Advances in Botanical Research, Volume 19

Advances in BOTANICAL RESEARCH VOLUME 19 Advances in BOTANICAL RESEARCH Editor-in-Chief J. A. CALLOW School of Bio...

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Advances in

BOTANICAL RESEARCH VOLUME 19

Advances in

BOTANICAL RESEARCH Editor-in-Chief J. A. CALLOW

School of Biological Sciences, University of Birmingham, Birmingham, England

Editorial Board M. E. COLLINSON H. G. DICKINSON R. A. LEIGH D. J. READ

Kings College, London, England University of Oxford, Oxford, England Rothamsted Experimental Station, England University of Sheffield, Sheffield, England

Advances in

BOTANICAL RESEARCH Edited by

J. A. CALLOW School of Biological Sciences University of Birmingham Birmingham, England

VOLUME 19

1993

ACADEMIC PRESS Harcourt Brace & Company, Publishers

London

San Diego New York Boston Sydney Tokyo Toronto

This book is printed on acid-free paper

ACADEMIC PRESS LIMITED 24/28 Oval Road, London NW17DX

United States Edition published by ACADEMIC PRESS INC. San Diego, CA 92101

Copyright 01993 by ACADEMIC PRESS LIMITED

All rights reserved No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

A CIP record for this book is available from the British Library ISBNCk12-005919-3

Typeset by Phoenix Photosetting, Chatham, Kent Printed in Great Britain by TJ Press, Padstow, Cornwall

CONTRIBUTORS TO VOLUME 19

S. ALDINGTON, Centrefor Plant Science, Universityof Edinburgh, Daniel Rutherford Building, The King's Buildings, Mayfield Road, Edinburgh EN9 3JH, Scotland, UK J. G. DUCKET", School of Biological Sciences, Queen Mary and Westfield College, University of London, Mile End Road, London El 4NS, UK S. C. FRY, Centre for Plant Science, University of Edinburgh, Daniel Rutherford Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JH, Scotland, UK M. B. JACKSON, Department of Agricultural Sciences, University of Bristol, AFRC Institute of Arable Crops Research, Long Ashton Research Station, Bristol BS18 9AF, UK R. LIGRONE, Dipartimento di Biologia Vegetale, Universita di Napoli, Via Forla 223, I-80139 Napoli, Italy G. I. McFADDEN, Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Parkville VIC 3052, Australia K. S. RENZAGLIA, School of Biological Sciences, Box 23590A, East Tennessee State University, Johnson City, TN 37614, U S A

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PREFACE

In this volume of Advances in Botanical Research we start with an article by Aldington and Fry on the “oligosaccharins”, the name given to that group of diverse oligosaccharides which exerts biological activity in plants, at low concentration. Oligosaccharins have been implicated in a wide range of physiological processes and are frequently described as being “hormonelike”. Some of these roles are by now fairly well established, most notably in defence response elicitation. Other roles are still speculative and controversial and this review attempts to explore some of these areas of controversy, to identify gaps in our knowledge of them and to provide pointers for future work. It brings together a wide range of topics, including methods for preparation and chemical characterization, the range of physiological effects, modes of action and transport properties. Intuitively, it seems fairly obvious that plants must possess strong controlling mechanisms to balance the growth of their various organs and a great deal of research does demonstrate that the growth and behaviour of shoots is coupled closely with that of roots, and that the internal controls are strongly influenced by environment. It has often been suggested that these environmental influences operate indirectly, by regulating the hormonal traffic between the two organs rather than through more direct influences following changes in water or mineral supply and the main thrust of Jackson’s article is to assess the evidence relating to this hypothesis. It would appear that no unequivocal conclusions can yet be reached because of limitations in the experiments that have sought to determine hormonal fluxes. The author identifies the need for more quantitative studies which take advantage of modern physicochemical and immunological methods and for computer-based modelling techniques which would enable a more comprehensive exploration of the hormone ‘economy’ of the whole plant. The general theory of endosymbiosis of photosynthetic prokaryotes as a basis for evolution of green algae and subsequently land plants is supported by a wealth of morphological, biochemical and molecular evidence. The origin of photosynthetic capacity in other groups of algae is less certain and the very diversity of algal chloroplasts has prompted speculation that they may have arisen from separate endosymbiotic events involving many different prokaryotes, or even the entrapment of photosynthetic [vii]

...

Vlll

PREFACE

eukaryotes. McFadden’s article reviews the morphological and molecular evidence relating to the origin of cryptomonad algae, reaching the conclusion that this group arose from an association between an unknown predatory phagotrophic flagellate and a chloroplast-containing eukaryote, probably a red alga, and thus involves no less than four different evolutionary lineages. The phenomenon of “alternation of generations” and its relevance to the study of phylogeny, taxonomy and functional biology of land plants was discussed extensively by Bell in volume 16 of this series. In the present volume, the article by Ligrone, Duckett and Renzaglia takes this analysis a step further by considering one aspect of this in greater detail. In all land plants there is an embryonic phase, of variable duration, during which the sporophyte generation is in direct physical contact with the gametophyte and the interface between these two generations, the so-calledplucentu, thus plays a critical role in integrating the two phases of the life-cycle. In their review, the authors present a detailed and comparative anatomical and ultrastructural analysis of this interface, including the first detailed and systematic study of many groups of land plants. As usual, I would like to thank the authors for their excellent contributions, for their patience with the editor and their efforts to make his task easier.

JA CALLOW

CONTENTS

. . . . . .

V

. . . . . . . . . . . . . . . . . . .

vii

CONTRIBUTORS TO VOLUME 19 . . . . . . PREFACE

,

,

. .

,

Oligosaccharins S. ALDINGTON and S. C . FRY I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . A. Origin of the Oligosaccharin Concept . . . . . . . . . . B. Preparation of Oligosaccharins . . . . . . . . . . . . . C. Bioassays . . . . . . . . . . . . . . . . . . . . . . D. Purification and Chemical Characterization of Oligosaccharides . . . . . . . . . . . . . . . . . .

11. Physiology of Oligosaccharin Effects . . . . . . . . . . . . A. Fungal Oligo-p-glucans . . . . . . . . . . . . . . . B. Xyloglucan-derived Oligosaccharides as Growth Regulators C. Oligosaccharides of Pectins . . . . . . . . . . . . . . D. Oligo-P-xylansasPossibleOligosaccharins . . . . . . . E. Chito-oligosaccharidesandRelatedFragments . . . . . F. OligosaccharinsfromN-IinkedGlycoproteins? . . . . . G. Conclusions . . . . . . . . . . . . . . . . . . . .

.

2 2 3 5

6

. . . .

7 7 12 17 32 34 37 38

111. Mode of Action of Oligosaccharins . . . . . . . . . . . . . A. Evidence for Receptors . . . . . . . . . . . . . . . . B. Rapid Effects of Oligosaccharins . . . . . . . . . . . . C. DirectEffectsofOligosaccharidesonEnzymes . . . . . .

41 41 46 56

IV.

Natural Occurrence of Oligosaccharins . . . . . . . . . . . A. Natural Occurrence of Xyloglucan Oligosaccharides . . . B. NaturalOccurrenceofPecticOligosaccharides . . . . . C. Glycoprotein-derived Oligosaccharins . . . . . . . . . D. Conclusion . . . . . . . . . . . . . . . . . . . . .

t ixl

. .

.

. .

. . .

58 58 59 61 62

X

CONTENTS

V . Mechanism of Formation and Degradation of Oligosaccharins . . A . Xyloglucan Oligosaccharides . . . . . . . . . . . . . . B . Pectic Oligosaccharides . . . . . . . . . . . . . . . . C . The Role of Chitinases, p-Glucanases and Other Enzymes .

62 62 66 73

VI . Movement of Oligosaccharins within the Plant: True Hormones? . A . Possible Transport of Xyloglucan Oligosaccharides . . . . B . Non-transport of Wound Signals . . . . . . . . . . . . C . Transport of Elicitors . . . . . . . . . . . . . . . . .

74 75 75 76

VII . Concluding Remarks . . . . . . . . . . . . . . . . . . . .

77

. . . . . . . . . . . . . . . . . . . .

77

References . . . . . . . . . . . . . . . . . . . . . . . .

77

Acknowledgements

Are Plant Hormones Involved in Root to Shoot Communication? M . B . JACKSON I . Introduction

. . . . . . . . . . . . . . . . . . . . . . .

104

I1. The Hormone Message Concept . . . . . . . . . . . . . . .

111.

A . Different Kinds of Hormonal Message . . . . . . . . . . B . Quantifying Hormonal Messages in Transpiration Stream . C . Assessing Developmental Impact of Hormonal Messages . .

106 106 107 111

Evidence for Regulation of Root : Shoot Ratio by Roots . . . . A . Nutrient Control Theory . . . . . . . . . . . . . . . B . Shortcomings of Nutrient Control Theory . . . . . . . . C. Conclusions . . . . . . . . . . . . . . . . . . . . .

112 112 113 116

IV . Examples of Hormone-like Action of Roots on Shoots . . . . . A . Early Research . . . . . . . . . . . . . . . . . . . B . Leaf Senescence . . . . . . . . . . . . . . . . . . . C . Shoot Extension. Photosynthesis and Flowering . . . . . D . Conclusions . . . . . . . . . . . . . . . . . . . . . V . Cytokinins . . . . . . . . . . . . . . . A . Introduction and Early Research . . . B . Development in Unstressed Plants . . C . Root Excision Studies . . . . . . . . D . Responses to Mineral Nutrient Shortage E . Effects of Other Stresses Applied to Roots F. Conclusions . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

117 117 117 120 122

. . . .

123 123 125 128 131 133 138

VI . Gibberellins . . . . . . . . . . . . . . . . . . . . . . . A . Introduction . . . . . . . . . . . . . . . . . . . . B . Studies on Unstressed Plants . . . . . . . . . . . . . .

138 138 139

. . . . . . . . . . . . . . . . . . .

. . . . .

. . . . . . . . . . . . . . .

CONTENTS

xi

C . Effects of Root Excision and Environmental Stresses Applied . . . . . . . . . . . . . . . . . . . . . toRoots D . Conclusions . . . . . . . . . . . . . . . . . . . . .

142 143

VII . Ethylene . . . A . Introduction B . Flooding . C . Conclusions

. . . .

. . . .

. . . .

. . . .

. . . .

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. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

VIII . Abscisic Acid . . . . . . . . . . . . . . A . Introduction . . . . . . . . . . . . . B . Water Deficiency and Stomata1Closure C . Water Deficiency and Leaf Expansion . D . Soil Flooding . . . . . . . . . . . . . E . Various Other Stresses . . . . . . . . F. Conclusions . . . . . . . . . . . . . IX . Final Remarks

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

144 144 145 149

. . . . . . . .

149 149 150 159 160 164 166

. . . . . . . . . . . . . . . . .

. . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . .

Acknowledgements References

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166 168

. . . . . . . . . . . . . . . . . . . . . . .

168

Second-hand Chloroplasts: Evolution of Cryptomonad Algae G . I . McFADDEN I . Introduction

. . . . . . . . . . . . . . . . . . . . . . .

I1. Overview of Cryptomonad Features 111.

. . . . . . . . . . . . .

The Nucleomorph . . . . . . . . . . . . . . . . A . Nucleus-like Organelle . . . . . . . . . . . B . DNA in the Nucleomorph . . . . . . . . . C . Eukaryotic Ribosomes around the Nucleomorph D . Origin of the Nucleornorph . . . . . . . . . E . Isolation of the Nucleomorph . . . . . . . .

IV . The Chloroplast . . . . . . . . . . . . . . A . Chloroplast Membranes . . . . . . . B . Storage Product . . . . . . . . . . C . Photosynthetic Pigments . . . . . . D . Chloroplast Genome . . . . . . . . V.

. . . . . . . . . . . . . . . . . . . . . . .

. . . . .

. . . . . .

. . . . . . . . . . . . . . . . .

190 192 192 192 195 196 200 203

. . . . . . . . . . . . . . . . . . . . . . . . . . .

208 208 208 208 210

Cryptomonads as Endosymbionts: Parasites of Cryptomonads and Endosyrnbionts of Cryptomonads . . . . . . . . . . . . .

213

xii

CONTENTS

VI . Second-hand Chloroplasts in Other Algae VII . Role of the Nucleomorph

. . . . . . . . . .

214

. . . . . . . . . . . . . . . . . .

216

VIII . Summary . . . . . . . . . . . . . . . . . . . . . . . . . IX . Taxonomic Appendix Acknowledgements

218

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

219 220

References . . . . . . . . . . . . . . . . . . . . . . . .

220

The Gametophyte-Sporophyte Junction in Land Plants R . LIGRONE. J . G . DUCKEIT and K . S. RENZAGLIA I . Introduction

. . . . . . . . . . . . . . . . . . . . . . .

232

I1. Bryophytes . . . . . . . . . . . . . . . . . . . . . . . . A . Mosses (Bryopsida) . . . . . . . . . . . . . . . . B . Liverworts (Hepatopsida) . . . . . . . . . . . . . C . Anthocerotes (Anthocerotopsida) . . . . . . . . . .

. . . . . .

234 235 253 275

I11. The Taxonomic Significance of the Placenta in Bryophytes and Implications for Phylogeny . . . . . . . . . . . . . .

. .

283

IV . Pteridophytes . . . . . . . . . . . . . . . . . . . . V . Seed Plants

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Acknowledgements

. . . . . . . . . . . . . . . . .

295 301 306

References . . . . . . . . . . . . . . . . . . . . . . . .

307

AUTHOR INDEX

. . . . . . . . . . . . . . . . . . . . . . . .

319

SUBJECT INDEX

. . . . . . . . . . . . . . . . . . . . . . . .

337

Oligosaccharins

SUZANNE ALDINGTON and STEPHEN C . FRY

Centre for Plant Science. University of Edinburgh. Daniel Rutherford Building. The King’s Buildings. Mayfield Road. Edinburgh EH9 3JH. Scotland. U K

I . Introduction . . . . . . . . . . . . . . . . . A . Origin of the Oligosaccharin Concept . . . B . Preparation of Oligosaccharins . . . . . . C . Bioassays . . . . . . . . . . . . . . . . D . Purification and Chemical Characterization of Oligosaccharides . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

2 2 3 5

. . . . . . .

6

I1. Physiology of Oligosaccharin Effects . . . . . . . . . . . . . A . Fungal Oligo-P-glucans . . . . . . . . . . . . . . . .

7 7

. . . . . . . . . . .

12 17 32 34 37 38

. . . . . . .

B . Xyloglucan-derived Oligosaccharides as Growth Regulators . . . . . . . . . . . . . . . . C . Oligosaccharides of Pectins . . . . . . . . . D . Oligo-P-xylansasPossibleOligosaccharins . . E . Chito-oligosaccharides and Related Fragments F. Oligosaccharins from N-linked Glycoproteins? G . Conclusions . . . . . . . . . . . . . . . .

111.

. . . . . .

. . . . . .

. . . . . .

. . . . .

Mode of Action of Oligosaccharins . . . . . . . . . . . . . A . Evidence for Receptors . . . . . . . . . . . . . . . . B . Rapid Effects of Oligosaccharins . . . . . . . . . . . . C . Direct Effects of Oligosaccharides on Enzymes . . . . . .

41 41

46

56

IV . Natural Occurrence of Oligosaccharins . . . . . . . . . . . 58 A . Natural Occurrence of Xyloglucan Oligosaccharides . . . 58 B . Natural Occurrence of Pectic Oligosaccharides . . . . . . 59 C . Glycoprotein-derived Oligosaccharins . . . . . . . . . . 61 D . Conclusion . . . . . . . . . . . . . . . . . . . . . . 62 Advancesin Botanical Research Vol . 19 Copyright 01993 Academic Press Limited ISBN 0-12-005919-3

All rights of reproduction in any form rescrved

2

S. ALDINGTON AND S. C. FRY

V.

Mechanism of Formation and Degradation of Oligosaccharins . . A. Xyloglucan Oligosaccharides . . . . . . . . . . . . . . B. Pectic Oligosaccharides . . . . . . . . . . . . . . . . C. The Role of Chitinases, P-Glucanasesand Other Enzymes .

62 62 66 73

VI.

Movement of Oligosaccharins within the Plant: True Hormones? . . . . . . . . . . . . . . . . . . . . . . . A. PossibleTransport of Xyloglucan Oligosaccharides . . . . B. Non-transport of Wound Signals . . . . . . . . . . . . C. Transport of Elicitors . . . . . . . . . . . . . . . . .

75 75 76

Concluding Remarks . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .

77 77 77

VII.

74

I . INTRODUCTION A.

ORIGIN OF THE OLIGOSACCHARIN CONCEPT

The idea that biologically active oligosaccharides (called oligosaccharins) exist is about 17 years old (Ayers et al., 1976a,b.c; Albersheim and Valent, 1978), and is currently the subject of much imaginative research and speculation. Many exciting claims, some substantiated, have been made as to the significance of oligosaccharins. On the other hand, the phrase “believe in oligosaccharins” is still common enough to raise doubts. It thus seems appropriate to assess the current status of the oligosaccharin concept, and to evaluate objectively the biological roles ascribed to oligosaccharins. Other recent reviews include those by Dixon and Lamb (1990), Aldington et al. (1991) and Ryan and Farmer (1991). Oligosaccharins are particular oligosaccharides which, at low concentrations, exert biological effects on plant tissue other than as carbon or energy sources (Albersheim et al., 1983). Thus, while all oligosaccharins are oligosaccharides, not all oligosaccharides are oligosaccharins. “Elicitors”, in contrast to oligosaccharins, are any substances that evoke defence-related responses (especially phytoalexin synthesis) in plants. An elicitor does not have to be an oligosaccharide. An oligosaccharin does not have to evoke a defence-related response. Some, but by no means all, elicitors are oligosaccharins. Most of the known oligosaccharins are derived from cell wall polysaccharides, although it seems rather arbitrary to make this a necessary part of the definition. We would also count as oligosaccharins any biologically active oligomers that, although rich in sugar residues, also contained some non-carbohydrate material, e.g. phenolic, peptide or acyl groups. We would not count polysaccharides (say, molecular weight > 5000) as oligosaccharins, although some biologically active polysaccharides may act by virtue of possessing a particular oligosaccharin domain within their

OLIGOS ACCHARINS

3

larger structure. More precise definitions of “oligosaccharin” are neither possible nor desirable at this rapidly advancing stage in the development of the subject. The oligosaccharin concept grew out of plant pathology. The first oligosaccharins to be recognized were of fungal origin and their biological effects on Angiosperm tissues appeared to be related to the activation of defence responses (Albersheim and Valent, 1978). Soon afterwards it was found that oligosaccharins obtained from higher plant sources (so-called endogenous elicitors) can also evoke similar defence responses (Hahn et al., 1981; Lee and West, 1981a,b). Only later were plant-derived oligosaccharins shown to exert effects that appeared to be unrelated to disease resistance so that a role could be proposed in the life of the healthy plant (Albersheim and Darvill, 1985).

B. PREPARATION OF OLIGOSACCHARINS

Like cytokinins, which were first demonstrated in samples of autoclaved DNA, oligosaccharins were also first prepared by artificial means. The vast majority of research still uses such artificial oligosaccharins, a fact that may detract slightly from the credibility of oligosaccharins as biologically relevant signalling molecules. The limited evidence for the natural occurrence of free oligosaccharins is discussed in Section IV. Artificial oligosaccharins are prepared by the partial degradation of polysaccharides or whole cell walls, usually by one of four methods. Method 1. Degradation is brought about by partial acid hydrolysis (Nothnagel et al., 1983; Yamazaki et al., 1983; Broekaert and Peumans, 1988). Depending on the severity of the conditions used (acid concentration, temperature and time), this treatment cleaves a certain proportion of the glycosidic bonds in polysaccharides. Unfortunately, some glycosidic bonds (especially apiose, arabinofuranose and fucose) are much more acid-labile than others. Therefore, during partial acid hydrolysis, many of the theoretically possible oligosaccharide structures are not isolated. Nevertheless, acid hydrolysis has the advantages of cheapness and reproducibility. Dilute trifluoroacetic acid is often used because this volatile acid can readily be removed in vacuo, after hydrolysis. Also, sodium trifluoroacetate does not appear to be more damaging to plant cells than NaCl, so traces of residual trifluoroacetate that may remain after evaporation and neutralization with NaOH would not be expected to have any gross effects on metabolism. Method 2. It has been proposed that oligosaccharins could be prepared by treating the cell walls with alkali. This has been less extensively used as its effects are more difficult to define. The principal effect of cold alkalis on cell walls is solubilization of polysaccharides (especially hemicelluloses). Some

4


polysaccharides are partially degraded by cold alkali via a “peeling” of sugar units, one by one, from the reducing terminus of the chain (Kuhn et af., 1958; Whistler and BeMiller, 1958). The rate and extent to which this occurs depends on the nature of the polysaccharide. A second effect of alkalis (at least at higher temperatures) is to cause the cleavage, by any of several mechanisms, of a small proportion of the mid-chain glycosidic linkages. Thirdly, even very mild alkali treatment will hydrolyse ester-linked substituents, e.g. ferulate and methyl esters, that may be present on the polysaccharides. Method 3. Cell walls are fragmented by autoclaving or other heat treatments. These treatments will cause partial degradation of polysaccharides by several means including Hf-catalysed hydrolysis and OH-catalysed p-elimination (Barrett and Northcote, 1965), to yield fragments of a wide variety of sizes. Each of the first three methods is liable to yield fragments of material extraneous to the cell wall. Even the most highly purified cell wall preparations are likely to be contaminated with small amounts of other material, including membranes, nuclear proteins, RNA, DNA, and polyphenolics such as tannin bodies. Any of these polymers may yield oligomers upon treatment with acid, alkali or autoclaving. Some of these fragments possess biological activity: phenolic substances have diverse effects on plants (Isaiah, 1971; Corcoran et af., 1972; Danks et af., 1975; Blum and Dalton, 1985), and any contaminating DNA would yield cytokinins upon autoclaving (Miller et af., 1955). In addition, heating can cause carbohydrates to undergo chemical reactions producing substances such as maltol and isomalto1 (components of the aroma of freshly-baked bread) (Backe, 1910), and to react with proteins and amino acids to produce substances known to food scientists as “non-enzymic browning products” (Eble et af.,1983; Goodwin, 1983). Not always has sufficient consideration been given to the possibility that “oligosaccharin” activities associated with cell wall fragments produced by these methods may not be due to simple oligosaccharides. Method 4. The fourth and certainly the best method of preparing oligosaccharins is by partial enzymic degradation of polysaccharides or cell walls. We would assume that, if oligosaccharins are produced in vivo, it would be by enzymic degradation. Therefore, if the right enzyme(s) can be found, we have the ideal way to make “realistic” oligosaccharins. The enzymecatalysed reaction can be stopped at various stages, thus generating fragments of diverse sizes. After the digestion, the enzymes can be precipitated, removed chromatographically, or inactivated by boiling. Enzymes that have been used in this way include cellulase (York et af., 1984; McDougall and Fry, 1988), pectinase (Bishop et al., 1981; Branca et al. , 1988), pectate lyase (Davis et al., 1986a,c) and chitinase (Kurosaki et al., 1988). Research has until recently been hampered by unavailability of these enzymes in pure

OLIGOS ACCHARINS

5

form, but specialist suppliers are beginning to offer some of them so future prospects are good. Other methods for the preparation of oligosaccharins have been mooted, e.g. ultrasonication of polysaccharides, and the enzymic synthesis of oligosaccharides de novo via transglycosylation reactions (Bezukladnikov and Elyakova, 1988; Nilsson, 1988; Usui et al., 1990), but relatively little use has been made of these methods to date. The chemical synthesis of oligosaccha; et rides de novo is a rapidly developing field (Sharp et af., 1 9 8 4 ~Ossowski al., 1984; Nakahara and Ogawa, 1987,1990; Sakai et al., 1990; Torgov et al., 1990; Cheong et al., 1991) and provides a very powerful means of confirming the proposed structures of oligosaccharins as well as of exploring structureactivity relationships (for review, see Aldington et al., 1991). Oligosaccharides are among the very few natural products to be (1) hydrophilic enough not to partition from water into butanol (unlike many plant hormones, which are lipophilic weak acids) and (2) t o have molecular weights of about 600-3000 (larger than most intermediary metabolites; smaller than proteins, nucleic acids and polysaccharides). These features make oligosaccharides relatively easy to isolate. Separation methods commonly used include phase partitioning, gel-permeation chromatography to determine the size of the active molecules (Kobata et al., 1987), ion-exchange chromatography (Redgwell and Selvendran, 1986) or electrophoresis (Stoddart and Northcote, 1967) to determine charge, and highpressure liquid chromatography (HPLC) to effect final purification (Sharp er al., 1984a; McDougall and Fry, 1991a). C. BIOASSAYS

The definition of “oligosaccharin” demands some effect on plant tissue. Therefore, having prepared (or possibly isolated from natural sources) a mixture of oligosaccharides, the only way to demonstrate the presence of oligosaccharins is to perform a bioassay. Unfortunately, bioassays are notorious for their irreproducibility. There are many possible reasons for this lack of consistency: batches of plant material may vary genetically; one year’s harvest of seeds may differ phenotypically from the next; local conditions under which seedlings are grown may vary in subtle ways; different organs are used; scientists differ in the way they handle the plants; the physical stress inflicted on the plant by administration of the oligosaccharin may vary. Tissue cultures are particularly prone to change between one sub-culturing and the next and certainly change as they pass through the growth cycle. Oligosaccharin folklore is full of stories about differences between types of Petri dish, effects of volatile substances derived from particular plastics, auspicious corners of the greenhouse where the assay is always successful, and even of the benefits of “green fingers”!

6


Ultimately, bioassays are the only way to detect biological activities, and the present chapter illustrates the widespread support that has emerged for the existence of biological activities of oligosaccharides despite the experimental difficulties. The most reproducible bioassays are those in which the measured effect is relatively close to the initial action of the oligosaccharin. In contrast, with morphogenetic effects such as flowering (Gollin et al., 1984), the ultimate effect (flower development) may be far removed in space and time from the initial molecular action of the oligosaccharin, and thus the chain of events is particularly susceptible to interruption. At the other extreme, direct effects of oligosaccharins on enzyme action, or on membrane functions, are rapid and depend on few or no intervening steps. However, studies limited to this level would miss some of the possible systemic effects of oligosaccharins (cf. Hammerschmidt and KuC, 1982; Wildon et al., 1989). Bioassays are also at the heart of the methodology required to purify an oligosaccharin. The methods of polysaccharide fragmentation outlined in Section 1.B generate mixtures of oligosaccharides, most of which may lack oligosaccharin activity. The initial crude preparation can be subjected to a series of separation methods, but at each step a bioassay is required to indicate which fractions contain the activity. D. PURIFICATION AND CHEMICAL CHARACTERIZATION OF OLIGOSACCHARIDES

It is not possible here to discuss this topic in detail (see Fry, 1988; Dey, 1990), but the current status will be briefly reviewed. Oligosaccharides are often initially fractionated. according to their native charge, by ionexchange chromatography, e.g. on a column of QAE- (quaternary aminoethyl-) Sephadex (Baydoun and Fry, 1985). The second criterion is often size: a sub-fraction of the oligosaccharides, such as the neutral ones, is further fractionated by gel-permeation chromatography (gel filtration), e.g. on Bio-Gel P-2 or Sephadex G-25 (Sharp el al., 1984b). These gels are often set up in moderately large columns, e.g. of 1litre capacity, to which about 50ml of sample containing about 1g of oligosaccharides can be applied. Often the lowest molecular weight fraction with biological activity is taken for further purification because this will be the easiest to characterize chemically and because it may be the essential core structure conferring biological activity on larger molecules. Further fractionation can be achieved with a variety of chromatographic methods, e.g. preparative paper chromatography (which will accommodate about 10 mg per sheet; for reviews, see Bailey and Pridham, 1962; Fry, 1988), affinity chromatography on immobilized lectins (Blake and Goldstein, 1980; Green and Baenzinger, 1989), or semi-preparative HPLC (which will typically accommodate about 100 kg per run; McDougall and Fry, 1990,1991a). A recent development in

OLIGOSACCH ARINS

7

(analytical rather than preparative) HPLC is the use of anion-exchange chromatography on a pellicular resin (Dionex “CarboPac”) with 0.1 M sodium hydroxide plus sodium acetate as eluent: under such alkaline conditions even “neutral” oligosaccharides acquire a negative charge, by ionization of some of the -OH groups, and can thus be separated by anion-exchange chromatography. Excellent resolution of some closely related oligosaccharides is possible, providing evidence for or against homogeneity of an oligosaccharide preparation (Hardy and Townsend, 1989; McDougall and Fry, 1991a). The major limitation of this method for preparative purposes is the need to remove the NaOH from the sample very quickly after chromatography so as to minimize alkaline degradation. Another promising advance in HPLC of oligosaccharides is the use of graphitized carbon columns (Koizumi et al., 1991). Once purified, the oligosaccharide can be structurally characterized. Features that can readily be determined include monosaccharide composition (by acid hydrolysis and chromatography), anomeric configuration (by susceptibility to specific glycosidases), pyranose/furanose ring form (by susceptibility to mild acid hydrolysis), the occurrence of certain repeating motifs, e.g. Xyl-a-(1+6)-Glc (by enzymic release; Kooiman, 1961), and the reducing terminus (by NaB3H4-reduction to the corresponding [3H]alditol; Hall and Patrick, 1989). Precise linkages between other sugar residues in the oligosaccharide, as well as the presence of non-carbohydrate moieties, can often be determined by the rapidly advancing techniques of ‘H and 13C nuclear magnetic resonance spectroscopy (NMR) and fast-atombombardment mass-spectrometry (FAB-MS), as well as by conventional methylation analysis. Despite the many recent advances in carbohydrate chemistry, it remains considerably more difficult to sequence an oligosaccharide than an oligopeptide. This is because oligosaccharide structures have more variables D- versus L-isomers, pyranose versus than oligopeptides-including furanose ring-forms, a-versus P-anomers, (1+2)-, (1+3)-, (1+4)-linkages, etc. , and the presence of numerous modifications, e.g. deoxy-sugars, sugar acids, amino-sugars and O-acetyl-sugars. The automatic oligosaccharide sequencer is a long way off! It is also more difficult to apply the techniques of molecular biology to oligosaccharides than to peptides because the former are several steps further removed from transcription than the latter.

11. PHYSIOLOGY OF OLIGOSACCHARIN EFFECTS A.

FUNGAL OLIGO-8-GLUCANS

Oligo-P-glucans were the first oligosaccharins to be recognized; it is therefore appropriate to trace the work on them first and in some detail. It is well

8


established that plants, when challenged by microorganisms, can often resist becoming diseased by switching on any of a wide range of defence responses such as the accumulation of phytoalexins, lignin, silica, callose, extensin, peroxidase, chitinases and P-glucanases, and the activation of the hypersensitive response (EsquerrC-Tugaye and Mazau, 1974;Bell, 1981;Bird and Ride, 1981; Bailey, 1982; Hammerschmidt and KuE, 1982; Kratka and Kfidela, 1984; Mauch et al., 1988a,b). Studies in the mid-l970s, aimed at identifying the factor(s) by which plant cells can recognize the presence of foreign (fungal) cells, demonstrated that fungal cell wall components played a particularly important role. Specifically, it was shown that mixed-linkage P-( 1 4 ),( 1+6)-~-glucans, which are prominent components of the cell walls of many fungi but not of higher plants, were able to activate the synthesis of phytoalexins in uninfected plants (Ayers et al., 1976a,b,c; Ebel etal., 1976;for reviews, see Albersheim et al., 1981; West, 1981;Lamb et a f . , 1989). Commercial yeast extract (i.e. autolysate) was also found to contain ethanol-precipitable elicitor activity, which was due largely to the P-glucan component rather than to the more abundant a-mannan (Hahn and Albersheim, 1978). Phytoalexins are low molecular weight compounds with anti-microbial properties; they are virtually absent in healthy tissues but are synthesized and accumulated by the plant after exposure to microorganisms, at and fairly near the site of infection. Phytoalexins are chemically diverse-they include phenolics, terpenoids and polyacetylenes, the precise compound(s) formed depending on the plant species challenged (Grisebach and Ebel, 1978; Bailey and Mansfield, 1982). The production of phytoalexins by plants appears to be a widespread response which can aid in disease resistance. Elicitation of phytoalexins has, in a number of cases, been shown to depend on the synthesis of the rate-limiting enzyme in the biosynthetic pathway (Hahlbrock et al., 1981;Chappell et al., 1991). Plants may inhibit the growth of many microorganisms (both prokaryotes and eukaryotes) by accumulating high concentrations of phytoalexins, which have a very indiscriminate action. One of the differences between a successful and an unsuccessful infection may be that phytoalexins are not synthesized rapidly enough or in sufficient quantities (Darvill and Albersheim, 1984; KuE and Rush, 1985; Ebel, 1986). Detailed studies by Albersheim’s group, designed to determine the precise components of the fungal cell wall material responsible for the biological activity, established that quite small oligosaccharides of P-( 1 4 , (1+6)-~-glucan were effective (Albersheim and Valent, 1978). Partial acid hydrolysis of the fungal polysaccharide yielded a mixture of oligosaccharides; these were fractionated according to size by gelpermeation chromatography. The fractions obtained were bioassayed for their ability to elicit the synthesis of a flavonoid phytoalexin (glyceollin) in excised soybean cotyledons. After NaBb-reduction (in order to simplify

9

OLIGOSACCHARINS

the chromatography), it turned out that the smallest highly activated material was a heptasaccharide (strictly a hexasaccharidyl-alditol), which was purified to homogeneity by HPLC, and whose complete chemical structure was determined (Sharp et al., 1984a,b) (Table I). The structure of the active heptasaccharide was elegantly confirmed by chemical synthesis (Sharp et al., 1984c; Ossowski et al., 1984), and it was shown that seven other, closely related, heptasaccharides had much less activity (Sharp et al., 1984a). The active principal was a simple, pure oligosaccharide with no amino acid or other constituents. This discovery provided a very firm footing for the oligosaccharin concept, contradicting the view that cell wall carbohydrates were inert, purely structural and relatively uninteresting substances. Although much of this work was done with soybean cotyledons, it was established that P-( 1+3),( 1+6)-~-glucans also elicited the synthesis of different phytoalexins in a wide range of other plant species (Cline et al., 1978; Darvill and Albersheim, 1984). The oligosaccharin phenomenon therefore seemed to have a wide applicability. The active heptasaccharide was effective in very low dosesapproximately 0.lpmol per cotyledon (Sharp et al., 1984b). The doseresponse curve resembled a rectangular hyperbola, such as is frequently seen in enzyme kinetics; the concentration giving a half-maximal effect (equivalent to the K,) was about 10 nM (Cheong et al., 1991). A careful investigation of the structure-activity relationships of a wide range of related oligosaccharides (Table I) showed that, although several oligo-P-glucosides were able to elicit phytoalexin synthesis at high concentrations ((LM to mM), effectiveness in the 5-50 nM range depended on the presence of at least the following core hexasaccharide (Cheong et al., 1991): ~ - G l c -1+6)-~-Glc-( ( 1+6)-~-Gk-(1 - - + 6 ) - ~ - Gt) l~( (*I 3 3

t

1 D-GIc(*)

t

1 D-G~

where D-GIc is a P-D-glucopyranose residue. Larger oligosaccharides (e.g. compounds 1 and 2,Table I) were active if they possessed this motif within their structure.The two non-reducing terminal glucose residues marked (*) were essential for maximal activity (Sharp et al., 1984a), and activity was reduced if either of these residues was converted to glucosamine (compounds 6 and 7) or N-acetylglucosamine (compounds 8 and 9). Modification of the reducing terminus (t) had relatively little effect. Through this discriminating recognition system, the plant is presumably provided with a means of detecting small amounts of fungal P-(1+3),(1+6)-glucans in the presence of the much larger amounts of other P-glucans that are found in all higher plants.

TABLE I Concentrations of oligo-p-glucosides required to elicit phytoalexin synthesis in vivo and to compete with the specific membrane-binding of a highly elicitor-active, radiolabelled oligo-/3-glucoside in vitro

Compound structurea

Concentration (nM) required for half-maximal biological effect . ) b or membrane binding ( TI .)" ..

