS
Pathogenesis-related (PR) proteins are major, inducibly formed products in plant tissues undergoing defence reactions. Several PR proteins exhibit antimicrobial properties (Broekaert et al., 1997), and some of them are similar to the defensins of insects and vertebrates (DeSamblanx et al., 1997; Fearon, 1997; Lemaitre et al., 1997; Cavallarin et al., 1998). Indeed, because they appear to exhibit a non-specific mode of action (MagetDana and Ptak, 1997), it may be possible to use animal or plant defensins as an approach to transgenic resistant plants (Huang et al., 1997; Cavallarin et al., 1998). Thevissen et al. (1997), however, have reported the occurrence of specific high-affinity binding sites for defensins on the microsomal membranes of Neurospora crassa, and DeSamblanx et al. (1997) identified key amino acid residues important for activity of a defensin from radish. It is therefore possible that pathogens might quickly confound a disease control strategy based on overexpression of single defensin genes. Yun et al. (1997), for example, showed that the defence response protein, osmotin, shows variable antifungal activity on a range of pathogenic fungi. They utilized yeast to study resistance to osmotin, and capitalized on the fact that yeast strains varied in their degree of resistance. Using such strains, Yun et al. demonstrated that cell-surface proteins, Pir proteins, are associated with the degree of osmotin resistance. It would seem reasonable to assume that pathogenic organisms have developed similar tolerance mechanisms. This speculation is supported by the work of Lopez-Solanilla et al. (1998), which showed that a locus of genes in Erwinia chrysanthemi for resistance to defensins is important for full virulence.
IV. APPROACHES TO THE USE OF NEW KNOWLEDGE IN DISEASE CONTROL With increasing pressure for the reduction and eventual elimination of pesticides, and the generally poor success of biological control approaches in agricultural disease control, transgenic plants will clearly become important in the future. Although many experimental approaches have been taken to develop disease-resistant plants by introducing single transgenes, relatively few such plants are currently employed in commercial agriculture. However, a substantial number of candidate genes have been identified for use in transgenic plants (for recent reviews see Beachy, 1997; Mourgues et al., 1998; Schuler et al., 1998). This bodes well for the future because of the theoretically improved efficacy and durability of resistance in plants carrying two or more transgenes with different modes of action. This section will briefly review some of the approaches currently being undertaken.
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A. TRANSGENIC PLANTS EXPRESSING FOREIGN DISEASE RESISTANCE GENES
One of the appeals of cloning plant disease resistance genes is that the cloned genes can, in principle, be transformed into heterologous plant species for disease control. Surprisingly, relatively little progress has been made, to date, in testing this idea. The Pto disease resistance gene was shown to function in tobacco plants against Pseudomonas syringae pv. tabaci carrying the cloned avrPto avirulence gene (Rommens et al., 1995; Thilmony et al., 1995). As such, however, the exercise does not offer the immediate prospect for control of the wildfire disease in tobacco, caused by P . s. pv. tabaci. More promising was the demonstration by Whitham et al. (1996) that transformation of the cloned N resistance gene against TMV virus into tomato plants resulted in resistance against the native virus. All of these demonstrations show that heterologous but related plants have all other necessary components for active resistance once the cloned resistance gene is introduced. Providing the pathogen in question carries the cognate avirulence gene, resistance would be expected to occur. Additional examples of the successful introduction of cloned resistance genes into heterologous plants may be expected. As discussed earlier, compelling evidence now indicates that LRR regions determine the specificity of resistance gene-mediated disease resistance. One of the appeals, therefore, of utilizing cloned disease resistance genes in transgenic plants is the prediction that their specificities can be changed by altering the LRR domains. It is even possible to envisage that LRR domains may eventually be synthesized or empirical mutant domains selected using rapid screening techniques, such as phage display to target particular pathogen metabolites. As mentioned earlier in this review, Lauge et al. (1998) screened tomato genotypes for resistance using a cloned gene from Cladosporium fulvum encoding ECP2, an essential virulence factor. This screening approach presages attempts to select resistance genes tailored to specific pathogen elicitors. For example, plant disease resistance genes are not known to be targeted to extracellular pectic enzymes from pathogens, particularly pectate lyases. The reason for this is not known, but it may be possible in the future to screen for resistance genes directed to virulence factors such as pectate lyases. Polygalacturonase inhibitors are plant LRR proteins (Desiderio et al., 1997) that recognize these fungal pectic enzymes. However, the inhibitors do not appear to activate defence responses, as is the case with LRR disease resistance genes. Oldroyd and Staskawicz (1998) constitutively expressed the LRR protein Prf from tomato in the same plant, and observed enhanced disease resistance to three bacterial pathogens. Evidence was also provided that the transgenic plants expressed constitutive systemic acquired resistance. As discussed earlier, Prf is required in tomato for the function of the Pto disease resistance gene. These workers speculated that Prf functions downstream from the Pto disease resistance gene in the activation of defence response genes. Although no
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negative effects were observed on growth of the Prf transgenic plants, their utility in agricultural situations remains to be evaluated. B. TRANSGENIC PLANTS THAT INACTIVATE TOXINS
This approach has the appeal that plant resistance through detoxification of an essential pathogen toxin will presumably be difficult for the pathogen to overcome. There are several experimental and naturally occurring uses of this approach, as discussed earlier, the best known being the Hml gene in corn that degrades the HC toxin of Cochliobolus carbonum (see Multani et al., 1998). C. TRANSGENIC PLANTS EXPRESSING REGULATORY OR DEFENCE RESPONSE GENES
There are several cases in which naturally occurring defence response genes have been constitutively or inducibly expressed in the native or heterologous plants (Hain et al., 1993; Dixon et al., 1996; Masoud et al., 1996; Huang et al., 1997; Wu et al., 1997). None of these have yet been commercially used on any scale. Lorito et al. (1998) recently suggested that chitinase genes from a mycoparasitic fungus might exhibit greater efficacy against pathogens when expressed in transgenic plants. This idea is worth exploring because plant pathogenic fungi might not previously have seen the mycoparasite enzyme or that it has adapted to confound evolution of resistance in pathogens. Cao et al. (1 998) reported that the Arabidopsis NPRl gene, discussed earlier, provided broad spectrum disease resistance when overexpressed. This is an avenue worth exploring for applied disease control. However, as is also true for Prf overexpression, discussed above, if simply overexpressing N P R l results in disease reistance, why did plants not do this millions of years ago? D . EXPRESSION OF PATHOGEN GENES IN PLANTS
Several reviews on this subject have appeared (e.g. Beachy, 1997; Dempsey et al., 1998). The dramatic resistance of plants transformed with viral coat protein genes has made this a prime target of transgenic research, and commercial successes have occurred on relatively small scales (see Beachy, 1997). The use of viral replicase genes and movement protein genes have also shown promise in transgenic plants. Recently, Waterhouse et al. (1998) have shown that coexpression of viral gene sequences in the sense and antisense orientations in transgenic plants result in gene silencing of the targeted viral genes. As such, this is a very promising rationale for the construction of virus-resistant transgenic plants. Limited numbers of experiments have been made in which genes from bacterial pathogens were expressed in plants with the objective of disease control. For instance, DeWit (1992) devised a clever approach in which
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the Cladosporium fulvum avr9 gene was transformed into tomato plants carrying the cognate resistance gene, Cfs. The avr9 transgene was expressed from a plant defence gene promoter so that expression was very low until the plants were attacked by a pathogen. Then Avr9 peptide would accumulate and an HR would occur in the infected area. While an intriguing idea, the approach requires that control of the avirulence gene promoter be very stringent. It is possible that expression of pathogen avirulence genes in plants lacking the cognate receptor or disease resistance genes may nonetheless confer resistance. For example, preliminary evidence indicates that avrD and avrRxv transgenic tobacco plants exhibit enhanced resistance to several fungal and bacterial pathogens (Y. Huang and N. Keen, unpublished observations). A similar approach to that with avr9 involves the use of a cloned minimal root-specific promoter introduced into plants ahead of a ribonuclease gene (Opperman and Conkling, 1994). These workers deleted a portion of a rootspecific plant promoter and found that the modified promoter only functioned in roots attacked by root knot nematodes. As such, the proposed strategy is clever, although the specificity must be extremely tight such that the promoter does not function in response to unknown physiological conditions that plants might be exposed to. E. OTHER RATIONALES
Several searches have been made for antagonistic genes to pathogens that can be expressed in plants. The well-known Bt toxins are a precedent for this approach, and some evidence indicates that other genes may function in a similar fashion. For example, Urwin et al. (1998) reported that expression of a protease inhibitor in Arabidopsis increased resistance to cyst and root-knot nematodes, presumably by inhibiting proteases in the digestive tracts. It is likely that other genes whose products are antagonistic to pathogens but not plants will be used in this manner. As genes encoding enzymes involved in plant cell wall synthesis are cloned and characterized, it will be possible to alter the composition and properties of cell walls (e.g. Sterky et al., 1998). This will probably have important consequences for disease control, especially with pathogens that rely on cell wall-degrading enzymes. While perhaps further in the future, genes will be identified that cause pronounced changes in general morphology and anatomy of plants. Plants transformed with such genes can be expected to confound pathogens that rely on specific anatomical features of their host plants for correct development (e.g. Hoch et al., 1987). Phage display and other rapid screening techniques will result in new peptides and other chemicals that specifically antagonize particular pathogens such as viruses. It is very likely that future transgenic plants will utilize genes leading to such tailor-made antagonistic molecules.
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V. CONCLUSIONS A N D FUTURE DIRECTIONS Despite the rather slow development of transgenic plants exhibiting disease resistance, many factors are converging to make this the approach of choice for disease control in the twenty-first century. While many countries fear transgenic plants and their produce a t present, the use of adequate testing for safety and efficacy, as well as their increased productivity, will lead t o the widespread use of transgenic plants exhibiting many characters, including disease resistance. The major task for plant biologists is to identify useful genes and devise clever schemes for their deployment. The future is also clear in this arena as the several genome-sequencing projects underway and proposed for plants a n d pathogens will present pathologists with a plethora of new genes. An important activity in the next century will be to understand how these genes function, and to select those with potential for improving the control of plant diseases.
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328
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AUTHOR INDEX
Numbers in italics refer to pages on which full references are listed
A Aarts, M. G. M. 300, 315 Aarts, N. 306, 315 Abad, L. R. 311, 327 Abel, W. 0. 255, 282 Ackerson, R. C. 19, 84 Acland, D. P. 311, 317, 326 Adachi, F. 145, 197 af Ugglas, M. 6, 52, 54, 77, 83 Afifi, F. 187,202 Aganval, S. K. 135, 194, 199 Ageorges, A. 30, 69 Agrawal, P. K. 164, 223, 274 Aguera, E. 14, 15, 18, 73 Ahmed, R. 302, 325 Ahn, J. H. 295, 315 Aitken, W.M. 123, 196 Akagawa, E. 24, 83 Akiyama, Y. 209, 211, 212, 214, 222, 230, 236, 274
Alain, M. 227, 228, 274 Albelda, S. M. 272, 278 Albersheim, P. 230, 245, 233, 271, 236, 277, 278, 286, 287
Albo Martin, B. 226, 227, 228, 283 Alborn, H. T. 104, 107, 109 Alexander, I. J. 3, 69 Alfano, J. R. 293, 298,299, 301, 315, 317, 319 Alford, A. R. 105, 109 Allard, S. 209, 215, 216, 247, 278 Alvarez, M. E. 305, 315 Amarasinghe, R. 11, 12, 17, 32, 69 Amarjit, , 11, 12, 17, 32, 69, 191 Amato, R. 11, 12, 17, 32, 69, 218, 287 Amblard, P. 10, 82 Amini, B. 314, 325 Amino, S, 186, 191 Amor, Y. 257,277 Ampong-Nyarko, K., 112 Anderson, D. M. W. 222, 228, 229, 274 Anderson, J. A. 46, 85, 86 Anderson, L, 126, 145, 161, 191 Anderson, M. A. 212, 215,216,221,230,231, 232, 235, 242, 271, 274, 282, 285
Anderson, P. 300, 318 Anderson, R. L.,208, 209,211, 214, 217, 219, 222, 223, 226, 227, 228, 229, 230, 236, 243, 252, 259, 273, 274 Anderson, V. 308, 316 Andre, B. 46, 50, 51, 80, 81 Andreae, M. 225, 274 Andreau, D. 311,317 Andriesse, X. 23, 83 Aneshansley, D. J. 102, 109 Anzai, H. 296, 315 Aparicio, P. J. 24, 69, Apel, P. C. 296, 315 Aplin, R. T. 99, 107 Apostolakos, P. 186, 198 Aracri, B. 312, 317 Arai, N. 23, 83 Arlat, M. 296, 299, 316 Arnold, G. M.6, 72, 81 Arst, H. N. 2, 11, 25, 71, 72 Asano, Y. 175, 192 Ashfield, T. 300, 319 Ashihara, H. 117, 119, 120, 122, 124, 125, 126, 127, 128, 131, 133, 134, 135, 136, 139, 140, 141, 143, 145, 146, 147, 148, 151, 153, 154, 155, 156, 158, 159, 160, 161, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 177, 179, 181, 183, 185, 186, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 200, 202, 203, 204, 205
Ashton, A. R. 201 Adam, M. 5, 9, 10, 12, 14, 52, 56, 69, 70 Aspinall, G. 0. 208, 211, 219, 226, 236, 259, 274
Assmann, S. M. 30, 85 Astruc, S. 47, 78 Athanassapoulos, E. 298, 325 Atilio, B. J. 17, 70 Atkin, 0. K. 5, 56, 67, 79 Atkins, C. A. 63, 79 Atkins, 126, 136, 193,201,202 Atkinson, A. H. 212, 215, 216, 221, 231, 232, 242, 285 Atkinson, D. E. 124, 193
330
AUTHOR INDEX
Atkinson, H. J. 314, 326 Ausubel, F. M . 293, 301, 306, 309, 319, 321, 324 Avila, J. 28, 70, 83 Ayliffe, M.,300, 318 Ayliffe, M.A. 308, 321 Azuma, J . 4 . 209, 213, 240, 242, 243, 280
B Bacher, A. 97, 106, 110 Bachmann, M. 2, 11,68, 77 Bacic, A. 208,209,210, 211, 212,213,214,215, 216,217, 218, 219,221, 222, 223, 225, 226, 227,230, 231,232, 233, 234,236, 238,239, 240,241, 242,243, 247,255, 261, 264, 268, 271,272,273, 274, 275,276,277, 278,282, 283, 285, 286, 289 Back, K. 306, 327 Backer, A. 126, 201 Backer, A. I . 124, 126, 131, 135, 159, 201, 204 Baeuerle, P. A. 308, 316 Bak, S. 97, 98, 111 Baker, B. 293, 299, 308, 312, 327 Baker, C. J. 304, 316 Baker, T. C. 105, 107 Bakken, A. K. 15, 70 Balandin, T. 24, 69 Balderrama, J. R. 229, 279 Baldwin, I. T . 92, 93, 111 Baldwin, S. A. 33, 70 Baldwin, T. C. 209, 211, 212, 214, 221, 222, 226, 227, 228, 236, 243, 275, 283 Balentine, D.A. 119, 193 Baht-Kurti, P. J . 300, 326 Ball, 0. J.-P. 99, 107 Balogh-Nair, V. 98, I12 Baltimore, D. 308, 316 Barankiewicz, J. 134, 163, 193, 196, 197 Barash, I. 296, 326 Barber, S. A. 45, 60, 74 Barg, C. 225, 274 Barhanin, J. 27, 70 Barker, G. M . 99, 107 Barnes, E. M . 50, 74, 77 Barnett, N. M . 19, 88 Barny, M.-A. 298,319 Baron-Epel, 0. 222, 275 Barr, R. 13, 70 Barraclough, P. B. 4, 60, 70 Barthes, L. 19, 20, 70, 76 Basile, D. V . 209, 214, 238, 252, 275, 278 Basile, M . R. 209, 238, 252, 275 Basse C.W. 303 316 Basset, M. 47, 78 Bate, N. J . 294, 322 Bauer, D. W . 298, 299, 301, 316, 319, 321 Baulcombe, D. C. 312,317
Baumann, T. W. 119, 122, 123, 143, 144, 145, 146, 150, 151, 152, 153, 154, 155, 171, 172, 175, 186, 187, 193, 194, 195, 197, 198, 199, 201, 202, 204 Bazan, J. R. 308, 324 Beachy, R. N . 294, 311, 313, 316, 323 Beale, M . H . 94, 115 Becker, J. M. 31, 32, 87 Beckhove, U. 94, 110 Beer, S. V. 298, 299, 316, 321 Beevers, H. 133, 198 Beevers, L. 30, 82 Behl, R. 9, 70 Beilharz, V. C. 63, 79 Bell, E. 69, 70 Belvin, M. P. 308, 316 Benn, M. H. 99, 107 Bennett, M. A. 299,298, 322, 325 Bent, A. F . 299, 304, 316, 327 Ben-Zioni, A. 66, 70 Berenbaum, M.R. 92, 93, 107, 114 Berestovsky, G. N. 53, 78 Berg, D. E. 50, 82 Berger, S. 69, 70, 306, 324 Bergersen, F. J . 49, 77 Bergey, D. R. 93, 94, 107, 108 Bergfeld, R. 238, 239, 245, 246, 255, 267, 269, 285 Bernasconi, P. 127, 204 Bernays, E. 100, 107 Bertl, A. 47, 70 Beynon, J. L. 300, 315, 325 Bhalerao, R. 314, 325 Bhatt, G. 310, 318 Bhattacharjee, A. K. 226, 265, 279 Bi, J. L. 93, 107, 294, 316 Biller, A. 99, 100, 107, 112 Birch, R. G. 296, 328 Bird, P. B. 227, 228, 284 Bisgrove, S. R. 300, 316 Bismuth, E. 10,82 Bisseling, T . 215, 285 Bissell, M. J . 273, 275, 282 Blackwell, A. 98, 112, 113 Blanco, G. 24, 84 Blatt, M.R. 6, 7, 24, 69, 71, 81 Blechert, S . 94, 107 Bleeker, M.300, 325 Blevins, D. G. 19, 88, 127, 136, 138, 139, 197, 201, 204, 205 Blight, M.M . 94, 100, 1101, 105, 107, 108, II3 Bliska, J . B. 298, 323 Blomquist, G. J . 99, 114 Bloom, A. J . 3, 13, 14,43, 53, 58, 59, 71, 76, 86 Blumwald, E . 306, 327 Bobalek, J. F. 209, 266, 275 Boerjan, W. 314, 325 Bogdanove, A. J . 298, 316
AUTHOR INDEX Bogner, F. 100, 108 Bol, J. F. 310, 321 Boland, M. J. 126, 139, 194, 202 Boland, W. 99, 104, 108, 109, 113 Boller, T. 302, 303, 305, 306, 316, 318, 319 Bolwell, G. P. 208, 224, 225, 275, 285 Bonas, U. 301, 326 Bonig, I. 232, 235, 282 Bonnefoy, V. 23, 71 Bonnema, G. 303,321 Bonnemain, J. L;. 65, 75 Booij, H. 208, 238, 239, 250, 252, 277, 284 Bopprb, M. 99, 100, 107, 112 Borremans, F. 311, 317 Boswell, H. D. 156, 204 Botella, M. A. 300, 316 Bottger, M. 7, 13, 70, 71 Botton, B. 3, 81 Boucaud, J. 6, 78 Boucher, C. A. 296, 299,316 Bouchez, D. 300, 325 Boudreau, N. 273, 275 Bouquelet, S. 217, 218, 283 Bournoville, R. 65, 75 Bousser, A. 19, 20, 70, 76 Bouthyette, P. Y . 30, 81 Bowers, M. D. 99, 108 Bowyer, P. 294, 295, 316, 323 Boyd, C. 296, 299, 304, 320, 321 Boyd, R. L. 163,201 Braddon, M. 11, 12, 15, 17, 32, 69 Bradley, D. J. 209, 213,230, 257, 258, 260,266, 283 Brady, D. J. 60, 71 Braekmann, J. C. 100, 113 Breeze, V. G. 61, 90 Bregante, M. 46, 76 Brennan, P. J. 208, 277 Brenner, M. 302, 316 Bressan, R. A. 239,243,252,253,279,289,311, 312, 326, 327 Breteler, H. 13, 14, 71 Briggs, S. P. 97, 106, 110, 295, 313, 316, 322, 323 Brikun, I. 50,82 Brillouet, J.-M. 218, 219, 221, 223, 239, 249, 275, 283, 285 Brishammar, S. 65, 90 Brisset, M.-N. 311, 323 Brito, N. 28, 32, 37, 70, 83 Brix, H. 41, 74 Brodbeck, U. 262, 275 Brodelius, P. 188, 196 Brodschelm, W. 94, 107 Broekaert, W. F. 311, 316, 317, 326 Brommonschenkel, S. H. 299, 322 Brouwer, Y . 210, 237, 281 Brow. M. A. D. 25. 77
33 1
Brown, J. K. M. 295, 323 Brown, K. S. 100, 108 Brown, W. 293, 316 Brownlee, A. G. 25, 71 Browse, J. 310, 326 Bruun, L. 248, 249, 282 Buch, M. 99, 112 Buchala, A. 302, 325 Bucher, M. 31, 38, 45, 51, 79 Buczek, J. 13, 78 Bunn, R. C. 38, 72 Bun-Ya, M. 33, 71 Burkovski, A. 50, 86 Burns, I. G. 62, 71 Busby, S. 24, 83 Busby, S. J. W. 24, 76 Busch, M. A. 7, 71 Butikofer, P. 262, 275 Butowt, R. 256, 275 Buzzell, R. I. 301, 303, 320
C Caboche, M. 2, 3, 10, 12, 15, 17, 32, 67, 72, 76, 78, 81, 84, 85 Calderon De La Barca, A. M. 229, 279 Cambron, S. 301, 318 Cameron, K. C. 4, 71 Cammue, B. P. A. 31 1, 316 Campbell, E. I. 25, 88 Camps, F. 98, 108 Canas, L. 300, 326 Cantoni, G. L. 158, 196 Canvin, D. T. 63, 79 Cao, H. 307, 313, 317 Caporale, L. H. 106, 108 Cirdenas, J. 1 1, 51, 75 Carland, F. M.,298, 327 Carlson, W.C. 238, 279 Caro, L. H. P. 264, 275 Carozzi, N. 106, 108 Carpita, N. 222, 276 Carpita, N. C. 208,222,224,230,239,241,243, 257, 271, 273, 276, 279, 286, 288 Carr, M. C. 134, 201 Cartier, N. 236, 276 Carvajal, M. 19, 20, 71 Carvalho, A. 122, 199 Casadei, E. 228, 283 Casero, P. J. 245, 269, 271, 276 Casimiro, I. 245, 269, 271, 276 Caul, S. 60, 85 Causin, H. 17, 70 Cavallarin, L. 31 1, 317 Cavicchioli, R. 13, 71 Century, K. S. 306, 317 Cervone, F. 312,317 Chaisson. A. 38. 72 Chambat; G. 236, 276
332
AUTHOR INDEX
Chamberlain, K. 93, 103, 108, 114 Chamnongpol, S. 305,317 Champagne, D. E. 98, 108 Champigny, M. L. 10, 82 Chang, J. H., 205, 301,303, 325 Chapin, F. S. 20, 71 Chappell, J. 306, 327 Chapya, A. 98, 112 Charkowski, A. 0. 298,299, 316, 317 Charubala, R. 175, 204 Chasan, R. 260, 276 Cheesman, J. M. 52, 76 Chen, C.-G. 209, 213, 218,226, 231, 232, 233, 241, 261, 276, 282 Chen, C.M. 134, 143, 194 Chen, J. 13, 71 Chen, Z. 312, 326 Cheng, F. 120, 205 Cheung, A. Y. 210,211,212,215,216,218,222, 226,231,232,233,234,235, 242, 247,272, 273, 276, 288 Chevreau, E. 311, 323 Chiang, C. S. 9, 29, 31, 38, 39, 40, 77 Chiang, P. K. 158, 196 Chiang, R. C. 13, 71 Chiang, V. L. 156, 197 Chikemai, B. N. 228, 283 Chilcott, T. C, 47, 78 Chiliswa, P. 103, 104, 106, 112 Chinault, A. C. 50, 74 Chittor, J. M. 301, 328 Cho, T.-J. 94, 108 Choi, D. 308, 327 Chomet, P. 97, 106, 110 Chong, J. 243, 285 . Chory, J. 300,322 Chrispeels, M. J. 225, 278 Chu, H.4. 94, 108 Chua, N.-H. 253, 273, 287 Chung, K. R. 310,321 Chunwongse, J. 299, 322 Churms, S. C. 209,212,214,218,219,221,222, 225, 228, 236, 275, 276, 278 Citovsky, V. 294, 321 Ciufetti, L. M. 310, 317 Clark, B. C. 227, 228, 230, 231, 284 Clarke, A. 230, 281 Clarke, A. E. 208,209,211,214,217,219,222, 223, 226,227,228,229, 230, 231,238, 243, 252, 256,259, 273,274, 275,276,277, 278, 279, 280. 281. 282,. 283.. 285, 286. 288 Clarke, B. 295, 324 Clarke, B. R. 294, 316 Clarke, J. D. 307, 317 Clarkson, D. T. 1, 3, 5,6, 11, 13, 17,20,40,42, 53, 55, 57, 58,61,62, 63,64, 71, 72, 78, 79, 88 Clay, R. P. 245, 278