(ml.. 101

102

103

104

I

I

I

I

105 I

I

I

I

I

I

I

I

I

I

I

I

I

1

I

I

.......... I I

............. ......... .............:...... :.: ..: ..I.. :::::: :.. .:.I:.. ... ............ . . .I.... . .....~.;~.-...$1;...... .:, ................... ..... ..: ;...:]...I... 2.1.. .I::.t~.:t.:rl..-.:l:t:t:,~-.~ ...I..] .>%

12

I

I

2.1

13 14

Symbols used in structures: 0 , propyl group; 0 , ally1 group; 0, reducing terminal glucose unit; 0, glucose residue; +, P-(1+6)-linkage; t , P-(1+3)linkage; =),other glycosidic linkage; 0, glucitol unit; 6, glucosamine residue; 0 , N-acetylglucosamine residue. All the sugar residues are in the P-D-pyranose form. .) shows the approximate concentration of each The soybean cotyledon bioassay devised by Ayers et al. (1976a) was used; the upper bar oligo-P-glucoside required to elicit half-maximal accumulation of glyceollin. The “maximal” response was the amount of glyceollin elicited by saturating implies that the concentration indicated gave less than a half-maximal response. levels of compound 3. The symbol ‘The concentration of the same oligo-P-glucosides required to inhibit (competitively) 50% of the specific binding of a radioiodinated derivative of compound 3 was also tested (lower bar, CIII]...;see Section III.A.l). Data summarized from Cheong et al. (1991) and Cheong and Hahn (1991).

(m..

m+

12

S . ALDINGTON AND S. C. FRY

The above investigations have provided a clear indication of the chemical nature of the active principle from fungal cell walls. In addition, numerous other studies have probed the diverse biological effects of various carbohydrate-rich fragments derived from fungal cell walls. These preparations have often been less well defined chemically. Effects reported include: (1) the accumulation of lysozyme and chitinase (Bernasconi et al., 1986), (2) induction of an arabinosyltransferase (Bolwell, 1984, 1986) and a prolyl hydroxylase (Bolwell et al. ,1985b;Bolwell and Dixon, 1986)involved in glycoprotein synthesis, (3) evocation of quantitative and qualitative changes in the phenolic components of the plant cell wall (Bolwell et al. , 1985a) and in the levels of an enzyme (phenylalanine ammonia-lyase) partly responsible for these (Bolwell et al., 1 9 8 5 ~ ) ~ (4) the synthesis of diverse low molecular weight secondary metabolites in plant tissue cultures, including several commercially valuable alkaloids, e.g. berberine in cultured cells of Thalictrum rugosum (Funk et al., 1987; see also Constabel and Eilert, 1986), ( 5 ) a burst of respiration (Funk etal., 1987) and ethylene synthesis (Piatti et al., 1991) in cultured cells. Nigeran, a different mixed-linkage fungal polysaccharide [a-( 1-+3), (1+4)-~-glucan], is also capable of eliciting the synthesis of phenylalanine ammonia lyase, in Petunia cell suspension cultures, although at the relatively high concentration of 400 mg 1-’ (Hagendoorn et al. , 1990). Perhaps surprisingly, it seems that p-( 1 4 ),( 1+6)-~-glucans can block the induction by other fungal components of another defensive reactionthe hypersensitive response (Section II.C.4)-in potato protoplasts (Doke and Tomiyama, 1980a,b). It is unclear why the plant should respond “less defensively” to a combination of two fungal components than to one. For further discussion of possible synergism between oligosaccharins, see Section II.G.3.

B. XYLOGLUCAN-DERIVED OLIGOSACCHARIDES AS GROWTH REGULATORS

Xyloglucan was first described as a storage polysaccharide of certain seeds (Kooiman, 1961). Only later was it shown to be present in large amounts in the primary cell walls of higher plants, although here, as a structural component, is where it undoubtably plays its major role (Bauer et al., 1973). It is a hemicellulosic polysaccharide, i.e. it cannot readily be extracted from the cell wall in hot water or by chelating agents, but can be solubilized by cold concentrated alkali-although even NaOH at 6.0 M, the optimum

F = a-L-fucose G = P-D-glucose L = P-D-galactose X = a-D-xylose

fi , il = (1+2)-glycosidic linkages

t,4

+ +

= = =

A

Fig. 1.

(1+6)-glycosidic linkages (1+4)-glycosidic linkage (1+4)-glycosidic linkage susceptible to hydrolysis by cellulase.

General arrangement of major sugar residues in xyloglucan.

14


concentration, may take many days for efficient extraction of xyloglucan at 25°C (Edelmann and Fry, 1992a). Xyloglucan from the primary cell wall is composed of the sugar residues D-glucose, D-xylose, D-galactose, L-fucose and L-arabinose, in decreasing order of abundance. It is readily distinguished from the other main xylosecontaining hemicelluloses, the xylans, by the fact that the xylose residues are a-linked rather than (3-linked. The arrangement of the major sugar residues of xyloglucan is shown in Fig. 1. All the sugar residues shown in Fig. 1 are in the pyranose ring form; a small amount of arabinofuranose may also be present. The galactose residues of xyloglucan are often acetylated (York et af.,1988). The structure and functions of xyloglucan have been reviewed (Hayashi, 1989; Fry, 1989a). Xyloglucan can be fragmented into a limited number of major oligosaccharides by exhaustive digestion with pure cellulase [EC 3.2.1.4; endo-p(1-+4)-~-glucanase]. This enzyme attacks the (3-( 1+4)-~-glucan (celluloselike) backbone of xyloglucan wherever there is a non-xylosylated glucose residue (marked * in Fig. 1; Bauer et af., 1973). Since these tend to occur every fourth residue along the backbone, the major oligosaccharides generated (XG7, XG8, XG9, XG9n, and XG10) are based on a G-+G-+G+G (cellotetraose) core, which may bear a variety of substituents (Valent et af.,1980; Kato and Matsuda, 1980; Matsushita et af.,1985). Other fragments such as XG5 may arise because the spacing of non-xylosylated glucose units is not completely regular and because of partial breakdown of some of the initially formed cellotetraose-based oligosaccharides by the action of contaminating enzymes present in commercial cellulase preparations. In addition, a few non-xylosylated glucose residues may carry arabinofuranose residues and therefore be protected from the action of cellulase; this leads to the production of an oligosaccharide of degree of polymerization (DP) 17 (Kiefer et al., 1990). York et af. (1984) were the first to show that one particular xyloglucanderived oligosaccharide, XG9, can regulate plant growth. XG9 can, at an optimal concentration of 1nM, partially block the promotion of growth ~ acid (2,4-D, an artificial auxin). caused by 1 p , 2,4-dichlorophenoxyacetic At higher concentrations, e.g. 100 nM, XG9 was much less effective; this was surprising because growth inhibitors are usually more effective at higher concentrations. These observations have been reproduced and extended in three other laboratories (McDougall and Fry, 1988; Emmerling and Seitz, 1990; Hoson and Masuda, 1991). It was confirmed that highly purified XG9 was active, showing that the activity resided in the structure of the oligosaccharide itself, rather than in a contaminant (McDougall and Fry, 1988, 1989a,b, 1991a). It was shown that the activity was critically dependent on the a-L-fucose residue present in XG9 since XG8 (McDougall and Fry, 1989a) and a mixture of fucose-free oligosaccharides from pine (Nealey et

-

15

OLIGOSACCHARINS

al., 1989) were inactive. The XYl

1

Glc+Glc-.

..

t XYl unit from the non-reducing terminus of XG9 was apparently irrelevant to the growth-inhibiting activity because XG5 [Fuc+Gal+Xyl+Glc+Glc] and the commercially-available 2’-fucosyl-lactose [Fuc+Gal+Glc] (Kuhn et al., 1958) were also active at low concentrations (McDougall and Fry, 1989b). On the other hand, the L-fucose was not sufficient for activity, as shown by the lack of effect of free L-fucose or methyl-a-L-fucopyranoside (McDougall and Fry, 1989b). No effect of XG9 on the growth induced by indoleacetic acid (IAA) was seen in several bioassays, e.g. using pea internodes, Azuki bean epicotyls, cucumber hypocotyls and oat coleoptiles (Hoson and Masuda, 1991). It was also found that the inhibitory effect of XG9 on the action of 2,4-D in pea stem segments could not be reversed by increasing the 2,4-D concentration, showing that the effect was uncompetitive (Hoson and Masuda, 1991). Since 1nM XG9 also blocked the stimulatory action of low pH on the elongation of pea stem segments (Lorences et al., 1990), it seems likely that XG9 interferes with some basic process common to the action of both H+ and 2,4-D on elongation, such as the generation of turgor or the production of a wall-loosening enzyme. There is an intriguing report, possibly related to the above, that 1nM XG9 will antagonize the beneficial effect of auxin on the regeneration of isolated carrot protoplasts; again, the activity was lost at 1 nM XG9 (Emmerling and Seitz, 1990). It will be of great interest to see whether any other auxin responses are antagonized by 1nM XG9. Priem et al. (1990) have reported that an L-fucose-containing oligosaccharide (see Section 1I.F) is able to regulate the growth of flax hypocotyls. It seems possible that this effect could operate via a mechanism similar to that of the L-fucose-containing XG9. Questions need to be answered about the importance of the (3-D-galactose residue to which the fucose is attached in XG9 but which is missing in Priem’s oligosaccharin. Tran Thanh Van and Mutaftschiev (1990) reported a stimulation of elongation in wheat coleoptiles by surprisingly low concentrations (- 1-10 p ~ of) unspecified heptaand nonasaccharides of xyloglucan obtained from Rubus culture filtrates. The surprising loss of ability of XG9, at higher concentrations, to block the response of pea stem segments to 2,4-D appeared to be due to a second, growth-prompting effect of this oligosaccharide. This second effect was not exhibited by XG5 or 2’-fucosyl-lactose, both of which lack the cellotetraose core (McDougall and Fry, 1989b). In agreement with this, it was found that XG9, added to stem segments in the absence of 2,4-D, was able to promote

-

-

16


1.o

XG 7

Fig. 2. The effect of HPLC-purified xyloglucan-oligosaccharides on the elongation of pea stem segments. The difference in elongation between untreated and treated segments ( A L ) is plotted against concentration for each oligosaccharide. The letters a-e indicate the statistical significance of the apparent deviation of AL from 0: a,p 30 000), which both elicit glyceollin synthesis. In both cases, the ligand quickly accumulated at the cell surface before becoming internalized into the vacuole or bound to the tonoplast. The GalA12-fluorescein complex required less time for internalization (2 h) than the larger fungal elicitor (4 h). Two fluorescein-labelled non-elicitor polypeptides-bovine serum albumin (molecular weight 68 000) and insulin (molecular weight 6000)-were not able to enter the cells even after an 8 h incubation, nor did they bind to the cell surface. A GalA12-[’251]iodotyro~ine conjugate was taken up whereas an ovomucoid[1251]iodotyrosineconjugate was not (Fig. 5 ) . The GalAI2derivatives were internalized intact (rather than after breakdown to the monosaccharide), by an energy-dependent, temperature-dependent process. Uptake of the GalA12-[1251]iodotyrosine conjugate was reduced about 10-fold by addition of a 10-fold molar excess of non-radioactive GalA12. It was suggested that uptake was receptor-mediated. Filippini et al., (1992) have shown that oligogalacturonides can interfere with the specific binding of indoleacetic acid (IAA) to isolated membranes. Thus, it seems possible that oligogalacturonides and IAA may have a common binding site in the plasma membrane. Chitosan oligosaccharins, which affect plant cell membranes rapidly in suspension cultures and more slowly in intact tissues, can apparently bind to the plant cell wall, membrane and nucleus. When [3H]chitosan was applied to the surface of pea endocarp tissue, the label was detected in the cytoplasm and nucleus within 15 min (Hadwiger et al., 1981; Kendra and Hadwiger, 1987b). On the other hand, 3H-labelled oligosaccharides of xyloglucan (including XG9) failed to enter cultured spinach cells to any appreciable extent even after an incubation period of 72 h (Baydoun and Fry, 1989; Smith and Fry, 1991). This lends support to the idea that the uptake of oligosaccharins, where it does occur, is mediated by specific binding sites in the plasma membrane and is not simply a consequence of “accidental” trapping during endocytosis. The mechanism by which 1nM XG9 inteferes with the growth-promoting action of 2,4-D and H + in pea stem segments is unknown. The possibility should be considered that it acts by interfering with the transport or binding of auxin, a plasma membrane phenomenon (Barbier-Brygoo et al., 1991). The specific structural requirements for the growth-inhibiting activity of XG9-especially the dependence on an a-L-fucose residue-suggest a specific receptor for XG9 and related fucose-containing oligosaccharides

-

-

45

OLIGOSACCHARINS

20

15 r

rl

X -1 J

w

x2

10

t : 2 0

2c

5 A c I

1

2

3

4

5

TIME (HWRS) Fig. 5. Time-course of internalization of 'Z51-labelled conjugates of oligogalacturonides and ovomucoid in cultured soybean cells. 0 , '251-labelledoligogalacturonides at 23°C; A , l r s I labelled oligogalacturonides at 4°C; 0,'2sI-labelled oligogalacturonides at 23°C in the presence of non-radioactive oligogalacturonides; 0, '2SI-labelledovomucoid at 23°C. From Horn er al. (1989).

(McDougall and Fry, l988,1989a,b). Since XG9 is not significantly taken up by cells, the putative receptor would have to be located on the plasma membrane or (less probably) in the cell wall. The very low concentration optimum (-nM) of XG9, some orders of magnitude lower than the K , values of typical enzymes, is also compatible with a receptor, similar to a hormone receptor. However, no work has yet been reported to show XG9-binding sites, either in membranes or in any other subcellular location. A binding site for a less well characterized lignification-inducing glycoprotein from Puccinia graminis (see Section 1I.F) has also recently been reported (Kogel et al., 1991). Specific binding sites of molecular weight 30000 were found in the plasma membrane of wheat and barley leaf cells.

46


Finally, a novel approach to the demonstration of putative membranelocalized oligosaccharin-receptors is to demonstrate biological effects on isolated protoplasts of carbohydrates that have been conjugated to molecules or particles so large that they cannot possibly penetrate the plasma membrane. Thus, the finding of effects of silica-immobilized sugar residues (Lienart et al., 1991) is circumstantial evidence for receptors in the plasma membrane. B. RAPID EFFECTS OF OLIGOSACCHARINS

If, as suggested in Section III.A, there are oligosaccharin receptors in the plasma membrane, what do these receptors do after binding an oligosaccharin? Some very fast, almost instantaneous responses by plant cells have been reported to occur after the addition of oligosaccharins. These responses tend to be related to effects which the oligosaccharins have on the plant cell membrane, suggesting some sort of direct link between the two. 1. Membrane depolarization Some oligosaccharins can cause rapid membrane depolarization. Fungal elicitors prepared from the ethanol-soluble material of mycelia from two plant pathogens (Colletotrichum lagenarium and Phytophthora parasitica var. nicotianae) were applied to the roots of host plants-melon and tobacco, respectively (Pelissier et al., 1986; see Fig. 6b). Depolarization occurred within seconds in both systems; in melons, a new steady state was reached after lmin, which remained stable for at least a further 25min. Removal of elicitors caused repolarization. Elicitors from C. lagenarium also caused depolarization in maize roots; this effect therefore seems unrelated to pathogen specificity. After elicitors from P. parasitica had been fractionated by gel-permeation chromatography on Bio-Gel P-2, those of intermediate and high molecular weight induced a marked membrane depolarization and were very active inducers of ethylene biosynthesis. The elicitors caused depolarization by reducing the electrogenic component. The ethylene-inducing oligosaccharins derived from melon cell wall pectins (see Section II.C.7) also induced rapid membrane depolarization in roots of host and non-host plants (Esquerre-Tugaye et al., 1985). Mayer and Ziegler (1988) treated soybean tissue with a highly purified glucan elicitor preparation (molecular weight 4000-8000) from the cell walls of the fungus Phytophthora megasperma f.sp glycinea. At 1 mg l-', the elicitor induced depolarization within 2 min of contact with the plant cells. However, after a further lOmin, the membrane hyperpolarized. The concentration of the glucan which induced nearly maximal phytoalexin synthesis (0.1 mgl-') did not induce depolarization, but did cause the hyperpolarization. This effect was maximal at pH 6, which was also the

47

OLIGOSACCHARINS On A

-201

B

-185

1

NaCN

Low DP

C Off D High DP

-100

f

I

I

I

I

I

,

off

I

I

Time (5-min intervals)

(4

1

z2

f .parasitica E.

1C.

lagenarium E.

a

-15017

i

I \

rc

/Melon t-E

-loo/

f

-E

5 min

Fig. 6. Effects of oligosaccharins on plant cell membrane potential. The effectors were added at and then rinsed away at t . (a) Depolarization of tomato leaf mesophyll cells by NaCN (1 mM; curve A), small oligogalacturonides (mean DP = 4) (1 g I-'; curves B, C), and large oligogalacturonides (mean DP = 15) (1 gl-'; curve D). The units for the y-axis are indicated on the individual curves. The gaps (-I )-I were 10 min. From Thain et al. (1990). (b) Depolarization of melon and tobacco root hairs by elicitors (-0.1 g l - ' ; E.) from Colletotrichum lagenarium and Phytophthora parasitica, respectively. From PClissier et al. (1986).

48


optimum pH for induction of PAL. It was suggested that hyperpolarization played a role in the induction of phytoalexin synthesis but that depolarization did not. Results reported by Tomiyama et af. (1983) suggested a much slower effect of living Phytophthora infestans on the membrane potential of infected potato cells. Infection by compatible races did not affect the membrane potential within 24 h; infection with incompatible races resulted in depolarization one to several hours after fungal penetration of the cell. It is understandable that exogenous purified oligosaccharins should have quicker and more widespread effects than fungal infection. More recently, Thain et al. (1990) studied the effects of oligogalacturonides on the membrane potential of tomato leaf cells. The membrane potential (about - 200 mV) that is normally present in these cells is thought to be maintained by the operation of an electrogenic H+-pump; it can be approximately halved by application of 1mM cyanide (Fig. 6a). At 181-' (- 1.4 m ~ )small , oligogalacturonides (DP 1-7; mean 4) caused a large and rapid depolarization (by -50 mV within -0.5 min), which was reversed within 5-10 min when the oligosaccharins were removed. Similarly, larger oligogalacturonides (DP 10-20; mean 15) at 1 gl-' ( - 0 . 4 m ~ ) caused a depolarization which was initially rapid (- 25 mV within 1min) and then followed by a slower continued depolarization (by a further -25 mV within 10min); this effect was also reversed within 10 min after removal of the oligosaccharins (Fig. 6a). The difference between the two oligosaccharin preparations may have been related to the different defence responses evoked by them: both the preparations induced protease inhibitor synthesis; phytoalexin synthesis was elicited by the larger fragments only (see Sections II.C.2 and 3). How pectic oligosaccharins evoke the transcription of protease inhibitor genes is unknown; indeed, as discussed in Section VI.C, it is unclear whether they do evoke it at all, or whether their action is limited to initiating the dispatch of the mysterious second messenger, the true PIIF. The effect of oligogalacturonides (and of injury) on protease inhibitor induction in tomato can be reversibly blocked by pre-treatment of the plant with acetylsalicylic acid and related benzoic acid derivatives (Doherty et af., 1988), although in cultured rice cells salicylic acid induced a protease inhibitor (Masuta et af., 1991). However, in view of the major uncertainty about the target of the oligogalacturonides, there is little that can yet be concluded from this about the mode of action of wound signals. So what are the consequences of oligosaccharin-induced depolarization? Is electrolyte leakage enhanced? Is membrane permeability affected allowing internalization of oligosaccharins? Addition of polycations (e.g. chitosan) to cell cultures rapidly resulted in electrolyte leakage owing to an increase in membrane permeability (Young et al., 1982; see Section II.E), although polyanions (e.g. polygalacturonic acid and poly-L-aspartic acid)

-

-

-

OLIGOSACCHARINS

49

that induced the release of Ca2+from whole cells did not affect membrane permeability (Young and Kauss, 1983). The major electrolyte leaked by cultured cells on addition of chitosan was K + , with 5 1 5 % of cellular K+ exiting within the first 30 min; by the time this efflux had diminished, callose synthesis had reached a constant rate. Ca2+ may possibly enter the cell by the membrane depolarization occurring owing to the K+ efflux but there was no correlation between the degree or time course of electrolyte leakage and callose formation (Kauss, 1987). Atkinson et al. (1985) provided evidence that a K+ efflux/H+ influx is an important preliminary to the hypersensitive response of tobacco to Pseudomonas syringae. Net K+ efflux began 1.0-1.5 h after inoculation of tobacco cells with bacteria and it reached a maximum in 2.5-3.0 h, dropping within 5 h. Purified pectate lyase from Erwinia chrysanthemi induced a similar K + efflux/H+ influx in suspension-cultured tobacco cells (Atkinson et al., 1986). This may point to a mechanism by which pectic enzymes (or perhaps their oligosaccharide products) induce the hypersensitive response. Peever and Higgins (1989) also reported a rapid enhancement of electrolyte leakage in tomato-Cladosporium fulvum interactions. Specific elicitors isolated from apoplastic fluid induced electrolyte leakage only in cultivars resistant to the race of C.f u f v u mused. A glycoprotein non-specific elicitor induced electrolyte leakage in both resistant and susceptible cultivars. Dow and Callow (1979) had previously shown that culture filtrate of three races of C.fulvum elicited rapid electrolyte leakage from isolated tomato leaf mesophyll cells, the active elicitors in this case being glycopeptides. Uptake of [''C]leucine across the plasma membrane in cultured sycamore cells was found to be inhibited by the same acid-solubilized plant cell wall fragments that caused cell death (Fry et al., 1983; cf. Section II.C.4). This again indicates an interference by oligosaccharins with the activities of membranes.

2. Oxidative metabolism The generation of the superoxide anion radical ( 0 2 - - ) appears to act as a trigger for several defence responses when inflammatory (animal) cells are exposed to microbial challenge (Badwey and Karnovsky, 1980). One effect is to produce more reactive oxygen species such as hydroxy radicals (OH.) (Halliwell, 1978; Halliwell and Gutteridge, 1984), which can attack many biological molecules, especially polyunsaturated fatty acids, which are thereby peroxidated. Hence the lipids in membranes are particularly vulnerable to free radicals of oxygen: RH+OH* * R*+H20 Re + 0 ROO.

2

+ R'H

+

(1)

ROO- (peroxide radical)

(2)

+ ROOH (peroxide)

(3)

-+ R'.

50


where R are R’ are fatty acids. This chain reaction can be terminated at eqn (2) by reduction of the peroxide radical with glutathione (G-SH) and glutathione peroxidase: 2 ROO.

+ 2 G-SH + 2 ROOH + G-S-S-G

(4)

Epperlein et al. (1986) attempted to assess the involvement of reactive oxygen species in phytoalexin (glyceollin) accumulation in soybeans responding to an abiotic elicitor, AgNO3. The cotyledons were treated with acetaldehyde), excessive a 02---generating system (xanthine oxidase being prevented with superoxide dismutase which accumulation of 02-* catalyses the dismutation of 0 2 - * to H202

+

2 02-.

+ 2H+ + H202 + 0 2

(5)

supplied in this way did not mimic AgN03 in eliciting glyceollin was not directly involved in phytoalexin synthesis, suggesting that 02-* elicitation. However, OH. appeared to have a role: scavengers of OH. such as mannitol, dimethylsulphoxide, benzoate and methionine inhibited the AgN03-induced accumulation of glyceollin. This suggested a possible role for lipid peroxidation in phytoalexin accumulation. As this type of reaction was shown to occur in plant cells with an abiotic elicitor, the possibility of biotic elicitors initiating events by free radical generation has been investigated. Reactive oxygen species have now been detected in host-pathogen interactions and as a rapid response to elicitors. For example, suspensioncultured soybean cells exhibited a greatly enhanced luminol-dependent chemiluminescence 20-30 min after the addition of P-glucan elicitor from Phytophthora megasperma; the response increased for at least a further hour. The increase in chemiluminescence was inhibited by exogenous superoxide dismutase and catalase, and also by peroxidase inhibitors, suggesting . H 2 0 2were involved in generating the luminescence and that both 0 2 -and that peroxidase played a role in the formation of these active oxygen species (Lindner et al., 1988). The rapid production of H202 by soybean cells, and its use by endogenous cell wall peroxidases following the addition of elicitors (crude autoclaved cell walls/membranes; Apostol et al., 1987) from Verticillium dahliae and oligogalacturonides, caused vigorous peroxidatic activity within 5 min. A variety of water-soluble exogenous test compounds, including IAA and certain fluorescent dyes, were rapidly destroyed by oxidation (Apostol et al., 1989) (Fig. 7). IAA has been shown to inhibit the induction of chitinase (a pathogenesis-related enzyme) in Nzcotiana tubacum; the enzyme could not be detected in the upper leaves where the IAA concentration was relatively high, but represented 1 4 % of total soluble protein in the lower leaves and roots where there was less IAA (Shinshi et al., 1987). Hence, the destruction of IAA as a consequence of oligosaccharin treatment may be related to 02-*

OLIGOSACCHARINS

51

NADH t Hi

Fig. 7. A model of the arrangement of components involved in the oxidative burst evoked by elicitors. According to this model, any component which interferes with receptor-reductase coupling, or which consumes electrons [e.g. Fe(CN),'-] or reduces H2O2 [e.g. Fe(CN),"], or which blocks the oxidase (e.g. KCN) would be expected to inhibit both the bleaching and transmembrane signalling functions of the oxidative burst. From Apostol et al. (1989).

evocation of certain defence responses. The same response might also account for the ability of oligogalacturonides to antagonize many of the effects of IAA (see Section II.C.l);if so, we would predict that oligogalacturonides should not affect the response of plants to "non-oxidizable" auxins such as 2,4-D. The generation of 0 2 - - may be involved in the hypersensitive response (Doke, 1983a,b). Potato tuber disks inoculated with spores of an incompatible race of Phytophthora infestans, which evoked the hypersensitive response, increased in ability to reduce extracellular cytochrome c 1-4 h after inoculation (Doke, 1983a); the increase was also evoked by fungal wall components (Doke, 1983b). [Spores, which germinated on the disks, started to penetrate cells 1h after inoculation.] Infiltration of the disks with superoxide dismutase delayed both the hypersensitive response and phytoalexin accumulation in the incompatible interaction. The conclusion was that the reducing activity was dependent on 0 2 - * cyt c (Fe")

+ 0 2 - * + cyt c (Fe2+) + 0 2

(6) Another hypersensitive-like response, the browning and death of cultured rice cells induced by chitosan, was blocked by catalase, suggesting a

52


role for H202, although exogenous H 2 0 2itself did not induce browning and death (Masuta et al., 1991). The response was also blocked by vitamins C and E (free-radical scavengers) but not by superoxide dismutase. Lipid peroxidation, which can cause dramatic alterations to membrane permeability, has been reported in several plant tissues on exposure to elicitors. It appears to be a characteristic response to mechanical damage and microbial infection (Chai and Doke, 1987);it has been reported to occur during the hypersensitive response and to be initiated by 0 2 - * (Adam et al., 1989). Keppler and Novacky (1987) found an increase in lipoxygenase activity in cucumber cotyledons infected with incompatible bacteria. Lipoxygenase (EC 1.13.11.12) catalyses the reaction: ,

..--CH=CH-CH,-CH=CH--.

.. + 0

2

+ ...-CH-CH=CH-CHSH-.

.. (7)

I

0-OH

The target group (. ..-CH=CH-CH2-CH=CH-. ..) occurs in several common fatty acids, e.g. linoleic, linolenic, arachidonic (= eicosatetraenoic) and eicosapentaenoic acids, some of which are themselves elicitors (Bostock et al., 1981) and precursors of traumatin and jasmonic acid. One way in which lipoxygenase may be beneficial during infection is to scavenge harmful free fatty acids, so any increase in lipoxygenase activity may be a response to free radical-initiated membrane damage. Increased levels of lipoxygenase activity were induced in the system studied by Peever and Higgins (1989), in which lipid peroxidation was also induced. Preparations from Colletotrichum findernuthianurn (including a galactoglucomannan) elicited phytoalexins and stimulated the accumulation of products indicative of lipid peroxidation in bean tissues 6 hours after treatment. These responses were also stimulated by generators of activated oxygen species, although whether they were stimulated more quickly than by the polysaccharide was not stated (Rogers et al., 1988). The accumulated evidence suggests that elicitors of defence responses, especially elicitors of fungal origin, do play some role in generating activated oxygen species, and that the presence of such species can have rapid effects on plant cell membranes. Hence, membrane damage could be an early signal to the plant of a potential pathogen, and this could be beneficial in allowing the release of autolytic enzymes that generate oligosaccharins from the plant’s own cell wall (Lyon and Albersheim, 1982).

3. Protein phosphorylation Another interesting rapid oligosaccharin effect rather different from those described above was reported by Grab et al. (1989). Following the addition of a P-glucan elicitor from Phytophthora megasperma, there were rapid changes in phosphate turnover in several phosphoproteins. The effect of the

OLIGOSACCHARINS

53

elicitor on protein phosphorylation was tested after in vivo labelling with [32P]orthophosphate. Decreases and increases in the labeling of several phosphoproteins occurred about 5 min after elicitor application. One particular polypeptide (molecular weight 69000) showed a decrease in 32Pin response to elicitor treatment but was strongly phosphorylated in vitro in the presence of a low molecular weight factor ( M , = 1000) from soybean cell cultures. This “effector” was partially characterized and found to be negatively charged at pH 7.3; its activity was reduced after treatment with alkaline phosphatase and with crude preparations of pectinase and cellulase. Farmer et al. (1989, 1991) have shown that plasma membranes isolated from tomato and potato are promoted in their ability to incorporate label from [ Y - ~ ~ P I A into T P proteins in vitro by the addition of 0.1-1.0mM oligogalacturonides of D P 20. Specific protein phosphorylation was also reported to occur by Dietrich et al. (1990) in parsley cells elicited by a fungal P-glucan. A 45 kDa protein, found in microsomal and cytoplasmic fractions, was phosphorylated as early as 1min after treatment. A 26 kDa nuclear protein was also phosphorylated rapidly. Changes in protein phosphorylation correlated with the biological response of the cells and appeared to depend on the presence of Ca2+ in the medium. Phytoalexin accumulation was reduced in Ca2+-deprivedcells. It was hypothesized that signal transduction may involve the activation of a Ca2+-dependentprotein kinase.

-

4. Second messengers D o levels of putative second messengers, such as intracellular Ca2+,change during oligosaccharin treatment, as predicted by the work of Dietrich et al. (1990)? Kurosaki et al. (1987b) found that addition of verapamil (a Ca2+channel blocker) to carrot cell cultures, 30 min (but not 60 min) after fungal elicitors, inhibited subsequent phytoalexin accumulation, suggesting that intracellular Ca2+ concentration was important in elicitor action. They also found that intracellular levels of cAMP increased on addition of an elicitor. This suggested that Ca2+ and cAMP may participate as second messengers in the regulation of phytoalexin production. Hahn and Grisebach (1983), however, denied any role of CAMP. Direct measurements, using plant cells transformed with the gene for a Ca2+-sensitive fluorescent protein, aequorin, have demonstrated a rapid elevation of intracellular Ca2+concentration by P-glucan elicitors (Knight et al., 1991). Recent work in the same laboratory (Messiaen et al., 1992) has shown that A4~s-oligogalacturonideslarge enough to form an oligosaccharide-Ca2+ “egg-box’’ conformation (see Jarvis, 1984)-and to elicit phytoalexins-also caused substantial increases in intracellular free Ca2+, especially near the plasma membrane. This effect was blocked by verapamil. It thus appears plausible that some oligosaccharins act primarily by interacting with the plasma membrane, secondarily inducing a change in

54


intracellular free Ca2+. Certain protein kinases are Ca2+-dependent,so the tertiary effect could be on protein phosphorylation. Ca2+ also regulates the phosphatidylinositol phosphate signalling system in animals, and so it is interesting to note that Kurosaki et al. (1987a) have demonstrated a turnover of phosphatidylinositol during the elicitation of phytoalexins in carrot cell cultures. 5. R N A synthesis Following infection by a potential pathogen, the transcription of defencerelated genes is activated as part of a massive switch in the pattern of host gene expression (Cramer et al., 1985). Ryder et a f . (1986) treated bean cell cultures (Phaseolus vulgaris) with elicitors released by heating walls of the fungus Colletotrichum lindemuthianum. This inhibited the synthesis of several polypeptides whilst stimulating the synthesis of at least 60 others. Stimulation was usually observed only 1h after elicitor treatment. The pronounced change in the pattern of translation was due to a switch in the pattern of mRNA synthesis. Marked changes were also observed in bean hypocotyls infected with an incompatible race of C. lindemuthianum. PAL and chalcone synthase (CHS) mRNA accumulated within 35 h of inoculation; this may actually represent a rapid response as it takes 3 0 4 0 h following inoculation for the spores to germinate and come into contact with the first host cell. PAL is the first enzyme on the pathway from primary metabolites to phenolic phytoalexins and lignin; CHS is the first enzyme on the branch leading to flavonoid synthesis. Somssich et al. (1989) isolated numerous cDNA clones corresponding to genes which were rapidly activated in parsley cells by fungal elicitors. Transcription of 18 different genes was rapidly and transiently activated. The genes encoding PAL and CHS were activated within 2-3min of elicitor treatment (K. Edwards etal., 1985; Lawton and Lamb, 1987). Ryder et al. (1986) reported that the elicitor stimulation of cinnamyl alcohol dehydrogenase (CAD) mRNA in suspension-cultured bean cells was more rapid than that observed for PAL and CHS. CAD is the enzyme that diverts metabolic flux from general phenolics towards lignin synthesis. The sequence for mRNA accumulation was (1) CAD, (2) PAL and CHS, (3) HRGP. The increase in HRGP mRNA occurred much more slowly, usually requiring a lag period of at least 1h after elicitation (Showalter et al., 1985; Templeton and Lamb, 1988). Transcripts encoding chitinase also accumulated very rapidly and with kinetics similar to those of C A D mRNA (Hedrick et al., 1988). Somssich et al. (1986), using the elicitor from Phytophthora megasperma f. sp. glycinea which induces phytoalexin accumulation in parsley, reported a rapid increase in mRNAs for certain pathogenesis-related (PR) proteins. There was a four-fold increase in the transcription rate of the PR1 gene within 5 min and a three-fold increase for the PR2 gene within 20min (Fig. 8).

OLIGOSACCHARINS

200

55

-I

Time after addition of elicitor (min)

Fig. 8. Rapid effects of an elicitor (P-glucan from the fungus Phytophthoru megasperma) on transcription of the genes for two pathogenesis-related proteins (PR1 and PR2), pcoumarate: CoA ligase (4CL), and phenylalanine ammonia lyase (PAL), relative to that of an elicitor-insensitive gene (LF14). in cultured parsley cells. Nuclei were isolated at the times indicated and allowed to incorporate [IY,-~’P]NTP into “run-off” RNA, which was then hybridized to the relevant cDNA and assayed for 32P.From Somssich et ul. (1986).

Templeton and Lamb (1988) have described the early transcriptional events of several enzymes in bean-Colletotrichum lindemuthianum interactions. The rapidity of these responses suggested that there were very few steps between oligosaccharin recognition and activation of defence-related transcription. The genes were activated much more quickly in incompatible interactions than in compatible interactions. Furthermore, the transcription of these genes was also induced some distance from the infection site. The induction of protease inhibitors in tomato leaves by pectic fragments also appears to depend on transcription, but the protease inhibitor mRNAs do not accumulate very rapidly (Nelson et al., 1981). The mechanism by which gene transcription is modulated by oligosaccharins is unknown. In the case of oligosaccharides of chitosan, which can enter the plant cell and accumulate in the nucleus, it could be proposed that they interact directly with the chromatin. Indeed, it was shown that, not surprisingly for a polycation, chitosan will bind in vitro to DNA molecules and hence change their physical properties (Hadwiger et al., 1981). It was also shown that chitosan inhibited the incorporation of [’Hluridine into mRNA (Kendra and Hadwiger, 1987b). I t was proposed that defence

56


responses initiated by the host were due to structural changes in the host’s nuclear material, with the hypersensitive response and host membrane deterioration being consequences of these responses. However, it seems improbable that other, non-cationic, oligosaccharins act in this way, and even oligosaccharins of chitin would presumably have to act by a different mechanism. It seems clear that plant cells exhibit numerous diverse rapid responses to oligosaccharins. There would appear to be little real evidence on which to decide objectively which, if any, of these are required for the ultimate physiological effects observed (Section 11), and in what cause-and-effect order they may operate.

C. DIRECT EFFECTS OF OLIGOSACCHARIDES ON ENZYMES

1. X y Zoglucan endotransgly cosylase Although XG9 at 1 nM inhibits the growth-promoting effect of 2,4-D in pea stem segments, at concentrations 3 100nM it does not (York et al., 1984; McDougall and Fry, 1988). This suggests that, at the higher concentrations, a second (growth-restoring) effect of XG9 comes into play, nullifying the growth-inhibiting effect (see Section 1I.B). The fact that XG5 and 2‘fucosyl-lactose do not become less effective inhibitors of auxin-promoted growth at 3 l 0 O n ~suggests that they lack this second function of XG9. Direct tests of this hypothesis led to the discovery of the ability of XG7, XG8, XG9 and XG9n to promote elongation in the absence of auxin (McDougall and Fry, 1990). What is the mode of action of XG9 as a growth restorer/prornoter? An important clue was the finding by FarkaS and Maclachlan (1988) that oligosaccharides derived from xyloglucan can apparently stimulate the activity of “cellulase” in a cell-free system. The effect was only observed when xyloglucan was used as substrate (not carboxymethylcellulose), only when the “cellulase” was obtained from plants (not from fungi), and only in viscometric (not reductiometric) assays of substrate degradation. Since xyloglucan oligosaccharides are an end-product of the action of cellulase, it would be unusual if they did indeed stimulate cellulase activity-a case of positive feedback. In fact, the enzyme responding to the oligosaccharides now appears likely to have been xyloglucan endotransglycosylase (XET; see Section 1I.B) (Fry et al., 1992) rather than cellulase. The oligosaccharides XG7, XG8, XG9 and XG9n (which all have four glucose units in the backbone) are substrates for XET, whereas XG5 and 2’-fucosyl-lactose (which have two and one glucose units, respectively) are not. The stimulation of xyloglucan degradation (decrease in viscosity) by the larger oligosaccharides would be due to their ability to compete with other xyloglucan molecules as glycosyl acceptors in the XET reaction. Endotransglycosyl-

57

OLIGOSACCHARINS

ation between twopolysaccharide molecules (0000... and o. ...) results in no change in the mean molecular weight of the reactants since for every bond broken a new bond is re-formed:

................................................................ ................

00000000000000000000000000000000000000000000000000000000000000000000

+

+

0000000000000000000000000000000000000000

......

However, when this principle is applied to endotransglycosylation between .) equimolar amounts of xyloglucan (say, molecular weight 200 000; and XG9 (molecular weight 1500; a), the products should have a mean molecular weight of 100 000

-

-

00000000000000000000000000000000000000000000000000000000000000000000

+. + 0000000000000000000000000000.

+

0000000000000000000000000000000000000000

and, since XG9 contributes only negligibly to viscosity, the viscosity of the reaction mixture would be strongly reduced by the occurrence of this reaction. Thus, there is a correlation between the ability of xyloglucan oligosaccharides to promote (and/or restore) growth and their effectiveness as substrates for XET. Could this be a causal relationship? The action of X E T in vivo, in the absence of xyloglucan oligosaccharides, may promote the extensibility of the cell wall by cleaving load-bearing, intermicrofibrillar xyloglucan chains-thus allowing incremental growth (Fry, 1989b)-and then re-joining the cut ends to different acceptor xyloglucan chains, thereby restoring the original strength of the cell wall. When FM XGOs are experimentally added into the system, however, the XET can frequently transfer the cut xyloglucan chains on to these short “stubs”, using them, instead of the neighbouring structural polysaccharides, as acceptors; the strength of the cell wall would thus fail to be restored, and cell extension would continue for longer than usual.

2. Other enzymes Several polyvalent anions, e.g. pyrophosphate, oxalate, oxomalonate and citrate, dramatically promote the oxidation of IAA by plant peroxidases in vitro (Pressey, 1990). The optimum concentration of these anions was 100 FM. Pressey (1991) has also shown that a similar effect is exerted on peroxidase by oligogalacturonides that have been enzymically modified by oxidation of the reducing terminal galacturonate unit to galactarate. Galac, oligomers of the form taric acid itself was active above about 300 p ~ but

-

[D-Galacturonic acid-a-( 1-+4)],-Galactarate

58


were active at considerably lower concentrations. For example, the penta. (1991) also saccharide appeared to be effective at about 50 p ~Pressey states that several plant tissues were shown to contain a uronate oxidase that converted normal oligogalacturonides (the products of pectinase digestion) to these oxidized derivatives. Promotion of IAA oxidation in vivo by an enzymic oxidation product could be a further factor explaining the fact that oligogalacturonides antagonize several of the effects of IAA on plants (see also Sections III.A.2 and B.2). A plant pectinmethylesterase has also been reported to be inhibited by oligogalacturonides of D P 3 8 (Termote et al., 1977). It is interesting to consider whether this effect may be related to growth regulation.

IV. NATURAL OCCURRENCE OF OLIGOSACCHARINS If a biological signalling role is to be ascribed to oligosaccharins, it is essential to demonstrate their natural occurrence at the right time, place and concentration to carry out the biological functions envisaged for them. Clearly the appropriate polysaccharides and glycoproteins (from which oligosaccharins may arise) occur in vivo, but surprisingly few studies have addressed the question of whether the oligosaccharins themselves occur. This is particularly true for the oligo-P-(1+3),(1-+6)-glucans, which, despite the detailed information available about the action of artificial oligosaccharins (Section II.A), do not appear to have been demonstrated to occur naturally at all. Although the mere existence of biological responses to artificial oligosaccharins, some extremely potent and specific, may suggest that they do play a natural role, this kind of evidence is not proof. Other reasons for developing techniques for studying the natural occurrence of oligosaccharins are: (1) so that changes in their endogenous levels within plant tissues can be monitored, e.g. in responses to environmental and hormonal stimuli or to infection; and (2) so that potential natural sources can be explored for possible novel oligosaccharins that might never be generated by the in vitro methods currently used (Section 1.B). A . NATURAL OCCURRENCE OF XYLOGLUCAN OLIGOSACCHARIDES

Acetylated and non-acetylated forms of XG9 and related oligosaccharides of xyloglucan (DP 5-10) have been detected in vivo in the spent medium of suspension-cultured spinach cells (Fry, 1986). The culture medium can be regarded as an extension of the apoplast of these cells, and the discovery of XG9 in the spent medium indicates that it might also occur extraprotoplasmically in intact plant organs. The concentration of XG9 plus its acetates was estimated at 0.4 p ~ . The method used to enable detection of this low concentration of XG9

-

OLIGOSACCHARINS

59

involved the prior in vivo feeding to suspension-cultured spinach cells of high activity ~ - [ ~ H ] f u c oand s e ~-[~H]arabinose, which are incorporated into the L-fucose and D-xylose residues, respectively, of xyloglucan. The culture filtrates were then collected and fractionated on the basis of molecular weight by gel-permeation chromatography on Bio-Gel P-2. This simple method is the most sensitive means currently available for detecting trace levels of specific carbohydrates, permitting the detection of picogramme quantities. The nonasaccharide-enriched, 3H-labelled material was further fractionated by two-dimensional paper chromatography, and a spot was detected that appeared to be an acetylated derivative of XG9. After mild alkaline hydrolysis to remove the acetyl group(s), evidence that the material was indeed [3H]XG9 and not another oligosaccharide of similar size and RF was obtained by use of acid- and enzyme-catalysed hydrolysis, paper electrophoresis in the presence of borate or molybdate (Weigel, 1963), and related techniques (Fry, 1986; for a review of the methods, see Fry, 1988). No evidence has yet been obtained that spinach cell cultures are responsive to xyloglucan oligosaccharins. Thus, the biological significance l ~in spinach culture filtrates is difficult to assess. of the finding of 0.4 l ~XG9 This concentration is supraoptimal for the inhibition of 2,4-D-stimulated growth in pea stem segments, and closer to that required for growth promotion in the absence of auxin (Section 1I.B).

B. NATURAL OCCURRENCE OF PECTIC OLIGOSACCHARIDES

1. Wound signals The natural occurrence of pectic fragments, possibly acting as wound signals in mechanically injured plant tissue, has been considered. Ryan (1974) found that when fresh tomato leaves were ground in a mortar and pestle at 2°C and then squeezed through a hand-operated garlic press, the juice possessed wound-signal activity. This is evidence for the natural occurrence of wound signal in wounded tissue, although, unfortunately, this particular material was not studied further. The detailed characterization of a tomato wound signal was conducted on extracts of autoclaved leaves (Ryan, 1974, and subsequent papers). Autoclaving of plant homogenates would certainly generate soluble pectic fragments from wall-bound pectins; this would occur both by acid hydrolysis (even at pH4-6; see Smidsrod et al., 1966) and, especially in the presence of certain ions such as would be found in crude tomato leaf juice, by p-elimination degradation (Barrett and Northcote, 1965; Aspinall and Cottrell, 1970). The wound signal found in autoclaved preparations may therefore not have been the same as that found in the cold homogenate. The finding of high concentrations of soluble pectic substances in autoclaved tissue is therefore not evidence for their natural occurrence. If the wound signal solubilized at 2°C also turns out to be pectic, this will

60


support the hypothesis that such substances play a natural role in the activation of protease inhibitor synthesis upon injury, but it will not be sufficient evidence. It poses the difficult question of whether or not uninjured leaves contain the same amounts of soluble pectic fragments. No-one has yet reported the presence of soluble pectic oligosaccharides in uninjured tissues, although this might not be as difficult as it sounds; for example apoplastic fluid could be collected by the vacuum-infiltration/ centrifugation method (de Wit and Spikman, 1982), or the tissue could be homogenized under conditions carefully chosen to prevent both the chemical and the enzymic degradation of pectins. It is difficult to estimate what increase in concentration of endogenous pectic fragments would be needed in order to provide credible evidence for their natural involvement in the wound-signalling system. This is because there is no precise estimate of the quantity of exogenous pectic fragments necessary for the induction of protease inhibitor synthesis. Exogenous pectic fragments ( M , 5000-10 000) have generally been administered through the transpiration stream of excised leaves, and would thus have had to pass along the petiole before arriving in the lamina. The minimum dose that it is necessary to supply to the petiole to obtain a response in the lamina is -0.1 g I-' for 30 min (Ryan et al., 1981); the transpiration rate under the conditions used in earlier work was p1 min-' (Ryan, 1974). These figures suggest that the minimum dose for induction of protease inhibitor is of the order of 1-10 pg of pectic material per leaf. However, it is unclear whether the oligosaccharides need to travel as far as the lamina; the proportion of the pectic material that completes the journey into the lamina has not been adequately investigated. Preliminary studies using ['4C]pectins and oligo-[14C]galacturonideshave indicated that a substantial proportion becomes immobilized at certain points within the xylem of the petiole (E. A.-H. Baydoun and S. C. Fry, unpublished); the formation of pectic plugs in the xylem has also been reported in other circumstances, e.g. as a cause of premature wilting in cut rose flowers (Burdett, 1970). It has long been known that the media of plant cell suspension cultures accumulate soluble polysaccharides, some of which are pectic, during cell growth (Aspinall etal., 1969; Fry, 1980). Some of these, e.g. from tomato, tobacco and lucerne cultures, have wound-signal activity (Walker-Simmons and Ryan, 1986). There has been no evidence for an increase in their concentration in response to injury (e.g. caused by stirring instead of shaking; Brett, 1978), nor does there appear to be any evidence for pectic oligosaccharides (cf. evidence for xyloglucan oligosaccharides-Section 1V.A). Levels of the polysaccharides did tend to increase when the sucrose supply was exhausted, so their accumulation may be a response to starvation (Walker-Simmons and Ryan, 1986).

-

OLIGOSACCHARINS

61

2. Other pectin-related oligosaccharins Hargreaves and Bailey (1978) demonstrated the presence of elicitor-active substances (which may possibly have included pectic fragments) in damaged Phaseolus hypocotyls. Benhamou et al. (1991) used a polygalacturonic acid-binding lectin, complexed to gold, to study the distribution of pectin during the infection of bean tissues with Colletotrichum lindemuthianum. At the same time a PGIP (Section V.B.2) which binds to C. lindemuthianum pectinase, was also tagged with gold and used to localize the site of enzyme accumulation in infected host tissues. The level of pectinase activity increased several-fold when the fungus developed in host plant tissues. The enzyme was found to diffuse freely in the host cell wall and apparently caused degradation of pectins of the primary cell walls and middle lamella. Pectic material appeared to become solubilized and to accumulate at specific sites such as adjacent to the intercellular spaces and in the coagulated cytoplasm of infected host cells. This may reflect the natural production of oligosaccharins of the oligogalacturonide class, which might contribute to the activation of defence responses (Section 1I.C). However, in common with many cytological approaches, the method used does not give unequivocal information about the chemical nature (especially DP) of the material localised, and thus it cannot be definitely concluded that biologically active oligogalacturonides were generated. Details of the methodology are provided by Benhamou etal. (1988), and a similar application of the techniques to the case of the tomato-Fusarium oxysporum interaction has been described (Benhamou et al., 1990). Another difficulty to be overcome is the measurement of the concentration of pectic oligosaccharins in vivo: as much as 0.1-1.0 mM is required for many of the reported biological effects of pectic oligosaccharides and it is far from certain that such high concentrations are generated in vivo.

C. GLYCOPROTEIN-DERIVED OLIGOSACCHARINS

The lignification-inducing glycoprotein of Puccinia walls (see Section 11.F) has been reported to be present in the apoplastic fluid of rust-infected wheat leaves (Kogel et al., 1988), suggesting natural occurrence. Culture filtrates from Silene cell suspension cultures were found to contain a growthregulating heptasaccharide, possibly related to the carbohydrate moieties of N-linked glycoproteins (Priem etal., 1990; Section 1I.F). This, together with the embryogenesis-regulating glycoproteins which also accumulate (de Vries et al., 1988) and the growth-regulating XG9 (Fry, 1986), is another example of the value of the spent media of cell cultures as a source from which to seek novel oligosaccharins. Culture media also accumulate

62


arabinogalactan proteins-a group of polymers currently considered likely to play a role in the control of morphogenesis (Stacey et al., 1990). A systematic survey of the oligosaccharides that accumulate in culture media, to see how many of them are oligosaccharins, would be valuable. D. CONCLUSION

The study of the natural occurrence of oligosaccharins is in its infancy. This is surprising because the techniques required are simple enough (in vivo feeding of radioactive sugars followed by paper chromatography of the oligomer fraction is a good start!) and the question of natural occurrence is pivotal to the biological relevance of the whole oligosaccharin concept.

V. MECHANISM OF FORMATION AND DEGRADATION OF OLIGOSACCHARINS A. XYLOGLUCAN OLIGOSACCHARIDES

1. Synthesis of xyloglucan oligosaccharides The machinery theoretically required for the production of oligosaccharides from xyloglucan is present in vivo (Fry et al., 1990). It has been shown that xyloglucan is degraded in the stems of many plants during auxin-promoted elongation. This degradation can be shown by pulse-chase experiments in which [14C]sugars are briefly fed to the tissue to generate a cohort of radioactive xyloglucan molecules, which can be extracted and analysed. Some of the initially-formed [ ''C]xyloglucan subsequently vanishes, presumably by degradation to products which are lost upon dialysis (Labavitch and Ray, 1974; Gilkes and Hall, 1977). An apparent solubilization of wall-bound xyloglucan, with little hydrolysis, to yield xyloglucan freely soluble in the apoplast, may also occur (Terry et al., 1981). In addition, the average molecular weight of xyloglucan molecules that remain wall bound may decrease in response to treatment with auxin or H + (Nishitani and Masuda, 1982). Although these studies of xyloglucan turnover were carried out with a view to explaining mechanical wall-loosening during plant growth, the same xyloglucan-degrading machinery could also be responsible for the production of oligosaccharins. The proportion of total wall-bound labelled xyloglucan degraded in vivo is small, at least in rapidly growing Rosa cell cultures (Edelmann and Fry, 1992b), but xyloglucan is so abundant in the cell wall (-10% w/v) that even a small percentage conversion to oligosaccharides would easily lead to the nM and perhaps also p~ concentrations required for oligosaccharin activity (Section 1I.B). All these changes in xyloglucan could theoretically be catalysed by cellu-

63

OLIGOSACCHARINS

lase. Pea stems contain small amounts of cellulase constitutively, and synthesize much greater quantities in response to auxins, especially 2,4-D, at concentrations high enough to promote lateral swelling rather than elongation of stems (Verma et af., 1975). The preferred cell wall substrate of pea cellulase is xyloglucan; cellulases cleave the P-glucan backbone of xyloglucan at specific sites (Section 1I.B). Thus, at least some plant cellulases, as well as fungal cellulases, can cleave xyloglucan in vitro to its constituent oligosaccharide units, mainly XG7 and XG9 (Bauer et af., 1973; Hayashi et al., 1984). Other plant cellulases, e.g. one from tobacco callus, appear to require more than one non-xylosylated glucose residue at the site of attack (Truelsen and Wyndaele, 1992) and will therefore only hydrolyse xyloglucans that have a low xylose :glucose ratio (e.g. that of tobacco; Kato and Noguchi, 1976). To release XG9, XG7 etc. from xyloglucan, cellulase would have either (1) to attack the first non-xyosylated glucose residue away from the reducing terminus of the polysaccharide ( * in the diagram) or (2) to attack two contiguous non-xylosylated glucose residues of the P-glucan backbone (e.g. T, 7 in the diagram) [in the following diagram, the polysaccharide chain has been grossly shortened and galactose and fucose omitted for clarity]: x

x

x

x

x

x

x

x

x

x

x

x

x

1

1

1

1

1

1

1

1

1

1

1

1

1

x i

x .

x

1

1

G-G-G~G-G-G-G~G~G-G-G-GIG-G-GCi-GfG-G-G-G-G-ti-G-(i-G-G-G-G~G~G-G

T

T

T

T

X

X

X

X

T

X

1

T

T

X

X

X

Although some endo-acting glycanases have been shown to carry out multiple attacks on a single chain, thus converting the polysaccharide directly to di- and trisaccharides (English et af., 1972; Bezukladnikov and Elyakova, 1990), this has not so far been demonstrated with a plant cellulase. Nevertheless, even stochastic cleavage of xyloglucan by cellulase would release some XG9, and this might be adequate for an oligosaccharin that is active at concentrations as low as 1 nM. Thus it is reasonable to propose that cellulase catalyses the post-synthetic degradation of xyloglucan to generate oligosaccharins such as XG9. Alternatively, however, it could be suggested that XG9 is synthesized within the protoplast as a pre-formed nonasaccharide, and secreted as such. The mere detection of XG9 in spent media of cell cultures (Section 1V.A) does not distinguish between these two possibilities. Kinetic in vivo labelling studies have recently indicated that the XG9 arises by polysaccharide breakdown. ~-['H]Fucose was fed to suspensioncultured spinach cells and the kinetics of labelling of XG9 and xyloglucan were contrasted (McDougall and Fry, 1991b). After a short lag period (- 0.5 h; presumably the time required for newly synthesized polysaccharide molecules to transit the Golgi vesicular system), [3H]xyloglucan started to appear extracelularly. Its accumulation in the culture medium

64


continued at a constant rate for at least 7 h. Extracellular [3H]XG9 and [3H]XG9-acetate also appeared in the culture medium after a short lag, but accumulated at a constantly accelerating rate for several hours (indicated by a linear relationship between concentration of [3H]XG9 and ?-Fig. 9).

0

10

20 30 40 Square of incubation time

50

60

(h2)

Fig. 9. Kinetics of accumulation of [3H]XG9 and of soluble [3H]xyloglucanin the culture Values are cpm per 4-ml culture. The medium of spinach cells after addition of ~-[~H]fucose. abscissa shows t2: the fact that the XG9 curve plotted in this way is linear indicates that the accumulation of [3H]XG9exhibits a constant acceleration.

This indicates that the free [3H]XG9 was not synthesized directly from NDP-[3H]sugars and rapidly secreted via the Golgi vesicular system, but that it arose from a gradually labelled pool of precursor material. The steady acceleration in the rate of formation of [3H]XG9would most likely be due to an enzyme (e.g. cellulase) acting at a constant rate on a large, relatively stable, pool of substrate (apoplastic xyloglucan) that was itself increasing in specific radioactivity at an almost constant rate. This mode of origin of XG9 is that proposed in the classic oligosaccharin hypothesis-i.e. that soluble signalling molecules arise by partial enzymic cleavage of wall polysaccharides (Fig. 10). Although it may be reasonable to suggest that cellulase is the enzyme that liberates XG9 from xyloglucan, a new enzyme has recently been found that could also play this role. This is xyloglucan endotransglycosylase (XET) (Smith and Fry, 1991; Fry e t a l . , 1992; Section III.C.l), which cuts (t) the backbone of a xyloglucan molecule, releasing one portion (containing the original reducing terminus) and possibly forming a reactive polysaccharideenzyme (E) intermediate:

65

OLIGOSACCHARINS

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

l

l

l

l

l

l

l

l

l

l

i

l

l

l

l

l

G-G-G-G-C-G~G-G-G-G-G-GIC-G-G-G-G-G-G-~~-G-G-G-G-C~-G-G~ T r T T r r T T X

X

X

X

X

X

X

X

i€ +

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

1

1

1

1

1

1

J

l

l

l

l

l

l

l

l

l

G-Ci-G-G-G-G-G-G-tG-G-G-G-G-G-G-G-G-G-G

~G-G-G-G-G-G-CwG-G-G-G-€

I

t X

t X

X

r

t

T

X

X

X

T X

T

X

from which the polysaccharide can be transferred on to the non-reducing terminus of a different xyloglucan molecule (bb b b b b b b b b b b b b ), x

x

1

1

x 1

x 1

x 1

x J

.

+

G - r G - r G - G - G - G - G - t G - r G - t G - G - t G - t ~ ~ ~ ~ ~ ~ E~ ~ ~ ~ ~ ~ ~ ~

r

X

r

X

T

X

thus effecting transglycosylation, rather than being transferred on to H 2 0 (as in hydrolysis) XET was shown to be a different enzyme from cellulase (Fry etal., 1992). If XET attacks xyloglucan close to the reducing terminus,

-

:.\

[3H]fucose

&*

xyloglucan

*;:*

. oligosaccharides (

.-t ,

Fig. 10. Two models to show how ["H]XG9 could theoretically accumulate in the culture medium (cf. Fig. 9). In hypothesis (a), 3H from GDP-[3H]fucose is incorporated into xyloglucans, some of which are later broken down to oligosaccharides. In hypothesis (b), some 3H is incorporated directly into XG9, which is then secreted ready-made. The data in Fig. 9 support hypothesis (a).

66


it can release X G 9 (just as it can use X G 9 as an acceptor substrate), although, as with cellulase, such sub-terminal attacks do not appear to be significantly preferred over other potential cleavage sites in the substrate xyloglucan molecule (P. R. Hetherington and S. C . Fry, unpublished). There is, however, some evidence suggestive of multiple attacks on a single xyloglucan molecule (Smith and Fry, 1991) and this might suggest a special role for XET in generating X G 7 , X G 9 etc. in vivo. 2. Degradation of xyloglucan oligosaccharides If X G 9 is a natural signalling molecule, it might be expected to have a relatively short half-life within plant tissue: when it had evoked the appropriate response, the signal would be removed from the system. However, X G 9 seems to be remarkably stable in the presence of cultured plant cells. When IfucosyZ-’H]XG9, [xyZosyl-’H]XG9 or [reducing terminal-’H]XG9 was fed to actively-growing spinach cell cultures, only a neglible proportion of the ’H was taken up o r bound by the cells (Baydoun and Fry, 1989; Smith and Fry, 1991). A small amount of the [’H]XG9 was broken down, but much free [‘H]XG9 remained in the culture medium after 7 2 h. Thus, at least this particular plant tissue does not readily degrade this oligosaccharin. The principal means of removing X G 9 in the spinach system appeared to be by a form of sequestration rather than hydrolysis. By 7 2 h, in the above experiments, an appreciable amount (15-70%) of the total ’H had become sequestered by covalent binding to a soluble, extracellular polymer. Subsequent work has shown that this polymer was xyloglucan, and that the “sequestration” was catalysed by XET (Section V . A . l ) ,for which [’H]XG9 is capable of acting as an acceptor substrate (Smith and Fry, 1991). Plants do contain enzymes theoretically capable of achieving the complete hydrolysis of X G 9 to monosaccharides (Koyama et al., 1983; Edwards et al., 1985, 1988; Fanutti et aZ., 1991; FarkaS et al., 1991). The a-L-fucosidase described by FarkaS et al. (1991)is able to hydrolyse the fucose residues from X G 9 but not those of xyloglucan itself. It is unclear why this battery of enzymes, if present in cultured cells, fails to bring about any appreciable degradation of exogenous [‘H]XG9. B. PECTIC OLIGOSACCHARIDES

1. Formation of pectic oligosaccharides as wound signals In their high molecular weight, wall-bound condition, pectins are unlikely to act as signalling molecules since they are present constitutively. It is only when they are solubilized that they are likely to be effective “alarm calls”. It should be emphasized that, although artificial pectic oligosaccharides have numerous well-documented biological effects, there is to date no clear evidence for the natural occurrence of biologically active pectic oligosac-

OLIGOSACCHARINS

67

charides (Section 1V.B). However, we may reasonably ask what could solubilize them at times of stress. There seem to be two plausible hypotheses: (1) Chelating agents, e.g. citrate and oxalate, are released from the cytosol or vacuole of injured cells and, by breaking Ca2+ bridges in the cell wall, could solubilize some pectins. Chelating agents at room temperature will solubilize small amounts of pectin from most primary cell walls and larger amounts from certain specialized cell types, e.g. the parenchyma of ripe fruit (Jarvis, 1982, 1984). However, pectins are constantly being secreted in soluble form during wall growth (Hanke and Northcote, 1974; Boffey and Northcote, 1975), so the outside of the plasma membrane will normally be coated with newly-secreted, soluble, “periplasmic” pectic molecules; therefore, mere solubilization of wall pectins may be insufficient as a stress signal. Partial degradation to oligosaccharins is probably required. (2) Cellular injury could induce the plant to synthesize pectin-degrading enzymes, release them from a compartmentalized condition, or activate them. Certain plant tissues, especially ripening fruits and abscission zones, have indeed been reported to contain pectinases (Rexova-Benkova and MarkoviE, 1976; Rombouts and Pilnik, 1980), but as yet there is no definite evidence for their action on the primary cell walls of other tissues in vivo, let alone for an enhancement of their concentration or effectiveness upon injury. Addressing the possibility of in vivo conversion of pectic polysaccharides to oligosaccharides, Bishop et al. (1981) attempted to demonstrate the shortening of [3H]pectic fragments (initial mean DP 30-60) when fed, via the transpiration stream of the hypocotyl, into expanded tomato cotyledons. Water-soluble material subsequently extracted from the cotyledons (boiled for 5 min) was analysed by gel-permeation chromatography on Sephadex G-25: the majority of the recovered 3H now appeared to co-elute with di- or trigalacturonides (Ve/Vo=1.9). However, the authors do not state what percentage of the [3H]pectin supplied to the hypocotyl actually reached the cotyledons and thus became available for analysis: if this was a small proportion, it could already have been enriched in the smallest components of the labelled pectic preparation-for example owing to an inability of larger pectic substances to migrate up the xylem-and the results would therefore be misleading. The production of pectic oligosaccharides by contact between aphid saliva and plant cell walls has been demonstrated (Campbell, 1986). The accumulation of pectic fragments in honeydew is due to the presence of pectinase in the saliva (Ma et al., 1990), and may be part of the wound-signalling system that culminates in the synthesis of protease inhibitors (Section II.C.2). It would not, however, be part of the response to simple mechanical injury,

68


where no saliva is usually present. There seems to have been no convincing evidence for the partial hydrolysis of pectins to oligosaccharides during the mechanical wounding of vegetative plant tissues. 2. Formation of pectic oligosaccharides as elicitors In several cases where pectic oligosaccharins have been found to induce biological responses, initial observations were made with pectic enzymes rather than with pectic oligosaccharides. For instance, (1) pectinase from Rhizopus stolonifer elicited phytoalexins in castor bean (Lee and West, 1981a; Bruce and West, 1982); (2) endo-pectate lyase from Erwinia carotovora induced phytoalexins in soybean (Davis et al., 1984); (3) purified pectinase from Aspergillus niger was implicated in hypersensitivity (Cervone et al., 1987a). In each case, the biological effect was induced by the purified enzyme. Only later was activity found to be associated with oligosaccharides generated by the (in vitro) action of these enzymes. The hypothesis is reasonable but unproven that the enzymes act in vivo to generate sufficient of the right oligosaccharides to induce a biological effect. Pathogenic fungi and bacteria are known to produce a variety of enzymes that degrade the plant cell wall (Cooper, 1976). Pectic enzymes are usually the first to be synthesised when fungi are grown axenically with isolated cell walls of Dicotyledons as the carbon source, and the same may also be true in planta (Cooper, 1984; Cervone et al., 1986; Collmer and Keen, 1986). This is understandable since the first layer of the plant cell met by a pathogen, the middle lamella, is particularly rich in pectins. In particular, maceration of tissue in rot diseases is believed to be due to pectic enzymes. Mutants deficient in pectic enzymes tend to be impaired in their pathogenicity (Mussel1 and Strand, 1976). However, it is difficult to correlate the production of pectic enzymes in axenic cultures with that in planta. Enzyme production in cultures depends on numerous factors such as the isolates used, the composition of the culture medium and the age of the culture. Enzyme activity in planta may differ from that seen in cultures owing to the added effects of plant products (e.g. PGIP-see below). Culture filtrates of two races of Cladosporium fulvum have been shown to contain enzymes active on plant cell walls (S. Aldington and S. C. Fry, unpublished), although this fungus does not penetrate the host cell wall and there is thus no obvious requirement for such enzymes in planta. Wijesundera et al. (1989) compared the enzyme activities produced by Colletotrichum lindemuthianum in axenic culture and in infected bean hypocotyls. The culture filtrate contained a-arabinofuranosidase, a- and P-galactopyranosidase, protease, pectinase and two forms of pectin lyase. The first four of these were also recovered from infected hypocotyls, but there was no extractable

OLIGOSACCHARINS

69

pectinase activity and only one form of pectin lyase. Extraction of pectic enzymes from infected tissues may also be hampered by binding to the cell walls (Skare et al., 1975). Furthermore, the amounts of enzymes produced in planta may be very low and require the use of highly sensitive assays for their detection. Bashan et al. (1985) detected pectinase activity in leaves of susceptible and resistant tomato cultivars within 48 h of infection with Pseudornonas syringae, suggesting that this enzyme may be involved in the primary stages of disease development (see also Bateman and Basham, 1976). Pectic enzymes are also secreted by mutualistic mycorrhizal fungi (Garcia-Romera et al., 1991); it will be interesting to discover how these fungi avoid eliciting the plant’s defence responses. Other enzymes produced later appear to relate to the progressive degradation of the cell wall. As more wall components become accessible, so the pathogen produced corresponding degradative enzymes. Page1 and Heitefuss (1990) found that pectinase, pectate lyase, cellulase, protease and xylanase appeared sequentially 10, 14, 16, 19 and 22h, respectively, after inoculation of potato with Erwinia. Pectinase peaked at 12-14 h whereas pectate lyase peaked at 22 h. In contrast, pathogens of the Gramineae produce xylanase as a key enzyme early during infection (Cooper, 1984; see Section V.C.3), agreeing with the predominance of xylans in grass cell walls. Pectinase (EC 3.2.1.15) catalyses the mid-chain hydrolysis of a-(1-+4)-~-galacturonic acid residues within those parts of a pectin molecule that consist of several contiguous non-esterified residues. Although they are endo-hydrolases, some pectinases have a marked tendency to catalyse multiple attacks close together on a single polysaccharide chain and thus to release large amounts of di- and trisaccharide once one mid-chain glycosidic bond has been hydrolysed (English et al., 1972). Other pectic enzymes may also play a role in pathogenesis. For example, pectin lyase is produced by Aspergillus niger (Albersheim and Killias, 1962) and pectate lyase by Erwinia carotovora (Davis et al., 1984); these both catalyse an elimination reaction resulting in an unsaturated bond between C4 and C5 at the new non-reducing end (A4,5-galacturonicacid). A specific rhamnogalacturonase has also been reported (Schols etal., 1990). Pectin methyl esterases may also be induced (Miller and Macmillan, 1971) whose action can facilitate the action of pectinase. Many pectic enzymes have been found to be inducible by pectins and sometimes by D-galacturonic acid and its derivatives (Cooper, 1976). The release of oligogalacturonides may therefore be simultaneously advantageous to the pathogen (for continuing induction of these enzymes and thus wall degradation) and disadvantageous to it, amplifying the oligosaccharin signal to the host cell. Certain proteins which were isolated from plant cell walls were found to be capable of inhibiting pectinase activity secreted by phytopathogens (Albersheim and Anderson, 1971; Albersheim and Valent, 1974). These

70


pectinase-inhibiting proteins (PGIPs) have since been found in the cell walls of a variety of plants (see Cervone et a f . , 1987b). PGIPs appear to inhibit only pectinases of fungal origin; plant and bacterial pectinases were not affected although a plant a-D-galacturonidase (EC 3.2.1.67) was inhibited (Cervone et a f . , 1989b, 1990). PGIPs may retard the pectinase-catalysed hydrolysis of polygalacturonic acid (Cervone et a f . , 1987b) leading to the accumulation of oligomers with DP > 4, which can evoke necrosis (Cervone et a f . , 1987a) and induce PAL (De Lorenzo et a f . , 1987). Cervone et a f . (1989a) found that oligogalacturonides (DP > 10) capable of eliciting phytoalexin synthesis were produced after incubation of polygalacturonic acid with pectinase for 1 min. After 15 min, the active fragments had been depolymerized to inactive oligosaccharides (DP < 6). With the same substrate and enzyme concentrations, but in the presence of PGIP, the level of elicitor-active oligogalacturonides continued to increase for 24 h (Fig. 11); only by 48h did oligosaccharides with D P < 6 become predominant. Hence, by increasing the length of time that the active oligogalacturonides are present at the host-fungal interface, PGIPs may convert fungal pectinase from an elicitor destroyer to an elicitor enhancer. The prolonged accumulation of elicitor-active oligosaccharides would also have been assured by the simple expedient of using less pectinase (Cervone et a f . , 1989a). The above picture of the situation may thus be over-simplified. If a localized site of fungal colonization were to emit fungal pectinases, a concentration gradient of enzyme would be created with the lowest concentrations towards the periphery. At some point in this gradient, it might be expected that the pectinase concentration would be just right to hydrolyse wall pectins to moderate sized oligosaccharides without unduly rapidly degrading these further to elicitor-inactive fragments. Might PGIP be more sophisticated in its effects than so far envisaged? It would be advantageous to the plant for the PGIP to permit pectinase to act efficiently on pectic polysaccharides (say, DP> 30), but to inhibit the action of the enzyme on elicitor-active oligogalacturonides (DP == 8-14). If, on the other hand, PGIP efficiently inhibited all action of fungal pectinase, including the hydrolysis of pectic pofysaccharides, few soluble elicitor molecules would be generated in the first place. One way in which PGIP might achieve this goal would be to abolish the propensity of fungal pectinases to catalyse multiple attacks on a single polygalacturonic acid chain (see English et a f . , 1972), i.e. to cause the enzyme to detach from the substrate poly- or oligosaccharide molecule after each hydrolytic event and search for a new substrate molecule rather than simply moving two or three residues along the same chain as normally occurs (English et a f . ,1972). Apparently no work has yet critically tested this possible action of PGIP. De Lorenzo et a f . (1990) showed that the PGIPs from four different cultivars of Phaseolus vufgaris inhibited equally the pectinases from three different races of Coffetotrichumfindemuthianum. The abilities of these

OLIGOSACCH ARINS

1.6

71

Untreatedpolygalacturonic acid

24 h digestion with pectinase and PGIP

o.!31

0.61

Fractions (1 ml)

Fig. 11. Effect of a pectinase inhibitor (PGIP) from Phuseolus on the hydrolysis of polygalacturonic acid (PGA) by Aspergillus pectinase. (a) Untreated PGA; (b) PGA treated for 15 min with pectinase; (c) PGA treated for 24 h with the same concentration of pectinase but in the presence of PGIP (5mol PGIP per mol pectinase). The products were analysed by anionexchange chromatography on “Mono Q” (Pharmacia) with a 0 . 2 -1 . 0 ~NH4HC03gradient and assayed for uronic acid residues by the rn-hydroxybiphenyl method. The elution of authentic mono-, tri- and decagalacturonic acid is indicated by the arrows labelled 1, 3 and 10. From Cervone er ul. (1989b).

fungal enzymes to generate oligogalacturonides capable of eliciting phytoalexins in soybean were equally affected by PGIPs from different cultivars of P . vulgaris whether PGIPs from compatible or incompatible hosts were used. These results suggest that pectinase and PGIP are not involved in race-cultivar specificity although, as the authors point out, they used an in

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vitro system, generating active oligogalacturonides from polygalacturonic acid rather than from the more complicated pectins of the primary cell wall. No clear evidence exists showing the presence of both pectic enzymes and pectic oligosaccharins in vivo during the course of infection. Nevertheless, circumstantial evidence suggests that pectic fragments may well arise through the action of pectic enzymes produced by potential pathogens during infection. 3. Formation of pectic oligosaccharides in ripening fruit Various pectic enzyme preparations induce ethylene biosynthesis and pectic oligosaccharides have similar effects (Section II.C.7). Many fruits synthesize pectinase during ripening at about the same time as they produce large amounts of ethylene (Fischer and Bennett, 1991). This has been seen for example in the fruits of avocado (Awad and Young, 1979), peach (Hinton and Pressey, 1974), and tomato (Babbitt et al., 1973), though not in the receptacles (“fruits”) of strawberry (Abeles and Tadeka, 1990) or apples (Abeles and Biles, 1991). Despite the exceptions, it is attractive to suggest that the initial production of small amounts of pectinase early in the ripening process leads to the release of pectic oligosaccharins which evoke ethylene synthesis, which induces the climacteric production of larger amounts of pectinases and other fruit-softening enzymes-an amplification system. This hypothesis is supported by the fact that soluble products of the in vitro autolysis of isolated tomato fruit cell walls can evoke ethylene synthesis in unripe fruit (Brecht and Huber, 1988). 4. Degradation of pectic oligosaccharides in plant tissue The fate of pectic oligosaccharins in vivo has been remarkably little studied. Horn et al. (1989) showed that oligogalacturonides can be taken up by plant cells and recovered intact 40 min later; however, their longevity in the cell is unknown. A priori it would seem advantageous for the plant to have a disposal system for any biological signal so that the response could be switched off when no longer beneficial. In the case of wound signalling, oligogalacturonides would need to be degraded to the monosaccharide for complete inactivation (Bishop et al., 1984), whereas in the case of phytoalexin elicitation, ethylene induction, inhibition of IAA-induced growth etc., degradation to D P < 8 would be adequate. Degradation to D P < 8 could be catalysed by a pectinase, although whether this actually occurs in vegetative tissue in vivo has apparently not been critically studied. Pectinases do not usually yield much galacturonic acid (Rombouts and Pilnik, 1980), so degradation to the monosaccharide would probably require the action of an a-D-galacturonidase (EC 3.2.1.67; “exopolygalacturonase”). Such enzymes occur in plants (Konno and Tsukumi, 1991), but their action on oligogalacturonides in vivo has not been demonstrated. The demonstration of the existence of an enzyme in certain plant tissues does not

73

OLIGOSACCHARINS

establish that it catalyses any particular reaction in vivo, still less that it acts in all plant tissues: witness the stability of XG9 in living spinach cell cultures despite the existence of plant a-D-xylosidases and a-L-fucosidases (Section V.A.2).