Clement, A. 127, 134, 195 Clement, C. R. 15, 60,72 Clutterbuck, A. J. 25, 77 Cohen, P. B. 209,212, 214,218,219,221,222, 225, 236, 275 Cole, G. E. 25, 77 Cole, J. 24, 83 Cole, J. A. 25, 76 Cole, 76, 77, 83 Coleman, M. J. 300, 316 Coll, J. 98, 108 Colling, C. 304, 320 Collins, N. 300, 318 Collmer, A. 293, 296, 297, 298, 299, 301, 315, 316, 317, 319, 321,324 Colucci, G. 313, 322 Comeau, J. 293, 314, 319 Condemine, G. 297,320 Conejero, G. 47, 78 Conejero, V. 300,326 Conkling, M. A. 314, 323 Conlin, A. 298, 316 Constabel, C. P. 93, 108 Conzelmann, A. 263,278 Cook, R. J. 310,326 Cooke, D. T. 19, 20, 71 Cooksey, D. 297- 298,322 Cooper, C. 245, 246, 267,269,271,281 Cooper, H. D. 62, 63, 64,72 Copping, L. G. 106,108 Cordewener J. 238, 277 Cornelissen, B. 300, 325 Corr, C. 308, 327 C o d , G. M. 2, 78 Coschigano, K. T. 2, 78 Cove, D. J. 25, 88 Covert, S. F. 310, 318 Cowles, R. S. 106, 112 Craig, J. W. T. 211, 219, 236, 274 Craik, D. 212, 219, 221,222, 223, 278 Cram, W. J. 55, 72 Crane, F. L. 13, 70 Crawford, N. M. 2,9, 11, 12,28,29, 30, 31, 38, 39,40, 69, 72, 77, 88, 89 Crawford, D. V. 4, 60, 76 Cresti, M. 247, 248, 249, 282, 284 Cronk, Q.C. B. 209,272 Crouzet, J. 24, 83 Crozier, A. 117, 139, 143, 145, 146, 147, 148, 151, 153, 154, 155, 158, 159, 161, 165, 171, 172, 173, 174, 175, 177, 178, 181, 183, 184, 185, 189, 190, 191, 192, 193, 194, 195, 197, 199 Crute, I. R. 309, 319
D Daffe, M. 208,277
AUTHOR INDEX Dahlbeck, D. 297,298,301,304, 306,317,320, 325, 327 Dalling, M. J. 63, 79 Daloze, D. 100, 113 Damsz, B. 3 11, 327 Dancer, J.E. 138, 194 Dangl, J. L. 297, 300, 319,324 Daniele-Vedele, F. 11, 12, 15, 32, 84 Daniels, M. 295, 323 Daniels, M. J. 294, 295, 306, 315, 316, 323 Daniel-Vedele, F. 2, 3, 1 1, 12, 15, 17, 72, 76, 78 Darvill, A. 230, 277 Darvill, A. G. 223,230,236,245,271,278,286, 287 Das, J. 138, 199 Dauterive, R. 38, 72 David, G. 272, 277 Davies, C. 215, 284 Dawson, G. W. 98, 102, 105, 108, 109, 113 Day, D. A. 49, 77, 88 Day, S. 209,230, 236, 245,265, 266, 269, 281 de Bolano, L. M. 228, 229, 277 de Both, M. 300, 325 de Bruxelles, G. L. 11, 12, 15, 17, 32, 69 De Feyter, R. 301, 308, 317 de Gotera, 0. G. 229, 277 de Groot, K. E. 229, 302,320 De Jong, A. J. 238,277 de la Haba, P. 14, 15, 18, 73 de Moncada, N. P. 229, 277 De Moraes, C. M. 104, 109 de Pinto, G. L. 228, 229, 274, 277 de Vries, 0. M. H. 293, 325 de Vries, S . 238, 277 de Vries, S . C. 208,237,238,239,250,251,252, 255, 277, 284, 287, 288 de Willigen, P. 3, 73 De Wit, P. J. G. M. 301, 303, 312, 321 Dea, I. C. M. 228, 274 Dean, R. A. 293, 317 Deane-Drummond, C. E. 14,41, 52, 55, 56, 73 DeBary, A. 296,317 Deboer, A. H. 57, 90 DeBolle, M. F. C. 311,316 Debray, H. 217,218, 283 Defaye, J. 211, 223, 269, 277 Deji, A. 13, 85 Delaney, T. 94, 112 Delaney, T. P. 293, 299, 307, 31.5, 317, 324 Delauney, A. J. 128, 130, 131, 194, 198 Deleens, E. 19, 20, 70, 76 Delhaize, E. 33, 86 Delhon, P. 65, 66, 67, 73 Dell, A. 223, 226, 230, 233, 245, 246, 250, 265, 267,269, 27 1, 289 Delmer, D. P.,222, 230, 257, 276, 277 DeLorenzo, G. 312,317 Demarco, A. 13, 73
333
Demoss, J. A. 23, 71, 73 Dempsey, D.A. 293, 313, 317 Denholm, I. 311, 325 Dennis, L. 308, 301, 317 Dennis, D.T. 119, 194 Denny, T. P. 297, 317, 319 DeSamblanx, G. W. 311,316,317 Desiderio, A. 312, 317 Desponds, C. 263, 278 Dettner, K. 99, 113 Devienne, F. 18, 73 Devreux, J. 34, 73 DeWit,P. J. G. M. 293, 301,303,313,318,321 Dey, P.M. 119, 194, 205 Deyrup, M. 102, 109 Dhugga, K. S . 30, 73 Di, M. 311, 313, 319 Dicke, M. 92, 103, 104, 109, 112, 114 Dickens, J. C. 105, 109 Dickman, M. B. 295, 318 Diergaarde, P. 300,325 Dietrich A. 306, 318 Dijkman, H. 104, 109 DiMilla, P. A. 272, 278 Dincher, S. 94, 110 Dinesh-Kumar, S. P. 293,299, 304, 308, 316, 327 Ding, L. 210, 252,253, 254, 255, 273, 277 Dixon, R. 304, 321 Dixon, R. A. 93, 112, 294, 304, 305, 306, 310, 313, 315, 316, 318, 321,322 DMello, J. P. F. 93, 109 Doddema, H. 28, 74 Doerner, P. 306, 318 Doi, Y. 187, 204 Dolan, L. 245, 265, 267, 269, 277 Donath, J. 104, 109 Dong, B. 33, 86 Dong, K. 300,318 Dong, X. 307, 308, 313, 317 Dopico, B. 271, 277 Doremus, H. 165, 194 Doughty, K. J. 94, 109 Downey, R. J. 24, 74 Drew, M. C. 5, 6, 9, 12, 13, 14, 15, 45, 55, 62, 74, 77, 79 Dreyer, I. 46, 76 Driessen, A. J. M. 23, 85 Droge-Loser, W. 306, 318 Dry, I. B. 263, 287 Du, H. 209, 210,214, 215, 216, 217, 226, 231, 232, 255, 261, 272, 273, 275, 277 Du. S. 209, 215.. 216., 247.. 284 Du; Y. 104, 109 du Penhoat, C. H. 219, 223, 285 Dubcovsky, J. 47, 85 Dubus, A. 18,84 Duffus, C. M. 93, 109
334
AUTHOR INDEX
Duffus, J. H. 93, 109 DUrzo, M. P. 31 1, 327 Dussourd, D . E. 99, 100, 109 Dweikat, I. 301, 318 Dwivedi, R. S. 303, 320 Dyhr-Jensen, K. 41, 74
E Ealing, P. M. 33, 86 Earle, E. D. 299, 322 Earnshaw, M. J. 62, 72 Eastwell, K. C. 165, 195 Ebel, J. 303, 323 Eckard, K. J. 235, 280 Eda, S. 209, 21 1, 214, 230, 274 Edashige, Y. 251, 252, 281 Eddy, A. A. 24, 74 Edelman, G. M., Edelman, 272, 273, 278 Edgar, J. A. 100, 107 Edwards, D. G. 60, 74 Edwards, G. 155, 195 Edwards, R. 146, 155, 195 Egertsdotter, U. 210, 236, 237, 238, 278 Ehmke, A.,100, 111 Eidenbock, M. P. 19, 84 Eikmanns, B. J. 50, 86 Eikmanns, M. 50, 86 Eisenreich, W. 97, 106, 110 Eisner, M. 102, 109 Eisner, T. 99, 100, 102, 109 Ek, B. 20, 77 Elkind, Y. 294, 322 Eller, B. M., 195 Elliott, M. 93, 109 Ellis, D. 307, 324 Ellis, J. 300, 318 Ellis, J. G. 308, 321 El-Omer, M. A. 187, 202 Emmerling, M. 256, 283 Emran, A. M. 12, 15, 77 Engels, C. 61, 62, 74 Engen, T. 214, 218, 279 Enkerli, J. 310, 318 Epstein, W. 50, 77 Erickson, L. 209, 215,216, 247, 284 Ericsson, A. 61, 81 Erner, Y. 3, 44, 46, 48, 75 Escoubas, P. 97, 109 Esquerre-Tugaye, M.-T. 305, 324 Evans, L. V. 187, 196 Ezeta, F. N. 19, 74 Ezra, D. 296, 326
F Fabiny, J. M. 50, 74 Fahn, A. 226,278 Faleri, C. 247, 248, 249, 282
Falk, A. 306, 323 Falkengren-Grerup, U. 4, 74 Falkow, S. 298, 323 Fan, X. 302, 325 Fankhauser, C. 263, 278 Fant, F. 311, 317 Fares, Y. 12, 15, 77 Fast, B. 24, 74 Fath, A. 303, 316 Fauquet, C. 300, 326 Faye, F. 134, 195 Fearon, D. T. 311, 318 Feenstra, W. 28, 83 Feenstra, W. J. 28, 28, 74, 86 Feierabend, J. 138, 200 Feixas, J. 102, 110 Feldmann, K. A. 9, 1 I , 12, 30, 31, 38, 40, 88 Felix, G. 302, 306, 318, 319, 325 Felle, H. H. 42, 76 Felsenstein, J. 34, 74 Felton, G. W. 93, 107,294,316 Feng, J. N. 58, 75 Feng, Z. 104, 108 Fenical, W. 92, I l l Ferguson, L. R. 229, 238, 279 Ferguson, M. A. J . 261, 263, 278, 285 Fernandez, C. 308, 325 Fernandez, E. 11, 12, 15, 18, 26, 27, 32, 39, 51, 24, 25, 27, 69, 75, 82, 84 Fernandez, I. G. 313,322 Fernando, M. 6, 12, 15,86 Feuillet, C. 300, 318 Filippone, E. 313, 322 Fillery, I. R. P. 60, 71 Filleur, S. 3, 72 Fincher, G. B. 208,209,212,214,218,219,221, 222, 223, 224,225, 229, 236, 241,243, 259, 273, 275, 278, 279, 282, 285, 288 Fink, M. 27, 70 Finnegan, E. J. 308, 321 Finnegan, J. 300, 318 Finnegan, P. M. 49, 77 Firn, R. D. 92, 109, 111 Fischer, U. 20, 77 Fischerschliebs, E. 13, 73 Fisher, J.R, 107, 139, 203, 205 Fitzsimmons, K. C. 313, 327 Flaishman, M. A. 295, 324 Flegg, L. M. 295, 323 Florencio, F. J. I I , 75 Fluhr, R. 238, 278 Fong, C. 21 1,212,214,218,221,222,227,228, 284
Forde, B. G. 1, 11, 12, 13, 15, 17, 25, 32, 33, 34, 37, 39, 62, 68, 69, 84, 88, 90 Forsberg, L. S. 209, 250, 251, 272, 282, 284 Fournier, J. 305, 324 Fouts, D. E. 301, 319
AUTHOR INDEX Fox, J. E. B. 273, 278 Fox, R. L. 60, 75 Foyer, C. H. 155, I95 Fragu, P. 64, 76 Fraisier, V. 12, 15, 17, 78 Franco, A. R. 51, 75 Frary, A. 299, 322 Frederick, R. D. 301, 302, 303, 318, 326 Frensch, J. 20, 87 Freshour, G. 245, 278 Frey, M. 94, 106, 110 Frey, P. A. 134, 201 Friedrich, L. 94, 110, 112, 307, 317, 318, 324 Frischknecht, P. M. 122, 171, 186, 187, 193, 195 Frommer, W. B. 3, 4, 27, 31, 38, 42, 45, 46, 47, 48, 51, 75, 79, 82, 89 Frost, D. 300, 318 Frost, L. N. 306, 323 Fry, S. C. 225, 255, 278, 279 Fujii, T. 251, 252, 281 Fujimori, N. 120, 122, 145, 147, 151, 158, 159, 166, 168, 170, 171, 174, 175, 195 Fujimura, T. 145, 151, 153, 154, 155, 156, 189, 197 Fujisawa, Y. 127, 130, 197 Fujita, Y. 24, 82 Fujiwara, S. 139, 193, 195, 203 Fujui, T.,236, 279 Fuller, M. S. 245, 278 Funkhouser, E. A. 11, 83 Furtula, V. 249, 287 Furusaki, S. 187, 198 Furuya, T. 187, 195 Fyte, F. 140, 198
G Gaber, R. F. 46, 85 Gabler, A. 104, 108 Gabriel, D. W. 294,296, 301, 326, 327 Gabriel, H. 172, 193 Gaffney, T. 307, 317,318 Galan, J. E. 298, 323 Galatis, B. 186, 198 Gallagher, J. T. 272, 278 Gallois, R. 127, 134, 195 Galvan, A. 12, 18, 26, 27, 39, 75, 82, 84, Gambale, F. 46, 76 Gammon, D. W. 228, 278 Ganal, M. W. 299, 322 Cane, A. M. 211, 219, 221, 222, 223, 238, 239, 268, 278 Ganjian, I. 214, 278 Garciagonzalez, M. 23, 85 Garcia-Olmedo, F. 31 1, 322 Gardiner, M. 225, 278 Garg, R. P. 297,319 Garvin, D. F. 47, 78
335
Gasch, A. 253, 273, 287 Gaudriault, S. 298, 319 Gaut, B. S. 300, 326 Gavish, H. 238, 278 Gaymard, F. 47, 78 Gazzarrini, S. 3,89 Gedeon, C . A. 24, 74 Geering, K. 27, 85 Geitmann, A. 247, 248,249, 282 Cell, A. C. 209, 231, 278 Gellisen, G. 28, 75 Genevieve, S. 217, 218, 283 Geraeds, C. C. J. M. 243, 285 Gershenzon, J. 92, 110 Gerster, J. 209, 215, 216, 247, 278 Gessler, A. 64, 86 Gharyal, P. K. 222, 275 Ghori, N. 298, 323 Ghoshroy, S. 294, 321 Giamoustaris, A. 94, 110 Gianoli, E. 94, 110 Gibbon, M. J. 293, 298, 302, 326 Gibbs, M. 126, 145, 161, 191 Gibeaut, D. M. 208, 211, 223, 224, 230, 236, 239,241,243,256,257,271, 276, 279, 286 Gierl, A. 94, 97, 106, 110 Gilbert, M. 104, 108 Gill, M. C. L. 228, 274 Gillespie, R. 243, 285 Gillies, F. M. 139, 145, 146, 147, 148, 151, 154, 155, 159, 161, 171, 172, 173, 174, 175, 177, 178, 179, 181, 190, 192,193, 195, 197, 199 Gilson, P. 210, 215, 255, 264, 272, 273, 285 Girousse, C. 65, 75 Glass, A. D. M. 3, 4, 5, 6, 8, 9, 12, 13, 14, 15, 18, 19, 25, 29, 30, 38,41,42,44,45,46,48, 53, 57, 58, 69, 73, 75, 78, 86, 88, 89 Glaudemans, C. P. J. 226, 265, 279 Glawischnig, E. 97, 106, 110 Glazebrook, J. 293, 307, 309, 317, 319, 328 Gleeson, P. 21 1, 230, 276 Gleeson, P. A. 209,211,212,214,223,230,236, 238, 256, 259, 276, 279 Glogau, U. 13,87 Goderis, I. J. 311,317 Godiard, L. 300, 319 Godon, C. 17, 76 Goggin, F. L. 300, 324 Goh, K. M. 3, 13, 43, 76 Gojon, A. 6, 12, 15, 17, 64, 65, 66, 67, 72, 73, 76, 78 Gols, G. J. Z. 99, 110, 112 Golstein, C. 300, 323 Gomez, M. D. 300, 326 GonzLlez, C. 28, 70, 83, GonzLlez-Fontes, A. 67,85 Goodwin, P. H. 301, 312, 321 Gopalan, S. 301, 319
336
AUTHOR INDEX
Gorlach, J. 94, 110 Gotz, F. 24, 74 Goulter, K. C. 305, 320 Govers, F. 302, 320 Goyal, S. S. 12, 45, 76, 87 Goycoolea, F. M. 229, 279 Graf, L. 214, 289 Graham, M. W. 313,327 Graham H.N. 119, 193 Grant, A. J. 100, 108 Grant, M. R. 300, 319 Gray, J. 313, 323 Greaves, P. 25, 77 Gregory, P. J. 4, 60, 71, 76 Grieve, C. 25, 88 Grifiiths, D. C. 93,97,98, 105, 109, 110, 113 Griffiths, L. 24, 76 Grignon, C. 45,41, 66, 76, 78,86 Grignon, N. 64,66, 76, 88 Grob, J. 238, 279 Groenen, J . 300, 325 Groenendijk, J. 300,325 Groisman, E. A. 299, 319 Grolig, F. 41, 78 Gronewald, J. W. 52, 76 Grosskopf, D. G. 306, 318, 319 Grouzis, J. P. 30, 69 Gruber, H. E. 163, 197 Griin, S. 97, 106, 110 Gu, Q. 212, 215, 216,218,226, 231,232, 233, 242, 276 Guclu, J. 20, 81 Guern, J. 20,81 Guerrero, A. 102, 110 Guerrero, M. G. 21, 23, 79, 85 Guggisberg, A. 175, 204 Guidotti, G. 258, 279 Guillemare, E. 27, 70 Guillot, A. 131, 198 Gundlach, H. 94, 110 Gunn, N. D. 306, 323 Gunsalus, R. P. 13, 71 Gupta, P.K. 238, 279 Guranowski, A. 134, 136, 158, 195, 196 Gurney, K. A. 187, 196 Gurr, S. J. 25, 77 Gustafsson, P. 314, 325 Gut Rella, M. 94, 110, 307, 317 Guthrie, R. 214, 280 Gutierrez, J. C. 24, 84