C. THE ROLE OF CHITINASES, P-GLUCANASES AND OTHER ENZYMES

1. Enzymes acting on chitin and chitosan Many plant tissues contain chitinases, both constitutively and in increased amounts after infection [they are therefore described as “pathogenesisrelated” (PR) proteins] or after hormonal treatments (Boller, 1985). Chitinases may be directly toxic to fungal cells (Pegg and Vessey, 1973; Schlumbaum et al., 1986), especially in conjunction with p-glucanases (Mauch et al., 1988b). The action of chitinases on the walls of invading fungal hyphae, especially at the growing hyphal tips where the chitin is most exposed, could also generate oligosaccharins of chitin which would elicit defence responses: whether this occurs in vivo has not been established, although the elicitor activity associated with chito-oligosaccharides would suggest that such an action would be very beneficial (Section 1I.E). Ride (1992) has shown that when [3H]chitinis applied to wheat leaves, 2% of the 3H is solubilized to oligosaccharides, including the oligosaccharin, chitotetraose. Prolonged action of plant chitinase could also degrade chitooligosaccharides to the disaccharide, which is too small to be an oligosaccharin (Barber et al., 1989). Ride (1992) has shown, however, that [3H]chitotetraose is relatively stable when applied to wheat leaf tissue. A chitin acetamidase has been detected in cucumber leaves infected by Colletotrichum lagenarium (Siegrist and Kauss, 1990). This enzyme might de-N-acetylate the chitin, making it resistant to chitinase and so suppressing the formation of oligosaccharins. In addition, chitin oligomers might have oligosaccharin activity destroyed (in N-acetyl-dependent bioassaysSection 1I.E.1) or conferred (in bioassays dependent on the free amino group-Section II.E.2) by the action of acetamidase.

-

2. P-D-Giucanases Fungi growing in artificial media often slough p-( 1 4 ),( 1+6)-~-glucans into the culture medium, and some of these polysaccharides are elicitor active (Ayers et al., 1976a,b,c). In addition (and perhaps required for elicitor activity of the polysaccharides), it seems likely that endo-acting p-D-glucanases, which are present in many plant tissues, attack the soluble and wall-bound p-( 1+3) ,( 1+6)-glucans of invading fungi and thereby liberate soluble oligosaccharides such as those with elicitor activity (Section

14

S. ALDINGTON AND S . C. FRY

1I.A). Indeed, Yoshikawa et al. (1981) and Keen and Yoshikawa (1983) have demonstrated that soybean endo+-( 1+3)-glucanases will solubilize elicitor-active P-glucan fragments from hyphal walls of Phytophthora megasperma. The continued action of P-glucanases might (depending on the branching patterns of the substrate) progressively reduce the size of any oligosaccharides initially formed until they were too small to be oligosaccharins. This process would be enhanced by the action of P-glucosidases (i.e. exoglucanases), as has been demonstrated in vitro (Cline and Albersheim, 1981a,b). However, the fate of oligo-P-glucans in vivo has not been investigated experimentally. P-Glucanases could also have a direct damaging effect on fungal hyphae, e.g. by attacking their walls (Mauch et al., 1988a,b); they can also act as antiviral proteins (Edelbaum et al., 1991).

3. Other polysaccharidases Novel endogenous elicitors might arise through the action of the variety of enzymes known to be produced during fungal infection of plant tissues (Section V.B.2). Tomato leaves infected with the fungus Cladosporium fulvum accumulated in their apoplastic fluid a number of enzyme activities which, in vitro, can partially hydrolyse plant cell walls to liberate soluble oligosaccharides. These enzymes can be assayed by a very sensitive method in which the substrate consists of uniformly 14C-labelledwalls isolated from plant cells cultured in the presence of ~-[U-’~C]glucose as sole carbon source (Aldington and Fry, 1992). This assay will detect enzymes that attack any wall polymers (even hitherto unknown polymers) and that yield soluble fragments as products. It was found that tomato leaves infected with an incompatible race of the fungus accumulated within 3-5 days an enzyme activity (probably an endoarabinanase) that solubilized arabinose-rich oligosaccharides from plant cell walls. Leaves infected with a compatible race accumulated this activity later in the infection process (Aldington and Fry, 1992). It will be interesting to determine whether the arabinose-rich oligosaccharides solubilized by this enzyme in vitro possess any oligosaccharin activity in vivo-perhaps activating a defence response.

VI. MOVEMENT OF OLIGOSACCHARINS WITHIN THE PLANT: TRUE HORMONES? Oligosaccharins have a wide range of biological effects, some of which are exerted by exceedingly low concentrations, and they have been associated with systemic responses. They have for this reason frequently been described as “hormone-like,’. A hormone is a substance that acts at a site distant from its site of production, and this implies intercellular transport within the plant. Does this occur?

OLIGOSACCHARINS

7s

A . POSSIBLE TRANSPORT OF XYLOGLUCAN OLIGOSACCHARIDES

It has been speculated that XG9, formed in response t o the auxin that is present in the main shoot, is transported down the main stem to the axillary buds, where it is responsible for preventing their growth, i.e. for imposing apical dominance (Albersheim and Darvill, 1985). This hypothesis would ascribe a classical hormonal role to XG9. Exogenously supplied nonasaccharide must reach its site of action in the pea stem segment for its effect to be noted. The target cells for the action of XG9 are unknown but it seems reasonable to suggest that they might be those of the tissue most responsive to auxin-stimulated elongation, i.e. the epidermis. If it is further assumed that the relatively large, hydrophilic, XG9 molecule cannot readily penetrate the cuticle and is excluded from the symplast by the plasma membrane (Baydoun and Fry, 1989), and yet can inhibit the growth of 6-10mm pea stem segments (York et af., 1984; McDougall and Fry, 1988; Lorences et al., 1990), it can be suggested that XG9 is capable of moving distances of at least a few millimetres through the apoplast of a stem. However, direct studies of the movement of exogenous XG9 have not been reported.

B. NON-TRANSPORT OF WOUND SIGNALS

The discovery that pectic oligosaccharides possess wound-signal activity, evoking the biosynthesis of protease inhibitors, prompted Bishop et af. (1981) to conclude that these oligosaccharides are the protease inhibitor inducing factor (PIIF), previously defined as the long-distance wound hormone (Green and Ryan, 1973). However, while it is plausible that endogenous pectic oligosaccharides might increase in concentration at injury sites, and well-established that exogenous pectic oligosaccharides can evoke protease inhibitor synthesis in excised leaves, the missing link in the argument is whether pectic oligosaccharides can be transported from injured to uninjured leaves. Such transport would require the oligosaccharides to move both basipetally (out of the injured leaf, presumably in the phloem) and acropetally (into the uninjured leaf, possibly in the xylem). Even the smallest pectic oligosaccharides are too large to traverse such distances in the time required purely by diffusion. To provide an estimate of the velocity at which PIIF moves within the tomato plant, Green and Ryan (1972, 1973) inflicted a standardized injury on the lamina of one leaf and then, after an additional time t (0-8 h), they excised the injured leaf close to the base of its petiole, thus preventing further export of PIIF. The response (accumulation of protease inhibitor in a neighbouring uninjured leaf) at 48 h was taken at a measure of the amount of PIIF that had moved the few centimetres from the injury site on the

76


lamina past the excision point in the petiole within time t. Half-maximal response was observed when t was -45min. It was concluded that PIIF covered this basipetal leg of its journey at 3-5 cm h-’. However, this is a minimum estimate: the 45 min delay may have been due to the time taken by the injured lamina to produce enough PIIF to evoke a response, each individual PIIF molecule being instantly exported at a velocity much greater than 3-5 cm h-I. To investigate whether pectic oligo- and polysaccharides can indeed move from leaf t o leaf, as would be required if they were PIIF, three different preparations of soluble, relatively low molecular weight [U-14C]rhamnogalacturonans were applied to small injury sites on the leaves of potted tomato seedlings and the re-distribution of the radioactivity was monitored by autoradiography (Baydoun and Fry, 1985). Most of the 14Cremained at the injury site. A small amount of it moved acropetally towards the tips and margins of the treated leaf, in a pattern characteristic of xylem transport (Canny, 1990). No detectable I4C (corresponding to S 1ng of pectic material) moved basipetally. The lack of export of applied pectic fragments was confirmed with [3H]oligogalacturonides of DP 6 9 and 10-14 (Baydoun and Fry, 1985). The treated lamina tissue had been injured so that the 14C-and 3H-labelled pectic substances were certainly brought into contact with cell surfaces. Wounds are clearly valid application sites for testing the transport of a putative wound hormone (even if not of other hormones!). The acropetal movement of some of the labelled pectic material via the xylem shows that the applied pectic substances came into contact with vascular tissue. [‘4C]Sucrose applied in the same way was efficiently translocated away from the injury site, both acropetally and basipetally, so that phloem loading must have been possible under the experimental conditions used. We conclude that pectic oligosaccharides cannot move basipetally in tomato plants, and that, if they are involved in the induction of protease inhibitor biosynthesis in response to injury, they act locally in the immediate vicinity of the wound rather than as long-distance wound hormones. It is for this reason that we use the term “wound signal” rather than “PIIF” to describe pectic oligosaccharins; PIIF is the long-distance hormone by which injured leaves communicate their plight to neighbouring uninjured leaves and its identity remains a mystery.

C. TRANSPORT OF ELICITORS?

Since the hypersensitive response tends to be highly localised, any oligosaccharins involved in its evocation seem, apriori, likely to remain close to the site of infection and thus not to be hormones in the normal sense of the word. However, not all defence responses are as localized as the hypersensitive response. For example, phytoalexin synthesis often occurs over a

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77

large proportion of the area of an infected leaf (Holliday et al., 1981; Bailey and Mansfield, 1982) or of locally elicited tissue (Dixon et al., 1983b), and localized infection can sometimes trigger systemic responses, e.g. the accumulation of peroxidase (KuC, 1982), HRGP-mRNA (Showalter et al., 1985) and chitinase (Roby et al., 1988): in these cases, more mobile signals would be implicated. It is possible that some oligosaccharins may be mobile enough to serve this role (though probably not the oligogalacturonides-see Section V1.B); alternatively, oligosaccharides may prompt signals which can be rapidly transmitted to more distant parts of the plant. Ethylene is arguably one such signal; Ecker and Davis (1987) suggested that locally produced ethylene is suited to activate defence-response genes both nearby and at a distance.

VII.

CONCLUDING REMARKS

Oligosaccharins have been implicated in a wide range of botanical processes. Some of their proposed roles, especially as elicitors of phytoalexin synthesis, are well established, even if there are still conspicuous gaps in our knowledge of the oligosaccharins responsible-such as their mode of action, whether they occur naturally and (if so) how they are generated, transported and degraded. Some other roles are still much more speculative and even the existence of the biological response in artificial bioassays requires definitive confirmation or negation. However, despite all the gaps, the oligosaccharin concept is clearly here to stay, and, because of the gaps, the coming decade will no doubt be an exciting period in the history of the oligosaccharin concept.

ACKNOWLEDGEMENTS The authors are very grateful to the EEC for the award of a “BRIDGE” contract enabling them to work in this area.

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pisatin in pea pods and antinutrient proteinase inhibitors in tomato leaves. Biochemical and Biophysical Research Communications 110, 194-199. Walker-Simmons, M., Jin, D., West, C. A., Hadwiger, L. and Ryan, C. A. (1984). Comparison of proteinase inhibitor-inducing activities and phytoalexin elicitor activities of a pure fungal endopolygalacturonase, pectic fragments and chitosans. Plant Physiology 76, 833-836. Walker-Simmons, M. and Ryan, C. A. (1984). Proteinase inhibitor synthesis in tomato leaves. Induction by chitosan oligomers and chemically modified chitosan and chitin. Plant Physiology 76, 787-790. Walker-Simmons, M. and Ryan, C. A. (1986). Proteinase inhibitor I accumulation in tomato suspension cultures. Plant Physiology 80, 68-71. Weigel, H. (1963). Paper electrophoresis of carbohydrates. Advances in Carbohydrate Chemistry 18, 61-96. Wessels, J. G. H. (1986). Cell wall synthesis in apical hyphal growth. International Review of Cytology 104, 37-79. West, C. A. (1981). Fungal elicitors of the phytoalexin response in higher plants. Naturwissenschaften 68, 447457. West, C. A., Moesta, P., Jin, D . F., Lois, A. F. and Wickham, K. A. (1984). The role of pectic fragments of the plant cell wall in the response to biological stress. In “Cellular and Molecular Biology of Plant Stress” (J. J. Key and T. Kosuge, eds), pp. 335-350. Alan R. Liss, New York. Whistler, R. L. and BeMiller, J. N. (1958). Alkaline degradation of polysaccharides. Advances in Carbohydrate Chemistry 13, 289-329. Wijesundera, R. L. C., Bailey, J. A., Byrde, R. J. W. and Fielding, A. H. (1989). Cell wall degrading enzymes of Colletotrichum lindemuthianum: their role in the development of bean anthracnose. Physiological and Molecular Plant Pathology 34, 403-414. Wildon, D. C., Doherty, H. M., Eagles, G., Bowles, D. J. andThain, J. F. (1989). Systemic responses arising from localized heat stimuli in tomato plants. Annals of Botany 64,691-695. Yamazaki, N . , Fry, S. C., Darvill, A. G. and Albersheim, P. (1983). Host-pathogen interactions. XXIV. Fragments isolated from suspension-cultured sycamore cell walls inhibit the ability of the cells to incorporate [‘4C]leucine into proteins. Plant Physiology 72, 864-869. York, W. S., Darvill, A. G . and Albersheim, P. (1984). Inhibition of 2,4dichlorophenoxyacetic acid-stimulated elongation of pea stem segments by a xyloglucan oligosaccharide. Plant Physiology 75, 295-297. York, W,S., Oates, J. E., Van Halbeek, H., Albersheim, P., Tiller, P. R. and Dell, A. (1988). Location of the 0-acetyl substituents on a nonasaccharide repeating unit of sycamore extracellular xyloglucan. Carbohydrate Research 173, 113-132. Yoshikawa, M., Matama, M. and Masago, H. (1981). Release of a soluble phytoalexin elicitor from mycelial walls of Phytophthora megasperma var. sojae by soybean tissues. Plant Physiology 67, 1032-1035. Yoshikawa, M., Keen, N. T. and Wang, M.-C. (1983). A receptor on soybean membranes for a fungal elicitor of phytoalexin accumulation. Plant Physiology 73,497-506. Young, D. H. and Kauss, H. (1983). Release of calcium from suspension-cultured Glycine max cells by chitosan, other polycations, and polyamines in relation to effects on membrane permeability. Plant Physiology 73, 698-702. Young, D. H., Kohle, H. and Kauss, H. (1982). Effect of chitosan on membrane permeability of suspension-cultured Glycine max and Phaseolus vulgaris cells. Plant Physiology 70, 1449-1454.


Are Plant Hormones Involved in Root to Shoot Communication?

M . B . JACKSON Department of Agricultural Sciences. University of Bristol. AFRC Institute of Arable Crops Research. Long Ashton Research Station. Bristol BS18 9AF. UK

I.

Introduction

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I1. The Hormone Message Concept . . . . . . . . . . . . . . . A . Different Kinds of Hormonal Message . . . . . . . . . . B . Quantifying Hormonal Messages in Transpiration Stream . C . Assessing Developmental Impact of Hormonal Messages . . . . I11. Evidence for Regulation of Root :Shoot Ratio by Roots A . Nutrient Control Theory . . . . . . . . . . . . . B . Shortcomings of Nutrient Control Theory . . . . . . C . Conclusions . . . . . . . . . . . . . . . . . . . .

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. . . IV . Examples of Hormone-like Action of Roots on Shoots A . Early Research . . . . . . . . . . . . . . . . . B . Leaf Senescence . . . . . . . . . . . . . . . . . C . Shoot Extension. Photosynthesis and Flowering . . . D . Conclusions . . . . . . . . . . . . . . . . . . . .

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V . Cytokinins . . . . . . . . . . . . . . . A . Introduction and Early Research . . . B . Development in Unstressed Plants . . C . Root Excision Studies . . . . . . . . D . Responses to Mineral Nutrient Shortage E . Effects of Other Stresses Applied to Roots F . Conclusions . . . . . . . . . . . . .

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VI . Gibberellins . . . . . . . . . . . . . . . . . . . . . . . A . Introduction . . . . . . . . . . . . . . . . . . . . Advances in Botanical Research Vol . 19 ISBN Ck12-005919-3

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Copyright 01993Academic Press Limited All rightsof reproductionin any form reserved

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Studies on Unstressed Plants . . . . . . . . . . . . . . Effects of Root Excision and Environmental Stresses Applied to Roots . . . . . . . . . . . . . . . . . . . . . D. Conclusions . . . . . . . . . . . . . . . . . . . . .

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Ethylene . . . A . Introduction B. Flooding . C. Conclusions

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Abscisic Acid . . . . . . . . . . . . . A. Introduction . . . . . . . . . . . . B. Water Deficiency and Stomata1 Closure C. Water Deficiency and Leaf Expansion D. Soil Flooding . . . . . . . . . . . . E. Various Other Stresses . . . . . . . F. Conclusions . . . . . . . . . . . .

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Final Remarks . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References

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I. INTRODUCTION In plants the growth and behaviour of the shoots are coupled closely to growth and behaviour of the roots. This is reflected in root : shoot dry weight ratios, which are predictable at various stages of development (Davidson, 1969) implying the existence of a controlling mechanism balancing the growth of above- and below-ground parts. This impression of regulation is reinforced by evidence that particular root :shoot ratios are genetically linked (Monyo and Whittington, 1970; McMichael and Quisenberry, 1991) and that they return towards earlier values after physically constricting the roots (Richards, 198l), droughting or defoliation (Blaikie and Mason, 1990), or after excision of parts of the shoot (Buttrose, 1966) or large portions of the root (Biddington and Dearman, 1984; Fig. 1). Superimposed on this in-built regulation of the root : shoot ratio is a marked, and sometimes dominating, influence of environmental conditions around the roots, for example, increases in the ratio caused by salinity (Kuiper et al., 1990), or by restricting external nitrogen or phosphorus concentrations to levels that inhibit shoots more than roots (Hunt, 1975). Stressful soil conditions are also known to bring about many rapid morphological changes to shoots such as leaf epinastic curvature, foliar senescence, adventitious rooting and stomata1 closure, in addition to a loss of growth in dry matter. The way in which roots sense shortcomings in the soil and transmit a response to the shoot, where growth patterns are changed in consequence, is of obvious botanical interest and important in the context of agricultural crop performance.

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t No roots removed o 76% roots removed at start 0

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Fig. 1. Effect of removing 76% by weight of the root system of 2-week-old barley plants (Hordeum vulgare L. cv. Midas) on subsequent changes in root: shoot dry weight ratio. By the end of 14 days the initially small root:shoot ratios of the root-pruned plants once more approached those of unpruned plants. Similar effects were seen at both warm (16°C) and cool (8°C) root temperatures. From M. B. Jackson and J . V. Lake (unpublished results).

This review considers evidence that roots regulate the root :shoot ratio and other aspects of shoot development in stressed and unstressed plants by influencing the passage of plant hormones [auxin, gibberellins (GAS), cytokinins, ethylene, abscisic acid (ABA)] or their precursors between roots and shoots. A broad review is useful at this time because of a resurgence of experimental work that explores hormone involvement in root-shoot relationships (Jackson, 1985a; Kuiper and Kuiper, 1988; Davies and Jeffcoat, 1990) as an alternative to regulation directly through changes in water or mineral supply. The review may also help assess, in full bibliographic context, the remarks of Trewavas (1986) condemning the idea that hormone traffic between roots and shoots can reliably carry morphogenetic information. Trewavas suggested that any regulatory information thus carried would be destroyed by “noise” generated by environmental irregularities.

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Thus, on the one hand we have the view that environmental change may transmit morphogenetically active hormonal-like messages from roots to shoots, and on the other, the view that such environmental changes degrade information any messages might otherwise convey. Much of the present chapter assesses the evidence for the generation of hormonal messages by roots experiencing environmental change.

11. THE HORMONE MESSAGE CONCEPT A . DIFFERENT KINDS OF HORMONAL MESSAGE

In principle, roots can influence hormone levels in the shoot either by exporting one or more hormones or precursors (e.g. cytokinins or the ethylene precursor 1-aminocyclopropane-1-carboxylicacid), or by acting as sinks for phloem-mobile hormones produced in shoot tissues (e.g. ABA). On this basis, roots may generate or modify several kinds of hormonal message (Cannell and Jackson, 1981; Jackson, 1981). Firstly, they could increase their output of hormones (positive message) or decrease their output (negative message), or they may become less active sinks for hormones made by shoot tissues, thereby causing an accumulation in source tissue (accumulative message). It is also possible for roots to become more active sinks for hormones from the shoots (debit message). An example of the latter may be found in the stimulation of root growth resulting from infection with the parasitic weed, S t r i p hermonthica (Parker, 1984). Research into the hormonal basis of root-shoot relationships has largely ignored most of these different kinds of operational message, and usually incorporates only one of them-sometimes twc-in the design o r interpretation of experiments. Because the contents of the xylem and phloem are not entirely isolated from one another (Canny and McCully, 1988), there is a strong possibility of hormonal transfer between the two. In some plants, certain zones may exist where such exchange is facilitated by an especially close juxtaposition of xylem and phloem (McCully, 1990). Thus, hormonal messages may be recycled between root and shoot, as shown experimentally by Hoad (1978) for ABA in droughted plants, and discussed recently by Wolf e f al. (1990) and Hartung and Slovik (1991). In addition, xylem sap may become more alkaline (Hartung et al., 1990) and phloem less alkaline (Baier and Hartung, 1991) in stressed plants. Since several hormones, particularly ABA, are weak acids they become increasingly ionized as the pH rises, thus maintaining the inward diffusion gradient for undissociated molecules that are membrane permeable (Hartung et al., 1988). Xylem sap, if alkalinized by stress, may therefore act as a trap for hormones such as ABA in phloem or produced by cells surrounding the xylem along its entire length. These


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findings warn us that change in the amount of a hormone in xylem sap does not necessarily mean that it originated in the roots.

B. QUANTIFYING HORMONAL MESSAGES IN TRANSPIRATION STREAM

Many experiments assess the concentration of hormones in the transpiration stream of intact plants. Unfortunately, it is extremely difficult to sample the transpiration stream directly, since under-pressures driving transpiration are destroyed if xylem elements are punctured for sampling. Thus, estimates are usually made indirectly, using measurements made on sap that flows from root systems under osmotic pressure, or arbitrarily chosen external pressures, after the shoot is removed (de-topped plants). The flow of osmotically-driven sap is rarely more than 10% of daytime transpiration. Surprisingly, much experimentation has ignored the effect this large reduction in sap flow rate will inevitably have on solute concentration, through a lessening of dilution. The effect of a varying flow rate of sap through the xylem of root systems on hormone concentration can be readily demonstrated using a pressure vessel (Fig. 2a). In unstressed tomato plants, greater pressures give faster sap flow which, over the range approximating to transpiration rates of whole plants, dilute endogenous ABA in a linear fashion. It follows that concentrations of hormones in slowly flowing sap from de-topped plants cannot be equated with those of the intact plant and will be overestimates of the true concentration present in the transpiration stream. Furthermore, relative differences in concentration between sap from de-topped roots previously exposed to various treatments are likely to be distorted by the differences in sap flow rates these treatments must often cause through altered hydraulic resistance and root metabolism. Authors sometimes calculate the likely delivery rate of a hormone from roots to shoots of whole plants by multiplying sap flow rate from de-topped roots by the hormone concentration (e.g. Beever and Woolhouse, 1973; Bradford and Yang, 1980a; Heindle et af., 1982; Carlson et al., 1987; Neuman et af.,1990; Meinzer et af.,1991). In relating this value to the whole plant they are, axiomatically, assuming that the faster sap flow of the intact plant would dilute hormone strictly in proportion to the rate of transpiration, with no effect on total delivery. This seems to be a reasonable assumption, and for flooded and well-drained tomato plants delivery rates remain approximately the same over a range of flow rates similar to those of whole plant transpiration (Fig. 2b). In support of this, Radin et af. (1982) found delivery of exogenous ABA into detached Gossypium hirsutum leaves to be similar at different leaf conductances (i.e. different rates of water flow). However, there are at least two reports of delivery increasing with faster sap flow (Wagner and Michael, 1971; Meinzer et al., 1991), suggesting that delivery rates are best worked out using sap flows

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16C m -

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Sap flow (mm3SS’) Fig. 2 . Effect of increasing flow of sap through detached root systems of tomato plants grown in well-aerated soil or in soil flooded for 12 h on (a) concentration of abscisic acid in xylem sap and (b) rate of delivery of abscisic acid from cut stumps. Arrows show transpiration rates of comparable intact plants. Means of four replicates. From M. A. Else, W. J . Davies and M. B. Jackson (unpublished results).

approaching those of intact plants. When calculating delivery rates, some workers make the mistake of assuming a no-dilution effect, and consider hormone delivery from roots to be increased in proportion to whole plant transpiration flow (e.g. King, 1976; Smith and Dale, 1988). This can lead to multiplying the concentration measured in slow-flowing sap by the faster flows of whole plant transpiration in calculating hormone delivery. This misunderstanding will inevitably lead to gross over-estimations of hormone delivery from roots to shoots (e.g. Skene, 1967). To avoid making this mistake, it is imperative to multiply concentration by the flow rate of the sap used for hormone measurement, and not by some other flow rate. Clearly, there is unnecessary confusion and contradiction in the literature concerning the interpretation of measurements of hormones in xylem sap. Authors’ claims, therefore, always require careful scrutiny. A related concern originates in the well-known observation that in experimentally or environmentally perturbed plants, transpiration is often slower than normal because of stomata1 closure. Thus, even if an increase in concentration in the transpiration stream is correctly estimated in the


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stressed plants, it could merely indicate less dilution of the same amount of hormone, with no real change in message or hormone delivery to the shoots. Consequently, there is a risk that claims of increased hormone transport from roots to shoots based on concentration measurements may be untenable if all that is really happening is decreased dilution caused by stomata1 closure. Thus, it is of paramount importance that increases in delivery rates are demonstrated before claims of changes in a hormonal message can be accepted with confidence. Recording increases or decreases in concentration are, by themselves not sufficient evidence of changes in the passage of hormones into the xylem sap from roots, or elsewhere. The potentially confounding effects that different sap flow rates can have on estimates of hormone concentration in xylem sap of intact plants and delivery rates from roots to shoots may be addressed in several ways. One possibility is to measure hormones in sap samples obtained after pressurizing detached roots to generate sap flows close to those of comparable whole transpiring plants. Although this approach is highly desirable and has been endorsed recently (Incoll and Jewer, 1987; Meinzer et al., 1991), no results obtained in this way have been published for hormones, except in flooded and well-drained tomato plants (Fig. 2). An alternative approach has been to pressurize the roots to a level equivalent to the leaf water potentials of intact plants. However, results obtained this way have assumed similar leaf water potentials in control and stressed plants (e.g. Neuman et al. , 1990; Smit et al., 1990). This assumption will often be incorrect. A more straightforward method is to take the first few microlitres of sap issuing from the surface of a cut stump, since this may represent true transpiration fluid issuing from the roots. The method appears to be satisfactory in maize (Zhang and Davies, 1990a) but in tomatoes, the first 5&100 ~1 of sap are heavily contaminated by extra ABA generated as a result of applying a collecting tube over the cut stump (Else e f al., 1991). Several authors have expressly avoided these first droplets of sap because they suspected that they would be contaminated (Henson and Wareing, 1976; Heindle et al., 1982; Cahill et al., 1986). Clearly there are obvious risks inherent in this simplest of methods. Alternative techniques include expelling small volumes of sap from cut leaf veins by over-pressurizing the roots of whole plants with a specially designed pressure bomb (Passioura and Munns, 1984; Munns and King, 1988; Schurr and Gollan, 1990). This seems to be the best way of measuring what is arriving at a particular leaf (Munns, 1990; Schurr and Gollan, 1990). However, it is less helpful for assessing what is initially delivered to the shoot from the root system since there will be losses or gains on the way up to leaf, and also a differential partitioning of the delivered hormone between leaves in proportion to their individual rates of transpiration. Another possible approach is to extract the contents of the xylem vessels of excised stem segments by applying increased or reduced pressures or centrifuging in the hope of obtaining a sample with hormone content

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similar to that of the transpiration stream of the source plant (Loveys, 1984a,b; Tromp and Ovaa, 1990; Trejo and Davies, 1991). However, some components of this sap have been shown to differ from those of the transpiration stream (Ferguson, 1980). This may be because the small volumes involved are highly susceptible to either enrichment or depletion in the time between excision and extraction by the biochemical activities of the relatively large mass of surrounding living cells (Nonhebel et al., 1985; Griggs et al., 1988), by partitioning between pools of different acidities, or by metabolism within cell walls (Loveys and Robinson, 1987). Crushed cells at the ends of such stem segments may also introduce artifacts (Zhang and Davies, 1990a). Yet another method for trying to obtain an authentic sample of the transpiration stream is to place individual leaves in a Scholander-type chamber and pressurize until sap issues from the xylem (Hartung et al., 1990; Tardieu et al., 1992~).Some of the expressed fluid will undoubtedly be captured transpiration stream. However, a variable proportion of this fluid will have been expressed from cell interiors rather than just from xylem vessels. This is because xylem water will inevitably have been withdrawn into leaf cells when underpressures were released on cutting the leaf from the intact plant. This movement of sap into cells, and then out again under the pressure of the Scholander bomb, may therefore result in a misleading hormone enrichment since sap re-emerging from the cells may have become contaminated with hormones and other solutes. Also, the volume of sap expressed for analysis (50-100 p1 minimum) may exceed the volume of leaf xylem (Hartung et al., 1988), at least when small leaves are used. In these circumstances, the sap sample will always contain material expelled from leaf cells. In all work with sap flows from de-topped root systems, it is highly desirable that hormone measurements are made as quickly as possible after wounding artifacts are over, that is, before root biochemistry is altered by a shortage of assimilates from the shoots. Many workers use sap delivered over long periods (3 days-Salama and Wareing, 1979; 24 h-Sattelmacher and Marshner, 1978) during which time root biochemistry will have changed considerably with inevitable consequences for hormone production. Modern assay procedures for hormones are highly sensitive and preclude the need for such long collection times. Possibly the most useful estimates of hormone levels in the transpiration stream would comprise the following elements: (1) a concentration in sap flowing at rates of whole plant transpiration at the time of sampling; (2) a delivery rate calculated by multiplying the sap flow rate and hormone concentration derived in (1) above; (3) a “specific” delivery rate that takes into account the delivery rate calculated as in (2), together with the size of the root system supplying the hormone message (e.g. Bradford and Yang, 1980a; Coleman etal., 1990) and the weight or area of shoot tissue into which it will be delivered. This would generate expressions such as mol of hormone

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delivered per g root per m2 of leaf per min. There a r e , as yet, no published data of this kind, even though such data have the great advantage of allowing meaningful and direct comparisons t o be made between plants of different sizes and root :shoot ratios, growing under different experimental treatments and transpiring a t different rates. C. ASSESSING DEVELOPMENTAL IMPACT OF HORMONAL MESSAGES

Demonstrating a change in hormone delivery in sap, or in tissue concentration is in itself insufficient evidence t o establish any physiological significance. T h e several different kinds of additional information needed t o d o this convincingly are summarized in Table I and based on previously TABLE I Criteria for implicating a hormone in the regulation of a naturally occurring develoumental uhenomenon Principal criteria (1) Correlation Joint occurrence between the timing of developmental changes and alterations to the endogenous hormone concentration. The ideal is precedence by the hormonal change, commensurate with its speed of action. ( 2 ) Duplication Reproduction of the phenomenon by re-creating quantitatively, changes in the internal concentrations of hormone measured when the process occurred naturally. ( 3 ) Deletion and re-instatement (a) Prevention or inhibition of the phenomenon by removing or decreasing the internal hormone titre by chemical means or by molecular genetics. (b) Reversing the effect of (a) by demonstrably re-instating quantitatively the original internal hormone levels. (c) Inhibiting the phenomenon by interfering with the action of the hormone, preferably using a non-toxic, competitive inhibitor, or by molecular genetics or mutation. (4) Chemical specificity Evidence in (1)-(3) should not apply to other substances found in plants, other than precursors. (5) Relevance to higher levels of organization Developmental process studied should occur beyond the confines of the laboratory and relate to performance in environments to which organs, whole plants or populations are naturally subjected to. ( 6 ) Relevance to lower levels of organization Association between the action of exogenously supplied hormone and the naturally occurring phenomenon is retained at cellular, subcellular and biochemical levels. Ideally criteria (1)-(4) should apply to each aspect examined. (7) Generality Extent to which the proposed hormonal controls apply to other taxonomic groups with similar developmental traits should be established. From Jackson (1987).

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published rules of evidence (Jackson, 1987) that are modifications to those of Jacobs (1959) and Koch (1876). Unfortunately, it is extremely rare to find any experimental system where all these requirements have been met. The rules in Table I form a useful basis for judging the thoroughness of a piece of research and of the claims made for hormonal intervention in root-shoot relationships. Important among the requirements are tests of physiological activity using the most appropriate hormone dose. This could be one that matches quantitatively the concentrations in xylem sap close to the target tissues of intact plants. Preferably, it would be the amount of hormone delivered from the root by transpiration to known amounts of target tissue in the shoot. This exemplary approach has been rarely adopted (e.g. Loveys, 1984b). Obviously, such tests cannot be made meaningfully without first obtaining realistic estimates of hormone levels in the transpiration stream. As discussed above, achieving this is not straightforward, concentrations in sap issuing from de-topped root systems at rates well below those of transpiration being especially misleading. Unfortunately, several workers have used concentrations in sap obtained in this way as a guide to choosing the concentrations of hormones for testing physiological potency (e .g. Smith and Dale, 1988; Nooden et al., 1990a,b).

111. EVIDENCE FOR REGULATION OF RO0T:SHOOT RATIO BY ROOTS As discussed in Section I, the size relationship between root and shoot is controlled by a combination of intrinsic and external factors. The questions here are whether roots themselves regulate the root:shoot ratio, and the extent to which any effect they have is based on factors other than nutrient or water supply (i.e. hormonal factors). A. NUTRIENT CONTROL THEORY

The division of labour between roots and shoots, arising from their very different environmental resources and absorbing properties, suggests that each might control the size of the other by limiting the supply of one or more constituents to the dependent part (i.e. negative control). Clearly, roots could not expand in size beyond the quantity of carbon assimilates available from the shoot, while it is inconceivable that shoot growth could proceed entirely unchecked by a finite supply of water or minerals available from the root. The root:shoot ratio of plants acclimatized to a given environment may thus be little more than the size ratio at which supply and demand of basic constituents from the environment are in equilibrium as roots and shoots continue to grow. The root :shoot ratio may, thus, be underpinned by a balance between the functional efficiency of roots and shoots along the


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lines expressed arithmetically by Davidson (1969) as: root mass x rateabsorptionx k = shoot mass x ratephotosynthesis, where k is a constant representing the amount of mineral nutrient or water used per unit amount of carbon increment. Thornley (1972) has extended this approach by considering that shoot and root growth each separately depend on the concentration of carbon and minerals (mainly nirogen), which in turn is determined by how much of each is received from the supplying part. The amounts root and shoot receive presumably reflects the relative competitive abilities of root and shoot and their individual absorption capacity, e.g. for nitrogen (McDonald et ul., 1986). Wilson (1988) concluded that the Thornley model largely accounted for decreases in the root :shoot ratio seen in many plants as they grow larger, and for increases in the ratio in the face of deficits in water and major inorganic nutrients. An example of the latter effect is seen in the experiments of Hunt (1975), where the functional efficiency of roots for absorbing and supplying nitrogen was depressed by diluting the nitrogen concentration in the rooting medium. In the context of an overall decrease in plant growth, nitrogen shortage slowed root growth less than shoot growth, thereby increasing the root : shoot ratio. This is to be expected if the amount of available nitrogen falls away steeply with distance from the site of uptake, thus starving the shoot more than the root. The power of mineral nutrition to affect the root:shoot ratio is highlighted by a report that the ratio of root:shoot of individual but joined tillers of Curex can be made to vary 10-fold simply by contriving a different level of soil fertility around each tiller (Harper, 1977). The sophisticated regulation of mineral availability in solution cultures in the experiments of Ingestad and Lund (1986) also illustrates the controlling influence that nutrient supply can have on the absolute and relative growth rates of plant parts. Thus, in reviewing the problem, Wilson (1988) had little difficulty in rejecting hormonal controls in favour of mineral supply as the principal means by which roots influence the root :shoot dry weight ratio.

B. SHORTCOMINGS OF NUTRIENT CONTROL THEORY

There are problems with the idea that mineral supply from roots sensitively maintains a size of shoot that is in a predictable proportion to the size of the root. The notion necessarily rests on the principle that the mineral supply rate per unit weight of roots remains reasonably constant. Only if this holds true can mineral supply be a reliable indicator of root size, and thus a sensitive regulator of the root : shoot ratio. However, under conditions where mineral ions are readily available to roots, as in a complete nutrient solution, specific supply rates of nitrogen phosphorus and potassium (i.e. mg transported to the shoot per g dry weight of root) are greatly decreased if a large proportion of the roots (75-80%) is removed (Table 11). Thus, delivery

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TABLE I1 Effect of pruning 7 5 4 0 % of roots from 7-day-old plants of barley (Hordeum vulgare), maize (Zea mays) or oilseed rape (Brassicd napus) on the specific supply rate of mineral nutrients to the shoot over the following 7 days. The final concentration of the nutrients in the shoot are also Riven

Parameter

Nitrogen

Phosphorous

Potassium

Control Pruned Control Pruned Control Pruned Specific supply rate (mgg-' root day-') Barley Maize Oilseed rape

18.3 18.7 53.0

13.0 8.5 13.0

2.8 3.8 4.4

1.5 0.9 0.7

19.6 25.7 37.2

17.7 16.7 9.9

62.4 47.4 65.1

55.7 38.8 63.3

8.7 10.2 5.9

6.5 7.4 5.7

68.0 61.7 48.6

68.2 55.1 50.7

Concentration in shoot (mg g-' dry wt)

Barley Maize

Oilseed rape

Means of 8 plants. 75% of roots were pruned from maize, 77% from barley and 80% from oilseed rape (dry weight basis). From J . V. Lake, K. Brown and M. B. Jackson (unpublished results).

rates of minerals per unit amount of root are not a reliable basis by which shoots may gain some measure of root size, even when external nutrient concentrations remain unchanged. Under such circumstances the fall in specific uptake rates by roots remaining and re-growing after substantial root pruning suggests that this parameter is governed more by a disproportionate decrease in shoot growth and, thus, shoot mineral demand rather then being a property of the roots themselves (Table 11). Such a feedback mechanism is well established (Pitman, 1972). This reasoning leads to the conclusion that the disproportionate decrease in shoot growth must have some cause other than mineral shortage. The dominance of the shoot in determining mineral absorption is illustrated by the relative consistency of mineral nutrient concentration in the shoot despite root pruning (Table 11), even though the mineral supply per unit weight of root is depressed greatly in the three species examined. A further difficulty is that surprisingly large reductions in root size, achieved by pruning, can be sustained with little change in shoot growth. For example, Tschaplinski and Blake (1985) reduced the weight of the root system of alder (Alnus glutinosa) seedlings from 1.73 g to 0.70 g by constricting them for 96 days using perforated polypropylene tubes submerged in aerated nutrient solution. This had almost no effect on growth in shoot weight, other aspects of shoot development or shoot-water relationships. Similarly, Das Gupta (1972) removed half the roots in the vertical plane from 28-day-old seedlings of sugar beet (Beta vulgaris) without reducing

PLANT HORMONES A N D COMMUNICATION

115

significantly the relative growth rate of the shoot over the subsequent 21 days. Buttrose and Mullins (1968) pruned the roots of grape vine (Vitis vinifera) to approximately 75% of the weight of unpruned control plants every week for 8 weeks without any statistically significant effect on shoot growth, while Humphries (1958) removed up to half the roots from barley or rye with no effect on shoot growth. There are other reports where shoot growth is restrained by pruning 50% or less of the roots (Humphries, 1959; Buttrose and Mullins, 1968; Carmi, 1986) or by severely constricting roots with small containers (Krizek et a f . , 1985; Ruff ef a f . , 1987) or cages suspended in nutrient solution (Richards and Rowe, 1977; Richards, 1981). However, recent careful re-examination of the latter approaches suggests that such effects may have been mediated by gross interference with water relations (Hameed et a f . , 1987; Tschaplinski and Blake, 1985) or root metabolism through restricting oxygen availability (Peterson et a f . , 1991), thus complicating interpretation. The few published examples of reduced growth in shoot dry weight from more modest root pruning (Humphries, 1959; Buttrose and Mullins, 1968; Carmi, 1986) are unlikely to be the result of water deficits since hydraulic conductance is increased rather than decreased by removing roots (Milligan and Dale, 1988). Thus, in certain situations or species, root size can be more sensitively measured than in others, but generally, size perception by the shoot is poor. We may conclude that while mineral supply is an important mechanism by which roots limit shoot growth, it is not necessarily a sensitive indicator of root size and thus an unlikely basis for the sensitive regulation of the root :shoot ratio. Indeed, it may be that shoots exercise a stronger control over the root:shoot ratio than the roots. This is suggested by grafting experiments between dwarf and normal sized tomato cultivars (van Staden ef al., 1987), by the influence of the shoot on specific uptake rates by roots (Pitman, 1972), and by reports that considerable amounts of shoot growth can sometimes occur in the absence of roots (Killingbeck, 1990). Presumably, in the short term, growing parts of the shoot can obtain minerals from non-growing shoot tissues, thereby displaying a limited degree of independence from the roots. It now remains to ask if there is any evidence that roots can influence growth of shoot dry mass in ways other than through the supply of minerals per se. Of course, failure to match demand for water by evaporation from the foliage will be one rather crude mechanism. Clues that other more subtle mechanisms exists include the simple observation by Vanden Driessche and Wareing (1966) that tree seedlings growing in a range of nutrient solution concentrations exhibited very different relative growth rates, despite having closely similar internal mineral nutrient concentrations. Thus, species differences in responses to a wide range of mineral nutrient concentrations cannot be attributed to differing requirements for minerals themselves as part of the biochemical and structural requirements for growth. Similar

116

M. B. JACKSON

conclusions were drawn by Kuiper and Staal(l987) from work with different species of Plantago, which were found to have very different responsiveness to changes in mineral supply. Thus, factors are at work that influence what Ingestad has termed “nutrient productivity”, i.e. the amount of growth sustained by a given availability of minerals. In his experiments, “nutrient productivity” is shown as the slope of relative growth rate against the internal nutrient concentration expressed as a percentage of the optimum concentration for growth (Ingestad and Lund, 1986). This expression is roughly analogous to the constant in Davidson’s equation (Section 1II.A; Davidson, 1969) expressing the size relationship between root and shoot. Application of hormone analogues such as benzyladenine has been found to increase the amount of growth in shoot dry mass per unit of absorbed mineral nutrient or of shoot mineral content (Richards and Rowe, 1977; Richards, 1978), suggesting that endogenous hormones could influence “nutrient productivity”. Furthermore, there are reports of growth responses in shoots to mineral deprivation that may be too fast or too slow to be explained easily in terms of direct mineral shortage in growing shoot cells (Kuiper and Staal, 1987).

C . CONCLUSIONS

Overall, it appears that root activities may have less to do with regulating the root :shoot dry weight ratio than the shoot. This may explain the apparent insensitivity of shoots to quite large reductions in the size of root systems well-supplied with minerals and water. This has serious consequences for the commonly held notion that reductions in growth in shoot dry mass caused by stress in the root environment are a result of decreased output of growth promoting hormones from the roots (negative messages). Shoot growth (in dry weight at least) is clearly often insensitive to a loss in putative output of hormonal growth promoters (e.g. cytokinins) equivalent to that emanating from at least 25% of the root system. Roots can of course influence shoot through mineral supply, especially where amounts in the rooting medium are already limiting overall plant growth. However, there is little direct support for believing that roots sensitively regulate the root :shoot ratio with minerals or with hormones. Some indirect evidence suggests that hormones could influence the extent to which growth in dry mass by shoots responds to a given mineral input from the roots. But, this is far from indicating a mechanism for regulating closely the root :shoot ratio. There remains a large body of evidence that particular aspects of shoot behaviour (e.g. stem extension, senescence, flowering, stomata1 apertures and leaf expansion) can be affected by roots in ways not readily explained in terms of water or mineral supply. Evidence for this is assessed in the subsequent sections.


117

IV. EXAMPLES OF HORMONE-LIKE ACTION OF ROOTS ON SHOOTS A. EARLY RESEARCH

In the “Silliman Lectures” to Yale University in 1937, Chibnall (1939) was “. . . tempted to suggest that some influence of the root system, possibly hormonic, is responsible for the regulation of protein levels in leaves”, although it was not until 1954 that his experimental results supporting such an idea were published (Chibnall, 1954). By that time other evidence supporting this hypothesis had appeared. For example, Went (1938a) reported an elongation-promoting effect on dark-grown pea shoots by roots extending into demineralized water. When shoots were grafted onto root systems in the dark, they re-commenced extension only when cell continuity across the graft union was established (Went, 1938b). He assumed that the role of the roots was to supply a graft-transmissible growth factor (“caulocaline”) rather than to drain the shoot of growth inhibitor. Working with light-grown tomato plants, Went (1943) demonstrated that an adventitious root system growing in nutrient-free peat and comprising approximately 10% of the mass of the main root system below, was capable of doubling the rate of stem extension. Similar tests were done with plants with vertically divided root systems. When one of the two “half” root systems was grown in well-aerated nutrient-free peat, gravel or pumice instead of nutrient solution, leaf senescence was retarded and stem extension promoted. Similar but simpler experiments have also been published by D e Ropp (1946), Galston (1948), Jordan and Skoog (1971) and Sabanek et al. (1985). Detached leaves of some species such as Phaseolus vulgaris or Nicotiana rustica can be induced to root in water. Mothes and Engelbrecht (1956) noted a coincidence between the arrest of protein decline in the lamina and the emergence of roots from the petiole. Humphries (1963) and Humphries and Thorne (1964) measured very slow rates of photosynthesis until just before the new roots emerged, when rates increased coincidentally with a halt in the decline of leaf chlorophyll. Along similar lines, Parthier (1964) showed that the ability of isolated leaves to incorporate l4C-rnethionine into protein declined steadily until the precise time that new adventitious roots first appeared when, from this point on, ‘‘C-incorporation rates increased markedly. The presence of roots clearly has influences on shoot behaviour that cannot easily be explained in terms of mineral nutrition. B. LEAF SENESCENCE

When a large proportion of a root system is removed so that the root :shoot ratio is suddenly very much reduced, shoot growth may not be inhibited

118

M. B. JACKSON

proportionately to the reduction in the supply of minerals from the root (Humphries, 1959). Similarly, when Topa and Mcleod (1986) withdrew oxygen for 15 days from roots of flooding tolerant Pinus serotinu and P. tuedu, mineral uptake into the shoots was depressed but with no associated reduction in growth of shoot dry weight. It is likely that in both these cases minerals used to sustain growth by the younger parts were provided by senescing leaves. In support of this interpretation, Darrall and Wareing (1981) showed that shoots of birch growing in nitrogen-deficient sand continued to grow with smaller than usual whole-shoot mineral concentrations. This appeared possible because younger growing parts obtained nitrogen from older senescing leaves. Of course, leaf senescence is atypical of more vigorous, healthy plants, even though their shoot growth may still be restrained by nutrient supply. The reason for the lack of senescence in the older leaves may be connected with the presence of a sizeable and healthy root system per se and less with the continued supply of minerals, for which mature leaves have in any case little or no requirement. The principle is made clear by the work of Ingestad (1982), which showed that shoot growth can be constrained by slow mineral supply over long periods without symptoms of leaf senescence, provided that the external supply is increased regularly to stay in proportion to the growth rate. Thus, total shoot size can be constrained by mineral supply in the absence of leaf senescence, but in association with the presence of an enlarged root system (Fig. 3). How do these roots prevent leaf senescence in the face of less than optimal mineral nutrition? The possibilities are that a full complement of healthy roots drains the shoot of some senescence-promoting influence, or alternatively, that a complete and healthy root system supplies one or more senescenceretarding metabolites, possibly hormones. Thus, one explanation for leaf senescence when roots are stressed is that the export of some senescence-promoter from the shoot system is slowed sufficiently enough for it to accumulate in leaves to damaging levels. It is well known that removing metabolic sinks increases sucrose in photosynthesizing leaves (Neales and Incoll, 1968; Farrar and Farrar, 1985). This in turn might inhibit photosynthesis (Herold, 1980) and damage chloroplasts through an excessive build-up of starch. However, Lawrence and Strangeways (1985) have shown the injurious effects of exogenously supplied sucrose to be artifactual and that endogenously generated increases in the sugar are innocuous. Similarly, Humphries (1963) followed the accumulation of assimilate in detached, rooted leaves over several weeks. Net assimilation rate (dry weight increase cmP2 week-’) remained unchanged despite the steady accumulation of dry matter in the lamina, resulting from the absence of growing sinks in the “shoot”. Weights of up to 7.4 mg cmP2were found, which is much greater than those in intact plants. However, leaf yellowing was seen only towards the end of the experiment, indicating how tolerant non-growing leaves can be of accumulated photosynthate and presumably

119


Leaves Stem X Root

0

E

60

.-cn

s??

0 K cn ._

5 40

H

. I -

-0m c

0

c

;Iz

20

0

I

I

I

1

5

10

15

Sub-optirnum (RNYO)

I

I

2 0 .E 5 1 c

0"

1

z2

.c

(1

0

Fig. 3. Dry weights of roots, shoots and stems of seedlings of birch grown in nutrient solutions replenished by frequent additions of mineral nutrients, with nitrogen given at different rates. After Ingestad and Lund (1979).

other metabolites. The latter may well have included the hormone abscisic acid, which accumulates in detached leaves (Jackson and Hall, 1987) or de-rooted seedlings (Smith and Dale, 1988), but not when they are senescent (Osborne et al., 1972). Another way to test if accumulations of substances in the leaf prompts senescence is to kill the phloem connection between root and leaf by girdling. This allows the inward movement of water and solutes from the root in the xylem while preventing outflow of materials. Kulaeva (1962) showed convincingly that girdling using steam does not simulate the senescence-promoting effects of de-rooting in Nicotiana. She also showed that roots are able to exert a senescence-retarding effect when connected to the leaves by xylem alone (see also the girdling experiments of Carmi and Koller, 1979). It seems unlikely that in plants with environmentally damaged or partially removed root systems, leaf senescence is neither explicable by mineral shortage nor by the accumulation of some senescence promoter. Changes in the output of senescence regulators from the roots seems more probable.

120

M. B. JACKSON C. SHOOT EXTENSION, PHOTOSYNTHESIS AND FLOWERING

Wareing and Nasr (1961) and Smith and Wareing (1964) examined the possibility that roots exercise control over shoot extension by means other than inorganic nutrition or by draining the shoot or growth inhibitors. They exploited the potential of shoot cuttings of willow (Safix virninafis),rooted at their basal end, to sprout axillary shoots on top of a loop formed by bending the stem (zone 1 in Fig. 4). It proved possible to induce axillary shoots to sprout in a second location along the stem if a second set of adventitious roots was induced to form further up by embedding a short length of stem in damp vermiculite. For example, axillary shoots at the tip of the stem (zone 2, Fig. 4) grew only if an additional set of adventitious roots were induced at the bottom of the loop (position B, Fig. 4). The effect was not explicable in

Total Iongth of sideshoots (cm)

Zone2

I

/ /

zone 1 1

zone 2

Roots at A only

1240

140

Roots at A & B (- minerals)

060

400

Roots at A & B (+ minerals)

000

I

660

Fig.4. Effect of inducing a second adventitious root system on cuttings of willow (Saliw viminalis) trained in a circle. With only basal roots present (position A), axillary shoots emerged mainly from zone 1. When a second set of roots was induced to grow out into a mineral-free medium, axillary buds were also induced to grow in zone 2. Based on results of Smith and Wareing (1964).


121

terms of enhanced mineral nutrient supply since roots were highly active even when grown in deionized water. That the roots acted as a sink for a phloem-mobile growth inhibitor was also discounted because the roots remained effective even when the stem between the roots and potentially responsive lateral buds was bark-girdled. In contrast, removing the roots, as they formed, prevented axillary bud outgrowth. Smith and Wareing (1964) concluded, quite reasonably, that roots release into the xylem some hormonal influence that is transported by transpiration or root pressure flow to sites of action upstream of the roots. Similar evidence for hormone-like effect of roots on shoots is to be found in the sequential rise and fall in net carbon dioxide fixation associated with the outgrowth of whorls of nodal roots of Sorghum saccharum (Jesko et al., 1971; Jesko, 1972) and Zea mays (Jesko and Vizarova, 1980). The effect occurs prior to the emergence of roots through the covering of leaf bases, and thus precedes their development into major sinks, or as organs of mineral or water uptake. Jesko and colleagues concluded that the young roots donate a stimulus for photosynthesis. Surprisingly, the results show that leaf expansion slows during these brief surges in photosynthesis. Roots can also influence flowering in ways that strongly suggest a hormone-like influence. Chailakhian (1961) showed that removing roots from Rudbeckia tricolour, a long-day plant, prevents flowering in response to long days. Roots also influence flowering in Scrofularia arguta,a long-day plant, or in the short-day species Perilla ocimoides and Chenopodium polyspermum. In each case, isolated vegetative buds, cultured in vitro, flower quickly even in non-inductive daylengths, provided the explants remain free of roots (Miginiac and Sotta, 1985, and references cited therein). In Silene, the need for long days can be obviated by first exposing only the roots to high temperatures (Wellensiek, 1985). In related work, with Cichorium intybus, a vernalizing treatment is needed before long days can induce flowering (Joseph, 1984, cited Miginiac and Sotta, 1985). From grafting experiments (Fig. 5) it is clear that it is the roots which respond to the chilling not the potentially flowering bud. O n the other hand, non-vernalized roots can inhibit flowering of a vernalized bud to which it is grafted. These examples point to a supression, by cold or by heat, of flowering inhibitor production in roots. The rooting behaviour of stem cuttings of blackcurrant (Ribes nigrum) also points to a flowering inhibitor produced by roots. Schwabe and Al-Doori (1973) found that cuttings cut down to less than 20 nodes long before being rooted will not flower in normally inductive short days. The effect was related to the distance between the root system and the shoot apex where flowering occurs. On longer shoots, with more than 20 nodes, flowering occurred in the upper buds in short days. However, this could be prevented by inducing a second set of adventitious rooting closer to the shoot tip. Other examples of hormone-like influences of roots on flowering are given by Bernier and Kinet (1986).

122

M. B. JACKSON 18°C Photoperiod:16h

f

Yo of vering bumS A

B

0

0

85

92

0

0

I oa

00

- -

Crossedgrafts

Autografts

c 1 e-

- -

Fig. 5. Flower initiation in apical buds of Cichoriurn inlybus cultured in vifro. To form a flower in a 16 h photoperiod the original explant comprising root ( V ) and shoot ( A ) requires 8 weeks vernalization at 3°C. Grafting combinations show that it is roots that sense chilling and transmit the response to buds across a graft union. Redrawn from Miginiac and Sotta (1985).

D. CONCLUSIONS

Even if the known plant hormones had remained undiscovered, the examples and arguments presented would still constitute a strong case for believing that hormone-like influence are involved and that mineral and water supply or assimilate accumulation are not the sole means by which roots influence shoots. The mere presence of growing roots would seem to exert a promoting influence on shoot extension and photosynthesis, and to stimulate or prevent flowering, although these phenomena are less thoroughly researched than that of the senescence-retarding effect roots have on leaves. The latter effect is difficult to explain exclusively in terms of mineral nutrient supply. Girdling experiments suggest that roots exert their effect by changes in the export of one or more senescence-retarding hormones (positive or negative messages) rather than modulating the withdrawal of a senescence promoter produced by the shoot (accumulative and debit messages). However, if the latter mechanism does operate, sucrose is unlikely to be the senescence promoter involved. Subsequent sections will explore the extent to which the influences of roots on shoots can be explained by changes in amounts and actions of one or more of the five main classes of plant hormone (cytokinins, gibberellins, abscisic acid, auxin, ethylene). Each will be dealt with separately, except for auxin, which although present in both xylem and phloem sap (Hall and


123

Medlow, 1974) has attracted relatively little attention. The most common experimental approach has been to apply a stress to the roots in attempts t o alter qualitatively or quantitatively the input or output of hormones by the roots and to link these with developmental effects of the stress.

V. CYTOKININS A . INTRODUCTION AND EARLY RESEARCH

Chemical and physiological characterization of these purine hormones is not straightforward. Most possess a side-chain of five carbons at the N6 position that can become conjugated by glycosylation. Amongst several biological properties, cytokinins must promote cell division, e.g. in soybean callus, to satisfy the definition. Other recognized effects of exogenously applied cytokinins include the retardation of leaf senescence and the promotion of stornatal opening, lateral bud outgrowth, shoot extension and seed germination (Horgan, 1984). Chemically, most cytokinins can be classified into three groups, each based on one of three bases, namely zeatin, dihydrozeatin and isopentenyl adenine. The bases exist free and as ribosides (i.e. ribofuranosyls), glucosides (i.e. glucopyranosyls of base o r riboside) o r ribotides (i.e. phosphate derivatives of ribosides). At least 30 such compounds have been identified by gas chromatography/mass spectrometry (GC-MS), although only a few plant species have been comprehensively analysed (McGaw, 1987). Positive identification of zeatin riboside and other cytokinins in xylem sap by GC-MS has been reported for Acer (Horgan et al., 1973), Lycopersicon esculentum (van Staden and Menary, 1976) and Phaseolus vulgaris (Palmer and Wong, 1985), amongst other plant species. The demanding technology needed for such analyses, coupled with the difficulty of obtaining internal standards for GC-MS, and the problem of deciding which one out of so many cytokinins to measure, may explain the decline in the number of papers published on cytokinins and root-shoot relationships in recent years. Radio- or enzyme linked-immunoassays are a convenient and rapid alternative to GC-MS but kits can be obtained commercially only for zeatin-based cytokinins. Furthermore, crossreactivity with related conjugates requires their prior separation by chromatography before quantification is reliable (Incoll et al., 1990). Xylem sap can contain a range of cytokinins (Hall et al., 1987); this increases the complexity of the analyses considerably. In the preceding era of the bioassay, plant science laboratories could obtain the requirements for cytokinin measurements easily and relatively cheaply. This accessible technology underpins a sizeable literature (van Staden and Davey, 1979) inspired by the idea that cytokinins are carried by transpiration in physiologically meaningful amounts from the many

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M. B . JACKSON

thousands of root tips present on a typical root system (Miller, 1916) to recipient shoot tissues. Much of the published work contains measurements of cytokinin activity in unpurified or crudely purified samples of xylem sap, most often obtained from de-topped plants, and in ways criticised unfavourably in the preceding Section 1I.B. Such measurements cannot distinguish between the presence of cytokinin precursors and fully active cytokinins. At best they provide estimates of net cytokinin-like activity remaining after the contrary effects of any inhibitory substances present. These are useful physiological measures, in principle, but they are likely to overestimate net cytokinin concentrations because the diluting effect of the transpiration stream has, for the most part, been ignored. As early as 1943, Went deduced that it was unlikely that auxin from roots sustains the shoot. By the late 1950s it had become accepted that other hormones, most notably the cytokinins and gibberellins, with different physiological properties from auxin, were probably active in plants. The ability of applications of kinetin, a synthetic cytokinin, to retard senescence in detached (i.e. rootless) leaves (Richmond and Lang, 1957; Mothes et al., 1959; Mothes and Engelbrecht, 1963) and to stimulate stem extension, was reminiscent of the effects of roots on shoots. Earlier, Went and Bonner (1943) had shown that coconut milk (liquid endosperm of Cocos nuciferu) could overcome the inhibition of stem extension caused by removing the roots from dark-grown tomato seedlings. Coconut milk is now known to contain zeatin and zeatin riboside (Loeffler and van Overbeek, 1964; van Staden and Drewes, 1975) and to promote cell division in tissue cultures, one of the physiological tests for the presence of cytokinins. These early results suggested that cytokinins might be formed in roots and influence shoots after transport in xylem sap. Accordingly, in 1962, Kulaeva (1962) reported cytokinin activity, measured by the ability to retard leaf senescence, to be present in crude xylem sap from Nicotiana rusrica. This paper was followed by a similar report of senescence-retarding and cell divisionpromoting activity in two fractions of xylem sap of sunflower partially purified by paper chromatography (Kende, 1964,1965). At about the same time, extracts from root rips of sunflower were found to be especially rich in cytokinin activity (Weiss and Vaadia, 1965), and decapitated root systems were shown to be capable of net production of cytokinins over 24 h (Henson and Wareing, 1976). Later, aseptically cultured roots were shown to be capable of making cytokinins independently of shoots (Griffaut, 1977; Chen and Petschow, 1978; van Staden and Smith, 1979; Butcher et al., 1988). Jesko (Jesko and Vizarova, 1980; Jesko, 1981) detected pulses of cytokinin output from successive nodal roots of Zea mays as they emerge, in association with temporary increases in photosynthesis. Thus, roots became to be recognized as rich sources of cytokinins, and it is widely believed they are the main source of these hormones in plants. Of course, roots are not uniquely capable of making cytokinins. Shoot tissues can also produce these


125

hormones (Wang and Wareing, 1979) and even enrich xylem sap with them (van Staden and Dimalla, 1980). Many experiments have used environmental stress applied to roots as a means of decreasing their cytokinin production and assessing its importance in shoot development. An early paper along these lines was that of Itai and Vaadia (1965), who found that drought stress promoted leaf senescence in association with a marked fall in the cytokinin activity of sap bleeding from de-topped sunflowers. However, possible links between cytokinin output from roots and normal events in shoots, such as spring bud break or leaf senescence in monocarpic plants, has also been investigated. This is reviewed next. B. DEVELOPMENT IN UNSTRESSED PLANTS

1. Spring sap in woody plants

Osmotically driven xylem sap flowing vigorously from the cut surface of freshly de-topped trees in late winter or early spring is the so-called spring sap. The flow precedes shoot growth and leaf emergence and thus is undiluted by transpiration and consequently rich in cytokinin activity (Luckwill and Whyte, 1968; Reid and Burrows, 1968). This activity is thought to promote bud burst, stem extension and early leaf expansion. In support of this possibility, Jones (1973) found cytokinin activity in spring sap of apple that co-chromatographed with zeatin riboside. When sap was applied to rootless shoots in vitro, at concentrations estimated at 0.5 p ~it ,substituted for the missing roots by stimulating extension and delaying leaf senescence. Authentic zeatin riboside had similar effects at 0.5 p ~In.more recent work, cytokinin concentrations in sap, obtained from excised shoots by air displacement, have been found to increase in February, before buds start to grow (Tromp and Ovaa, 1990), suggesting a causal role for the cytokinins. These levels (100-300 n ~ were ) sustained until leaf emergence in May, before declining substantially in July, and remaining low until the following spring. It is by no means certain that the cytokinins originated in the roots and Tromp and Ovaa suggest bark as a source, where hydrolysis of storage proteins also commences in February. Dilution rather than reduced production by roots may explain the decreases in concentration seen once the leaves emerged and transpiration commenced. Immunoassays of HPLCpurified samples for isopentenyl-adenine, zeatin, zeatin riboside and ribotide showed zeatin to predominate except in April, when the riboside was dominant (Tromp and Ovaa, 1990). There are other reports confirming the presence of cytokinins in xylem sap of woody species detected by GC-MS (Purse et al., 1976; Waseem et al., 1991) or bioassay (Ahokas, 1984) but measuring cytokinin delivery rates from roots and testing the physiological significance of the cytokinins has been non-existent. Thus, the case for believing that cytokinins in spring sap play a role in controlling early shoot

126

M. B. JACKSON

growth is based almost entirely on the fact that they are present in the sap at high concentrations at the time buds start to grow in the spring. 2. Leaf senescence and related processes Sitton et al. (1967a) reported that cytokinin activity bioassayed in sap exuding from de-topped sunflower plants increased during exponential plant growth, but dropped by 90% when roots stopped growing and leaves began to senesce, thus suggesting a causal relationship. Similarly, Davey and van Staden (1976) reported a fall in zeatin riboside concentration in tomato plants during flower formation and thus prior to leaf senescence. Nooden and co-workers have sought stronger evidence of causality in studies of leaf senescence in soybean (Clycine max L.) (reviewed in NoodCn et al., 1990a). Four cytokinins active in retarding leaf senescence (zeatin, zeatin riboside, dihydrozeatin and dihydrozeatin riboside) were quantified using thin-layer chromatography, column chromatography and radioimmunoassays, before and during the time leaves senesced (NoodCn et a l . , 1990b). Concentrations measured were compared to those needed to retard senescence in an explant assay in which the cytokinins were administered, in the presence of minerals, at concentrations found in the xylem sap (Mauk et al. , 1990). Concentrations of zeatin riboside and other cytokinins in xylem sap from pressurized roots of de-topped plants were shown to fall dramatically at the time pods were fully extended (a reduction of 89%) and to remain at this low value (approximately 10-20n~) until leaves became yellow, before rising again slightly (Table 111). The early fall in cytokinin concentration was thought to contribute to the leaf senescence because of its precipitous nature, because it preceded senescence, and because concentrations of zeatin fed through the transpiration stream in the senescence TABLE I11 Effect of stage of development on zeatin riboside in xylem sap of de-topped soybean plants. A comparison is made of concentrations in sap and calculated concentrations after adjusting to a common flow rate. Delivery rates from roots are also shown Stage of development Pods 1 cm long Pods fully extended Early-mid pod fill Late pod fill Leaves and pods yellow

Concentration Delivery rate Sap flow" Original" (nmol per concentration at 518 p1 per (k.1per 50 min) (kM) 50 min (FM) 50 min) 518 1925 1162 550

63.04 7.2 11.2 16.0

63.04 26.8 25.1 17.0

32.7 13.9 13.0 8.8

400

20.0

15.4

7.9

"Results from NoodCn et al. (1990b). Sap was obtained by pressurizing detached roots for 50 min at 100 kPa.