H Haanstra, J. P. W. 301, 312, 321 Haeberli, P. 34, 37, 73 Hahlbrock, K. 293,304,305,306,318,320,325 Hahn, M. G. 209, 213,223,230, 245,257,258, 260,266, 271,278,283, 286, 293, 302, 303, 319
Hain, R. 106, 110, 309, 313, 319, 326 Haldimann, D. 188, 196 Halkier, B. A. 97, 98, 110, 111, 306, 318 Hallahan, D. L. 106, 110 Halpern, S. 64, 76 Halterman, D. A. 301, 303, 326 Ham, J. H. 299, 301, 303, 319, 321 Hamer, J. E. 293, 325 Hammer, B. 139, 196 Hammerschmidt, R. 309, 319 Hammerstone Jr, J.F. 123, 196 Hammond Kosack, K. E. 300, 319 Hanemann, U. 64,86 Hanni, K. 123, 194 Hanson, J. B. 52, 53, 76,84 Hansson, B. S. 101, 111 Hara, Y. 94, 115 Haraguchi, K. 263,283 Harashima, S. 33, 71, 90 Harborne, J. B. 93, 99, 111, 119, 194, 196 Harborne, N. 24, 83 Harborne, N. R. 24, 76 Harbowy, M.E. 119, 193 Harder, D.E. 243, 285 Hardie, J. 99, 100, 102, 111, 113, 114 Hardiman, G. 308, 324 Hari, V. 238, 279 Haring, M. 300, 325 Harman, G. E. 313,322 Harris, P. E. 302, 322 Harris, P. J. 208, 229, 230, 238, 239, 275, 278, 279 Harrison, S. 211,230, 276, 281 Harrison, K. 300, 303,319, 323, 326 Hartmann, T. 92, 99, 100, 107, 111, 112, 165, 166, 205 Hartung, W. 64, 83 Harty, J. T. 302, 325 Harvey, C. C. 293, 316 Harvis, C. 99, 100, 109 Hascall, V. C. 265, 288 Hasegawa, H. 30, 76, 89 Hasegawa, P. M. 252, 253, 279, 289, 311, 327 Hashiguchi, S. 156, 196 Hashimoto, T. 156, 196 Hashimoto, Y.209, 218, 221, 222, 223, 225, 226, 229, 230,238, 265, 271,279,281, 283, 287, 288 Haslam, E. 92, 111 Haslam, S. M. 223,226,230,233,245,246,250, 265, 266, 267, 269, 211, 289 Hassanali, A. 98, 110, 112, 113 Hatch, M. D. 127, 196 Hatzixanthis, K. 300, 326 Hauser, K. 212, 215, 216, 221, 231, 232, 242, 285 Hawker, K. L. 25,88 Hay, M. E. 92, I l l , 114
AUTHOR INDEX Hayashi, T. 224, 225, 279 Haynes, R. J. 3, 4, 13, 43, 71, 76 He, S. Y.299, 301,317,319,324 Hediger, M. A. 21, 76 Hedin, P. A. 106, 109, 111 Hedrich, R. 46, 76 Hehl, R. 308,327 Heiskell, M. E. 298, 327 Heitman, J. 51, 80 Hekkert, B. T. 300, 315 Heldt, H.-W. 119, 1% Henderson, J. F. 124, 126, 127, 139, 142, 196 Henderson, P. J. F. 33, 76 Hendriks, T. 250, 287 Hengy, G. 94, 110 Henk, A. 300,326 Hennig, J. 310, 321 Henriksen, G. H. 43, 58, 59, 76 Henrissat, B. 296, 321 Henzler, T. 20, 87 Hepler, P. K. 210,211,226,233, 238, 249,253, 282, 285 Herman, E. 304, 320 Herman, E. M. 303, 320 Hermann, A. 42, 76 Herrera-Estrella, L. 296, 319 Herschbach, C. 64,84 Hert, H. 306, 322 Hertzbert, M. 314, 325 Hesse, M. 175, 204 Hetherington, P. R. 255, 279 Hibi, N. 156, 1% Hick, A. J. 102, 106, 111, 113 Hidalgo, J. 26, 84 Higashi, Y.94, 115 Higgins, 21, 23, 76 Higgins, V. J. 306, 327 Hillestad, A. 214, 218, 279 Hirano, A. 23,83 Hirano, Y.271, 279 Hirel, B. 2, 81 Hirose, F. 126, 127, 128, 134, 136, 140, 173, 196, 197, 204 Ho, J. 294, 321 Hoarau, J. 19, 20, 70, 76 Hobbie, S. 298, 323 Hocart, C. H. 186, 199 Hoch, H. 293, 314,319 Hockenhull, R. 299,322 Hoff, T. 2, 11, 76 Hoffman, B. 41, 78, 321 Hoffmann, J. A. 308, 311, 321 Hofstra, J. J. 28, 74 Hoggart, R. M. 230,279 Holden, F. R., 315 Holder, S. 94, 107 Hole, D. J. 12, 15, 77 Hollenberg, C. P. 28, 75
337
Hollingworth, R. M. 106, 111 Holmberg, A. 314, 325 Holmes, E. W. 163, 2OI Holub, E. 300, 306 Holub, E. B. 300, 306, 309 Holub, 315, 317, 319, 323, 325, 326 Homans, S. W. 263, 278 Honda, K. 101, 111 Honee, G. 303, 321 Hopke, J. 104, 108 Hopkins, P. G. 24, 74 Hopper, M. J. 3, 15, 40,42, 55, 60, 72, 80 Horak, J. 68, 77 Hori, H. 102, 111, 236, 279 Horie, N. 21, 87 Horst, W. J. 60, 84, 90 Houtz, R. L. 156, 198, 205 Hove-Jensen, B. 128, 198 Howe, G. A. 94, 107 Howles, P. A. 294, 316 Howlett, G. J. 212, 219, 221, 222, 223, 278 Howlett, J. F. 228, 274 Hsu, P. Y.23, 73 Huang, J. 297, 319 Huang, N. C. 9, 29, 30, 31, 38, 39, 77 Huang, Y.311, 313, 314, 319 Huber, 3. L. 2, 11, 68, 77 Huber, S. C. 2, 11, 68, 77 Huet, J. C. 296, 299, 316 Huffaker, R. C. 5, 9, 10, 11, 12, 13, 14, 45, 52, 69, 70, 76, 87, 89 Hughes, D. E. 300, 316 Hughes, R.G. 138, 194 Hugouvieux-Cotte-Pattat, N. 297, 320, 325 Huisman, G. 252,277 Hulbert, S. H. 300, 320 Hummel, S. 4, 31, 75 Humphreys, M. 15, 70 Hunt, M. 94, 112 Hunt, M. D. 293, 307, 324 Hurst, W.J. 119, 204 Hurwitz, R. 13, 78, 263, 281 Hutcheson, S. W. 301, 324
I Iacobucci, G. A. 227, 228, 284 Iitaka,Y. 164, 200 Imagawa, H. 124, 135, 138, 145, 147, 151, 154, 156, 159, 161, 163, 164, 200, 203 Imsande, J. 63, 65, 77 Ingemarsson, B. 3, 6, 31, 52, 54, 77, 79, 82, 83, 90 Ingestad, T. 15, 54, 61, 77 Innes, R. W. 300, 316, 319, 326 Innis, M. A. 25, 77 Inoue, T. 145, 197, 198 Inze, D. 305,317 Iraki, N. M. 239, 243, 252, 279
338
AUTHOR INDEX
Ine, I. 145, 151, 153, 154, 155, 156, 189, 197 Isaacs, R. 102, III Ishida, I. 303, 326 Ishii, T. 225, 269, 251, 252, 280, 281 Ishikawa, A. 305,315 Islam, A. M. 222,227, 228, 280 Ismailbeigi, F. 38, 86 Isman, M. B. 98, 108, 112 Ito, E. 160, 163, 172, 174, 175, 186, 197 Ito, 303, 320 Itoh, T. 222, 280 Ives, D. H.,134, 201 Iwai, K. 130, 197 Iwama, M. 145, 151, 153, 154, 155, 156, 189, 197 Iwamatsu, A. 303, 326 Iwata, N. 300, 327
Johnston, A. W. B. 208, 285 Johnstone, I. L. 25, 77 Jones, C. G. 92, 109, 111 Jones, D. A. 299, 300,320, 323,326 Jones, D. J. G. 300,316 Jones, D. R. 263,280 Jones, J. B. 298, 327 Jones, J. D. G. 300, 303, 319, 323, 326 Jones, L. H. P. 3, 15, 40, 42, 55, 60, 61, 72, 90 Jones, T. H. 104, 107 Jongeneel, R. 211, 285 Joosten, M. H. A. J. 301, 312, 321 Joseleau, J.-P. 214, 236, 276, 283 Joshi, C. P. 156, 197 Jung, C. Y. 38, 77,80, 84, 86 Jung, J. 67, 77 Jungk, A. 0. 4, 60, 77 Jurasek, P. 228, 283
J
Jabs, T. 304, 320 Jackson, S. B. 15, 40, 42, 52, 56, 61, 80 Jackson, W. A. 3, 13, 19,46, 44,45,46, 48,49. 51, 54, 58, 66, 74, 75, 77, 82, 85, 87, 89 Jacob, J.-L. 127, 134, 195 Jagendorf, A. 165, 199 Janeway, C. A. 308, 322 Jang, E. B. 105, 109 Jang, J.-C. 67, 77 Janney, N. 156, 205 Janniche, L. 208, 209, 230, 237, 239, 250, 251, 257, 260, 265, 266, 268, 271, 284 Jauh, G. Y. 210, 211, 226,233, 235, 238, 248, 249,253, 280, 285 Jauniaux, J. C. 42, 46, 47, 48, 51, 82 Jayakumar, A. 50, 74, 77 Jenkins, G. I. 151, 172, 195 Jenkins, W. 45, 74 Jenner, C. 299,322 Jermy, T. 97, 111 Jermyn, M. A. 209,211, 214, 222, 236, 238, 243, 256, 259, 274, 276, 280, 288 Jeschke, W. D. 64, 83 Jesse, T. 307, 324 Jhun, B. H. 38, 77, 84 Ji, C. 293, 301, 303, 304, 320 Jia, C. 13, 73 Jia, Y. 301, 303, 326 Jimenez, R. 163, 197 Jin, S. 301, 324 Johal, G. S. 295, 313, 316,322,323 Johansson, E, 15, 81 Johansson, I. 20, 77, John, M. E. 215, 246, 247, 280 Johnson, J. 307, 326 Johnson, L. B. 301, 328 Johnson, M. A. 209, 266, 275 Johnson, S. 94, 113 Johnson, T. B. 122, 197
K Kagan, I., 319 Kahn, R. A. 97, 111 Kaiser, A. 306, 318 Kaiser, B. N. 49, 77 Kaiser, W. M. 5, 83 Kaji, A. 249, 287 Kakitani, M. 303, 326 Kaku, H. 303,320 Kalberer, P. 174, 175, 197 Kalinski, A. 303, 320 Kalkkinen, N. 301, 324 Kalman, L. V. 13, 71 Kaloshian, I. 300, 324 Kamiya, Y. 143, 158 Kammerer, L. 94, 107 Kammerloher, W. 20, 77 Kamoun, S. 302,320 Kamprath, E. J. 3, 77 Kamyab, A. 214,221, 280 Kanatani, H. 119, 197 Kanehara, T. 145, 148, 151, 154, 155, 197 Kaneko, Y. 103,221, 223, 225, 226, 229, 230, 238, 265, 281, 283 Karban, R. 92, 93, I l l Karlsson, J. 314, 325 Karmoker, J. L. 19, 78 Kastelein, R. A. 308, 324 Kataev, A. A. 54, 78 Katagiri, F. 301, 321 Katayose, Y. 300, 327 Katchalski, E. 225, 243, 289 Kato, A. 150, 197 Kato, K. 209, 211, 214, 222, 230, 236, 274 Kato, M. 141, 145, 148, 151, 153, 156, 165, 167, 173, 185, 189, 193, 197 Katz, S. N. 188, 197 Kaueko, H. 93, 108 Kawaguchi, K. 225, 269,280
AUTHOR INDEX Kawasaki, S. 218, 219, 221, 224, 225, 257, 280 Kawata, E. E. 212,215,216,218,226,231,233, 242, 276 Kazan, K. 305, 320 Kearney, B. 297,320 Keen, N. T. 293, 294, 296, 297, 298, 299, 301, 302,303, 304,305,314,316,320,321,322, 324, 327 Kelemu, S. 297, 321 Keller, B. 216, 280, 300, 318 Keller, G. 215, 246, 247, 280 Keller, H. 122, 172, 187, 197 Kelley, W. N. 124, 197 Kendall, A. C. 30,89 Kerguelen, V. 101, 113 Kernstine, K. H. 163, 201 Kerr, P. S. 138, 197 Kerry, B. R. 3 1 1, 325 Kershaw, M. J. 293, 325 Kessmann, H. 94, 110, 112, 307,317, 318 Khambay, B. P. S.,103, 108 Khan, Z. R. 103, 104, 106, 107, 112 Khan, R.A. 97, 98, 111 Kiddle, G. A. 94, 109 Kido, S. 209, 240, 242, 243, 280 Kieliszewski, M. J. 214, 216, 218, 221, 280 Kihlman, B. 186, 197 Kihlman, B. A. 119, 186, 198 Kikuchi, A. 226, 229, 230, 238, 265, 281 Kikuchi, S. 251, 252, 281 Kim, H. S. 299, 301, 304, 315, 325 Kim, J. F. 298, 299, 316, 321 Kim, J. H. 128, 130, 131, 194, 198 Kim, S. 38, 84 Kim, Y.263, 283 Kimani, S. 103, 104, 106, 112 Kindl, H. 106,110, 309, 313, 319 King, B. J. 5, 18, 19, 78, 86 King, J. E. 127, 204 Kingan, T. G. 105, 114 Kinghorn, J. R., 75, 77, 79, 88, 89 Kingsley, M. T. 296, 326 Kinoshita, T. 224, 281 Kjellbom, P. 20, 77, 209, 213, 230, 257, 258, 260,266, 267, 272, 281, 283, 284, 287 Klein, R. R. 156, 198 Kleiner, D. 49, 78 Kleinhofs, A. 12, 87 Klessig, D. F. 293, 305,306, 308, 309, 313,317, 327, 328 Klis, F. M. 211, 218, 264, 275, 285, 288 Klobus, G. 13, 78 Klocke, J. A. 93, 97, 112 Knauf-Beiter, G. 94, I10 Knight, M. 53, 88 Knoester, M. 310, 321 Knogge, W. 301,321
339
Knox, J. P. 208, 209, 210, 211, 212, 219, 223, 226,227,230,233,236,244,245,246, 250, 251, 252, 253,254, 255, 257, 260, 265, 266, 261, 268, 269,271, 272,276, 281, 284, 286, 287, 288, 289 Knox, R. B. 211, 230, 236,238, 243, 256, 259, 274, 276 Kobayashi, D. 248, 291, 322 Kobayashi, K. 13, 85 Kobayashi, M. 23,78 Kobayashi, Y. 24, 82 Koch, B. 97, 110 Kochian, L. V. 5, 6, 9, 42, 47, 75, 78, 81, 89 Kodukula, K. 264, 288 Koetz, R. 145, 151, 187, 193 Koge, K. 187, 195 Kogel, K. H. 94, 110 Kolattukudy, P. E. 295, 318, 324 Kolchinsky, P. 298, 316 Koller, K. 302, 323 Kolwyck, D. 302, 325 Komalavilas, P. 209, 214, 236, 240, 253, 257, 258, 259, 260, 262, 281 Komamine, A. 239, 256, 287 Komatsu, H. 102, 111 Kombrink, E. 133, 198 Komeda, T. 175, 192 Komor, E. 41, 64, 86 Kondo, S. 164,200 Konings, H. 56, 79 Konings, W. N. 23, 85 Konishi, S. 145, 198 Kono, I. 300, 327 Kooman-Gersmann, M. 303, 321 Kosegarten, H. 41, 78 Koul, 0. 98, 108 Kovac, P. 223, 226, 230, 245, 271, 286, 288 Koyama,G. 164, 200 Koyro, H. W. 5, 8, 10, 13, 18, 53, 90 Koziel, M. 106, 108 Kraehenbuhl, J. P. 27, 85 Kramer, R. 50, 86 Krapp, A. 11, 12, 15, 17, 32, 76, 78, 84 Krath, B.N. 128, 198 Krause, E. 106, 110, 309, 313, 319 Kreitinger, M. 238, 279 Kretschmar, J. A. 155, 196, 199 Kreuger, M. 209, 210, 226, 236, 237, 238, 252, 255, 272, 281, 287 Krishnan, B.R. 50, 82 Kroj, T. 306, 322 Kronzucker, H. J. 3,4,9,44,45,46,48, 58, 75, 78 Kubo, I. 93, 97, 98, 112 Kubota, H. 120, 122, 155, 166, 167, 192 Kubota, K. 148, 198 Kiihne, R. 61, 85 Kulakauskas, S. 50, 82
340
AUTHOR INDEX
Kunze, M. 13, 78, 263, 281 Kurata, H. 187, 198 Kurata, N. 300, 327 Kurokawa, T. 119, 197 Kutchan, T. M. 94, 107, I10 Kwan, J. S. 239, 282 Kwiatkowski, S. 247, 248, 286 Kwik, K. D. 66, 77
L Labrador, E. 271, 277 Labrie, S. T. 2, 72 Lacelle, M. 24, 83 Lachaal, M. 38, 77, 80, 84 Lafleuriel, J. 131, 134, 136,194, 198 Lagarde, D. 47, 78 Laink, P. 6, 78 Lajide, L. 97, 109 Lakkenborg-Kristensen, H. 4, 74 Laloue, M. 128, 131, 135, 199, 202 Lam, E. 304,323 Lam, H. M. 2, 78 Lamaze, T. 18, 73 Lamb, C. 93,112,304,305, 306, 313,315,318, 321, 322 Lamb, C. J. 209,213, 230,257,258, 260, 266, 283, 294, 310, 313,316,318, 322 Lambers, H. 5, 56, 57, 63, 67, 72, 73, 79 Lamport, D. T. A. 211,212,214,216,218,221, 222, 227, 228, 280, 284 Landolt, P. J. 105, 112 Lang, W. C. 225, 274 Langan, K. J. 210, 236,238,239, 253,254,282 Lange, U. 13,28, 78, 263, 281 Langebartels, C. 305, 317 Langebartels, R. 106, 110, 309, 313, 319 Lara, C. 21, 23, 78, 79, 85 Larkin, P. J. 243, 256, 282 Laroux, S. 38, 72 Larsson, C. 20, 77 Larsson, C.-M. 3, 6, 15, 41, 42, 44, 52, 54, 63, 64, 77, 79, 83, 88 Larsson, M. 15, 20, 41, 42, 44,63, 64, 79, 81, 88, 314, 325 Lartey, R. 294, 321 Lauerer, M. 67, 85 Lauge, R. 301, 312,321 Lauren, D. R. 99, 107 Lauter, F. R. 31, 38, 45, 51, 79 Lavelle, D. T. 301, 303, 304, 325 Lavoie, N. 4, 40, 79 Lawrence, C. B. 313, 322 Lawrence, E. B. 313, 327 Lawrence, G. 300, 318 Lawrence, G. J. 308, 32f Lawton, K. 94, 110, 307, 324 Lawton, K. A. 94, 112 Layzell, D. B. 63, 79, 119, 194