127

retardation assay (10 nM needed for minimum activity, with strong effects at 4.6 FM) were not dissimilar from those found in sap from de-topped plants ( 1 1 - 6 3 n ~ ,Table 111). The character of this evidence is in line with the requirements outlined in Table I for establishing a physiological role for a hormone. It is supported by other results from plants with pods removed at strategic times to influence both senescence and sap cytokinins. In all, the work of Nooden and colleagues is some of the most precise and quantitative available to support the view that cytokinins from roots can control the timing of natural leaf senescence. Unfortunately, when these results with soybean are re-examined in the light of guidelines for analysing hormones in xylem sap given in Section II.B, they appear less compelling. Problems arise because of dilution effects. If it is assumed that sap from de-topped roots will dilute the hormone in proportion to sap flow, cytokinin concentrations are more fairly compared after re-calculating for the same sap flow rate. In the experiments of NoodCn e t a f . (1990b) sap flows changed markedly between different stages in plant development when the sap was collected for assay. Thus, concentrations can only be compared validly after such re-calculations. This can readily be done using the original data of NoodCn etaf. (1990b). The outcome shows that the fall in concentration of zeatin riboside at the time pods are extended fully and prior to leaf senescence is less abrupt (by 57.5% rather than 88.5%Table 111) than originally thought and falls only gradually thereafter, to about 24% of the original value, by the time the plants are highly senescent. Calculations for zeatin, dihydrozeatin and dihydrozeatin riboside give a similar picture (data not shown). A further difficulty is that even by taking this approach, the diluting effect of whole-plant transpiration is ignored. According to Nooden et al. (1990b), transpiration averaged over 24 h was approximately 13 times faster than sap flow from de-topped plants used by the cytokinin analyses. Thus, in whole plants, the expected concentration of zeatin riboside in the transpiration stream prior to the time pod extension was complete (i.e. before senescence began) would be 13 times smaller, i.e. of the order of 5nM rather than 6 3 n ~ Five . nanomolar is only half the minimum concentration needed to show any retardation of senescence in the explant assay and almost 1000 times less than that needed to slow down senescence to the level seen on intact plants (Mauk et a f . , 1990). Thus, the likely concentrations of cytokinins in the xylem sap of intact plants are probably insufficient to retard leaf senescence based on tests with detached leaves. Such tests would, in any case, have been sounder if based on reproducing the delivery rates of cytokinins. If delivery rates of zeatin riboside per whole shoot are calculated for the plants used by Nooden et al. (1990b), i.e. concentration x sap flow rate, the amounts delivered from the roots are almost unchanged at the time original sap concentrations fell so dramatically (Table HI). Instead, delivery of zeatin riboside and other cytokinins decreased later during development, and gradually, with delivery

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at terminal senescence being about 24% of that from roots of young plants. Overall, these studies still have some way to go to before a convincing case is established for cytokinin regulation of leaf senescence on soybean. In contrast to the above studies, Heindle et al. (1982) have calculated fluxes of major cytokinins during soybean development in the field. In absolute terms the amounts were much smaller than those calculated from Nooden et al. (1990b) given in Table 111. However, they show a general decline with time, especially between full bloom and seed set, i.e. somewhat too early to be obviously implicated in leaf senescence control. Furthermore, the results also show a very small output of cytokinins from the roots of young pre-flowering plants when leaves were young and far from senescent. This observation, and the measurements of cytokinin flux and of responses to applied cytokinins published by Carlson et al. (1987), suggest that high hormone output from the roots of soybean may be more closely connected with successful seed set than with retarding senescence. Removing pods when fully expanded delays leaf senescence in soybean plants and also increases the levels of some cytokinins in xylem sap no matter how they are calculated (NoodCn et al., 1990b). Others have also reported that changes in reproductive development can affect sap cytokinins. For example, Beever and Woolhouse (1973) found delivery rates of cytokininlike activity to increase many-fold following flower induction by short days in Perilla frutescens, even though root growth was inhibited. Later, cytokinin delivery fell to a very low level when the leaves became senescent. In contrast, a very striking 75-90% decrease in the delivery rate of bioassayed cytokinin from roots of Xanthium stromarium was recorded by Henson and Wareing (1976) after just one flowering-inducingshort day. However, a lack of attempts to probe the physiological significance of these various and varied findings makes it difficult to make confident conclusions concerning physiological significance.The target requirements are measurements of (1) hormone delivery rates from roots to shoots, (2) delivery rates per gram of source roots to a unit weight or area of recipient shoot tissue, and (3) tests of physiological activity based on supplying the appropriate cytokinins to leaves at realistic delivery rates per unit leaf area. Recent work with sugar cane (Saccharum spp. hybrid) points the way forward (Meinzer etal., 1991). Delivery rates of zeatin riboside per unit leaf area from roots of unstated size decayed in an exponential manner as the plants grew larger and stomata closed. The closing of stomata is an early indicator of leaf senescence. This approach is clearly a marked improvement on all that has gone before.

C . ROOT EXCISION STUDIES

The assumption underlying such work has been that cytokinin output is proportional to the bulk of the root system. Thus, is should prove possible to


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link decreased cytokinin output with any changes in shoot behaviour caused by pruning or volume restriction. There are several substantial studies linking the formation of new roots on de-rooted leaf or shoot cuttings with long-term survival and increases in exudation of cytokinin-like activity. Using a senescence-retarding assay, Wheeler (1971) found increases in cytokinin activity in the water in which rooting of bean leaves was taking place at about the time new roots were forming. Similarly, Engelbrecht (1972), using the tobacco callus assay, showed increases in cytokinin activity in petioles 4 days after detaching bean leaves from the plant. At this time, new roots had formed but had not emerged from the petiole. Wang et al. (1977) confirmed by GC-MS that root formation in excised bean leaves does coincide with increased cytokinin content of the leaf, notably of zeatin glucoside. Bollmark et al. (1985) also reported only small amounts of zeatinand isopentenyl adenine-like cytokinins in detached leaves until adventitious roots emerged. Because applying dilute benzyladenine (10-8-10-9 M) inhibited the formation of adventitious roots, Bollmark et al. (1985) suggested that endogenous cytokinins from the new roots may explain why further root initiation ceases once the first set of roots has formed. These four pieces of experimental evidence show that the presence of roots is conducive to high levels of extractable cycokinin. However, convincing measurements of delivery rates of cytokinins from roots to shoots in xylem sap following removal and regeneration of roots are notably absent from the literature. There has been much testing of the possibility that applying cytokinins to shoots can replace the effect of missing roots. The earliest example of such an approach is that of Kulaeva (1962). Although her results are mostly anecdotal, the experimental design is a model of conceptual clarity and completeness. Excised leaves of Nicotiana rustica placed in water senesced rapidly, while steam girdling of leaves on whole plants to prevent outward phloem transport while retaining xylem links between the leaf and the roots, did not promote senescence. Thus, the promoting effects of leaf excision were related to severing inputs from the roots (negative message) rather than the result of any accumulation of senescence promoter normally exported in phloem (accumulative message) and from which excised leaves might suffer. Thus, xylem sap appeared to carry a senescence inhibitor. This inhibitor was found, by Kulaeva, to possess the properties of a cytokinin since the senescence-delaying influence of retaining xylem connections between roots and leaf could be reproduced either with kinetin or with applications of crude xylem sap. Support for a senescence-inhibiting and cytokinin-like role for xylem sap was provided by Mothes and Engelbrecht (1963), who found that rapid chlorosis, caused by shading the distal portion of detached tobacco leaves, or by heating them to approximately 49°C for 3 min, was prevented if the leaf was first allowed to root at the petiole, o r was treated with kinetin. The absence of roots also interferes with flower

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development, e.g. in stored cuttings of Vitis vinifera, where the absence of roots has been linked with the failure of flower buds to develop. This failure to form flowers can be prevented either by warming the medium to encourage early adventitious rooting, or by supplying kinetin (Mullins, 1967). A somewhat complicated example of the possible impact of cytokinins from roots was provided by Treharne et al. (1970), who showed that a halving of the root system in Phaseolus vulgaris strongly diminished the stimulation in photosynthesis per unit leaf area that could be induced under a wide range of light intensities by partial defoliation; applying 20 mg 1-' kinetin fully compensated for the missing roots. Similarly, indoleacetic acid (IAA) and gibberellic acid treatments induce aerial stolons of Solanurn andigena on underground shoots, but only if roots are present. However, applying the cytokinin benzyladenine can substitute for the missing roots in this system (Wooley and Wareing, 1972). More straightforwardly, Richards and Rowe (1977) found that the inhibiting effect on leaf expansion and leaf production or lateral shoot production caused by removing 50% of the roots from peach seedlings (Prunus persica) could be more than compensated for by applying benzyladenine. Several other papers have reported that inhibition of shoot growth o r photosynthesis caused by removing a sizeable proportion of the roots can be overcome with exogenous cytokinin treatments (Carmi and Koller, 1978; Carmi and Heuer, 1981). Unfortunately, the persuasiveness of this sort of evidence is seriously eroded by observations that plants not suffering a loss of roots also respond strongly and positively to applications of cytokinins (e.g. Richards and Rowe, 1977; Jackson and Campbell, 1979; Carmi, 1986). It is argued by Carmi (1986) that the exogenous synthetic cytokinin is active because it compensates for a fall in endogenous cytokinin output believed to result from the smaller root system of plants given exogenous cytokinin! Carmi (1986) has also made similar deductions from measurements of photosynthesis, which increased in plants of Phaseolus vulgaris with whole roots systems that were given benzyladenine. However, the explanation that it occurs because the applied cytokinins reduced output of root cytokinins as a result of inhibition of root growth has not been demonstrated by direct measurement. The explanation is particularly difficult to accept in Carmi's experiments since the shoot systems of his plants possess an artificially contrived excess of roots, and thus of putative cytokinin input, which was achieved by removing all shoot tissue except for the primary leaves. Apply benzyladenine to these plants would be unlikely to return the root :shoot ratio to that of ordinary unpruned plants, and yet growth promotion was obtained. A more likely explanation for the positive responses of shoot systems to exogenous cytokinins is simply that responsiveness to cytokinins is an intrinsic property of shoots and has little to do with how much natural cytokinin is supplied by roots. Therefore, in species where plants with an initially full complement of roots can respond positively to exogenous cytokinin, responsiveness following root excision is


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a poor test of the physiological importance of any associated decrease in root cytokinins. That cytokinin output from roots may be of little consequence is suggested by the observation in oak seedlings (Quercus rubra) that removing root tips, the most likely sites of intensive cytokinin biosynthesis, increased rather than decreased cytokinin in xylem sap (Carlson and Larson, 1977), and that similar treatment to Lolium perenne stimulated rather than slowed shoot growth (James and Hutto, 1972). In the experiments of Carmi and van Staden (1983), a large decrease in cytokinin levels in leaves and petioles measured 8 days after removing two-thirds of the roots of decapitated bean plants was not accompanied by any statistically significant change in leaf area, weight or soluble protein. Misgivings are compounded by a report that the decline in endogenous zeatin and zeatin riboside which takes place in de-rooted leaves of Helianthus annuus kept in water for 3 days, may be reversed by supplying nitrate-containing nutrient solution directly to leaf tissue, thus by-passing the need for roots (Salama and Wareing, 1979). In similar work, Wang and Wareing (1979) found not only that lateral buds on decapitated shoots of Solanum andigena could grow for up to 30 days in the absence of roots, but also that during this time cytokinin levels in the shoot did not decline and may even have increased during this period of rootless growth. Overall, the promise of early papers has not been fulfilled and de-rooting experiments have not succeeded in demonstrating convincingly that cytokinins from roots explain the positive effects of roots on shoot development, leaf longevity and photosynthesis. In future experiments, transgenic plants that overexpress genes for cytokinin biosynthesis (see Smart et al., 1991) may help, particularly if reciprocal grafts between roots and shoots are made with the wild-type, or expression is regulated through linking the structural cytokinin genes to a root-specific promoter.

D. RESPONSES TO MINERAL NUTRIENT SHORTAGE

A shortage of mineral nutrients in the rooting medium inhibits shoot growth and promotes senescence. Several authors have tried to attribute these responses to decreased cytokinin production by the stressed roots. Kulaeva (1962) showed that leaf senescence, induced in 55-day-old Nicotiana rustica plants by nitrate shortage at the roots, could be overcome with a foliar application of kinetin, but not with a range of other organic compounds including adenine and IAA, or with minerals applied directly to the leaves. These results imply that senescence of older leaves is promoted by a lack of cytokinin production by mineral-starved roots (negative message), although mediation by an accumulation message cannot be ruled out. Subsequent research has repeatedly shown decreased concentrations, o r delivery rates

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of bioassayed cytokinin activity in xylem sap, from de-topped roots systems when nutrient deficiency, especially of nitrogen, is imposed for several days (Cucurbita pep-Goring and Mardanov, 1976; Helianthus annuusWagner and Michael, 1971; Salama and Wareing, 1979; Solanurn tuberosurn-Sattelmacher and Marshner, 1978). An exception to these findings has recently been shown in root exudates from de-topped Urtica dioica plants grown in sand given various amounts of nitrate ferilization (Fubeder et al., 1988). In sunflower, much of the cytokinin activity cochromatographs with zeatin and zeatin riboside and has been tentatively identified as such by single ion current GC-MS (Salama and Wareing, 1979). Unfortunately, Salama and Wareing (1979) found that depleted concentrations of cytokinins within the foliage of nitrate-starved plants could be rectified simply by supplying nitrate directly to the leaves, rather than through the roots, by-passing the need for root cytokinins as second messengers. Thus, diminished supplies of root cytokinins to the shoots of mineral-starved plants may not contribute significantly to slow shoot growth, enhanced senescence and decreased foliar cytokinin levels (Horgan and Wareing, 1980; Darrall and Wareing, 1981), even where external applications of cytokinins have given some symptom relief (e.g. Horgan and Wareing, 1980). Clearly, detailed time-course studies over hours rather than days are needed in this kind of work. In any future experiments it would be important to establish when nutrient shortage at the roots reduces cytokinin flux in the xylem sap in relation to the time when (1) the physiology and cytokinin levels change in the leaves and (2) when mineral levels decrease in the foliage. Kuiper and Staal(l987) and Kuiper et al. (1989) have gone some way towards producing such information by showing, in Plantago major, that diluting the mineral supply to the roots reduces both shoot growth and extractable zeatin and zeatin riboside levels in roots and shoots, within a day and before mineral concentrations changed in the shoot as a whole. They also showed that shoot growth could be restored almost to normal for several days by applying benzyladenine, which presumably compensated for a low cytokinin output from the mineral-starved roots. When Plantago plants were put back into mineral-sufficient solutions, mineral concentrations returned to normal in the shoots several days before growth rates and endogenous cytokinin concentrations increased (Kuiper et al., 1989), implying that shoot growth was not limited by mineral levelsperse. Analyses of cytokinin and minerals in growing parts rather than in the whole shoot, and tests on the effect of benzyladenine on nutrient levels and on the behaviour of unstressed plants, would have bolstered this interpretation. These results obtained by withholding mineral nutrients have given support to the notion that cytokinin export from roots is depressed by the stress. A shortage of nitrogen seems to be more effective than a lack of phosphorus (Coleman et al. , 1990). Whether this decrease in cytokinins is of much importance in maintaining normal shoot growth and leaf longevity is


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less certain. Much of the available information remains rudimentary and benefits little from the opportunities created by modern analytical methods. Salutary papers by Radin and Eidenbock (1984) and Salim (1991) remind us of the importance of water in the root :shoot relationships of mineralstarved plants. In Gossypium hirsutum, phosphorus deficiency increased the hydraulic conductivity of the leaves to water intake. This in turn decreased daytime leaf water potentials that may have limited leaf expansion (Radin and Eidenbock, 1984). In all work that attempts to ascribe roles to cytokinins in stressed plants, more account should probably be taken of possible hydraulic influences.

E. EFFECTS OF OTHER STRESSES APPLIED TO ROOTS

When roots are subjected to drought, flooding, heat, cold, salinity, parasitism, etc., shoot growth slows, stomata may close and leaf senescence is promoted. Experimental approaches similar to those already discussed for mineral shortage and root excision or constriction have been used extensively to implicate diminished cytokinin supply from roots. Regulation of stomatal closure is of particular concern here because drought stress is often linked to stomatal closure. Exogenous cytokinins have been known to promote opening or inhibit closure of stomata in some but not all plants (Livne and Vaadia, 1965; Meidner, 1967; Luke and Freeman, 1968). Early results also indicated that exogenous application of cytokinin could ameliorate leaf senescence promoted by drought stress (Shah and Loomis, 1965).

1. Drought, salinity and biotic stresses Itai and Vaadia (1965) were perhaps the first to publish correlations between droughting and decreased cytokinin in xylem sap from de-topped plants. However, this first paper contained only very preliminary information, and described soybean bioassays of bleeding sap from sunflower, collected over 72 h, with little or no replication. Later papers substantiated the finding and extended it to salinity stress (Jtai et al., 1968) and heat stress (Itai et al., 1973). The measurements were of concentrations in xylem sap with no sap flow rates given to allow calculation of cytokinin delivery. However, it is probably safe to assume that, because sap flow from de-topped plants would have been slowed by drought, cytokinin delivery rates would have been even more strongly reduced than the 70% loss of concentration reported. Incoll and Jewer (1987) cited ten reports of water shortage or salt stress decreasing cytokinin concentrations in leaves or roots, but Itai and Vaadia (1971) was their most recent citing of analyses of xylem sap from droughted plants. However, the latter paper places considerable doubt on the necessary involvement of root cytokinins in drought-induced decreases in these

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hormones in xylem sap or in leaves. Itai and Vaadia (1971) showed that using a fan for only 30 min to wilt leaves on intact, well-watered plants reduced cytokinin concentrations in leaves and in bleeding xylem sap, in the absence of stress at the roots. Furthermore, droughting detached leaves had a similar effect and cytokinin levels recovered to 73% of their original concentration when the detached leaves were re-hydrated. The need for stress at the roots to mediate these effects was therefore obviated. Experiments by Aharoni et a f . (1977) and Ong (1978) also lead to the conclusion that cytokinin levels in leaves can change in response to water shortage independently of roots. A comparison of endogenous zeatin, zeatin riboside, isopentenyladenine and isopentenyladenosine concentrations in the roots and shoots of salt-resistant and salt-tolerant lines of barley (Kuiper et al., 1990) throw additional doubt on the notion that decreased cytokinin levels explain damage symptoms caused by root stress. In this work large decreases in cytokinins were restricted to the salt-resistant barley plants rather than salt-intolerant ones (Kuiper et al., 1990). One of the consequences of the diminished cytokinin content in leaves of droughted or osmotically stressed plants could be the promotion of stomatal closure. When leaves wilt, cytokinins may decrease (Itai and Vaadia, 1971), and this might enhance stomatal closure along with increases in abscisic acid (see Section VI1.B). However, such a mechanism cannot help explain how stomata close with little or no attendant decrease in leaf hydration (Bates and Hall, 1981). Here, transmission of the influence of drying roots to shoots is more likely to involve a hormonal message. The idea that this may be a negative message in the form of decreased export of cytokinins from roots received support from the split root experiments of Blackman and Davies (1985) and related work (Davies et al., 1986). These authors found that stomatal closure could be induced in the absence of shoot water deficits by drying only one of the two half-root systems. But, when Incoll et al. (1990) imposed drought stress that was gentle enough to induce some closing of stomata within 3-6 days, without any attendant fall in water potentials of leaves of Phaseolus vulgaris, there was no associated decrease in the concentration of zeatin, zeatin riboside or six other cytokinins in the xylem sap. The sap was obtained by pressurizing the roots of de-topped plants for 5 min and assaying by radioimmunoassay after HPLC purification (Ray, 1989). In other work, osmotic stress applied to roots failed to bring about a large decrease in the concentration of zeatin riboside in roots of a droughtintolerant tomato species, while a decrease in this cytokinin was seen in stressed roots of the most tolerant species (Solanurn pennelli) (Pillay and Beyl, 1990). Thus, a further uncertainty is added to the idea that droughting injury is associated causally with decreased cytokinin delivery by roots. There is some evidence that biotic stresses on roots results in decreased cytokinin levels in xylem sap. Drennan and El Hiweris (1979) reported a 90-95% decrease in the concentration of cytokinin activity in bleeding sap,


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using a tobacco pith callus assay, following infection of Sorghum vulgare with the parasitic root-infecting weed Striga hermonthica. Since the decrease would almost certainly have been accompanied by reduced sap flow rates and whole-plant transpiration rates, an extremely large decrease in cytokinin flux is indicated. The sap was partially purified by thin-layer chromatography prior to assay. Xylem sap from cultivars of Sorghum with greater tolerance to Striga contained the most cytokinin. Drennan and El Hiweris (1979) were able to show an increase in growth in dry weight and leaf area of parasitized Sorghum by applying benzyladenine at weekly intervals to the soil to compensate for the small cytokinin supply from the roots. Infection of the roots of Eucalyptus marginata by Phytophthora cinnamomi, a possible cause of Eucalyptus die-back, was shown by Cahill et al. (1986) to reduce concentrations of zeatin and isopentenyl adenine within 3 days in sap obtained from de-topped root systems pressurized to 10 kPa. After 14 days infection, concentrations in a susceptible type were 26% of those of controls, while in a field-resistant strain of Eucalyptus marginata, no decrease in cytokinin levels were seen. Root infection of Douglas fir (Pseudotsuga menziesii) by at least one type of ectomycorrhizal fungus (Thelephora) has been shown to reduce the flux of zeatin riboside in xylem sap (Coleman et al., 1990) from pressurized, de-topped root systems. However, on a root weight basis, no difference in cytokinin output was seen.

2. Soil waterlogging Waterlogging asphyxiates roots, thereby preventing growth, cell division and the aerobic respiration on which most root metabolism depends. This in turn influences shoot development, leading to a wide range of morphogenetic responses, such as epinastic curvature of petioles, hypertrophic stem swelling, adventitious rooting, aerenchyma formation, stomata1 closure, slow shoot extension and premature leaf senescence (Jackson and Drew, 1984; Jackson, 1990). Tomato and sunflower have been studied most by those wishing to understand how oxygen deprivation in roots leads to rapid developmental changes in the shoots. A diminution in cytokinin supply from roots to shoots caused by soil waterlogging was first shown by Carr and Reid (1969), who found smaller concentrations in bleeding sap of Helianthus annuus plants decapitated at intervals during 4 days of flooding. The decrease was rather small over the first 3 days (11-29%), with a much larger decrease on the fourth day of flooding (62%) in association with marked root death. Sap flow from decapitated plants also declined during flooding; the published rates allow cytokinin delivery to be calculated. These show that delivery declined by 22-25% during the first 2 days of waterlogging (by which time shoot growth had stopped). After 3 days, flooding delivery was 37% below normal (when lower leaves were starting to yellow). After 4 days (when lower leaves were clearly yellowing) cytokinin output to the shoots was 94% that of

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well-drained plants. Burrows and Carr (1969) showed that applying kinetin to young leaves taken from plants flooded for 96 h and kept in the dark delayed their senescence. Although this result suggests that leaves were suffering from a shortage of endogenous cytokinins supplied by roots, other results argue against this. For example, removing leaves from non-flooded plants failed to promote their rapid senescence, even though the leaves were obviously separated from a source of root cytokinins (Burrows and Carr, 1969). More recently, Neuman etal. (1990) and Smit etal. (1990), using immunoassays and a pressure of 0.3 MPa to generate sap flows from de-topped plants that are likely to have approached those of whole plant transpiration, confirmed that decreases in cytokinin flux result from oxygen deficiency. Zeatin riboside flux from roots of Phaseolus vulgaris and a hybrid poplar (Populus trichocarpa x P. deltoides) exposed to severe oxygen deprivation for 3,24 or 48 h decreased by at least two-thirds (Smit et al., 1990 and Fig. 6) in association with loss of root tip activity assessed in terms of nuclear

. .t

Sao concentration

Delivery rate

Leaf concentration

8ol , I-

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0.6

-&

0)

0)

0

f

E

0.4

8

$

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~

3c

c

I N 0.2

0

40

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3

48

0

3

24

4

4a

Time (h)

Time (h)

Time (h)

(a)

(b)

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Fig. 6. Effect of oxygen shortage at the roots of Poplar on (a) concentration of zeatin riboside in xylem sap, (b) delivery rate from the roots in xylem sap, and (c) concentration in growing leaves (means and standard errors of 3-6 replicates). Oxygen shortage was imposed by replacing the air supply to nutrient solution with nitrogen gas for up to 48 h. Xylem sap (5 ml) was obtained from de-topped plants by pressurizing roots to 0.3 MPa, equivalent to leaf water potential of plants of approximately -0.3MPa. The experiment shows a marked decrease in cytokinin delivery and xylem sap concentration after 3 h or more of oxygen deprivation but no associated change in leaf cytokinin levels. Redrawn from Smit el al. (1990).


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divisions. However, this decrease in delivery did not change the concentration of zeatin riboside activity in the leaves (see also Smit et a f . , 1990). Furthermore, dihydrozeatin levels in the leaves of Phaseofus vufgaris were doubled rather than depressed by 24 h of root hypoxia, even though sap concentrations were reduced by two-thirds (delivery rates were not stated). This evidence does little to strengthen the case for the involvement of cytokinins from roots in the stomata1 closure and slower leaf growth brought about by poor root aeration. The inability of leaf discs from Phaseolus plants with hypoxic roots to expand or re-open their stomata in response to exogenous zeatin riboside, while those from unstressed plants were able to give strong positive responses (Neuman et af., 1990), confirmed that other more active factors (i.e. positive messages) present in xylem are probably more influential than a decrease in root cytokinin. Other lines of research also suggest that decreases in cytokinin supply from roots are unlikely to be key elements in bringing about morphological changes in the shoot system. Drew et af. (1979) were unable to reproduce the senescence-promoting effects of root anaerobiosis in Hordeurn vulgare by removing root tips, the putative sources of root cytokinins. In these plants, inorganic nitrogen supply appeared to be more important than cytokinins, since senescence was more successfully reversed by nitrogen fertilization via a small number of aerated roots than by applying cytokinins. Cytokinins from roots also look unlikely regulators of epinastic curvatures that develop in waterlogged Lycopersicon esculenturn. Although applications of benzyladenine at rather high concentrations (10-15 mg I-l) can largely prevent the response, especially if gibberellins are also included (Selman and Sandanam, 1972; Railton and Reid, 1973; Jackson and Campbell, 1979), removing roots, and thus the source of root cytokinins, does not reproduce the effect of soil flooding (Jackson and Campbell, 1975b, 1976a). Furthermore, the presence of a second well-aerated root system growing above the flooded roots in damp peat, that is a potentially rich source of endogenous cytokinins, failed to inhibit epinasty (Jackson and Campbell, 1979). The opposite effect would have been expected if the upper root system was making good an endogenous hormone deficiency. In contrast to the results with epinastic curvatures, the slow rate of stem extension by flooded plants was successfully overcome by the presence of well-aerated upper roots. This effect could conceivably have been mediated by an output of root cytokinins since foliar sprays containing benzyladenine have a similar effect (Railton and Reid, 1973; Jackson and Campbell, 1979). However, since nonwaterlogged plants were even more responsive to hormone application than flooded ones, a safer conclusion is that flooding merely reduced the vigour and capacity of the shoots to elongate in response to cytokinin supply. Stomata1 closure in waterlogged plants is of particular interest since it can occur within 24 h, without the mediation of marked or prolonged water deficits in the leaves (Jackson et al., 1978; Bradford, 1982). Bradford (1983)

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and Zhang and Davies (1986) believe that a decrease in cytokinins is involved because applications of benzyladenine can open the stomata of flooded plants of Pisum sativum and tomato (Jackson and Campbell, 1979). However, Neuman et al. (1990) found that zeatin riboside and three other cytokinins over a wide range of concentrations were unable to re-open stomata of leaf discs taken from Phaseolus or Populus plants after 24 h root anoxia. This indicates that stomata were probably not closed by cytokinin shortage. Cytokinin treatments can overcome an inhibitory effect of soil flooding on the net rate of carbon dioxide fixation that is independent of stomata1 apertures. The effect is seen at various internal concentrations of carbon dioxide (Bradford, 1983). Maintenance of ribulose bisphosphate activity could explain the result since it has been known for many years that activity of this key photosynthetic enzyme can be increased with exogenously applied cytokinins (Treharne et al., 1970). This subject merits further work. Overall, there seems to be little doubt that oxygen-deficient roots export much less cytokinin to the shoots than do well-aerated ones. The evidence for believing this has a significant impact on shoot development remains unconvincing. F. CONCLUSIONS

The literature on cytokinins in xylem sap and its possible importance for shoot development is much larger than that for other hormones. It seeks to establish that cytokinin output from roots is a requirement for healthy shoot development and that decreases in output caused by root stresses (negative message) are detrimental to the shoot system. There are serious shortcomings in the way most of the measurements have been made. Bioassays rather than physicochemical methods or immunoassays have usually been used, and scant attention has been given to the effects of changes in sap flow rates on concentration. However, there seems little doubt that cytokinin levels in xylem sap are subject to large changes during normal development and that they alter considerably as a result of changes to the environmental conditions around the roots, and as a result of events in shoot development such as flowering. However, the physiological importance of these changes in cytokinins moving to the shoot in xylem sap has yet to be established, despite much experimentation.

VI. GIBBERELLINS A. INTRODUCTION

Gibberellins (GAS), in common with other hormones, elicit a wide range of responses when administered to plants, although considerable specificity of response is inherent in different species and tissues at various stages of


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development. Evidence of the kind advocated in Table I for hormonal roles is restricted primarily to stem or leaf extension and to germination of some cereal grains. There is also evidence that GAS are important in delaying leaf senescence, especially in concert with cytokinins (NoodCn, 1986). The gibberellins, defined by chemical structure rather than biological activity (cf. cytokinins), are diterpenoid acids and carry either 19 o r 20 carbon atoms associated with an ent-gibberellane four-ring structure (Sponsel, 1987). Approximately 60 different GAS have been found in higher plants, at least 16 of which are present in the seeds of Phaseolus vulgaris. It is fortunate for those wishing to study their physiology that only a small number of GAS possess biological activity p e r se, although these active forms are related to many others which occur earlier or later in the biosynthetic pathway. The pathway commences with mevalonic acid, which gives rise to many intermediates that include geranylgeranylpyrophosphate (GGPP), the C20 precursor for all plant diterpenes. GGPP is cyclized by ent-kaurene synthetase and a subsequent series of hydroxylations and oxidations to generate the first gibberellin (GA12aldehyde) from which all others are derived. These include biologically active G A I , GA3, GA4 and GA,. G A I is likely to be the primary active G A in vegetative tissue of many species. A hormonal role for endogenous gibberellins has been established with greatest certainty in stem extension. The evidence is firmly based on a thorough knowledge of the pathways of gibberellin biosynthesis, gained through the application of analytical chemistry and manipulation of G A levels using either dwarf mutants, with defects at certain steps in the biosynthesis of GAS (MacMillan, 1987), or specific inhibitors of gibberellin biosynthesis, most notably paclobutrazol (Lenton et al., 1987). Evidence implicating gibberellins from roots in shoot development is similar to that already outlined for the cytokinins. It suffers from many of the same deficiencies identified in the cytokinin work and has no new approaches to offer except for the use of gibberellin-deficient mutants in reciprocal grafting experiments between mutant and wild-type stocks and scions. Although the literature is too small to warrant the extensive subdivisions used to assess root cytokinins, it will be reviewed along similar lines. B. STUDIES ON UNSTRESSED PLANTS

The identification of G A activity in roots and in the media used to culture excised roots of Lycopersicon esculentum for over five years, and over many sub-cultured generations, provides some of the earliest evidence that roots are capable of producing GAS independently of the shoot (Butcher, 1963). Twenty-five years later, these GAS were identified by GC-MS as G A I and GA3 (Butcher et al., 1988). Concentrations of GAS in intact plants have been reported to be greater in roots than shoots (Michniewicz and Kriesel,

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1972; Faull et al., 1974), but the opposite has also been found (Kaufman et al., 1976). Some of the root gibberellins may find their way into the transpiration stream or spring sap flow (Dathe et al., 1982) and, thus, up to the shoots (Sitton et al., 1967b). Indeed, there may be considerable cycling of GAS between roots and shoots, with complex interconversions between active and inactive GAS taking place (Hoad and Bowen, 1968; Crozier and Reid, 1971). In the earliest work, G A activity was detected in unpurified bleeding sap and in paper chromatographed sap from de-topped Pisum sativum, Lupinus albus and Impatiens glandulifera plants using four different bioassays (Carr et al., 1964). The authors were careful to test sap at a range of dilutions and found growth-inhibitory activity in some undiluted samples and gibberellin activity in all samples at various sap concentrations. Rough calculations of gibberellin flux from roots to shoots of 10-30 pg per plant per day were judged sufficient to stimulate shoot extension when calibrated against responses of the shoot to GA3. Using the dwarf-pea epicotyl elongation bioassay and the barley endosperm test, Phillips and Jones (1964) also detected GA-like activity in extracts of 500 ml samples of bleeding sap from Helianthus annuus and found that the concentration decreased with time up to 24 h after removing the shoot. The danger inherent in bioassays of unpurified sap is illustrated in the analyses of Skene (1967). These showed that GA-like activity in the xylem sap of Vitis vinifera, measured by the barley endosperm test, was inhibitory to any GAS also present. The inhibitor was thought to be abscisin I1 (now abscisic acid, see Section VIII). This paper also illustrates the commonly held, but erroneous, notion that the concentration of GAS measured in slowly flowing bleeding sap of de-topped plants remains undiminished by the much faster flow of whole-plant transpiration. In assuming this, Skene (1967) over-estimated both the likely flux of hormone into the shoot and, thus, its probable impact on shoot growth. Although the presence of gibberellins in xylem sap has been shown many tmies (grape vine-Skene, 1967; sunflower-Sitton et al., 1967; tomatoSembdner et al., 1968; Douglas fir-Lavender et al., 1973; walnut-Dathe et al., 1982), assumptions concerning its physiological significance must be tempered by the strong likelihood that younger shoot parts, especially growing zones and developing seeds, may be self-sufficient in GAS. Jones and Phillips (1966) demonstrated that while roots (apical 3 4 mm only) were capable of producing GAS, young leaves and the apical buds of sunflower were also able to produce GAS, as assessed by the levels of GA-like activity in agar on which excised plant parts were placed. Indeed, almost all subsequent studies of G A biosynthetic pathways have been made on tissues other than roots (Phinney et al., 1986; MacMillan, 1987). In peas, GA1 is the most active endogenous G A in promoting stem extension. Mutants in which the conversion of GAzo to G A I by a 3p-hydroxylase enzyme is blocked (the le mutant) are dwarf (Ingram et al., 1984) unless given exogenous GA1. The

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3P-hydroxylase gene appears to be expressed mainly in shoot tips rather than in roots since grafting an le mutant shoot on to a non-mutant stock comprising roots and mature shoot tissue does not promote stem extension (Lockard and Grunwald, 1970; McComb and McComb, 1970; Reid et al., 1983). In pea, at least, this seems to rule out the ability of the normal root system to supply sufficient G A I , or its precursors, to influence stem extension. A different result has been obtained with dwarf tomato mutants, generated by ethylmethanesulphonate mutagenesis, which are defective at steps in the G A biosynthetic pathway either before or after ent-kaurene. Grafting onto wild-type stocks comprising roots and some stem reversed the dwarf growth habit of the scion (Zeevaart, 1983). This suggests that the root

1

I

1.5

I

2.0

I

1

I

2.5

I

3.0

Concentration of GA, (log,, pg g-’ fresh wt)

Fig. 7. Relationship between final length of first leaf of seedlings of wheat (Triricum aesfivurn, cv. Maris Huntsman) and different levels of endogenous gibberellin A l in the expansion zone obtained by supplying different amounts of the gibberellin biosynthesis inhibitor paclobutrazol. The linear response to loglo changes in endogenous GA1 shows that leaf extension would be much more responsive to small increases in GAI when existing endogenous GA1 concentrations are small. Endogenous levels of GAI would normally be similar to the highest concentration shown on this diagram, suggesting that shoot extension is normally well-buffered against modest changes in GA concentrations. From Lenton ef al. (1987).

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M. B. JACKSON

system can supply the shoots with physiologically significant amounts of GA. However, if shoots of non-mutant plants are dependent upon GAS from roots, non-mutant shoots should be dwarfed by grafting on to mutant roots; however, such dwarfing does not occur (Zeevaart, 1983). The explanation for this seemingly contradictory result is probably that wild-type shoots are self-sufficient in GAS. The GA-deficient shoots are, however, much more responsive t o very small increases in GA. In turn this is related to the very small amounts of endogenous G A in the mutant shoots. Using different amounts of paclobutrazol to manipulate endogenous G A levels in Triticum aestivum, Lenton et al. (1987) showed that the smaller the internal concentration of endogenous G A , the more sensitive the shoot is to G A supply (Fig. 7). This implies that the growth of shoots containing a normal content of GAS (e.g. 5-6ngg-' fresh wt) are strongly buffered against changes in G A levels of the magnitude that root systems can be expected to bring about. These findings also place in doubt claims that developmental events in shoot apices, such as monocarpic senescence in peas, or the switch from juvenile to adult leaf form in Hedera helix, are causally related to associated decreases in the flux of root GAS (Frydman and Wareing 1973; Proebsting et al., 1978).