Lazdunski, M. 27, 70 Lazof, D. B. 59, 79 LeFloc’h, F. 131, 134, 136, 138, 140, 194, 195, 198 Le Mttayer, M. 101, 108, 113 Leckie, F. 312, 317 Lee, D. 134, 198 Lee, H. 311,327 Lee, R. B. 5, 6, 9, 13, 14, 15, 18,42,43,45,48, 53, 55, 58, 71, 79, 80 Lee, S. W. 301, 324 Lee, S.-F. 127, 204 Lee, Y.-W. 98, 112 Lefebvre, F. 64,76 Lefebvre, P. A. 26,84 Lefebvre, D.D. 119, 194 Leggett, J. E. 48, 86 Leigh, R. A. 5, 8, 10, 13, 18, 53, 84, 90 Leister, R. T. 301, 321 Lemaitre, B. 308, 311, 321 Lemoine, J. 217, 218, 283 Lennon, K. A. 211, 233,282 Leon, J. 306, 313, 324, 327 Leonard, R. T. 17, 30, 73, 83, 85 Lepetit, M. 47, 78 Lesage, F. 27, 70 Lesch, S. 41, 42, 44,88 Letham, D. S. 186, 199 Leven, A. 186, 197 Levesque, C. A. 310, 326 Levine, A. 304, 321 Levine, E. B. 313, 327 Lew, R. R. 55, 80 Lewald, J. 300, 319 Lewis, G. P. 228,229, 274 Lewis, W. J. 92, 103, 104, 109, 114 Ley, S. V. 98, 112 Leydecker, M. T. 17, 76 Leyh, T. S. 50, 82 Leykam, J. 218, 280 Leykam, J. F. 214, 221, 280 Li, D. 310, 321 Li, J. 300, 322 Li, S.-X. 209, 215, 216, 217, 218, 282 Li, X. 120, 205, 307, 313, 317 Li, X.N. 124, 125 Li, Y.-Q. 247, 248, 249, 282, 284 Liang, Y. K. 301, 324 Lidell, M. C. 301, 324 Light, D. M. 105, 109, 172 Ligterink, W. 306, 322 Lilley, C. J. 314, 326 Lilley, G. G. 243, 288 Lin, C. Q. 273, 282 Lin, J. T. 11, 21, 23, 80 Lin, Y. 120, 205 Lind, J. L. 221, 230, 232, 235, 271, 282, 285 Lindell, S.D. 138, 194
AUTHOR INDEX Lindgren, P. B. 298,322 Lindgren, P.-E. 24, 74 Lindhout, P. 301, 312, 321 Lindigkeit, R. 99, 112 Lindsay, W. P. 306,318 Linehan, D. J. 60, 85 Linskens, H. F., 203, 249, 284 Linstead, P. 245, 265, 267, 269, 277 Linstead, P. J. 245, 246, 267, 269, 271, 281 Linthorst, H. J. M. 310, 321 Lipke, P. N. 264, 288 Lipps, R. C. 60, 75 Lips, S. H. 65, 70 Lis, H. 225, 243, 289 Liu, H. Z. 38, 77, 80, 84, 86 Liu, Y.308, 317 Llewellyn, D. J. 305, 320 Lloyd, C. W. 208, 285 Lo Schiavo, F. 238, 277 Loake, G. J. 306, 318 Lohammar, T. 61,81 Loniello, A. 0. 301, 319 Loopstra, C. A. 215, 216, 217, 218, 246, 282 Looser, E. 143, 144, 145, 153, 198 Lopez-Solanilla, E. 3 11, 322 Lorang, J. M. 297, 298, 322 Lord, E. M. 210,211, 226,233, 235, 238, 248, 249, 253, 280, 282, 285 Lorenz, M. C. 51,80 Lorito, M. 313, 322 Lottspeich, F. 303, 323 Louda, S. 94, 112 Loughrin, J. H. 92, 103, 104, 107, 114 Lovatt, C. J. 126, 127, 173, 174, 198,200 Lovell, H. 98, 112 Low, M. G. 261,263, 282 Lowery, D. T. 98, 112 Lucas, W. J. 46, 47, 63, 78, 80, 85 Luck, J. 300, 318 Lui, J. 139, 196 Lund, A.B. 15, 61, 77 Lundborg, T. 15, 81 Lundeberg, J. 314, 325 Lunness, P. 294, 295, 316, 323 Luque, F. 24, 84 Lush, W. M. 232, 233, 234, 247, 286 Luttge, U.11, 13, 72, 73 Lwande, W. 103, 104, 106, 112 Lyon, G. D. 306,322 Lyon, J. L. 243, 244, 256, 283
M
Maathuis, F. J. M. 51, 80 Macduff, J. 6, 15, 70, 78 Macduff, J. H. 15,40, 42, 52, 55, 56, 61,80, 90 MacIntosh, C. 306, 322 Mack, G. 10, 12, 45,80,86
34 1
Mackie, W. 223, 226, 230, 233, 245, 246, 250, 265, 266, 267, 267, 271, 288, 289 MacKintosh, R. W. 306, 322 Macklon, A. E. S. 6, 80 MacKown, C. T. 48, 86 Maclachlan, G. 224, 225, 279 MacVean, C. D. 187,204 Maeda, K. 22,80 Maeda, S. 164,200 Maffei, A. 302, 322 MagalhBes, C. 175, 199 Magasanik, B. 131, 198 Mager, J. 131, 198 MagetDana, R. 31 1, 322 Maher, E. A. 294, 322 Majewska-Sawka, A. 256, 275 Malandrin, L. 298, 319 Malcolm, S. B. 99, 112 Maldonaldo, J. M. 14, 15, 18, 73 Malloy, J. A. 211, 219, 236, 274 Manandhar, G. 186, 198 Manjula, B. N. 226, 265, 279 Manka, M. 38,84 Manners, J. M. 305, 320 Mansfield, J. 299, 322 Mansfield, J. W. 298, 325 Manulis, S. 296, 326 Marchalonis, J. J. 230, 281 Marchetti, N. 229, 230, 283 Marciniak, J. 13, 78 Marger, M. D. 33, 80 Margolis, H. A. 4, 40, 79 Marini, A. M. 46, 50, 51, 80, 81 Marion-Poll, F. 101, 108, 113 Marois, E. 301, 326 Marschner, H. 58, 60, 61, 62, 65, 74, 81, 85 Martin, F. 3, 81 Martin, G. 299, 318, 322 Martin, G. B. 301,302, 303,306, 312,318,326, 328 Martin, H. 209, 213, 226, 230, 233, 236, 257, 258, 260, 261, 286 Martinez, M. 228, 229, 277 Mary, B. 18, 73, 79 Marzluf, G. A. 2, 1 I , 17, 81 Mascara, T. 224, 225, 282 Master, E. P. 106, 111 Masner, P. 94, 110 Masoud, S. 310, 313, 318, Masoud, S. A. 313, 322 Massiot. P. 64. 76 Mathur,’ S. N.’I 19, 201 Matschke, M. 13, 87 Matsumura, S. 187, 198 Matsuo, N. 103, 108 Matsuoka, K. 218, 282 Matsushima, H. 226, 229, 230, 238, 265, 281 Mattei, B. 312, 317
342
AUTHOR INDEX
Matthews, B. F. 303, 320 Matthews, M. A. 19, 84 Mattiacci, L. 104, 112 Mattoo, A. K. 105, 114 Mattsson, M. 15, 81 Mau, S.-L. 209, 213, 214, 215, 216, 217, 218, 226, 231, 232, 233, 241, 261, 276, 282 Maurel, C. 20, 81 Maurousset, L. 7, 24, 71 May, B. 212, 215, 216, 218, 226, 231, 232, 233, 242, 276 Mayaux, J. F. 24,83 Mayda, E. 300,326 Mayer, J. E. 306, 318 Mayow, J. 67, 81 Mazzafera, P. 122, 123, 145, 151, 153, 156, 172, 175, 198, 199 McBeath, J. H. 311, 313, 319 McCabe, P. C. 25, 77 McCabe, P. F. 209, 250, 251, 258, 260, 266, 267, 272, 281, 282, 284 McCairns, E. 126, 193 McCall, P. J. 104, 114 McCann, M. C. 209, 212, 214, 221, 222, 225, 236, 246, 275, 282, 286 McClure, P. R. 5, 6, 30, 81 McConville, M. J. 263, 278 McCorrnick, K. D. 102, 109 McCormick, S. 312, 327 McDonald, A. J. S. 61, 81 McFadden, H. 301, 308,317 McGowan, M. 4, 60, 76, 89 McKenzie, R. J. 229, 238, 279 McKinney, E. C. 128, 131, 202 McMullen, J.-N. 227, 228, 274 McNab, C. G. A. 228, 274 McNally, J. G. 273, 284 McNamara, M. 209, 212, 214, 236, 259, 279 McNeil, M. 208, 230, 236, 277, 287 McPherson, M. J. 314, 326 McWhinnie, E. A. 135, 199 Meagher, R. B. 128, 131, 202 Mecsas, J. 298, 323 Medzhitov, R.308, 322 Meeley, R. B. 97, 106, 110, 295, 313, 322, 323 Meharg, A. A. 6, 7, 24, 71, 81 Mei, L. 306, 327 Meier, B. 94, 110 Meijer, P. J. 305, 315 Meinwald, J. 99, 100, 102, I09 Melchers, L. S. 312, 317 Melo-Oliveira, R. 2, 78 Melroy, D. L. 303, 320 Melton, R. E. 295, 323 Menzies, A. R. 211,214, 222,226, 221, 228,283 Merritt, L. A. 98, 110, 113 Mert, F. 309, 319 Mes, J. 300, 325
Messchendorp, L. 99, 110, I12 Metcalf, T. N. 230, 288, Mttraux, J.-P. 94, 110, 302, 325 Metz, M. 306, 315 Meyer, C. 2, 81 Meyer, R. 124, 199, 205 Meyer, Y. 255, 282 Meyerink, P. 252, 277 Meyers, S. 138, 204 Michalski, J.-C. 217, 218, 283 Michaut, L. 308, 321 Michelmore, R. W. 301, 303, 325 Michon, V. 219, 223,285 Midland, S. L. 301, 303, 304. 320 Mihara, T. 156, 196 Millenaar, F. 57, 79 Miller,A. J. 5 , 8, 9, 10, 13, 18, 25,26, 31, 32, 53, 81, 89, 90 Miller, D. M. 53, 81 Miller, J. F. 302, 325 Miller, J. R. 106, 112 Milligan, S. B. 300, 324 Mineyuki, Y. 186, 199 Minsavage, G. V. 298, 327 Minsnall, W. M. H. 19, 81 Misawa, H. 223, 225, 283 Miskiel, F. J. 229, 230, 283 Mistrik, I. 6, 7, 82 Mita, M. 119, 191, I99 Mithen, R. 94, 107, 110 Mithofer, A. 303, 323 Mitsui, K. 124, 131, 143, 192 Mittal, R. 138, 199 Mittler, R. 304, 323 Miyamoto, J. 106, 108, 111 Mizuno, K. 145, 151, 153, 154, 155, 156, 189, 197 Mizutani, J. 97, 109 Mocak, J. 228, 283 Mochizuki, N. 223, 226, 288 Mock, H.-P. 256, 283 Moeder, W. 305, 317 Moffatt, B. 135, 199 Moffatt, B. A. 135, 198, 199, 201 Mohan, S. 24, 83 Mole, S. 94, 112 Molenaar, D. 23, 85 Molina, A. 293, 307, 324 Moll, R. H. 3, 51, 54, 77, 87 Mollard, A. 214, 283 Msller, B. L. 97, 98, 110, 111 Monack, D. M. 298, 323 Montague, P. 25, 88 Monteiro, A. M. 139, 147, 159, 161, 171, 173, 174, 177, 178, 179, 181, 192, 193 Montezinos, D. 222, 276 Montreuil, J. 217, 218, 283 Moody, S. F. 211, 226, 227, 274, 283
AUTHOR INDEX Moorby, H. 58,82 Morath, P. 145, 146, 151, 152, 154, 187, 193, 202 Mordue (Luntz), A. J. 93, 112 Morel, M. H. 30, 69 Morgan, M. A. 44, 45, 82 Morita, N. 263, 283 Moritz, R. L. 209, 213, 214, 215, 216, 217, 226, 231,261, 276, 277, 282 Moritz, T. 139, 174, 177, 178, 179, 181, 193 Morotgaudry, J. F. 2, 81 Morre, D. J. 13, 70 Morris, H. R. 223,226,230,233,245,246,250, 265,266, 267, 269, 271, 289 Morris, S. 94, 115 Morvan, H., 239, 282 Mosli Waldhauser, S. S. 146, 151, 155, 199 Mothes, K. 139, 201 Motoyoshi, F. 300, 323 Mourgues, F. 31 I , 323 Moutounet, M., 275, 285 Mowery, P. 300, 326 Muccifora, S. 313, 322 Muchhal, U. S. 33, 82 Mudd, A. 98, 110, 113 Mueckler, M. 38, 82 Mueller, M. 94, 107 Mukerji, D. 119, 201 Mukoyama, H. 209, 222, 223, 226, 288 Muldin, I. 31, 82, 90 Muller, B. 3, 17, 55, 63, 64, 66, 82, 88 Muller, C. 302, 325 Muller, W. H. 264, 288 Muller, M. J. 94, 110 Muller-Rober, B. 67, 85 Mullet, J. E. 69, 70 Multani, D. S. 313, 323 Mumenthaler, C. 308, 325 Munch-Petersen, A. 124, 199 Munkle, L. 62, 74 Munro, S. L. A. 212, 219, 221, 222, 223, 278 Murakami, K. 138, 205 Murata, M. 300, 323 Murphy, J. B. 294, 316 Murray, F. R. 305, 320 Myers, C. 273, 275 Myers, R. L. 102, 109
N Nagahama, Y. 119, 199 Nagata, T. 120, 186, 191, 200 Nahrstedt, A. 93, 112 Naider, F. 31, 32, 87 Nakade, S. 33, 71 Nakamura, H. 164, 200 Nakamura, K. 218, 223, 282, 283 Nakanishi, K.,98, 112, 119, I97 Nakano, M. M. 24,83
343
Nakazato, H. 263, 283 Narasimhan, M. L. 311, 327 Nasser, W. 297, 320 Nathanson, J. A. 119, 200 Navarro, M. T. 12, 82 Nazario, G. M. 126, 173, 174, 200 Negishi, 0. 135, 145, 147, 151, 152, 153, 156, 159, 161, 163, 164, 200 Negrotto, D. 307, 317, 318 Nelson, R. R. 295, 325 Nennstiel, D. 303, 323 Neuenschwander, B. 186, 193 Neuenschwander, U. H. 293, 307,324 Neuhard, J. 124, 200 Neukom, H. 218,287 Neuwald, A.F. 50, 82 Newman, J. 306, 327 Ngambi, J. M. 10, 82 Nguyen, J. 139, 200 Ni, M. 30, 82 Ni, W. 294, 322 Nickisch-Rosengk, E. 100, 113 Nicolas, G. 271, 277 Nicolson, R. 24, 83 Niderman, T. 127, 204 Nielsen, H. L. 97, 110 Niemeyer, H. M. 94, 110 Niklas, A. 256, 275 Ninnemann, 0. 27, 31, 38,42,45,46,48, 51, 75, 79,82 Nishikoori, M. 263, 283 Nishimura, K. 160, 164, 200 Nishimura, M. T. 300, 316 Nishizawa, N. 253, 273, 287 Nizan, R. 296, 326 Nobel, P. S. 65, 89 Nobusawa, E. 133, 140, 192, 200 Nocolas, E. 308, 321 Nohno, T. 23,82 Noji, S. 23, 82 Nordeen, R. 0. 311, 313, 319 Norman, P. M. 209, 230, 257, 258, 260, 266, 283 Northcote, D. H. 143, 144, 187, 200, 225, 283 Nothnagel, E. A. 207, 208,209,210, 212, 214, 216, 217, 219, 221, 235, 236, 238, 239, 240, 241, 243,244, 249, 253, 254, 255, 256, 257, 258,259,260,262,263,267,273,274,280, 281, 282, 283, 286, 287 Nottingham, S. F. 102, 113 Novacky, A. 41,42, 44, 88 Novacky, A. J. 6, 88 Nurmaiho-Lassila, E.-L. 301, 324 Nurnberger, T. 303, 323 Nye, G. 307, 318 Nye, P. H. 58, 4, 82 Nygaard, P. 126, 128, 131, 143, 192, 200, 202
344
AUTHOR INDEX
0 Ocando, E. 228, 229, 277 Ochman, H. 299, 319 OConnell, R.J. 100, 108 Oechslin, M. 122, 193 Ogasawara, N. 24, 82 Ogawa, K. I. 24, 83, Ogawa, N. 33, 90 Ogawa, T. 23,83, 222, 280 Ogawa, 24, 83, 90 Ogura, K. 209, 222,223, 226, 288 Oguri, H. 156, 196 Ogutuga, D. B. A. 143, 144, 187, 200 Ohinata, A. 226, 229, 230, 238, 265, 281 Ohishi, K. 224, 281 Ohm, H. 301,318 Ohmori, M. 23,83 Ohmori, T. 300, 323 Ohno, M. 164,200 Okamoto, T. 263, 283 Okinaka, Y. 301, 303, 304, 320 Okuyama, H. 263,283 Oldham, N. J. 99, 113 Oldroyd, G. E. D. 301, 304, 312, 323, 324, 325 Oliveira, I. C. 2, 78 Oliver, R. P. 295, 323 Olsson, 0. 145, 151, 153, 154, 156, 172, 199, 314, 325 Omata, T. 2, 11, 21, 22, 23, 78, 80, 83, 87 Omholt, T. E. 5, 30, 81 ONeill, M. 218, 280 ONeill, M. A. , 239, 243, 283 Onstenk, J. 300, 325 Onyeocha, I. 11, 12, 13, 17, 25, 32, 37, 69, 88 Oostendorp, M. 94, 110 Oostindier-Braaksma, F. J. 28, 83 Oparka, K. J. 294, 323 Opperman, C. 314, 323 Opperman, C. H. 300,318 Orihara, Y. 187, 195 Orlandi, E. W. 304, 316 Orlando, R., 201, 280 Osborn, R. W., 316, 317, 326 Osbourn, A. 295, 323 Osbourn, A. E. 294, 295, 316, 323 Oscarson, P. 6, 52, 54, 77, 83 Oshima, Y. 33, 71, 90 Osman, M. E. 21 1,214,222,226,227,228,283 Otten, H. 28, 74 Ourry, A. 6, 78 Overholt, W. A. 103, 104, 106, 112 Owens, L. D. 311, 313, 319 Owens, R. J. 225, 283 Oxley, D. 210,215,217,221,255,261,264,272, 273, 285, 289 Ozawa, T. 135, 138, 145, 147, 151, 152, 153, 154, 156, 159, 161, 163, 164, 200
P Paans, A. J. M. 6, 72 Pace, G. M. 5, 30,81 Padgett, H. S. 294, 323 Padgett, P. E. 30, 17, 83, 85 Paiva, N. L. 310, 313, 318 Pajares, J. 102, 110 Palmer, L. E. 298, 323 Panaccione, D. G. 296, 315 Pang, M. K. I. 44,84 Pao, S. S. 33, 83 Papadopoulos, K. 302, 322 Pardo, J. M. 33, 82, 311, 327 Pare, P. 104, 109 Pare, P. W. 104, 113 Parker, D. M. 302, 323 Parker, J. 304, 327 Parker, J. E. 306, 315, 323 Parks, R.E. 164, 194 Parniske, M. 300, 323, 326 Passama, L. 65, 66, 67, 73 Pasteels, J. M. 100, 111, 113 Paszkowski, J. 134, 193 Pate, J. S. 3, 63, 79, 83 Patel, S. 311, 317 Paterson, A. H. 313, 323 Paterson, A. R. P. 127, 139, 142, 196 Patterson, F. 301, 318 Pau, W. L. 3, 77 Paulin, J. P. 298, 319 Paulsen, I. T. 33, 34, 83 Pawson, T. 304, 308, 324 Pazur, J. H. 229, 230, 283 Peakman, T. 24,83 Pearce, G. 94, 113 Pearson, C. J. 66, 83 Peart, J. 245, 246, 267, 269, 271, 281 Peart, J. M. 209, 230, 237, 250, 257, 260, 265, 266, 268, 271, 284 Peerless, A. C. J. 156, 204 Pellerin, P. 223, 239, 249, 283, 284 Pennell, R. I. 208, 209, 223, 230, 236, 237, 239, 244,250,251,257,258,260, 265,266,267, 268,271,272, 281, 282,284, 287, 305, 315 Pereira, A. 300, 315 PBrez, M. D. 28, 70, 83 Pernollet, J. C. 296, 299, 316 Pesakova, M. 175, 204 Petermann, J. 122, 175, 201 Pethe, C.,135, 199 Pettersson, J. 65, 85, 94, 103, 113 Peuke, A. D. 5, 64, 83 Pham-Deligue, M.-H. 101, 108, 113 Phillips, G. 0. 21 I, 214,222,226,228,280, 283, 284 Phillips, T. W. 105, 112 Pi, L.-Y. 300, 326 Pickard, B. F. 273, 284
AUTHOR INDEX Pickett, J. A. 91, 93, 94, 97, 98, 99, 100, 101, 102, 103, 105, 106, 107,108, 109, 110, 111, 112, 113, 114 Piechottka, G. P. 20, 77 Piel, J. 104, 108 Pierson, E. S. 248, 249, 282, 284 Pilnik, W. 243, 285 Pintor-Toro, J. A. 313, 322 Pirhonen, M. U. 301, 324 Pistorius, E. K. 11, 83 Plank-Schumacher, K.-H. 126, 201 Pnjak, A. 224, 285 Podila, G. K. 295, 318 Pogson, B. J. 215, 284 Polacco, J. C. 136, 138, 139, 205 Polle, A. 64, 84 Polya, G. M. 138, 201 Pont-Lezica, R. F. 273, 284, 285 Pope, A. J. 10,84 Pope, D. G . 218,284 Poppy, G. M. 103, 109, 114, 311,325 Porcelli, S . 302, 316 Posthumus, M. A. 104, 112, 114 Postma, E. 210, 237, 281 Powell, G. 99, 114 Powell, W. 100, 109, 113 Prasher, D. C. 134, 201 Prestidge, R. A. 99, 107 Preston, G. 299, 317 Preston-Hurlburt, P. 308, 322 Prevot, J.-C. 127, 134, 195 Prieto, R. 12, 18, 82, 84 Prior, D. A. M. 294, 323 Prioul, J. L. 19, 20, 70, 76 Proiser, E. 126, 161, 201, 202 Prusky, D. 294, 324 Pryor, T. 300, 318 Ptak, M. 311, 322 Pu, Z.-Y. 209,212,213,214,215,216,217,221, 226, 23 1, 232, 242, 26 1, 276, 282, 285 Pullman. G. 238. 279 Purves, J. V. 6, 14, 19,45, 55, 61, 63,64, 72, 78, 79, 80 Pye, B. J. 94, 103, 113
Q
Qi, W. 211, 214, 218, 221,222, 227, 228,284 Oiu. X. 209.215. 216. 247. 284 Quesada, A: 11, 12, 15, 17: 26, 27, 32, 39, 75, 78. 84 Quilici,’D; R. 99, 114 Quinones, M. A. 24, 69 Quiroz, A. 94, 103, I13
R Radin, J. W. 19, 84 Raemaekers, R. 31 1, 317 Raghothama, K. G. 33,82
345
Raina, A. K. 105, 114 Rainbird, R. M. 138, 201 Rains, D. W. 14, 52, 56, 70 Rains, W. D. 12, 87 Raistrick, N. 15, 70 Ram, A. F. J. 264,275 Raman, D. R. 58, 59, 76 Ramirez, J. M. 24, 69 Ramos, F. 24, 84 Rampal, A. L. 38, 77, 84 Rance, I. 305,324 Randall, D. D. 127, 136, 138, 139, 197, 201, 204, 205 Randall, R. C. 227, 228, 284 Randoux, T.,100, 113 Raper, C. D. 13,49,85 Rapp, B. J. 138, 197 Rapport, M. M. 214, 289 Raschke, K, 9, 57, 70, 89, 90 Raskin, I. 103, 114, 313, 327 Ratcliffe, R. G. 14, 42, 43, 45, 58, 80 Rathjen, J. P. 301, 303, 325 Ray, A. K. 227, 228, 284 Read, D. J. 3, 87 Redinbaugh, M. G. 59, 79 Reed, B. C. 38, 72 Regan, S. 314, 325 Regan, S . M. 135,201 Regenass, M. 306, 318 Reichhart, J.-M. 308, 311, 321 Reid, J. D. 47, 70 Reidenbach, G. 60, 84 Reif, H.-J. 106, 110, 309, 313, 319 Reijans, M. 300, 325 Reinbothe, H. 139, 201 Rennenberg, H. 64, 82, 84, 86 Rentsch, D. 4, 31, 75 Repetti, P. P. 306, 317 Resch, J. 99, 109 Resta, R. 13, 84 Reuzeau, C. 209, 258, 260, 266, 267, 273, 281, 285 Reverchon, S. 297, 320 Reynolds, P. H. S. 127, 201 Rhodes, M. J. C. 156,204 Rice, E. L. 119, 201 Richardson, A. 11, 12,32, 33, 34,37, 39,68,88, 278 Ridgway, D. 299, 320 Ried, J. L. 296, 297, 324 Riedel, J. 13, 78, 263, 281 Riesmeier, J. W. 31, 38, 45, 51, 79 Rincon, M. 53,84 Ripoche, P. 20, 81 Ripoll, C. 64, 76 Ritchie, A. 126, 193 fitter, C. 297, 324 Rivas, C. 228, 229, 277
346
AUTHOR INDEX
Rizvi, S. J. H. 119, 201 Robert, L. S. 209, 215, 216, 247, 278 Roberton, A. M. 229, 238, 279 Roberts, J. K. M. 44,84 Roberts, K. 208, 209, 212, 214, 221, 222, 223, 230,236,237,239,243,244,245,246,250, 251, 257, 260,265, 266,267, 268, 269,270, 271, 275, 277, 281,282, 284, 285, 286 Roberts, M. F. 145, 151, 166, 201 Robins, R. J. 156, 204 Robins D. J. 156, 204 Robinson, D. 60, 61, 62, 85 Robinson, D. G. 225, 274 Robinson, D. S. 187, 196 Rock, F. L. 308, 324 Rodgers, M. W. 225, 285 Rodriguez, R. 21, 23, 78, 79, 85 Rodriguez-Garcia, M. I. 256, 275 Rodriguez-Navarro, A. 47,85 Rodriguez-Palenzuela, P, 3 11, 322 Rogers, E. E. 293, 306, 309, 319, 324 Rogers, L. M. 295, 324 Rohde, A. 314,325 Rohring, L. 150, 193 Rohringer, R. 243, 285 Roine, E. 301, 324 Rojo, E. 306, 324 Romanczyk Jr, L.J. 123, 196 Romantschuk, M. 301, 324 Rombouts, F. M. 248, 285 Romero, J. M. 23, 79 Romey, G. 27, 70 Romheld, V. 58, 81 Rommens, C. M. T. 301, 304, 312, 324, 325 Ron, M. M. 6,80 Ronald, P. C. 297, 300, 320, 326 Ronquist, F. 65, 90 Rook, F. 67, 86 Rooney, J. M. 19, 78 Rose, U. S. R. 104, 114 Rosenberg, L. A. 30, 85 Rosenthal, G. A. 93, 108, 111, I12, 114 Ross, C. W. 123, 131, 201 Rossi, M. 300, 324 Rossier, B. C. 27, 85 Rothschild, M. 92, 99, 100, 107, 114 Rowe, J. J. 23, 85 Rowe, P. B. 126, 193 Rowell-Rahier, M. 100, 111, 113 Rowley, D. L. 301, 324 Roy, S., 79, 88, 210, 211, 226, 233, 234, 235, 236, 237, 238, 249,253, 282, 285 Ruan, D.-L. 300, 326 Rubio, F. 47, 85 Rudge, K. A. 48, 80 Ruess, W. 94, 110 Rufty, T. W. 6, 8, 9, 13, 15, 49, 59, 75, 79, 85, 86
Russell, E. W. 67, 85 Ruth, T. J. 6, 8, 9, 12, 13, IS, 18, 19,41, 42,44, 45, 46, 48, 53, 57, 58, 75, 78, 86, 89 Ryals, J. 94, 110, 112, 115, 307, 317, 318, 324 Ryals, J. A. 293, 307, 324 Ryan, C. A. 93,94, 107, 108, 113
S Saba, T. 190, 203 Sabina, R. L, 163, 201 Sabularse, D. 222, 276 Sadaie, Y. 24, 82 Sadka, A. 69, 70 Saier, M. H. 33, 34, 80, 83 Saito, H. 13, 90 Saito, T. 23, 82 Sakai, S. 120, 200 Sakaino, M. 102, 109 Sakakibara, H. 13, 85 Saker, L. R. 6, 14, 19, 45, 62, 72, 74, 78, 80 Salette, J. 6, 78 Salmeron, J. M. 301, 304, 312, 324, 325 Salvi, G. 312, 317 Samson, M. R. 211, 285 Samuel, S. J. 38, 80, 86 San Segundo, B. 311, 317 Sanchez-Serrano, J. J. 306, 324 Sandberg, G. 145, 151, 153, 156, 172, 175, 154, 199, 314, 325 Sandermann, H. 305, 317 Sanders, D. 51, 80 Sanders, L. C. 235, 285 Sandrin, M. S. 230, 274 Sandrock, R. W. 295, 325 Sandstrom, J. 65, 85 Saneyoshi, M. 119, 199 Sanson, T. 35, 72 Santa Cruz, S. 294, 323 Santa-Maria, G. E. 47, 85 Sasaki, T. 300, 327 Satoh, S. 251, 252, 281 Sattelmacher, B. 61, 85 Sattler, A. 300, 316, 319 Sauer, D. 126, 193 Saulnier, L. 218, 219, 223, 285 Sawers, G. 24, 87 Sawert, A. 126, 201 Scala, F. 313, 322 Schachermayr, G. 300, 318 Schachtman, D. P. 46,47, 51,85 Schaff, D. A. 135, 199 Schaffner, A. R. 20, 77 Schardl, C. L. 310, 321 Scheel, D. 303, 304, 306, 320, 322, 323 Scheffer, R. P. 295, 325 Scheible, W. R. 67, 85 Scheible, W.-F. 12, 15, 17, 78 Scheline, R. R. 119, 186, 201
AUTHOR INDEX Schell, M. A. 297, 319 Schenk, M. K. 60,87 Scherer, H. W. 48, 86 Scheres, B. 215, 285 Scheunvater, I. 5, 56, 57, 67, 79 Schibeci, A. 224, 285 Schiebel, H.-M. 99, 112 Schindler, M. 222, 275 Schindler, T. 238, 239, 245, 246, 255, 267, 269, 285 Schjoerring, J. K. 3, 44, 46, 48, 75 Schlee, J. 41, 86 Schmelzer, E. 106, 110, 309, 313, 319 Schmidt, E. D. L. 237,238, 250,287 Schmidt, H. 175,204 Schneider, D. 100, 113 Schneider, P. 261, 263, 285 Schneider, S. ,64,86 Schnell, R. A. 26,84 Schnorr, K. M. 131, 202 Schobert, C. 64,86 Schols, H. A. 243,285 Scholten, H. J. 28, 86 Schopfer, P. 209, 225, 238, 239, 245, 246, 255, 267, 269, 285 Schrader, L. E. 138, 204 Schreier, P. H. 106, 108, 110, 309, 313,319,326 Schroeder, J. I. 9, 11, 12, 30, 31, 38, 40, 46, 51, 85, 88 Schubert, K. R. 126, 139, 194, 202 Schuler, F. 13, 87, 263, 287 Schuler, T. H. , 311, 325 Schulthess, B. 152, 202 Schulthess, B. H. 123, 145, 146, 152, 154, 194, 202 Schultz, C. 210, 215, 255, 264, 272, 273, 285 Schultz, C. J. 212, 215, 216, 221,231, 232,242, 285 Schulze, E. D. 67, 85 Schweizer, P. 302, 325 Scofield, G. N. 208, 209, 212, 230, 236, 237, 239, 250, 251,257, 260,265, 266, 268, 271, 284 Scofield, S. R. 301, 303, 304, 325 Scott, J. D. 304, 308,324 Scott, J. W. 298, 327 Scudder, G. G. E. 98, 108 Sealy, J. R. 120, 202 Searle-van Leeuwen, M. F. 243,285 Sederoff, R. R. 215, 216, 217, 218, 246, 282 Sedgley, M. 231, 286 Seigler, D. S. 97, 114 Seitz, H. U. 256, 283 Selvendran, R. R. 209, 212, 236, 239, 243, 257: 260, 265, 266, 283, 284 Senanayake, U. M. 123,202 Senecoff, J. F , 131, 202 Sentenac, H. 45, 47, 70, 76, 86
347
Serenkov, G. P. 126, 161, 201, 202 Serpe, M. D. 207,209, 210, 238, 239, 240, 241, 243,253,254,255,258,259,260,262,267, 274, 286 Seskar, M. 304, 323 Sessa, G. 302, 318 Sewalt, V. J. H. 310, 313, 318 Seybold, S. J. 99, 114 Shaff, J. E. 5, 6, 9, 42, 75, 78, 81, 89 Shah, D. M. 303,327 Shah, J. 293, 305, 327 Shah, S. J. 98, 110 Shapiro, A. D. 306, 317 Sharma, C. B. 138, 199 Shaw, P. 208, 285 Shea, E. M. 211, 223, 236, 239, 243, 256, 276, 286 Sheen, J. 67, 77 Shelp, B. J. 127, 136, 193, 202 Shen, H. 297, 298, 302, 322, 325 Shervington, A. 187, 202 Shervington, L. A. 187, 202 Shevchik, V. E. 297, 325 Shi, Y. W. 38, 80, 86 Shibuya, N. 209, 218, 221, 222, 223, 225, 226, 269, 280, 287, 303, 320 Shikata, K. 33, 71 Shimazaki, A. 140, 202 Shimizu, H. 145, 148, 151, 154, 155, 175, 190, 192, 197 Shirai, H. 119, 197 Shishido, K. 124, 203 Shockey, J. 310,326 Shoji, T. 156, 196 Shortt, B. J. 313, 327 Showalter, A. M. 209, 215, 216, 217, 218, 282, 286 Shulaev, V. 103, 114, 304, 323 Shuster, L. 143, 202 Siddiqi, M. Y. 3,4, 5, 6, 8, 9, 12, 13, 15, 18, 19, 30,41,42,44,46, 48, 53, 57, 58, 75, 78,86, 89 Siebrecht, S. 12, 86 Siegerist, M. 13, 14, 71 Siehl, D. L. 127, 204 Sieris, S. 300, 326 Siewe, R. M. 50, 86 Sigon, C. A. M. 21 1, 285 Silverman, P. 103, 114 Silverstone, S. 301, 324 Sim, A. 6,80 Simcox, K. 97, 106, 110 Simonich, M. T. 300, 316 Simons, G. 300,325 Simpson, J. 296, 319 Simpson, R. J. 63, 79, 209, 213, 214, 215, 216, 217, 226, 231, 261, 276, 277,282 Sims, J. J. 301, 303, 304, 320
.