C . EFFECTS OF ROOT EXCISION AND ENVIRONMENTAL STRESSES APPLIED TO ROOTS

The balance of evidence presented in Section V1.B favours the view that the supply of GAS from the roots of healthy plants is not very important for shoot growth. If, indeed, this is the case, it must follow that effects on shoot elongation ,and possibly other processes brought about by stresses imposed on roots, cannot be consequences of a negative G A message. This could explain why Horgan and Wareing (1980) were unable to overcome the inhibiting effects of nitrogen or phosphorus starvation on shoot growth by administering GA3 to the shoots of Betula pendula or Acerpseudoplatanus, and why applications of GA1 or GA3 fail to reverse the inhibiting effects of de-rooting on shoot extension in juvenile Hedera helix (Frydman and Wareing, 1973). It is also clear that stress, such as water shortage, can depress endogenous G A levels when applied to detached leaves, thus bypassing the roots (Aharoni et al., 1977). Nevertheless, some papers have sought to implicate reduced output of root GAS in shoot responses to perturbations to the root system. For example, Holm and Key (1969) and Crozier and Reid (1971) reported that inhibition of shoot extension in seedlings of Glycine max or Phaseolus coccineus, caused by root excision, could be partially overcome by applying GA3 (lo-' M), although in the former case the hormone was only active when administered in the presence of a cytokinin. Soil flooding has also been thought to depress shoot growth


143

by interfering with GA production by roots. In support of this, concentrations of G A (GA3 equivalents) in partially purified xylem sap of de-topped tomato plants has been found to be substantially decreased by 1,2 or 3 days of soil waterlogging in association with a less dramatic decline in the concentrations in the roots and shoots and a slowing of stem extension (Reid et af., 1969; Reid and Crozier, 1971). However, although applying GA3 to shoots stimulated stem extension in waterlogged plants, well-drained plants were equally responsive during the first 2 days, suggesting they were not more GA-deficient than their non-flooded counterparts. There are several other disconcerting mismatches in the results of Reid and Crozier (1971) that detract from the notion that depressed G A levels in xylem sap are responsible for the slower shoot growth of waterlogged plants. For example, G A levels in leaves of waterlogged plants returned almost to control levels after 3 days flooding although xylem sap G A remained very low. Also, the responsiveness of stem extension to exogenous CAI was similar in control and flooded plants for 3 days, even though G A levels in sap and foliage and stem extension rates were all much smaller in flooded plants for most of this time. Selman and Sandanam (1972) and Jackson and Campbell (1979) found that stem extension-flooded tomato plants were actually less responsive to hormone treatments containing GA3, suggesting that the constraint on shoot extension may be on G A responsiveness rather than on G A levels. In Reid and Crozier’s work (Reid and Crozier, 1971) there was also a large difference between the amount of GA3 needed to be applied to the plants exogenously (1 pg per plant) to promote stem extension and the amounts of G A in shoots of untreated non-flooded plants (7.5 ng GA3 equivalents per plant). In more recent work with rooted Popufus cuttings (Neuman et al., 1990), 24 h in oxygen-deficient nutrient solutions inhibited cell division in roots and reduce stem extension by about 25% in association with 22-36% less C A I and GA3 in the shoot tip, when analysed by GC-MS. Neuman et af. (1990) considered these changes to be physiologically insignificant. On balance, evidence for implicating a lack of root GAS in the slow rates of shoot elongation in flooded plants is not very persuasive.

D. CONCLUSIONS

There can be little doubt that roots are able to make GASindependently of the shoot. But it is also apparent that shoot tissues, or at least young growing parts and also seeds, are vigorous producers of GAS, and that 3phydroxylase activity required to convert GA2o into physiologically active G A l may be largely restricted to the shoots. Shoots may, therefore, be self-sufficient in active GAS, although the position regarding older, nongrowing parts remains to be clarified. Grafting experiments using GAdeficient mutants also indicate that shoots are independent of root GAS, or

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their precursors, at least for the regulation of shoot elongation. Physiological studies of stressed plants indicate that the output of GAS by roots is suppressed by stress. Experimental support for the belief that this contributes in any significant way to slowing shoot growth under these conditions is poor at present.

VII. ETHYLENE A. INTRODUCTION

Ethylene is the only known gaseous plant hormone. It is produced by all plants, and, in common with the other hormones, has a wide spectrum of effects on development, with specificity of response being determined by the species or by the position, age or physiological state of the target cells. Evidence that satisfies the requirements given in Table I for hormonal action is strong for leaf, flower and fruit abscission, fruit ripening, accelerated underwater extension by many aquatic and semi-aquatic plants, swelling by roots and shoots in response to small mechanical pressures, development of intercellular gas space (aerenchyma) in poorly aerated roots, and epinastic leaf curvatures in flooded plants (Beyer etal., 1984; Jackson, 1985a,c, 1987). In most higher plants, ethylene is derived from methionine, which is regenerated in a four-stage sulphur-conserving cycle driven by ATP and a transamination step. One component of the cycle, S-adenosylmethionine (SAM), is converted to 5’-methylthioadenosine and l-aminocyclopropane1-carboxylic acid (ACC) by a rate-limiting step catalysed by the enzyme ACC synthase. ACC is the immediate precursor of ethylene and is oxidized to the gas by the highly expressed “ethylene-forming enzyme” (EFE), also known as ACC oxidase. The availability of substances that inhibit one or other of these last two steps in ethylene biosynthesis has allowed much testing of the involvement of ethylene in developmental processes. The most widely used inhibitors are aminoethoxyvinylglycine (AVG), which interferes with ACC synthase, and cobalt ions or anaerobiosis, which can block the oxidation of ACC. Research with ethylene also benefits from the availability of inhibitors that interfere with the action of ethylene, most notably silver nitrate and norbornadiene. The simple gas chromatographic equipment needed to measure ethylene with a sensitivity similar to that of the plant itself (95. The gametophyte-sporophyte junction in Anthocerotes. Light micrographs, longitudinal sections. Fig. 93. Megaceros flagellark; ovoidal foot. Fig. 94. Anthoceros formosae: bulbous foot. Fig. 95. Notothylas temperata; smaller bulbous foot than Anthoceros.

THE GAMETOPHYTE-SPOROPHYTE JUNCTION

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group and Jungermanniales than either the Marchantiidae or the Metzgeriales. This would imply a common origin for mosses and liverworts, from an ancestal form probably represented by an erect, radially symmetrical leafy gametophyte with terminal sporophytes. However, it should also be noted that the same distinctive wall substructure of dense radial bands also turns up in the sporophyte placental cells of Riccardia, a metzgerialean genus generally considered to be advanced (Schuster, 1984b), and in gametophyte placental cells of Sphagnum (Ligrone and Renzaglia, 1989). The possibility that this feature is merely associated with solute permeability rather than indicative of phyletic affinity should not be dismissed. In contrast to mosses and liverworts, the anthocerotes all exhibit the same, highly distinctive type of placenta, with minor variants mainly concerning plastid morphology and the presence, appearance and localization of protein deposits. This uniformity in placental organization emphasizes the homogeneity of this group and its separation from mosses and liverworts, as indicated by a wealth of anatomical, ultrastructural and developmental data (Crandall-Stotler, 1980, 1981, 1984; Duckett et a f . , 1982, 1984; Schuster, 1 9 8 4 ~ ;Carothers and Rushing, 1988; Duckett and Renzaglia, 1988a, 1989). The variations in placental morphology in anthocerotes appear to by very useful both for elucidating intergeneric affinities and for clarifying generic limits. The distinctive protein crystals found in Phaeoceros, Notothylas and Folioceros suggest close affinity between these genera. This contradicts the classification of anthocerotes recently proposed by Hasegawa (1988), where the family “Notothyladaceae”, comprising Notothyfas as the only genus, is separated from the family “Anthocerotaceae”, which includes Phaeoceros, Folioceros, Anthoceros and Megaceros. Recent ultrastructural studies of spermatozoid morphology and development have revealed close similarities between Phaeoceros and Notothyfus (Renzaglia and Duckett, 1988, 1989), although the lack of information on other genera presently precludes wider evaluation of the taxonomic relevance of male gamete microanatomy . The distinctive ultrastructural characteristics of the placenta in Dendroceros seemingly support the isolation of this genus in a separate taxonomical entity, e.g. the family Dendrocerotaceae as proposed by Hasegawa (1988).

IV. PTERIDOPHYTES By contrast to the wealth of comparative ultrastructural data on the gametophyte-sporophyte junction in bryophytes, knowledge of the embryology and the early stages in sporophyte differentiation in pteridophytes is limited exclusively to light microscope studies, the majority dating from the nineteenth century or the first half of the twentieth century and reiterated

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with little or no additional information in reviews and standard textbooks (Wardlaw, 1965; Bierhorst, 1971; Gifford and Foster, 1989). In these accounts it is usually tacitly assumed that the foot is the major site of exchange between gametophyte and sporophyte generations, but supporting experimental and or cytological data are lacking. More recent studies on the causal basis of the alternation of generations in pteridophytes (see reviews by Sheffield and Bell, 1987, 1989) have focused on the cytology of oogenesis and to a lesser extent on apospory and apogamy and do not consider the placental region. However, it is interesting to note that transfer cell morphology appears to be absent at the base of apogamous sporophytes in ferns whereas the cells initiating apogamous sporophytes in the moss Physcomitrium develop labyrinthine wall ingrowths even though a recognizable placenta is absent (Menon and Bell, 1981; La1 and Narang, 1985). Specific information on the placenta of pteridophytes is limited to a mention without micrographic details by Gunning and Pate (1974) of transfer cells in this region in Equisetum, two brief accounts of three ferns Polypodium, Adiantum (Gunning and Pate, 1969b) and Pteridium (Khatoon, 1986), and two very recent studies of Lycopodium (Peterson and Whittier, 1991; Duckett and Ligrone, 1992). Although these works clearly do not form a suitable basis for an incisive appraisal of the taxonomic and possible phylogenetic significance of the placenta in the different groups of pteridophytes and do not permit a comparison between these and other embryophytes, the fragmentary information they contain indicates a most fruitful area of enquiry for the future. In Polypodium and Adiantum wall ingrowths in both gametophyte and sporophyte develop very early, before the expansion of the first leaf, elongation of the root and differentiation of the first xylem: precisely the stage at which the sporophyte is most dependent on the gametophyte for nutrients (Gunning and Pate, 1969b). The ingrowths are more abundant and labyrinthine in the sporophyte in Adiantum and Pteridium, while the opposite occurs in Polypodium. The ingrowths are more highly developed on the tangential walls along the interface but also extend onto the lateral walls in the sporophytic cells in Adiantum and Polypodium. The sporophytic cells, but not those of the gametophyte, contain abundant starch, a situation probably reflecting sugar translocation from the gametophyte to the sporophyte. Unlike mosses and liverworts, where a clear-cut demarcation zone is generally interposed between the sporophyte and gametophyte, in ferns the

Figs. 96-9X. The garnetophyte-sporophyte junction in Anthocerotes (cont.). Fig. 96. Dendroceros tubercularis; gametophyte transfer cells and sporophyte haustorial cells. Note the chloroplasts in the former. Fig. 97. Dendroceros tubercularis; branched sporophyte haustorial cell. Note the undifferentiated plastids. Fig. 98. Megacerosjfagellaris;undifferentiated plastids in a sporophyte haustorial cell and crystals (arrowed) in the adjacent garnetophyte cell.

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ct

ul.

Figs. 99 and 100. The gametophyte-sporophyte junction in Anthocerotes (cont.). Fig. 99. Phaeoceros luevis; gametophyte transfer cells, sporophyte haustorial cells and placental lacunae (arrowed). Fig. 100. Dendroceros tuberculuris; plastid in a gametophyte transfer cell.


299

cells of the two generations interdigitate and mucilage-containing intraplacental species appear to be lacking. Further differences between bryophytes on the one hand and ferns and Lycopodium (see below) on the other are an absence of dead and collapsed cells along the interface and the lack of any evidence for the renewal of the placental cells. These differences are perhaps related to the transient nature of sporophytic dependence on the gametophyte in pteridophytes. Intercellular spaces are lacking in the placental region of Lycopodium uppressum (Chapm.) Lloyd & Underw: the cells of the two generations lie close together or are separated by electron-dense intercellular material (Peterson and Whittier, 1991). As in bryophytes and seed plants (see Section V), symplastic isolation, initially established during the early stages in the differentiation of the axial row in the young archegonium (Bell, 1989), characterize all stages of sporophyte development in pteridophytes. In the foot region of Lycopodium uppressum there is little or no interdigitation of the cells of the two generations and the contiguous walls of both sporophyte and gametophyte develop coarse labyrinthine ingrowths of low electron opacity very similar ultrastructurally to those in the marchantialean placenta. Subsequently the interstices become occluded by dense amorphous wall material. The sporophyte-gametophyte junction in Lycopodium cernuum L. is somewhat different. Here the interface between the two generations, which develops ingrowths but to a more limited extent than in L. uppressurn, is not a special lateral development of the early embryo (i.e. a foot region) as in other species of Lycopodium (Goebel, 1905), but rather the lower part of the primary embryonic axis derived from the suspensor (Duckett and Ligrone, 1992). The homologue of the foot in L. cernuum, in terms of its lateral position and early appearance in sporophyte differentiation, is the protocorm. This juvenile structure lies outside the confines of the parent gametophyte and presumably derives its nutrition partly from photosynthesis and partly from an endophytic fungus. Of particular interest in the present context is that within the protocorm there develop schizogenous, mucilage-filled intercellular spaces closely similar to those in the placental region of bryophytes. These lacunae are the principal habitat of the mycobiont. Although the protocorm cells do not develop wall ingrowths, their invasion by the fungus is associated with the production of massive overgrowths of host cell wall material with a texture similar to that forming the wall thickenings in the gametophytic placental cells of Sphagnum and Jungermanniales. The major differences between the sporophyte-gametophyte junction in two species of Lycopodium on the one hand reflect the considerable antiquity of this genus, and on the other perhaps anticipate diversity in placental morphology in pteridophytes parallelling that in bryophytes. It will now be particularly interesting to discover whether wall ingrowths are also present

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along the margins of the well-developed suspensor present in many species of Selaginellu (Bierhorst, 1971) and to investigate the position and sites of nutrient exchange between gametophyte and sporophyte in the haustorialike foot region of Tmesipferis (Holloway, 1918).

V. SEED PLANTS The classic reviews on transfer cells (Gunning and Pate, 1969a, 1974; Pate and Gunning, 1972; Gunning, 1977) underline the cytoplasmic discontinuities between sporophyte and megagametophyte, between embryo and megagametophyte and between sporophyte and developing microspores (also see Charzynska et al., 1990; Murgia ef al., 1991; Pacini, 1990 for discussion of structural and functional interrelationships between tapetum and microspores) as posing special problems for the transport of solutes. Gunning and Pate go on to point out that, in view of the complex nutritional relationships, plus the fact that the new gametophyte and sporophyte are both parasitic on the parent sporophyte, it is not surprising to find transfer cells in these situations. Indeed, transfer cell morphology has now been described at virtually every site of probable solute exchange via the apoplast in the female reproductive organs of angiosperms (Table VII). In a few situations the zones of transfer cells face each other along the intergenerational apoplastic gap, but more commonly only the receptor surface bears the ingrowths. Sometimes the boundary between the two generations is a common wall lacking plasmodesmata, but more often these are separated by a space containing remains of dead or dying cells. The diverse locations and temporal differences in the development of wall ingrowths during the ontogeny of the various tissues provide perhaps the strongest circumstantial evidence of transfer cell activity in solute transport. As in the placentas of most bryophytes and pteridophytes, it seems to be an anatomical necessity that nutrients directed to the growing sporophyte have to pass through cells with wall ingrowths. Rather than reiterate the substance of the earlier reviews, this account focuses on some of the more significant discoveries since 1977, compares the present stage of knowledge of the gametophyte-sporophyte junction in seed plants with that in bryophytes and pteridophytes and points to areas urgently needing further study. As in bryophytes, in seed plants there are also virtually no physiological data to validate inferences about routes and timing of solute transport based almost entirely on the formation of wall labyrinths. It is, however, Figs. 101 and 102. The gametophyte-sporophyte junction in Anthocerotes (cont.). Fig. 101. Notothylus orbicularis; sporophyte haustorial cells and gametophyte transfer cells. Fig. 102. Phoeoceros carolinianus; mitochondria1 aggregates and a pleornorphic plastid in a gametophyte transfer cell. A small pyrenoid is arrowed.

TABLE VII Occurrence and likely functions of transfer cells in the female reproductive organs of angiosperms. Modified and updated from Pate and Gunning (1972) and Gunning (1977) Organ or tissue

Embryo sac

Young embryo

Location

Probable function

Distribution

Recent references"

Egg cell, micropylar walls

Functions of synergids taken over by egg cell when synergids absent

Plumbag0

2,7,24

Antipodals, chalazal walls Synergids, micropylar walls The so-called filiform apparatus

Nutrition of embryo sac

Several genera

1,10,24


Several genera

Central cell, outer walls at micropylar pole chalazal region


Glycine, Scilla, Helianthus

5,6,7,16,17,23,26

Suspensor. outer walls

Nutrition of young embryo Nutrition of young embryo

Several genera

4,5,12,13,18,27

AIisma

3

Basal cell at micropylar pole

Chemotropic secretion, guidance of pollen tube

1,4,7,8,9,13,14, 15,19,20,24

Older embryo Nucellus Endosperm

Cotyledon epidermis, outer walls Epidermis near base of embryo Inner and outer faces Outer walls facing perisperm Aleurone, outer face

Integument

Inner face of inner

Nutrition of embryo

Some Leguminosae

21

Nutrition of embryo

Glycine, Triticum

5.22

Nutrition of embryo

Some Leguminosae and Cruciferae Mesern bryunthemuni

10,11,12

Nutrition of embryo Nutrition of embryo Facilitating viviparous germination and possibly salt exclusion Transfer to endosperm andlor embryo

21

Caryopses of Gramineae Rhizophoru

21

Some Leguminosae,

5

25

Glycine

"References: 1. Bhandari and Sachdeva (1983); 2. Bing-Quan Huang et al. (1990); 3, Bohdanowicz (1987): 4, Dute el al. (1989); 5 , Folsom and Cass (1986); 6. Folsom and Petersen (1984); 7. Kapil and Bhatnagar (1981); 8, Kennell and Horner (1985a); 9. Kennell and Horner (1985b); 10, Mansfield and Briarty (1990a); 11, Mansfield and Briarty (l990b); 12. Mansfield and Briarty (1991); 13. Mansfield er al. (1991); 14. Mogensen (1972); 15, Mogensen and Suthar (1979); 16, Newcomb (l973a); 17, Newcomb (l973b); 18, Newcomb and Fowke (1974); 19, Newcomb and Steeves (1971); 20, Olsen (1991); 21, Pate and Gunning (1972); 22, Smart and O'Brien (1983); 23, Tilton efal. (1984); 24. Willemse and Van Went (1984); 25, Wise and Juncosa (1989); 26, Yan eral. (1991); 27. Yeung and Clutter (1979).

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noteworthy that maize somatic embryos and embryos developing in vitro (Schel and Kieft, 1986; Fransz and Schel, 1991) appear to lack transfer cells. In Phoenix dactylifera L . differences in the acid phosphatases suggest that phosphate metabolism in the endosperm is independent of that in the cotyledon haustorium, and that acid phosphatases for endosperm phosphate metabolism are not secreted by the embryo nor by the cotyledon haustorium, but instead are stored in the endosperm (Sekhar and De Mason, 1989). There is now a pressing need to identify the specific nature of the metabolites undergoing import into young embryos via the various transfer cell zones (Table VII). Although relating to only two stages in development, the wealth of comparative information on the placenta in bryophytes forms the basis for wide-ranging taxonomic inferences. In angiosperms, by contrast, the situation is very different. A few very detailed developmental studies on a small number of genera, particularly Arabidopsis (Mansfield et a f . , 1991; Mansfield and Briarty, 1991), Capsefla (Schultz and Jensen, 1968a,b, 1969, 1971), Gfycine(Kennel1 and Horner, 1985a,b; Folsom and Cass, 1986; Dute et al., 1989) and Helianthus (Newcomb and Steeves, 1971; Newcomb, 1973a,b; Yan et al., 1991), reveal major differences in the extent and distribution of the wall ingrowths even between closely related genera. These relate partly to different patterns of embryo development and partly to differences in the location of seed storage reserves (e.g. cotyledons, perisperm or endosperm). Only one author (Mikeswell, 1990) attempts comparisons from a taxonomic standpoint. Mikeswell’s (1990) extensive survey of angiosperm families indicates that the differentiation of micropylar and chalaza1 haustoria from embryo sacs or endosperm is primarily confined to sympetalous plants with cellular endosperm and anatropous, unitegmic and tenuinucellate ovules. The presence of endosperm haustoria characterizes the subclass Asteridae, which includes the Plantaginales. The Plantaginaceae seems well aligned with families within the Scrophulariales. Mikeswell’s final conclusion, that the utilization of haustoria as an important embryological character in taxonomy would seem to be warranted, suggests the same could well be true for other transfer cell locations when data are available for more taxa. Ultrastructurally the vast majority of the wall ingrowths associated with the female reproductive tissues in angiosperms are coarse with a transparent matrix like those in the Marchantiidae. However, in rare instances, e.g. the suspensor of Gfycine(Dute et a f . ,1989) they are finer and electron-dense. A highly elaborate wall labyrinth occurs in the large basal cell of the suspensor Figs. 10>10h. The gametophyte-sporophyte junction in Anthocerotes (cont.). Crystals. Fig. 103. Folioceros fuciformis; intercellular. Fig. 104. Phaeoceros luevis; intercellular crystals after digestion with pepsin. Fig. 105. Nofothylasorbicularis; intercellular. Fig. 106. Folioceros fuciforrnis; intracellular.

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el

al.

of Afisma (Bohdanowicz, 1987).Thepresence of wall ingrowths is associated with cytoplasmic organization typical of transfer cells, namely abundant ribosomes, rough E R and numerous mitochondria. Very large megamitochondria have been described in young embryos of Capseffa(Schultz and Jensen, 1973) and Arabidopsis (Mansfield and Briarty, 1991). It is suggested that these may act as a reservoir for mitochondria1 DNA in relation to the rapid proliferation of mitochondria during embryo development. Although the cells along the gametophyte-sporophyte junction in angiosperms typically contain undifferentiated pleomorphic leucoplasts, with few reserve materials and rudimentary thylakoid systems, some highly unusual forms have also been described. Large plastoglobuli and prolamellar bodies characterize the endosperm plastids in Rhizophora (Wise and Juncosa, 1989). Prolamellar bodies also occur in the so-called “placental haustorium” plastids of Tropaeolum (Nagl and Kuhner, 1976). The suspensor by contrast contains plastids with an extremely dense stroma and scattered membranous vesicles. Much larger plastids with similar contents occur in the suspensor of Stelfaria (Newcomb and Fowke, 1974). Plastid tubules have been noted in the suspensors of Phaseolus and Pisum (Marinos 1970; Schnepf and Nagl, 1970). The current state of knowledge of the gametophyte-sporophyte interface in the gymnosperms is very similar to that for pteridophytes. In contrast to angiosperms, attempts to induce normal development of conifer zygotes and precotyledonary embryos in vitro have been unsuccessful (Gates and Greenwood, 1991, and literature cited therein) suggesting that a unique nutritional environment, probably involving continual variations in the physiological and chemical conditions, is required for embryo development. Although the considerable complexities of embryo development in gymnosperms are well documented at the light microscope level (Wardlaw, 1965), these have been totally ignored by electron microscopists. As far as we are aware the only report of wall ingrowths is in the basal plate wall between the oosphere cytoplasm and proembryo in Pinus (Gunning, 1977). For the future it would be interesting to discover whether or not ultrastructural differences between proembryos and embryos characterize the Gnetales, Cycads, Ginkgo and different families in the Coniferales in the same way that placental differences separate different groups of bryophytes.

ACKNOWLEDGEMENTS This review was made possible by a NATO Collaborative Grant to J . G . Duckett and K. S. Renzaglia and by a Guest Research Fellowship from the Royal Society of London enabling R. Ligrone to work at Queen Mary and Westfield College during 1989 and 1990. This financial support is most gratefully acknowledged. Collection of the specimens of Pogonatum neessii,


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Herberta spp. Zoopsis liukiuensis, Pallavicinia indica, Calobryum blumei, Dumortiera hirsuta, Folioceros fuciformis, Dendroceros javanicus and D. tubercularis used in this study was made possible by a travel grant to J. G. Duckett from the Royal Society of London and by laboratory facilities arranged by Drs M. A . H. Mohamed and A. Nasrulhaq-Boyce in the Botany Department of the University of Malaya, Kuala Lumpur. Cladophascum gymnomitrioides was collected in Lesotho by J. G. Duckett under a British Council LINK between Queen Mary and Westfield College and the National University of Lesotho. The authors also thank D. K. Smith for providing live specimens of Takakia and R. C. Brown and B.E. Lemmon for allowing the use of their embedded material of Carrpos and Monoclea.

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AUTHOR INDEX

A Abeles, F.B., 72, 77 Abou-Mandour, A., 156,173 Adfim, A., 52, 77 Adams, D.O., 147,168 Adams, M.J., 89 Addicott, F.T., 150,168 Adoutte, A., 227 Aharoni, N., 134, 142,168 Ahokas, H., 125,168 Albersheim, P., 2, 3, 8,9,21, 31, 40, 52,69, 74, 75, 77, 78, 79, 80, 82, 83,84, 85, 86, 87,88, 89, 91, 92, 94, 95, 96, 98, 99,100,101 Albert, F., 97 Aldington, S., 2, 5, 22,41, 68, 74, 78 Al-Doori, A.H., 183 Alejar, A.A., 186 Alexopoulos, C.J., 257, 263,307 Altman, D.W., 78 Anderson, A.J., 24,40, 69, 77, 97, 99 Anderson, E., 192,220 Anderson, J.D., 30, 78,85,88 Anderson, L.E., 287,316 Anderson, R.A., 220 Antia, N.J., 208, 220 Apostol, I., 50, 51, 78 Appell, G.S., 208,223 Appels, R., 201,220 Appleford, N.E., 169 Ariztia, E.V., 203, 220 Arteca, R.N., 147, 185 Asamizu, T., 23, 78 Ashton, N.W., 232,309 Asmundson, C.M., 183 Aspinall, G.O., 59, 60, 78 Asselin, A,, 80 Aston, M.J., 155, 168 Atkinson, C.J., 151, 157, 168 Atkinson, M.M., 26, 49, 78, 79 Audren, H., 225

Aurich, O., 183 Awad, M., 72, 79 Ayers, A.R., 2,8, 11,20,73, 78,86

B Babbitt, J.K., 72, 79 Bachellerie, J.P., 227 Backe, M.A., 4, 79 Badenoch-Jones, J., 173 Badwey, J.A., 49, 79 Baenziger, J.U., 6,89 Baer, H.H., 93 Baev, N., 37, 79 Baier, M., 106, 150, 168, 173 Bailey, J.A., 8, 61, 77, 79, 90, 99, 101 Bailey, R.W., 6, 79 Baker, C.J., 26, 79 Baker, K.K., 90 Baldwin, E.A., 31, 79 Bakes, S., 218,229 Banfalvi, Z., 79 Barber, M.S., 28,34,35,43,73,78,80,97 Barbier-Brygoo, H., 44,80 Barlow, B.A., 173 Barns, S., 223 Baroin, A., 227 Barr, M.L., 156,168 Barrett, A.J., 4, 59, 80 Barrett, J., 190, 225 Barry, D.A., 184 Barthe, J.P., 80 Bartholomew-Began, S.E., 269,307 Basham, H.G., 24,25, 69,80 Bashan, Y., 69,80 Bassi, P.K., 145, I68 Bateman, D.F., 24, 25, 69,80 Bates, L.M., 134, 155,168 Bauer, W.D., 12, 14, 63, 80 Bauw, G., 94 Baydoun, E.A.-H., 6,44,60, 66, 75, 76,80

319

320

AUTHOR INDEX

Baynes, J.W., 86 Beakes, G.W., 203,220 Beanland, T.J., 225 Beardmore, M., 34,80 Beckman, C.H., 285,313 Beckman, J.M., 35,89 Beever, J.E., 107, 128,168 Beissman, B., 92 Bell, A.A., 8,80 Bell, J.N., 82,84, 90, 98, 99 Bell, P.R., 232,297,299,307,313,316 Bellincampi, D., 18, 80, 84, 87 BeMiller, J.N., 4, 101 Bendayan, M., 196,220 Benhamou, N., 43,61,80 Bennett, A.B., 72,88 Benson, R.J., I68 Ben-Zioni, A,, I75 Bergami, M., 83 Berger, N., 79 Bergstrom, G.C., 90 Bernasconi, P., 12,81 Bernier, G., 121,169 Bertram, R.E., 80 Bethenod, O., 184 Bevan, M.W., 183 Bewley, J.D., 251,314 Beyer, E.M. Jr., 144,169 Beyl, C., 134,181 Bezukladnikov, P.W., 5, 63,81 Bhandari, N.N., 303,307 Bhatnagar, A.K., 303,312 Bhattacharya, D., 203,220 Biddington, N.L., 104,169 Bierhorst, D.W., 297,301,307 Biggs, K.J., 88 Biggs, R.H., 31, 79 Biles, C.L., 72, 77 Bing-Quan Huang, 303,307 Birberg, W., 83 Bird, P.M., 8,34,81 Bishop, P., 98 Bishop, P.D., 4, 19, 20, 67, 72, 75, 81 Bisseling, T., 89 Black, W.C., 94 Blackman, P.G., 134, 155,169 Blaikie, S.J., 104, 169 Blake, D.A., 6, 81 Blake, T.J., 114, 115, I84 Blank, J., 229 Blaschek, W., 92 Bledsoe, C.S., 171 Blom, C.W.P.M., 185

Blum, A., 159,169 Blum, U., 4,81 Blumenfeld, A . , I68 Boczar, B.A., 212,220 Boffey, S.A., 67,81 Bogemann, G.M., 185 Bogorad, L., 200,220,221 Bohdanowicz, J., 303, 306,307 Bold, H.C., 233,257,263,307 Boller, T., 30, 31, 73,81, 83, 90, 94, 96, 98,100 Bollmann, J., 99 Bollmark, M., 129, 169 Bolwell, G.P., 12,81,82,86 Bonig, I., 226 Bonnemain, J.L., 232,285,308,314 Bonnen, A.M., 90 Bonner, B.A., 100 Bonner, D.M., 124,186 Booij, H., 84 Boon, J.J., 89 Bostock, R.M., 20,40, 52,82 Bottger, M., 156,169 Bowen, M.R., 140,174,177 Bower, F.O., 233,307 Bowles, D.J., 18, 19,82,86,100,101 Bowman, Y.J.L., 87 Boyer, J.S., 169 Bradford, K.J., 107, 110, 137, 138, 146, 147, 149, 160, 161,169 BrameLCox, P., 94 Branca, C., 80,87 Branca, C.A., 4, l8,82 Brecht, J.K., 72, 82 Breck, E., 172 Bremer, K., 233,307 Brenner, M.L., 173,183 Brenzel, A., 228 Brett, C.T., 60, 82 Briarty, L.G., 285, 303, 305, 306,311, 313 Brinker, A.M., 178 Brinkerhoff, L.A., 90 Broadwater, S.T., 227 Brodelius, P., 88 Brodelius, P.E., 96 Broekaert, W.F., 3,82 Brown, R.C., 237,251,271,283, 287, 291,293,307,308 Browning, A.J., 232,234,237, 251, 271,273,308 Bruce, R.J.,21, 28, 68,82 Brun, W.A., 173,183

AUTHOR INDEX

32 1

Chailakhian, M.Kh., 121,170 Chamberland, H., 80 Chang, D.D., 200,221 Chapin, F.S. 11, 164,170 Chappell, J., 8, 30, 31,83 Charzynska, M., 285, 301,308,313 Chauhan, E., 237,251, 276,309,312 Chauhan, L., 237,308 Chelf, P., 100 Chen, C.-M., 124, 170 Cheng, J.Y., 220 Cheong, J.-J., 5 , 9, 11,42,83 Chibnall, A.C., 117, 171 Chihara, M., 229 Chuan-Jin, H., 179 C Cahill, D.M., 109, 135,170 Churchill, S.P., 233, 289,307,313 Cain, J.R., 208,226 Clapp, G.L., 253,309 Clarke, A.E., 43, 90, 226 Callow, J.A., 34, 43, 49,82,86, 94 Clarkson, D.T., 170,182 Camardella, L., 82 Clayton, D.A., 200,221 Cammack, R., 225 Campbell, A.D., 30,82 Cline, K., 9, 74,83 Close, T.J., 179 Campbell, A.K., 92 Campbell, B.C., 67,82 Clutter, M.E., 303,317 Cohen, D.B., 156,171 Campbell, D.H., 253,275,308 Campbell, D.J., 130, 137, 143, 144, Coleman, M.D., 110, 132, 135,171 Collmer, A., 23, 68, 78,83 146,175, 176 Conley, P.B., 223 Campbell, E.O., 253,255,279,291, Constabel, F., 12, 83 308,316 Cannell, R.Q., 106, 170 Coombe, B.G., I72 Canny, M.J., 76, 82, 106,170 Coombs, J., 202,221 Cantrell, M.A., 93 Cooper, R.M., 68,69,83 Caplan, A.B., 94 Corcoran, M.R., 4,84 Carbonneau, R., 185 Cordewener, J., 38,84 Corley, M.F.V., 235, 243,309 Carlson, D.R., 107, 128,170, I73 Carlson, W.C., 131,170 Cornforth, J.W., 168 Cornish, E.C., 226 Carmi, A., 115, 119, 130, 131,170, Cornish, K., 155, 156,171 177,185 Corredor, V., 201,222 Carns, H.R., 168 Cosio, E.G., 42,84 Carothers, Z.B., 271,287,291, 293, C6te, P., 185 295,307,308,309,314 Cotterman, D.C., 170 Carr, D.J., 135, 136, 140,169,170 Cottier, J.P., 182 Cass, D.D., 303, 305,310 Castaldo, R., 237, 249, 251, 276,310, Cottrell, I.W., 59, 78 Courtice, G.R.M., 232,309 312 Cattolico, R.A., 210, 211,220,221, Cousson, A., 100 Coutts, M.P., 160, 171 225,227,228 Caussin, C., 285,308 Cove, D.J., 232,309 Cavalier-Smith, T., 190,200,202,203, Cox, E.R., 215,229 Craig, J.W.T., 78 216,221 Craigie, J.S., 208,221 Cavers, F., 257,308 Cervone, F., 25, 68, 70,71,80,82,83, Cramer, C.L., 54,82,84,86, 98,99 Crandall-Stotler, B., 235, 253, 257, 84,87 263,275,291,293,295,309,316 Chai, H.B., 52,83 Bryan, I.B., 91 Bryant, J.E., 81 Bucheli, P., 25,26, 33,82,85 Bulpin, P.V., 87 Bunce, J.A., 177 Burdett, A.N., 60, 82 Burger-Wiersma, T., 229 Burrows, W.J., 125, 136,169,181 Burton, P., 200,225 Butcher, D.N., 124, 139,169 Buttrose, M.S., 104, 115,169 Byrde, R.J.W., 101 Byrne, H., 100

322

AUTHOR INDEX

Crane, P.R., 233,309 Creelman, R.A., 161, 171 Cresti, M., 285, 301,308,313 Cribbs, D.H., 96 Crocker, W., 187 Croker, S.J., 150,186 Crosby, M.R., 243, 245,247,287,309 Crozier, A., 140, 142, 143,171,180, 181 Crundwell, A.C., 235,243,309 Cueller, R.E., 225 Curtis, W.R., 40, 86 D da Costa, A.R., 171 Dale, J.E., 108, 112, 115, 119, 150, 164, 165,171, 179,183 Dalmon, J., 225 Dalton, B.R., 4,81 Daniell, H., 226 Danks, M.L., 4,84 Darrall, N.M., 118, 132,171 Darvill, A . , 82, 86 Darvill, A.G., 3, 8, 9, 17, 21, 40, 75, 78,83, 84,85,88,89, 91, 92, 95, 96, 98, 99,100,101 Das Gupta, D.K., 171 Dathe, W., 140,171 Davey, J.E., 123, 126,171,185 Davey, M.R., 285,309 Davidson, R.L., 104, 113, 116,171 Davies, L.A., 85 Davies, P.J., 181 Davies, W.J., 105, 109, 110, 134, 138, 151, 152, 154, 155, 156, 157, 158,159,160, 162,168,169, 171,172,180,183, 184,186,187 Davis, K.R., 4, 22, 39,40, 68,69, 78, 84 Davis, L.J., 229 Davis, R.W., 30, 31, 77,86 Davison, P.G., 235, 241,309,313,315 Davison, R.M., 156,171 Dea, I.C.M., 87 Deakin, A.L., 34,85 Dean, J.F.D., 32, 33,85 Dearman, A.S., 104,169 Degra, L., 82,83 De Greef, J.A., 179 de la Cruz, V.F., 224 Delaney, T.P., 211,220,221 Delevoryas, T., 257,263,307 Dell, A., 84, 95, 96,101