348
AUTHOR INDEX
Singh, N. 239, 243, 252, 279 Singh, R. 138, 191 Siverio, J. M. 28, 32, 37, 70, 83 Skerrett, M. 9, 86 Slaymaker, D. 301, 303, 304, 320 Slayman, C. L. 47, 70 Sljivo, A. 222, 227, 228, 280 Smallwood, M. 209, 226, 230, 233, 236, 257, 258,260, 261,281,286 Smart, D. R. 14, 86 Smart, L. E. 93, 97, 98, 99, 103, 110, 112, 113 Smedley, S. R. 102, 109 Smeekens, S. 67, 86 Smiley, D. W. M. 91, 106,111 Smith, C. M. 93, 114 Smith, D. A. 310, 321 Smith, F. 21 1, 286 Smith, F. W. 33, 86 Smith, J. W. 105, 109 Smith, M. C. 98, 105, 107, 113 Smith, N. M. 300, 316 Smith, P. 230, 281 Smith, S. E. 3, 87 Smith, S. J , 5, 9, 10, 13, 18, 53, 81, 89 Smith-Becker, J. 293, 303, 304 Smithies, 0. 34, 37, 73 Snogerup, L. 209, 258, 260, 266, 267,272, 281, 284, 286, 287 Snowden, M. J. 222, 227, 228, 280 Somerville, C. R. 135, 199 Somerville, S. 293, 308, 327 Somerville, S. C. 300, 316 Sommer-Knudsen, J. 210, 215, 218, 231, 232, 233, 234,240,241, 242, 243, 247, 286 Somssich, I. E. 293, 305, 325 Song, W. 31,87 Song, W.-Y. 300,326 Soole, K. L. 263, 287 Soussi-Boudekou, S. 50, 51, 81 Southworth, D. 247, 248, 286 Spangler, R. A. 38, 84 Spanswick, R. M. 6, 43, 58, 59, 76, 81 Spanu, P. 306, 318 Specker, N. 94, 110 Speulman, E. 300, 325 Spivey, R. K. 299, 322 Stacey, G. 31, 87 Stacey, N. J. 209, 246, 250, 286 Stachowicz, J. J. 92, 114 Stafford, A. 187, 202 Stahl, D. J. 309, 326 Stall, R. E. 298, 327 Staples, R. C. 293, 314, 319 Staskawicz, B. J. 293, 297, 298, 299, 301, 303, 304, 306, 312,315,316, 317, 320,323, 324, 325, 327 Staub, T. 94, 110, I12 Steer, B. T., 83
Steffan, W. 223, 230, 245, 271, 286 Stegwee, D. 21 1, 285 Steiner, H.-Y. 31, 32, 87, 278, 293, 307, 324 Steingrobe, B. 60, 87 Stenhagen, G. 104, 107 Stenzel, K. 106, 110, 309, 313,319, 326 Stephen, A. M. 209,211,212, 214, 218, 219, 221, 222, 225,226, 227, 228, 229,236,275, 276. 278, 286 Sterky, F. 314, 325 Stettner, C. 97, 106, 110 Steudle, E. 20, 87 Stevens, C. 297, 325 Stevenson, T. T. 236, 287 Stewart, P. A. 7, 87 Stewart, R. 299, 322 Stewart, V. 11, 21, 23, 80 Steyn, C. B. 228, 276 Stiekema, W. J. 300, 315 Stitt, M. 12, 15, 17, 67, 78, 85 Stocker, R. 309, 326 Stocker, R. H.,106, 110, 309, 313 Stoehr, C. 263, 287 Stohr, C. 13, 87, 209, 258,260, 266,267, 272, 281,284, 287 Stone, B. A. 208, 209, 210, 211, 212, 214, 215, 216,217,219, 222,223, 226, 227, 228, 229, 230, 236, 241, 243,252, 255,259, 273, 274, 275, 276, 278, 279 Storer, P. J. 127, 136, 193 Stowe, M. K. 92, 103, 114 Strahm, A. 218, 287 Straube, E. 300, 319 Strecker, G. 217, 218, 283 Strydom, D. 94, 113 Stryer, L. 124, 202 Stulen, I. 6, 72 Stumpf, P. K. 165, 195, 201 Subramanian, M. V. 127, 204 Sueyoshi, K. 12, 87 Sugden, A. 171, 202 Sugiyama, T. 13, 21, 85, 87 Sukrapanna, S. S. 13, 53, 71 Sun, S. 249,287 Sun, Z. W. 24,83 Sundberg, B. 314, 325 Suppmann, B. 24, 87 Suziedelis, K. 50, 82 Suziki, N. 127, 130, 197 s ~ ~ kr. i2 , 1 , ~ Suzuki, K. 156, 196 Suzuki, T. 119, 127, 135, 139, 140, 143, 144, 145, 147, 148, 151, 153, 154, 155, 158, 159, 161, 163, 168, 169, 170, 171, 172, 174, 175, 187, 190, 191, 192, 193, 194, 195, 197,202, 203, 204 Svendsen, I. 97, 98, 111 Svetek, J. 262, 263, 287
349
AUTHOR INDEX Swain, J. L. 163, 201 Sympson, C. J. 273, 275 Szyperski, T. 308, 325
T Tabata, H. 94, 115 Tacnet, F. 20, 81 Tagawa, K. 249, 287 Tajima, S. 138, 203 Takabayashi, J. 92, 103, 104, 114 Takahashi, E. 143,144,145, 151, 153, 155, 158, 198, 203 Takahashi, H.,24,82 Takahashi, T. 253, 273, 287 Takasawa, Y. 135, 139, 140, 147, 161, 193 Takeda, J. 224,281 Takeda, Y. 120, 145, 175, 190, 192, 203 Takeuchi, Y. 236,239,256,279, 287, 301, 303, 304, 320 Takhtajan, A. 121, 203 Takino, Y. 124, 203 Takos, A. M. 263,287 Talbot, N. J. 293, 325 Tamaki, K. 156, 196 Tanaka, T. 24, 82 Tang, S. J. 303, 319 Tang, X. 301,303, 306,326,328 Taniguchi, S. 23, 82 Tanksley, S. 299, 322 Tarka, S.M. Jr , 119, 204 Taylor, I. E. P. 222, 287 Taylor, J. D. 298, 325 Teen, T. T. 314, 325 Telkamp, G. P. 28, 74 Tenhaken, R. 304321 Tepfer, M. 222, 287 ter Steege, M. 52, 54, 87 Tens, A. 65, 89 Terrasaki, Y. 170, 204 Terzi, M. 238, 277 Tettelin, H. 264, 275 Teyker, R. H. 52, 54, 87 Thellier, M. 64, 76 Theodoulou, F. L. 31, 32, 90 Theuring, C. 100, I l l Thevissen, K. 311, 316, 317, 326 Thilmony, R. L. 302, 312, 318, 326 Thomas, C. M. 300, 323, 326 Thomas, R. J. 138, 204 Thomas, T. L. 252, 277 Thomas-Oates, J. E. 263, 278 Thompson, D. G.,106,111 Thompson, G. A. 263,283 Thompson, H. J. M. 251,253,254,287 Thompson, L. F. 13,84 Thomzik, J. E. 309, 326 Tigelaar, H. 312, 317 Tillard, P. 6, 15, 55, 64, 65, 66, 67, 7273, 76,82
Tillman, J. A. 99, 114 Timans, J. C. 308,324 Timmis, R. 238, 279 Timpa,, J. D., 287, 239 Tinker, P. B. 4, 82 Tischner, R. 9, 10, 12, 13,45, 70, 78,80,86,87, 89, 263, 281, 287 Titarenko, E. 306, 324 Tjallingii, W. F. 65, 89 Tobias, C. M. 301, 303, 325 Toki, S. 300, 327 Tomcsanyi, T. 50, 82 Tomos, A. D. 5, 8, 10, 13, 18, 53, 90 Tomsett, A. B. 25, 88 Toonen, M. A. J. 237, 238, 250, 287 Tootle, T. L. 309, 328 Tornero, P. 300, 326 Tortolero, M. 24, 84 Touraine, B. 3, 5, 13, 15, 17, 29, 38, 55, 64,65, 66, 77, 82, 88 Towers, G. H. N. 98, 108 Travis, R. L. 5 , 9, 10, 12, 14, 52, 56, 69, 70 Trewavas, A. 53,88 Triplett, B. A. 239, 287 Triplett, E. W. 19, 88, 138, 204 Trueman, L. J. 11, 12, 15,25, 32,33, 37, 39,68, 84,88 Truong, H. N. 2, 11, 76 Tsai, T. C. 134, 201 Tsay, Y. F. 9, 11, 12, 29, 30, 31, 38, 39, 77, 88 Tschope, M. 304, 320 Tsiamis, G. 298, 325 Tsuda, Y. 187, 195 Tsui, F. 309, 328 Tsumuraya, Y. 209, 218, 221, 222, 223, 225, 226, 229, 230,238, 265, 271, 279,281,283, 287, 288 Tsurushima, T. 301, 303, 320 Tsushida, T , 187, 204 Tull, J. 68, 88 Tumlinson, J. H. 92, 103, 104, 107, 109, 113, 114
Turlings, T. C. J. 92, 103, 104, 107, 114 Turner, J. C. 30, 89 Turner, J. F. 127, 204 Turpin, D.H. 119, 194 Tuzun, S. 313, 322 Tyerman, S. D. 9, 10, 49, 53, 54, 55, 77, 86, 88
U Ubbinkkok, T. 23,85 Ubik, K. 99, 109 Udenfriend, S. 264, 288 Udvardi, M. K. 11, 12, 17, 32, 49, 69, 77 Uhlen, M. 314, 325 Ukaji, T. 124, 125, 192, 204 Uknes, S. 94, 110, 115, 307, 317, 318, 324 Ullman, D. E. 300, 324
350
AUTHOR INDEX
Ullrich, C. I. 6, 7, 82 Ullrich, W. R. 6, 41, 42, 44,88 Ullstrup, A. J. 295, 325 Ulmer-Dufek, J. 122, 171, 195 Umemoto, N. 303, 326 Umezawa, H. 164, 200 Unkles, S. E. 25, 77, 88 Urrestarazu, A. 46, 50, 51, 80 Urwin, P. E. 314, 326
V Vaadi, Y. 65, 70 Vaalburg, W. 6, 72 Valdor, J.-F. 223, 226, 230, 233, 245, 250, 265, 266, 267, 269, 271, 288, 289 Vale, F. R. 46, 48, 89 Valentine, T. A. 250, 251, 282 Valenzuela, J. 229, 279 Valinsky, L. 296, 326 Van Aelst, A. C. 238, 247, 288 van Baarlen, P. 104, 109 van Beek, T. A. 65, 89 Van Camp, W. 305, 317 van de Wiel, C. 215, 285 Van den Ackerveken, G. 301,326 Van Den Ende, H. 264,275 van den Heuvel, J. 310, 321 van der Knaap, E. 215, 285 van der Lee, T. 300, 325 van der Leij, M. 13, 18, 89 van der Werf, A. 56, 79 van der Zandt, H. 238, 277 van Engelen, F. 215, 238, 277, 285 van Engelen, F.A. 238, 255, 288 Van Gijsegem, F. 296, 299, 316 van Helden, M. 65, 89 van Holst, G.-J. 209, 210, 218, 226, 236, 237, 238, 252, 255, 272, 281, 288 Van Kammen, A. 215, 237,238, 250,252,277 van Loon, ,J. J. A. 99, 110, 112 van Loon, L. C. 310, 316, 321 Van Montagu, M. 305, 314, 317, 325 Van Roekel, J. S. C. 312, 317 Van Went, J. L. 238, 247, 248, 288 van West, P. 302, 320 Vancanney, G. 306, 324 Vandekerckhove, J. 238, 277 Vanderburg, G . 38,86 Vanderwel, D. 99, 114 VanEtten, H. D. 295, 310,317, 325,327 Varanini, Z. 13, 73 Vardi, A. 238, 278 Varela-Nieto, I. 263, 280 Varga, S. 228, 283 Varner, J. E. 216, 217, 286 Veash, N. 123, 204 Vega, J. M. 11, 75 Veith, M. 99, 113
Vera, P. 300, 326 Verhoeven, H. A. 250, 287 Verma, D. P. S. 128, 130, 131, 194, 198 Vernooij, B. 94, 115, 307, 317, 318 Vizina, L. P. 4, 40, 79 Vidal, S . 221, 223, 239, 249, 284 Vijayan, P. 310, 326 Villaneuva, M. A. 230, 288 Villarroel, R. 314, 325 Vissers, S . 46, 50, 51, 80, 81 Vivian, A. 293, 298, 302, 326 Vleeshouwers, V. G . A. A. 302, 320 Vogel, J. 293, 308, 327 Volk, R. J. 46, 48, 51, 54, 58, 66, 75, 77, 87, 89 Volko, S . 307, 317 Volrath, S. 94, 110 von Arnold, S. 210, 236, 237, 238, 278 van Meyer, E. 67, 89 von Wiren, N., von Wiren, 3, 89 Vonsengbusch, D. 64, 86 Voragen, A. G. J. 243, 285 Vornam, B. 106, 110, 309, 313, 319 Vos, P. 300, 307, 324, 325 Voss, H. 11, 83 Vossen, J. H. 264, 275, 288
W Wadhams, L. J. 94, 98, 99, 100, 101, 102, 105, 114 Wagner, K. G. 124, 126, 131, 135, 159, 199, 201, 204, 205 Waines, J. G. 30, 73 Wakley, G. E. 293, 325 Waldeck, B. 12, 87 Walker, D. J. 5, 10, 18, 89 Walker, L. P. 58, 59, 76 Waller, D. A. 119, 155, 168, 170, 171, 172, 174, 175, 187, 194, 195, 201, 203, 204 Waller, G . R. 119, 145, 147, 151, 154, 163, 175, 194 Walling, L. L. 30, 85 Wallsgrove, R. M. 30, 89, 94, 106, 109, 110 Walter, C . H. S . 20, 71 Walters, E. W. 127, 204 Walton, J. D. 296, 295, 315, 322 Walton, N. J. 156, 204 Waney, V. R. 296, 326 Wang, G.-L. 300, 326 Wang, H. 210, 211,212,215, 218,222,226, 231,232,234, 235, 242, 247, 212, 276, 288 Wang, J. F. 298, 327 Wang, J. L. 230, 288 Wang, M. Y. 3,41,42,44,45,46, 48, 57, 58,89 Wang, M.-B. 313, 327 Wang, N. 65,89 Wang, R. 9, 29, 40, 89 Wang, X. C. 13, 71
35 1
AUTHOR INDEX Wang, Z.-X. 300, 327 Wanner, H. 122, 123, 143, 144, 145, 153, 171, 172, 175, 187, 193, 195, 197, 198, 201, 204 Ward, E. 94, 110, 307, 317, 318 Ward, J . L. 94, 115 Ward, M. R. 13, 87, 89 Warner, R. L. 11, 12, 18, 19, 78, 87, 89 Warren, R. F., 202, 300, 304, 326 Washio, K. 263, 283 Wasmann, C. C . 310, 327 Wasternack, C. 123, 204 Watanabe, N. 218, 282 Waterhouse, P. M. 313, 327 Weber, P. 64, 86 Wegner, L. H. 57, 89, 90 Wei, W. 301, 324 Wei, Z. 298, 316 Weibull, J. 65, 90 Weil, B. 50, 86 Weiner, I. M. 124, 197 Weinhandl, J. A. 238, 239, 278 Weiping, W. 228, 229, 274 Weir, E. 139, 205 Weisemann, J. M. 303, 320 Wells, B. 222, 246, 275, 282, 286 Welty, E . 238, 279 Werb, Z. 273, 275 Wessels, J. G. H. 293, 325 Wessels, R. 104, 109 Wettenhall, R. E. H. 209, 212, 214, 236, 259, 279 Weymann, K. 94, 112, 307, 317, 324 Whalen, M. C. 298, 300, 326, 327 Whistler, R. L. 21 I , 226, 227, 228,229, 273, 288 White, F. F . 300, 301, 321, 328 White, J . B. 229, 238, 279 White, P. J. 46, 62, 72, 90 White, R. E . 58, 82 Whitehead, B. 293, 314, 319 Whitehead, L. F . 49, 77, 88 Whitham, S . 308, 312, 327 Widholm, J. M . 139, 196 Wieneke, J. 52, 90 Wienke, J. 41, 78 Wiersema, P. K . 6, 72 Wiese, W. 106, 110, 309, 313, 319 Wiesler, F. 60, 90 Wijbrandi, J. 300, 325 Wijesekera, R. 0. B. 123, 202 Wild, A. 55, 61, 80, 90 Wilde, H. D . 252,277 Wilkinson, J. Q.2, 72 Willats, W. G. T. 209, 210, 213, 226, 230, 236, 252, 253, 254, 255, 257, 258, 260, 261, 286, 288 Willekens, H. 305, 317 Willemse, M. T. M. 249, 284 Williams, D. J. 98, 112
Williams, P. 221,223, 239, 249, 275, 283 Williams, P. A. 211, 214, 222, 226, 227, 228, 283, 284 Williams, P. J. 222, 227, 228, 280 Williamson, V. M. 300, 324 Willits, M. G. 293, 307, 324 Wilson, G. 41, 78 Wilson, I. 293, 308, 327 Wingsle, G. 145, 151, 153, 156, 172, 154, 199 Wink, M. 100, 113, 150, 165, 166, 205 Winkler, R. G. 139, 205 Winklmair, A. 97, 106, 110 Witham, T. F. 229, 230, 283 Witt, F. G. 24, 69 Witte, L. 100, 107, 111, 165, 205 Wold, J. K . 214, 218, 279 Wolf, E. D. 293, 314, 319 Wong, E. 21 I , 223, 269, 277 Woo, S. L. 313, 322 Woodcock, C. M. 91, 93, 94, 97, 98, 99, 100, 101, 102, 103, 105, 106, 108, 109, 110, I l l , 112, 113, 114 Woods, D. L. 99, 114 Woods, E. F. 243, 288 Woolhouse, H. W. 208, 285 Wootton, J. 24, 83 Wray, J. L. 2, 75, 79, 89, 90 Wright, A. F . 100, 113 Wu, G. 313, 327 WU, H.-M. 211, 212, 215, 216, 218, 222, 226, 231, 232, 233, 234, 235, 242, 247, 272, 273, 276, 288 Wu, T . 299, 322 Wu, Y. 209, 215, 216, 247, 284 Wulff, B. B. H. 300, 323 Wurgler-Murphy, S. M . 13, 90 Wuthrich, K. 308, 325 Wyatt, S. E . 208, 273, 288 Xia, Z. Q.94, 107 Xing, T. 306, 327 Xiong, S.H. 38, 80
x Y
Yabuki, N, 124, 131, 136, 140, 143, 159, 160, 164, 192, 205 Yadav, M. P. 221, 262, 263, 287 Yagisawa, N. 164, 200 Yamada, H. 175, 192 Yamada, Y. 156, 196 Yamaguchi, I. 296, 315 Yamaguchi, M. 139, 195 Yamaguchi, S. 121, 191 Yamamoto, S. 209, 218, 223,221, 222,223,225, 226, 283, 287, 288 Yamamoto, Y. 138, 203 Yamane, K. 24, 82, 83
352
AUTHOR INDEX
Yamanouchi, U. 300,327 Yamaoka, N. 301, 303,320,326 Yanagishita, M. 265, 288 Yang, B. 301, 328 Yang, Y. 293, 294, 296, 301, 305, 327 Yano, M. 300,327 Yariv, J. 214, 225, 243, 289 Yasufuku, H. 209, 213,240, 242,243,280 Yasumasu, I. 119, 199 Yates, E. A. 209, 213, 223, 226, 227, 230, 233, 236, 245, 246, 250,257, 258, 260, 261,265, 267, 269, 271, 286, 289 Ye, C. 120, 122, 205 Ye, C.-X. 141, 145, 153, 154, 166, 167, 185,193 Yeow, Y. M. 209, 211, 222, 243, 280 Yin, S. 306, 327 Yindeeyoungyeon, W. 291, 319 Ying, Z. 156, 205 Yokota, T. 143, 158, 191, 194 Yompakdee, C. 33, 71, 90 Yoneyama, K. 296,315 Yoshikawa, H. 24, 82 Yoshikawa, M. 301, 303, 320, 326 Yoshimura, S. 300, 327 Yoshino, M. 138, 205 Youl, J. 210, 215,255,264, 272, 273,285 Youl, J. J. 261, 289 Yu, I.-C. 304, 327 Yuan, J. 299, 301, 317, 324 Yuan, Q. 294, 296, 327 Yucel, I. 297, 327
Yukimune, Y. 94, 115 Yun, D.4. 311, 327
Z Zabeau, M. 300, 325 Zambryski, P. 293, 299, 304, 316 Zenk, M. H. 94, 107, 110 Zhan, X.-Y. 210, 234, 273, 276 Zhang, H. 32,40, 62,90 Zhang, H. Q. 247,248,249,282, 284 Zhang, L. 31, 87, 296, 328 Zhang, S. 300, 306,326,328 Zhang, Z. 300, 326 Zhang, Z.-N. 98, 113 Zhao, Y. K. 311,327 Zhen, R. G. 5, 8, 10, 13, 18, 53, 90 Zherelova, 0. M. 53, 78 Zhou, H. 120,205 Zhou, J. 301, 303, 306,326,328 Zhou, J.-J. 25, 31, 32, 90 Zhou, N. 309, 328 Zhu, J.-K. 209, 210, 212, 214, 236, 240, 252, 253,254,255, 257, 258, 259,260,262, 213, 277, 281 Zhu, Q.,313, 322 Zhu, W. 301,328 Zoina, A. 313, 322 Zook, M. 309,319 Zuber, P. 24,83 zur Nieden, U. 306, 322
SUBJECT INDEX
A ABC transporters 21-3 Acacia 228, 230 Acacia laetia 228 Acacia robusta 212, 221 Acacia senegal 21 1-13, 227, 228 Acacia seyal228 Acer pseudoplatanus 284 active oxygen species 304-5 adenine, metabolism of 140, 157-61 Agamous 234 alkaloids 93, 94 pyrrolizidine 96, 99-100 see also purine Allium cepa 80 amino acids as a source of nitrogen 3-4 feedback inhibition 14, 15-18 in phloem 63-5 transporters of 31, 39 ammonia diffusive uptake of 41-2, 43 ammonium channel in root nodules 49 efflux 57-8 inhibition 13-15 ammonium uptake 40-52 competitive interaction with potassium 48-9 intracellular, compartmentation of 43-4 kinetics of 44-9 physiology of 4 0 4 regulation by shoots 61-7 thermodynamic considerations 42 amtA 50 Anacystis nidulans 21, 2 5 4 , 28, 32, 33 antifeedants 97-9 aphids 65, 94, 99, 100, 102, 103, 104, 105, 300 Apis mellifera 101 aposematic coloration 99 aprt mutant 135 aquaporins 20 Arabidopsis 8, 9, 28, 29, 30-1, 32-3, 34, 38-9, 40, 42, 46, 47, 49, 51, 55, 62, 68, 94, 128, 130-1, 134, 135,215,233,245,247-8,253, 2545, 263, 264, 273, 300, 303, 304, 306, 307, 308, 309, 310, 313, 314
Arabidopsis thaliana 7, 94, 128, 130, 131, 134, 135, 215, 233, 245, 247, 254 arabinogalactanprotein (AGP) carbohydrate component 219-25 cell wall 238-56 culture medium and 2 3 6 8 epitopes 245-9, 25(tl, 265-6, 267-70 in gum 26630 gynoecium and 23&5 molecular shape 221-2 mucilages and 26630 plasma membrane 244-5, 25670 polypeptide component 21419 soluble 21 1-38 structural characterization of 212-13 Aspergillus nidulans 24, 25, 26, 28, 32, 33, 35 AtAMTl transporter 42, 51-2, AtNRT2 32 AtPTR2 31 Avena sativa 186 avirulence genes 297-9, 300-2 Azadirachta indica 98 azadirachtin 98-9
B Bacillus subtilis 23, 24 bacteria caffeine-degrading 189-90 cyanobacteria 21, 22, 23, 209 nitrifying 3 see also individual species bacterial virulence factors 2 9 5 4 BCHl32 BCH2 32 Ben-Zioni model 65-6 Botrytis species 293 Brassica carnpestris 247 Brassica napus 31, 94, 216 Brassica oleracea 213, 242 Brassicaceae, see individual species name Brassicae 94, 102, 215, 24748, see individual species name
3 54
SUBJECT INDEX
C caffeine 118-19, biosynthesis concentrations of intermediates of 148, 150 from purine nucleotides 15746 light and its effect on 172-3 methyl group donors in 145-7 routes of 143-5 sequence of methylation 147-8, 149, 150-1 tissue age and 167-72 catabolism of 174-5 variation in mechanisms 175-85 degradation I 8 6 8 plants containing 121-3 production 186-8 subcellular distribution of 150 synthase 1534, 173, 191 see also decaffinated beverages, Camellia SPP., coffea SPP. Camellia 118, 120-2 Camellia assamica 120, 122 Camellia irrawadiensis 120, 166, 175, 186 Camellia ptilophylla 154, 166, 167, 185, 187 Camellia sinensis 119, 145, 146, 150, 154, 158, 167-71, 176-7, 1834, 187, 189, 190 Cannabis sativa 218 Capsicum annuum 66 carbohydrate supply to root 66-7 carboxylates 6 5 6 Catharanthus roseus 124, 128, 134, 141, 159-60, 173, 186, 196, 200, 2 0 4 5 Cercospora nicotianae 294 Cfl300,303, 314 Chalara 3 10 Chara 55, 56 Chara corallina 41 Chara fragilis 209 chll 28, 29, 30, 38 CHLI 9, 29, 30 ch18 29, 39 Chlamydomonas 11, 12, 18,24, 25-7, 32,33, 35, 37, 39 Chlamydornonas reinhardtii 24, 25-7, 51 chlorate resistance 25, 28, 29, 30 Chlorella 11, 13, 41 Chlorella saccharophila 263 Citrus 238 Cladosporium fulvum 295, 301, 303, 312 Cochliobolus carbonum 295, 309, 313 Coffea 118, 119, 122, 167, 174, 175, 179, 183, 187-9, 1934, 199,203-4 low caffeine-containing species 179-83 Coffea arabica 119, 154, 171-2, 174, 175-9, 187, 188-90 Coffea bengalensis 179, 181, 183, 185, 188, 189 Coffea canephora 122, 188 Coffea dewevrei 122, 175, 188
Coffea eugenoides 179, 181, 183, 185, 187, 188, 189 Coffea liberica 122, 175, 187, 188 Coffea racemosa 122, 188 Coffea salvatrix 122, 179, 181, 183, 185, 188-9 coffee 118, 119, 145,149, 153, 154, 155, 188-90, see Coffea spp. Cola 120 Colosia esculenta 229 conditioned medium 2 3 6 8 Corynebacteriurn glutamicum 50 crnA 25, 28, 29 cutinases 295 cwh6/gpi3 yeast mutant 264 Cyclamen persicwn 237 cysG 24 cysQ 50 cytosolic nitrates 18-19
D Dactylis glomerata 57 Datura innoxia 143 Daucus carora 275 deamination 135 decaffeinated beverages 188-90 defence mechanisms in plants 2 9 4 5 similarities with animals 308, 31 I response genes 309-1 1 defensins 308, 309, 31 1 dephosphorylation 1 3 5 4 Deschanipsia flexuosa 57 dhurrin 97, 98 disease resistance genes 300, 303, 304, 306 dndl mutant 304 Drosophila 308
E E D 9 1 gene 306 elicitors 3 0 H enzymes biosynthesis-related 97 cell wall degrading 248-9 copper-containing 9 3 4 disease resistance and 294-7, 302, 305, 312, 314 glycan chain biosynthesis 224-5, 234 nutrient assimilatory 2, 11 purine metabolism-related 118, 124, 127, 128-32, 135-8, 139, 146, 151, 152, 153, 155, 15940, 161, 165, 167, 169, 173, 176-7, 189 salvage 1254, 133, 134, 135, 141, 142, 143, 157 stroma 155 see also individual enzyme names epi isomer 94, 95 Erwinia 291
355
SUBJECT INDEX Erwinia amylovora 298, 299 Erwinia chrysanthemi 297, 299, 3 1 1 Escherichia coli 13, 23, 24, 33, 34, 35, 50 mutants 130 etr 1-1 gene 310 eucaryotes ammonium transport and 50-1 nitrate transport in 24-8 see also individual species name
F Fagus sylvatica 64 fen gene 301-2 fungi 24, see also individual species name
G Gaeumannomyces graminis 294 glucosidases 104 glutamine synthetase (GS) 19,41,43, 44, 49 Glycine max 17, 130 glycosides 94, 97, 98, 301 Yariv phenylglycoside 214, 215, 225, 226, 228,238, 239, 242, 2434,249, 251, 253, 256,272 glycosidic bond cleavage 1 3 5 4 glycosyl-phosphatidylinositol 260 lipid anchors 260-5 G W O G A T cycle 41, 49 guanine, metabolism of 141, 161 guanosine, metabolism of 141, 161 guarana 123 gum arabic 227-8, 230 gums 266-30 Gymnocolea infrata 252 gynoecium 2 3 6 5
H Hansenula polymorpha 24, 27-8, 32, 37 HC toxin 2954, 313 Herrania 123 Heterodera 300 Higher plants, ammonium uptake in 51-2 honeybee, see Apis mellijiera hrp gene 298-9, 301, 302 Humulus lupulus 99 HvNRT2 32 hydraulic conductance 19-20 hydrogen cyanide 97 hypersensitive response (HR) 292,299-300,308 hypoxanthine 1368, 139, 142
I Ilex 118, 120, 122-3, 166, 167 Ilex paraguariensis 17 I inosine 142 insect adaptability 102 pheromone systems 105
isothiocyanates 94, 100, 102, 105
L Larix 229 leaf base temperature 61-2 LeAMTl45-6, 51-2 Lemna 4 1, 54 Lemna gibba 6 LeNRTl 3 1 Lepidoptera 94, 9P-100, 101, 103, 104 Leptinotarsa a'ecemlineata 99 leucine-rich-repeat (LRR) domains 292, 300, 303, 3 12 Lilium longijlorum 248 Limnobium stoloniferum 6 limonoids 98-9 lipid anchors 260 Listeria 302 Lolium multiflorum 56, 212, 218, 221-2 Lolium perenne 15, 99 Lotus japonicus 6, 12, 13, 17, 32
M Magnaporfhe grisea 293 Major Facilitator Superfamily (MFS) 33-8 mati, see Ilex Meliaceae 97 MEP genes 46, 50-1 metabolites, secondary 91-106, 123, 150 structure of 95-7 methyl salicylate 96, 102-3 methylation enzymes 150-7 Mi gen 300 mucilages 266-30 mutants 11, 18,25,27,28-30, 51, 130,264,273, 292, 294, 301, 3054, 307, 309, 310, 312
N nahG gene 307 NARl 24,26 Nar2 protein 26 narK genes 2 3 4 Nectria haemarococca 3 10 Neotyphodium lolii 99 Neurospora crassa 7 nia2 seedlings 29 niaD 25 Nicotiana 215, 218 Nicotiana alata 212, 213, 214, 215, 216, 221, 222, 231, 232, 233,240,241, 268 Nicotiana edwardsoniia 254 Nicotiana glutinosa 213, 257, 260, 266 Nicotiana plumbaginifolia 17, 32, 35 Nicotiana tabacum 216, 231, 232, 233, 242-3, 263 niiA 25 nir genes 2 1, 24 NiRC-FocA-FdhC family 24
356
SUBJECT INDEX
Nit1 gene 26 Nitellopsis obtusa 53 nitrate, cytosolic 18-19 nitrate efflux 52-7 effect on physical perturbation 52-4 measurement of 54 metabolic cost of nitrate absorption 56-7 role in uptake control 55-6 xylem loading and 57 nitrate induction 11-13 nitrate receptors 11-13 nitrate transport 11, 12, 16, 21-8, 30-8, 39, 40 genes 30-8 mutants 28-30 nitrate uptake energetics of 5 inhibition by ammonium 13-15 kinetics of 5-10 modelling 6CL1 regulation 11-19 shoots 61-7 see also transporter genes, uptake systems Nitrate-Nitrite Porter (NNP) family 27, 33-8 nitrite transport 2 1 4 , 34, 35 nitrogen deficiency 19 fixation 49 nutrition 3 4 supply, manipulation of 62-3 NLTl 39 N-methyl nucleosidase 156, 157 N-methyltransferases 150-3 Nostoc 209 Nrg mutants 18 nrt genes 22, 23 NRTI, see transporter genes NRT2, see transporter genes
0 oil-seed rape, see Brassica napus Oreina 100 Ostrinia nubilalis 94 oxidative bursts by plants 9 3 4
P parasitoids 103-4 pathogen entry and infection 2 3 4 genes in plants 313-14 resistance mechanisms to 294-5 pathogenesis-related proteins 31 1 Paullinia 118, 120, 123 Paullinia cupana 123 peribacteriod membrane (PBM) 49 Peronospora parasitica 309 Phaseolus 310 Phaseolus mungo 140 phloem delivery of amino acids 63-5
pho84 mutant 33 phylogenetic relationships 33-7 phytoalexins 106, 304, 306, 309-10 Picea d i e s 64 Picea glauca 4 Pieris brassicae 99 Pinus banksiana 4 Pinus taeda 246 plant resistance genes 17, 28-30, 106, 292, 293, 294-8, 299-300, 312-13 plant-insect interaction 91-106 polyphenol oxidase 9 3 4 polysaccharides, extracellular 2 9 6 7 potassium, interactions with ammonium 48-9 procaryotes ammonium transport and 49-50 nitrate transport in 2 1 4 see also individual species name proteolysis 3 protoplasts 57, 249, 255, 256, 298, 302 Pseudomonas 297 Pseudomonas cepacia 189, 190 Pseudomonas putida 175, 189, 307 Pseudomonas syringae 296, 298, 299, 308, 309, 312 pthA gene 296 Ptd 299-300, 301, 3034, 306, 312 PTR family 31-2, 33, 34, 35, 3 6 7 purine 118-19 bases, metabolism of 140-3 catabolism of nucleotides 13540 de novo nucleotide biosynthetic pathway 12631, 143 interconversion of nucleotides 131, 132 nucleosides, metabolism of 140-3 nucleotide metabolism in higher plants 1 2 3 4 occurrence in plants 120-3 ring 126 methylation of 143-50 salvage 131-5 specialized 143 see also caffeine, theobromine Pythium mastophorum 3 10
R Ralstonia solanacearum 297 rebl-l mutant 255 regulatory domains 37-8 Rhizopus 3 10 root systems, maximization of nitrogen capture 62 root zone temperature 61-2
S Saccharomyces cerevhiae 27, 33, 46, 50-1, 68, 263 Scilla maritima 120 semiochemicals 1034, 106
357
SUBJECT INDEX Senecio jacobaea 99 senecionine 96, 99 Serratia marcescens 189 shoot base temperature 61-2 shoot growth, manipulation of 61-2 shootxoot ratios 6, 61-2, 67 shoot-derived signals 63-7 signal transduction 305-7 soil acidic 3 effect on uptake 60-1 soybean 17, 32, 49, 65, 66, 67, 127, 128, 130, 138, 298, 303, 306 Spinacia oleracea 54 Spirodela oligorrhiza 263 Staphylococcus carnosus 24 Strong Ion Difference 7 Synechococcus 21,22,23,24 synomones 103-4 syringolides 301, 3 0 3 4 systemic acquired resistance 94, 307-8
T Taxus species 94 tea 118, 122, 125, 138, 143, 145, 146, 147, 148, 149, 151-3, 154, 1 5 5 6 , 157, 160, 161, 162-6, 190, see Camellia spp. tetranortriterpenoids 97 Theobroma bicolor 123 Theobroma cacao 123, 166, 187 Theobroma grandiflorum 123 theobromine 118, 120, 122, 123, 143, 144, 145, 146, 147-8, 15G1, 153-5, 158, 159, 160, 163, 164, 165, 1667, 169-72, 173-7, 179, 184, 185-6, 187-8, 189, 190 theophylline 118, 120, 122, 123, 154, 166, 167, 174-5, 1769, 180, 181-3 tobacco 20, 67, 125, 134, 222, 234, 247, 252, 257,294,296,302,303,304,305,306,307, 308, 309, 310, 312, 314 budwom 105,248 pollen tubes 248 see also Nicotiana tomatine 295 tonoplast membrane, role in nitrate adsorption 10, 18 toxic mechanisms of plant defence 93-7 toxicant sequestration 99-100 transgenic plants 67, 68, 118, 189-90, 191, 210, 234, 273, 292,294,2954,302, 303, 305, 307, 309, 310, 311, 312-13, 314, 315 transporter genes 68
ammonium 45-6,49-52 NarK 21, 2 3 4 , 33, 34, 35 nitrite reductase (NiR) 11, 21, 24, 25, 28 nrt genes 21-2 NRTl 9, 12, 29, 30-2, 37, 38-9, 40 NRT2 12, 13, 17, 24,25,26,29, 32-3, 37, 38, 39,40 YNTl 28, 32 Trifolium repens 56 Typha latijolia 41 Tyria jacobaeae 99 uptake activity along root system 5 8 4 1 uptake systems 4 high-affinity 5, 39, 40, 4 4 6 , 47 constitutive 5, 9-10 inducible 5, 6-8 low-affinity 5, 8-9, 3940, 4 6 7 role of tonoplast membrane 10
U uricate 139-9 Uromyces phaseoli 293 Utetheisa ornatrix 99
V Vigna 130 Vigna aconijolia 130 Vigna mungo 186 Vinca rosea 256 volatiles produced by plants 100-3
X xanthine 1368, 139, 142 Xanihomonas 301 Xanihomonas campestris 298 Xanihomonas cirri 296 Xanthomonas oryzae 300 xanthosine 142, 148, 157 Xenopus 25, 26, 30, 31, 32, 39, 47 xylem 19, 57, 61, 6 3 4 , 245, 246, 268, 269
Y yeast, 58, see individual species name Yersinia pseudotuberculosis 298 YNTl gene 28, 32
Z Zea mays 221 Zinnia 246 Zonocerus variegatus 100
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