De Lorenzo, G., 70,80,82,83,84,87 Delrot, S., 232, 285,309,314 Delseny, M., 97 de Lucia Sposito, M.L., 237,249,251, 259,271,310,312 Delwiche, C.F., 233,291,309,311 De Maggio, A.E., 232,309 DeMason, D.A., 305,315 DenariC, J., 93 De Proft, M.P., 179 Derocher, J., 227 De Ropp, R.S., 117,171 Desiderio, A., 80,87 Desjardins, A.E., 78 Despeghel, J.P., 285,308,309 Despeghel-Caussin, C., 232,285,314 Deuel, H., 17, 18,85 Deverall, B.J., 34,85 De Vries, S . , 84 de Vries, S.C., 27, 38,61,84 de Wit, P.J.G.M., 20, 24, 39, 60, 85, 98 Dey, P.M., 6,85 Diaz, C.L., 43,85 Dickerson, A.G., 32, 91 Dietrich, A., 53,85 Di Gregorio, S . , 94 Dildine, S.L., 98 Dilley, D.R., 146, 169 Dimalla, G.G., 125,185 Dixon, R.A., 2,12,21,23,39, 77,81, 82,85,86,93 Doares, S.H., 25, 78,82,85, 91 Dodge, J.D., 190, 201, 202,203, 209, 215,219,221 Doherty, H.M., 48, 86,100, 101 Doke, N., 12, 51, 52,83,86,88 Domard, A., 91 Doolittle, W.F., 190,223 Doubrava, N., 86, 95 Douglas, S., 208,209, 210, 216,228 Douglas, S.E., 200,201,202,207,210, 211, 212, 215,221,222 D’Ovidio, R., 83,84 Dow, J.M., 49,86 Downton, W.J.S., 178 Doyle, W.T., 281,313 Drennan, D.S.H., 134, 135,171 Drew, M.C., 135, 137, 145, 161,172, 175 Drewes, S.E., 124,185 Driguez, H., 93 Dron, M., 93

AUTHOR INDEX

Druel, L.D., 203,220 Duckett, J.G., 232, 237, 251,271,287, 291, 293, 295,297,299,307, 308,309,310,314,315 Duckham, S.C., 162,172 Dull, R., 235,243,309 Dumbroff, E.B., 171 Dunlop, D.S., 40,86 Dupler, A . W . , 255,310 Durand, E.J., 310 During, H . , 151,177 Durley, R.C., 170, 176 Durnford, D.G., 211,221,222 Dute, R.R., 303, 305,310 Duval, J.C., 225 Dwarte, D., 209,222 Dyer, D.J., 170 Dyer, T . A . , 183 E Eagles, G., 101 Ebel, J . , 8,42, 78,84,86,89, 98 Eberhard, S . , 27,86, 95 Eble, A.S., 4,86 Ecker, J . R . , 30, 31, 77,86 Edelbaum, O., 74,86 Edelmann, H.G., 14,27,62,86 Edwards, K . , 54,86 Edwards, M . , 66,87,223 Edwards, M . R . , 222 Edwards, S.R., 243,287,310 Egelhoff, T . , 217,222 Ehleringer, J.R., 182 Ehrenberg, C.G., 192,222 Eidenbock, M.P., 133,181 Eilert, U., 12, 83 Eisenberg, B.L., 223 Eklund, L., 146,172 El-Beltagy, A S . , 145, I72 Elbrachter, M., 214,228 El Hiweris, S.O., 134, 135,171 Eliasson, L., 169 Else, M . , 109,172 Else, M . A . , 173 Elstner, E.F., 96 Elwood, H . , 224 Elyakova, L.A., 5, 63,81 Emrnerling, M., 14, 15,87 Endre, G., 79 Enea, V., 201,222 Engelbrecht, L., 117, 124, 129,172, 179 English, P.D., 63, 69, 70,87

323

Ephritikhine, G., 80 Epperlein, M.M., 50, 87 Erhlich, H . A . , 228 Ericsson, A . , 178 Erni, S., 305,313 Eschbach, S., 201,204,206,207,222, 224 Esquerre-Tugaye, M.-T., 8, 29,46,80, 87, 96, 97 Etherton, B., 285,312 Ettl, H., 214, 222 Evans, L.V., 217,222 Ewings, D., 100 Eyme, J., 237,310 F Falk, H . , 224 Fantauzzo, F., 186 Fanutti, C., 66, 87 Farkas, T., 77 FarkaS, V., 56, 66, 87 Farmer, E.E., 2, 32, 53,87, 98 Farmer, M . A . , 214,222 Farr, M . E . , 232,316 Farrar, J.F., 118, 172 Farrar, S.C., 118, 172 Faucher, C., 93 Faucher, M., 285,308 F a d , K.F., 140,172 Fautz, E., 89 Feger, M., 89 Ferguson, A . R . , 110, 172 Ferraris, R., I78 Fielding, A . H . , 90, 101 Fields, S.D., 214,222 Filippini, F., 18, 27, 44, 87 Finelli, F., 79 Fischer, R.L., 72,88 Fletcher, J.S., 84 Fleurat-Lessard, P., 232,285,308 Flott, B.E., 95 Flower, D.J., 178 Fluhr, R . , 33, 94 Folsorn, M.W., 303,305,310 Fork, D.C., 209,224 Forsyth, C., 185 Foster, A . S . , 232, 297, 310 Fournier, J., 97 Fowke, L.C., 303,306,314 Foyle, R.A.J., 220 Fransz, P.F., 305,310 Freeling, M . , 181 Freeman, T.E., 133, 178

324

AUTHOR INDEX

Gingrich, J.H., 155,179 Giordano, S., 237,249,251,312 Giovannoni, S.J., 190,223,229 Glazener, J.A., 28,88 Glazer, A.N.?208,209,223 44,45,49,56,57,58,59,60,61, 62, 63, 64,65, '66,68, 74, 75, 76, Godovac-Zimmerman, J., 225 78,80,86,88, 94, 95, 99, I01 Goebel, K., 253,299,310 Golden, S.S., 190,212,227 Frydman, V.M., 142,172 Fubeder, A . , 132,172 Goldstein, I.J., 6, 81 Fuchs, Y . , 32,88 Gollan, T., 109, 151, 155, 156, 172,182 Fiigedi, P., 83 Gollin, D.J., 6,88, 95, 100 Goodwin, J.C., 4, 89 Fukuda, M., 92 Goring, H., 132, 172 Funk, C., 12,88 Furuichi, N . , 24,88 Goss, M.J., 166,172 Fushtey, S.G., 24,88 Govers, F., 37,89 Gowing, D.J.G., 159, 172 Grab, D., 52,89 G Gadelle, A., 97 Graham, L., 291,309 Gage, D.A., 171,186 Graham, L.E., 232,233,291,310,311 Gale, M.D., 177 Grand, C., 98 Gales, K., 176 Granger, J.W., 80 Galston, A.W., 117, I72 Grant, B.R., 170 Gambardella, R., 233,234,235,237, Grantz, D.A., I79 243,245,249,251,259,271, Gray, M.W., 190,222,223,228 273,276,279,281,283,310,312 Green, E.D., 6,89 Gamble, H.R., 85,88 Green, T.R., 19, 75,89 Greenwood, A.D., 192,193,195,202, Gantt, E., 192,209,222 221,223,227,228 Garcia-Garrido, J.M., 88 Greenwood, M.S., 306,310 Garcia-Romera, I . , 69,88 Griffaut, B., 124,173 Gardner, J.M., 24,88 Gardner, P.A., 159, 165, 166,180 Griffiths, H.B., 223 Griggs, P., 110, 173 Garegg, P., 98 Grignon, C., 96 Garegg, P.J., 83, 96 Grisebach, H., 8,42, 53,86,89, 94 Gaskin, P., 174 Grolle, R., 235,311 Gates, J.C., 306,310 Gross, K.C., 85 Gauhe, A . , 93 Grossman, A . R . , 209,217,218,222, Gautier, C., 93 223 Gehri, A., 81 Gruber, T.A.,95 Geissler, P . , 253,309 Geissman, T.A., 84 Grunwald, C., 141,177 Gelfland, D.H., 228 Grusak, M.A., 232,316 Guard-Friar, D., 208, 217,223 Geraeds, C.C.J.M., 98 Guerke, W.R., 257,263,309 Gerrish, C., 85 Guern, J . , 80 Gholson, R.K., 90 Giigler, K., 88 Ghosheh, N.S., 176 Guiamet, J.J., 180 Gibbs, S.P., 190, 192, 195, 201, 202, Guillemaut, P., 226 208, 209, 215,218,222,223, Guinn, G., 181 226,227 Gunderson, J.H., 201,203,224 Gidley, M.J., 87 Gunning, B.E.S.,232,233,234,237, Gifford, E.M., 232,297,310 251,271, 273, 283, 285, 297, Gilboa-Garber, N., 80 301,302,303, 306,308,311,314 Gilkes, N . R . , 62,88 Gutteridge, J.M.C., 49, 90 Gillott, M.A., 192,201, 208,223 Frey, T., 84 Friend, J . , 91 Fritsch, F.E., 233,310 Fry, S.C.,4, 5 , 6 , 7 , 14, 15,25,27,29,

AUTHOR INDEX

H Hadwiger, L., 100, 101 Hadwiger, L.A., 35, 36,44, 55, 89, 92, 94 Hafez, A.M.A., 97 Hagendoorn, M.J.M., 12,20,89 Hahlbrock, K . , 8, 22, 39, 40, 83, 84, 85,89, 99 Hahn, M.G., 3, 8, 11,21,31,42,53, 78,83,89 Hahn, R., 83 Hahne, G., 26,89 Hall, A.E., 134, 155,168 Hall, D.O.,225 Hall, K.C., 119, 148, 150, 161, 164, 173,175, 176, 179 Hall, M.A., 62,88, 145, 160,172,183, 186 Hall, N.A., 7, 89 Hall, P.J., 123, 173 Hall, S.M., 122, 173 Halliwell, B., 49, 90 Halverson, L.J., 39, 90 Hameed, M.A., 115,173 Hammerschmidt, R., 6, 8, 20,29, 90 Hanke, D.E., 67,90 Hanna, R., 87 Hansmann, P., 195, 196,203,204,206, 222,224 Hardy, M.R., 7, 90 Hargreaves, J.A., 61, 90 Harmsen, H., 89 Harper, J.L., 113,173 Harren, F.J.M., 185 Harrington, A., 190,224 Harrison, M.A., 185 Hartman, T., 229 Hartung, W., 106, 110, 150, 151, 152, 155, 156, 168,173,185,186 Harvey, B.M.R., I81 Hasegawa, J . , 235, 275, 279,291, 295, 311,316 Hatakeyama, N., 215,224 Haug, A . , 99 Haupt, A.W., 253,255,311 Haxo, F.T., 209,224 Hayashi, T., 14,43,63,90,92 Heath, T.G., 186 Hebant, C., 235,237,245,269,311 Hedden, P . , 169,177 Hedrick, S.A., 54, 90, 98 Heidstra, R., 89 Heilmeier, H., 173, 185

325

Heindle, J.C., 107, 109, 128, 173 Heinonen, T.Y.K.,228 Heinrichova, K., 176 Heinstein, P.F., 78, 90 Heitefuss, R., 69, 96 Helgeson, J.P., 32,87 Hendrix, D.L., 173 Henis, Y., 80 Henry, Y., 285,311 Henson, I.E., 124, 128, 164,173,177 Hermann, R.K., 177 Herold, A . , 118,173 Heuer, B., 130,170 Hevesi, M., 77 Hewett, E.W., 174 Hibberd, D.J., 213, 215, 217,224 Higgins, V.J., 49, 52, 96 Higuchi, R., 228 Hill, D.R.A., 192, 208, 219, 220,224 Hill, M.O., 235,243,309 Hiller, R.G., 225 Hillman, J.R., 174,180 Hinch, J.M., 43,90 Hinde, R., 217,224 Hinton, D.R., 72, 90 Hiron, R.W.P., 150, 155, 160,173,186 Hislop, E.C., 25,90 Hitchcock, A.E., 187 Hoad, G.V., 106,140, 157,164,174 Hocking, T.J., 164,174 Hodge, S.K., 88 Hoffman, C., 94 Hofmann, J.B., 222 Holliday, M.J., 77, 90 Hollingdale, M., 224 Holloway, J.E., 301,312 Holm, R.E., 142,174 Honeycutt, R.L., 201,220 Hong, N . , 83 Hooykaas, P.J.J.,85 Hopper, D.G., 24, 90 Horgan, J.M., 132, 142,174,181 Horgan, R., 123, 149,174,180,181, 185 Horn, G.T., 228 Horn, M.A., 44, 45, 72,90 Horner, H.T., 303,305,312 Hoson, T., 14, 15,90 Howard, J . , 38, 91 Howe, C.J., 209,225 Howe, T.J., 225 Hsaio, T.C., 149, 160, 161,169 Huang, J-S., 78

326

AUTHOR INDEX

Huber, D.J., 72,82 Hughes, R.K., 32, 91 Huisman, W., 19, 98 Humphries, C.J., 233,307 Humphries, E.C., 115, 117, 118,174 Hunt, R.C., 104, 113,174 Hutto, J.M., 131,176 I Iijima, M., 166, 174 Iizuka, A., 182 Incoll, L.D., 109, 118, 123, 133, 134, 174, I79 Ingestad, T., 113, 116, 118, 119,174 Ingold, A., 224 Ingram, D.S., 94 Ingram, T.J., 140,174 Inouye, D.W., 182 Inouye, I., 229 Isaiah, H., 4, 91 Ishii, S., 33, 91 Ishii, T., 33, 91 Isobe, K., 100 Itai, C., 125, 133, 134,175, 183 Ito, Y . , 83,84

J

Jackson, M.B., 105, 106, 111, 112, 119, 130, 135, 137, 143, 144, 145, 146, 147, 150, 160, 161, 162, 163, 164,170,172, 173,175, 176,179,180,183 Jackson, W.T., 160,177 Jacobs, W.P., 112,176 James, D.B., 131, I76 James, R., 179 Janssens, R., 84 Jarvis, M.C., 53, 67, 91 Jeblick, W., 91, 92 Jeffcoat, B., 105,171 Jeffrey, S.W., 203,225 Jenkins, J., 225 Jennings, A.C., 85 Jensen, W.A., 303,305,315,317 Jerie, P.H., 186 Jeschke, W.D., 186 Jesko, T., 121, 124, 176 Jewer, P.C., 109, 133,174 Jin, D., 101 Jin, D.F., 21, 22,23, 91 John, P., 229 Johnson, D.S., 257,312 Johnson, J.W., I69

Johnson, M.A., 96 Jolles, P., 81 Jones, H.G., 150,172,184 Jones, M.M., 155,168 Jones, O.P., 125,176 Jones, R.L., 100, 140,176,181 Jordan, W.R., 117,176 Jorgensen, R.A., 207,225 Juncosa, A.M., 303, 306,317 K Kado, C.I., 24,88 Kapil, R.N., 303,312 Karnovsky, M.L., 49, 79 Katerji, N., 184 Kato, K., 63, 91 Kato, Y . , 14, 91,92, 94 Katou, K., 100 Kauffman, S., 26, 91 Kaufman, P.B., 140,176 Kauss, H., 35, 36, 49, 73, 91, 92, 99, 101 Kavanagh, T.A., 225 Kawase, M., 145,176 Keegstra, K., 80,87 Keen, N.T., 23, 24, 68, 74,83, 90, 91, 101

Keenan, P., 24, 91 Keil, M., 19, 91 Kelley, C., 259,312 Kende, H., 124,176, 183 Kendra, D.F:, 35,36,44,55, 92 Kennell, J.C., 303,305,312 Keon, J.P.R., 90 Keppler, L.D., 52, 92 Kettemann, I . , 173 Key, J.L., 142, 174 Khatoon, K . , 297,312 Kiefer, L.L., 14, 92 Kieft, H., 305,315 Kijne, J.W., 85 Killias, U., 69, 78 Killingbeck, K.T., 115,176 Kindle, K., 224 Kinet, J.M., 121, I69 King, R., 109, 152, I79 King, R.W., 108,176 Kinraide, T.B., 285,312 Kiraly, Z . , 77 Kirk, T.K., 100 Klambt, D., 80 Knight, M.R., 53, 92 Knopp, J.A., 78

AUTHOR INDEX

Knox, J.P., 99 Kobata, A . , 5 , 92 Koch, Dr., 112,176 Kodde, E., 24,85 Kogel, G., 38, 61, 92 Kogel, K.H., 92, 95 Kohle, H., 36, 91, 92,101 Koizumi, K., 7, 92 Koller, D., 119, 130, I70 Kombrink, E., 99 Kondorosi, A . , 79 Konno, H., 72, 92 Kono, Y . , 166,174 Konze, J . R . , 96 Kooiman, P., 7, 12, 92 Kormelink, F.J.M., 98 Kowalewska, A.K.B., 160,163,176 Kowallik, K . , 211,225 Kowallik, K.V., 211,225 Koyama, T., 66, 92 Kozlowski, T.T., 145, 160,180, 184 Kramer, P.J., 160,177 Krassilov, V.A., 291,312 Kratka, J . , 8, 92 Krauss, A., 164,177 Kriesel, K., 139, 179 Krishna Rao, K., 190,225 Krizek, D.T., 115,177,181,182,185 Kuang, J.B., 159,177 Kubat, B., 169 Kubodera, T., 91 KuC, J., 6, 8,20,21, 26, 28, 39, 77,82, 90, 92, 93 Kddela, V., 8, 92 Kugrens, P., 192,214,225 Kuhn, R., 4, 15, 93 Kuhner, S., 306,314 Kuhshel, M . , 211,225 Kuiper, D., 104, 105, 116, 132,177 Kuiper, P.J.C., 105,177 Kulaeva, O.N., 119, 124, 129, 131,177 Kulajewa, O., 179 Kumamoto, J., 183 Kumar, D., 255,313 Kumar, S.S., 287,312 Kumpf, B .,228 Kurantz, M.J., 24, 40, 93 Kurosaki, F., 4, 22, 23, 35, 53, 54, 93 Kutacek, M., 182 L Labavitch, J . , 30, 62, 93 Labavitch, J.M., 30,82,100

327

Lachno, D.R., 156, 165,177 Lafitte, G., 80 Laine, R.A., 82 Lal, M., 231,251, 297,309,312 Lamb, C.J., 2, 8, 54, 55,82, 84,85, 86, 89, 90, 93, 98, 99 Lamport, D.T.A.,29, 90, 93 Lane, D.J., 223 Lang, A . , 124,182 Larkum, A.W.D., 190, 203,225 Larsen, J., 214,225 Larson, B . , 99 Larson, M . M . , 131,170 Lavender, D.P., 140,177 Lawrence, D.K., 118,177 Lawton, M.A., 54,85, 93, 98 Leach, J.E., 29, 93 Lee, J.J., 216,221 Lee, R.E., 192, 214, 225 Lee, S.-C., 3, 20, 21, 93 Leger, A . , 285,308 Lemaux, P.G., 223 Lemmon, B.E., 237, 251, 283, 287, 291,293,307,308 Lemoine, Y., 225 Lenton, J.R., 139, 141, 142, 156,169, 177 Leonard, J.-F., 182 Lerouge, P., 37,43, 93 Letham, D.S., 173, 180 Lewis, C.E., 255,312 Lewis, J . , 200,225 Li, N . , 210,225 Lian-Ju Mao, 303,307 Lichtle, C., 209,225 Lieberman, M . , 78 LiCnart, Y . , 46, 93 Ligrone, R., 233,234,235,237,241, 243,245,249, 251, 259,263, 271,273,276, 279, 281,283, 287,289,291, 295,297,299, 309,310,312,316 Lindberg, B . , 98 Lindberg, G., 96 Lindner, W.A., 50, 94 Linforth, R.S.T., 172 Ling, E., 179 Livne, A . , 133, 177 Lockard, R.G., 141, 177 Lockhart, D.J., 213,225 Lodge, T.A., 171 Loeffler, J.E., 124,177 Lohammer, T., I78

328

AUTHOR INDEX

Maclachlan, G . A . , 100 Maclean, D.J., 24, 94 McLeod, A., 158,178 McLeod, A.L., 159,179,180 McLeod, K.W., 118,184 McMichael, B.L., 104, 178 MacMillan, J., 139, 140,174,178 Macmillan, J.D., 69, 95 McNaught, H.L., 255,313 McNeil, M., 17, 78, 84, 95,96,98, 99, 100 Maerz, M., 224 Maglothin, A., 87 Maid, U., 212,226 Maier, K., 237, 312 Maier, U., 237,312 Maier, U.G., 222 Maillet, F., 93 Makus, D.J., 81 Mandoori, A., 223 Maness, N.O., 94 Manhart, J.R., 211,226 Mansfield, J.W., 8,77, 79 Mansfield, S.G., 303, 305, 306,313 Mansfield, T.A., 168 Mardanov, A.A., 132,172 Marechal-Drouard, L., 200,226 Marfa, V., 86, 95 Margulis, L., 190,226 Marinelli, F., 21, 25, 94 Marinos, N.G., 306,313 M MarkoviE, O., 18, 97 Ma, R., 67, 94 Markowicz, Y., 225 McBride, G.E., 291,311 Marsh, B.H., 281,313 McColl, R., 223 Marshner, H., 110, 132,182 McComb, A.J., 141,178 Martin, D.J., 88 McComb, J.A., 141,178 Martin, G.C., 160,183 McCormick, F.A., 253,313 Martinez-Molina, E., 88 McCracken, D.A., 208,226 Marx, G.A., 181 McCully, M.E., 106,170,178 Masago, H., 101 McCutchan, T.F., 224 Masia, A, 180 McDonald, A.J.S., 113,178 McDougall, G.J., 4, 5 , 6, 7, 14, 15, 45, Masle, J., 165, 166,178 Mason, W.K., 104,169 56, 75, 78,88, 94, 95 Masuda, Y . , 14, 15,62, 90,96 McDougall, G.M., 63, 95 Masuta, C., 24, 48, 52, 94 McFadden, B.A., 213,226 Matama, M., 101 McFadden, G.I., 196, 197, 199,215, Matsuda, K., 14, 91, 92, 94 226 Matsui, H., 100 McFarland, 19,20, 95 McFarland, K.D., 235,241,309,313, Matsushita, J., 14, 91, 94 Matthews, K.J., 88 315 Mattoo, A.K., 78 McGaw, B.A., 123,178 Mattox, K.R., 228 McKerracher, L., 195,227 Mauch, F., 8,32,35,73,74,81, 94, 98 Maclachlan, G., 43, 56, 87, 90

Lois, A.F., 101 Loiseaux-de Goer, S., 210, 211,220, 225 Long, M., 90 Longendorfer, D.H., 232,316 Longman, D., 43,94 Loomis, R.S., 133,183 Lorences, E.P., 15, 17, 38, 75,88, 94 Lorz, H., 26,89 Lo Schiavo, F., 87 Loschiavo, F., 84 Loschke, D.C., 35,55,89 Lotan. T.. 33. 94 Loveys, B.R., 110, 112, 151, 155, 157, 178 Low, P.S., 78, 90 Lowe, D.R., 190,229 Luckwill, L.C., 125,178 Ludlow, M.M., 159, 178 Ludwig, C.H., 28, 98 Ludwig, M., 195,209,215,226 Lugtenberg, B.J.J., 85 Lukacovic, A , , 176 Luke, H.H., 133,178 Lund, A.-B., 113, 116, 119,174 Lynn, D.H., 200,226 Lyon, G., 78 Lyon, G.D., 21,52,84, 94 Lyon, J.L., 168

329

AUTHOR INDEX

Mauch-Mani, B., 94 Mauk, C.S., 126, 127, 178 Maurel, C . , 80 Mayer, J.E., 85 Mayer, M.G., 46, 94 Mazau, D . , 8,29,87, 97 Medlow, G.C., 122, I73 Mees, G.C., 147, 160,178 Mehra, P.N., 255,313 Meidner, H., 133,179 Meins, F . , 99,100 Meinzer, F.C., 107, 109, 128,177 Melchers, L.S., 85 Melkonian, M., 214,228 Melton, L.D., 17, 95 Menary, R.C., 123,185 Menon, M.K., 297,313 Mereschkowsky, J . , 190,227 Mertens, R., 96 Messiaen, J., 53, 95 Metcalf, J., 171 Meyer, K., 255,313 Meyer, R.E., 155,179 Meyer, S.R., 192,227 Michael, G., 107, 132,185 Michaels, A.E., 229 Michielsen, P . , 89 Michniewicz, M., 139,179 Miginiac, E., 121, 122,179 Mignot, J.P., 192,227 Mikeswell, J., 305,313 Milborrow, B.V., 150, 156,168,179, 180 Miller, C.C.J., 271,287,293,295,309 Miller, C.O., 4, 95 Miller, E.C., 124,179 Miller, K.R., 209,229 Miller, L., 69, 95 Miller, M.H., 184 Milligan, S.R., 115, I79 Milliken, F.F., 285,314 Milon, H., 182 Mirecki, R.M., 182 Mirecki, R.N., 177 Mishler, B.D., 233,287,289,307,311, 313,316 Mitchell, W.A., 184 Moerschbacher, B.M., 34, 95 Moesta, P., I01 Moestrup, 8.,203,214,222,227 Mogensen, H.L., 303,313 Mohnen, D . , 27, 86, 95, 99 Moldau, H . , 160,179

Mollenhauer, D., 228 Molloy, J.A., 78 Moloshok, T . , 19, 95 Moloshok, T . D . , 87 Monge-Najera, J., 279,291,316 Monyo, J.H., 104,179 Morden, C.W., 190, 212,222,227 Mod, W., 176 Morgan, P.W., 145, 147,169,179 Morrall, S . , 193, 195,227 Morriset, F . , 228 Morschel, E . , 228 Mort, A.J., 94 Morvan, C., 97 Morvan, H., 97 Moss, G.I., 165, 179 Mothes, K., 117, 124,129, 179 Moyer, M., 86 Mueller, W.C., 285,313 Muldoon, E.P., 90 Mullet, J.E., 168 Mullins, M.G., 115, 130,169,179 Mullis, K.B., 228 Munns, R., 109, 152, 155,172, 179, 180 Mur, L.R., 229 Murashige, T., 91 Murfet, I.C., 174,181 Murgia, M., 285,301,308,313 Murphy, C.A., 222 Murphy, D . L . , 85 Murray, B.M., 235, 237, 241,314,316 Musgrave, A . , 145, I79 Mussell, H., 24, 68, 95 Mutaftschiev, S., 15, 27,100

N Nadakavukaren, M.J., 226 Nagl, W . , 306,314,315 Nakahara, Y., 5, 95, 98 Nakayama, N., 78 Nakosteen, L., I76 Narang, A . , 237, 297,312 Nasr, T., 120,185 Neales, T., 158,178 Neales, T.F., 118, 155,158, 159,179, 180 Nealey, L.T., 14, 96 Nechaev, O.A., 100 Nelson, C.E., 55, 96 Neuman, D.S., 107,109,136,137, 138, 143, 160, 161, 162, 163, 164,180 Neumann, D.S., 183

330

AUTHOR INDEX

Nevins, D.J., 33, 96 Newcomb, W., 303,305,306,314 Newman, LA., 285,312 Newman, S.C., 212,227 Nilsson, K.G.I., 5, 96 Nishi, A., 78, 93 Nishitani, K., 33, 62, 96 Noguchi, M., 63, 91 Noll, U., 95 Nonhebel, H.M., 110,180 Nooden, L.D., 112, 126, 127, 128, 139, 178,180 Noronha-Dutra, A.A., 87 Norris, R.E., 215, 217,224 Northcote, D.H., 4,5, 59, 67, 80,81, 90, 99 Nothnagel, E.A., 3, 18, 21,22, 23, 78, 96 Novacky, A., 52, 92 Novick, D., 86 Nozue, M., 88 Nunezbarrios, A,, 184 Nuri, W., 95 Nutt, H . , 228 0 Oakley, B.R., 192,213,227 Oates, J.E., 101 O’Brien, T.P., 303,316 Ocampo, J.A., 88 Ogawa, T., 5, 83, 95, 98 Ohjuma, K., 168 Okada, Y., 92 Okamoto, H., I00 O’Keeffe, L., 255,314 Oki, L., 91 Okon, Y . , 80 Oliver, M.J., 251,314 Olsen, G.J., 223,227 Olson, A.R., 303,314 O’Neill, M., 17, 96 Ong, H.T., 134,180 Ordin, L., 175 Osborne, D.J., 119,180 Ossowski, P., 5 , 9, 96, 98 Ovaa, J.C., 110, 125,184 Owen, H.A., 279,291,316 P Pace, N.R., 190,223,227,229 Pacini, E., 301,314 Pagan, F.M., 255,314 Pagel, W., 69, 96

Paleg, L.G., 172 Palme, K., 80 Palmer, J.D., 210, 211, 215,226,227 Palmer, M.V., 123,180 Palmer, R.G., 303,316 Palmer, S., 184 Panabibres, F., 97 Paradies, I . , 32, 96 Paranjothy, K., 184 Parker, C., 106,180 Parker, C.W., 173 Parker, L.L., 181 Parry, A.D., 149,180 Parthier, V.B., 117, 180 Passioura, J.B., 109, 155, 159, 165, 166,172,178,179, 180 Pate, J.S., 233,283, 285, 297, 301, 302, 303,311,314 Patrick, A.D., 7, 89 Patrone, L.M., 217,227 Patterson, M.E., 79 Paus, F., 99 Pauze, F.J., 80 Paxton, J., 96 Pearce, G., 81,87, 98 Peever, T.L., 49, 52, 96 Pegg, G.F., 73, 96 PClissier, B., 46, 47, 87, 96 Pefia-CortQ, H., 19, 96 Perasso, R., 201,227 Percival, E., 208,220,227 Pereira, J.S., 160,180 Peters, B.M., 96 Petersen, C.M., 303,310 Peterson, C.M., 303,305,310 Peterson, R.L., 297, 299,314 Peterson, T.A., 115,181 Petrovics, G., 79 Petschow, B., 124,170 Peumans, W.J., 3,82 Pharis, R.P., 176 Phillips, I.D.J., 140, 176, 181 Phinney, B.O., 84, 140,181 Phipps, J., 185 Piatti, T., 12, 31, 32, 96 Pienaar, R.N., 192,227 Pierce, M.L., 155,181 Pilet, P.-E., 81, 156,181, 182 Pillay, I . , 134, 181 Pilnik, W., 18, 67, 72,97,100 Pilotte, A., 96 Pilotti, A., 83, 98 Pitman, M.G., 114,181

AUTHOR INDEX

Popperl, H., 84 Poten, F., 92 Potts, W.C., 181 Powers, M.J., 79 Prat, S., 96 Pressey, R., 31, 57,58,72, 78,83, 90, 97 Pridham, J.B., 6, 79 Priem, B . , 15, 61, 97 Prins, M., 89 Pritchard, J., 182 Proctor, M.C.F., 232,234,314 Proebsting, W.M., 142,181 Prome, J.C., 93 Proskauer, J., 291,314 Provasoli, L., 222 Pughe, J., 184 Purse, J.G., 125,174, I81 Purwin, C., 89

Qu, L.H., 227

Q

Quisenberry, J.E., 104, 178

R

33 1

Rhia, S.J., 160, 164, 181 Rhiel, R.E., 209,228 Rhodes, R.G., 214,222 Ribaut, J.-M., 156,181 Ricci, A . , 80 Rice, E.L., 84 Richards, D., 104, 115, 116, 130,182 Richmond, A., 175,183 Richmond, A.E., 124,168,182 Rickauer, M., 67, 97 Ride, J.P., 8,28,34,35,73,78,80,81,97 Rivier, L., 156, 182 Robbins, M.P., 81 Roberts, K., 99 Roberts, K.R., 214, 218,222,228 Robertsen, B . , 28,29, 39, 97 Robertsen, B.K., I00 Robertson, D.S., 181 Robinson, D.R., 156,179 Robinson, H., 287,315 Robinson, S.P., 110, 178 Roby, D., 30,35, 77,87, 97 Rocha-Sosa, M., 96 Roche, P., 93 Rock, C.D., 186 Rogers, K.R., 52, 97 Rombouts, F.M., 18, 67,72,97,100 Ronchi, V.N., 94 Rood, S.B., 180 Roseboom, P.H.M., 20,85 Roth, D., 235,241,243,245,249, 287, 315 Rougier M., 285,301,313 Rowan, K.S., 208,224 Rowe, R.N., 115,116,130,173,182 Roy, M.A., 79 Rubinstein, M., 86 Ruff, M.S., 115,182 Rumeau, D., 29,87, 97 Rush, J.S., 8, 21, 39, 93 Rushing, A.E., 291,295,303,305,308, 310 Russell, R.S., 145, 166, 172,183 Russell, S.D., 303,307 Ryals, J., 86 Ryan, C.A., 2, 19,20,36,59, 60, 75, 81,87,89, 95, 96, 98,100,101 Ryback, G., I68 Ryder, T.B., 54,84, 98

Raa, J., 99 Radin, J.W., 107, 133, 165,173,181 Railton, I.D., 137, 181 Ramser, E.L., 169 Ranucci, A , , 84 Rashke, K., 155,181 Raven, P.H., 190,228 Ray, J.P., 134,174,181 Ray, P.M., 62, 93 Read, N.D., 95 Redgwell, R.J., 5, 97 Reece, C.F., 160, 164,181 Reese, J.C., 94 Reid, D.M., 125, 135, 137, 140, 142, 143, 150,170,171, I81 Reid, J.B., 141,173,174,181 Reid, J.S.G., 87 Reinsel, M.D., 181 Reisener, H.J., 92, 95 Reith, M., 208,209,210,211, 216,228 Renault, S., 232, 285,314 Renwick, K.F., 88 Renzaglia, K.S., 232, 237, 241,243, 251, 263, 265, 275,276, 279, 281, 287, 289, 291,293, 295, 310,312,314,315,316 S Reuss, J., 185 Saab, I.N., 157, 159, 160,182 Rexova-Benkova, L., 18, 97 Sabanek, J., 117,282

332

AUTHOR INDEX

Sachdeva, A., 303,307 Sagan, L., 190,228 Saiki, R.K., 200,228 Saka, H., 33, 91 Sakai, K., 5, 98 Saker, L.R., 172 Salama, A.M.S. El-D.A., 110, 131, 132,182 Salim, M., 133, 182 Salvi, G., 80,82,83,84, 87 Sanchez-Serrano, J., 91, 96 Sandanam, S., 137,143, I83 Sanderson, J., 156,182 Santore, U.J., 192, 193,223,227,228 Sargent, J.A., 94 Saris, L., 84 Sarjoni, G., 226 Sarkanen, K.V., 28,98 Sasa, T., 224,229 Satoh, S., 182 Sattelmacher, B., 110, 132, 182 Sattin, M., 171 Saunders, P.F., 177 Sawaguchi, T., 229 Saxena, A., 88 Saxton, M.J., 87 Schafer, E., 89 Scharf, S.J., 228 Scheer, U., 224 Schel, J.H.N., 305,310,315 Schell, J., 80, 91 Schertler, M.M., 253,315 Schlumbaum, A., 73, 98 Schmelzer, E., 99 Schmidt, W.E., 42,84, 98 Schnare, M.N., 201,228 Schneider, M.J., 180 Schnepf, E., 214,228, 306,315 Schofield, W.B., 245,275,289,315 Schols, H.A., 69, 98 Schopf, J.W., 190,228 Schottens-Toma, I.M.J., 24, 98 Schraudolf, H., 276,309 Schreiber, K., I83 Schuch, W., 82,86 Schuit, J., 177 Schultz, S.R., 305, 315 Schultze-Motel, W., 235,315 Schulz, D., 237, 245, 251,317 Schulz, W., 99 Schulze, E.-D., 173,182, 185 Schurr, U., 109, 151, 155,182,187 Schuster, R.M., 253,255, 257,263,

269, 271, 275, 289,291,293, 295,312,315 Schwabe, W.W., 183 Scofield, S.R., 183 Scott, J.L., 227 Searle-Van Leeuwen, M.F., 98 Seitz, H.U., 14, 15, 87 Sekhar, K.N.C., 305,315 Sela, I., 86 Selman, I.W., 137, 143,183 Selvendran, R.R., 5,86, 97 Sembdner, G., 140,171,183 Sepenswol, S., 192,228 Sequeira, L., 93 Setter, T.L., 164,183 Shah, C.B., 133,183 Shannon, L., 91 Sharp, J.K., 5, 6, 9, 78, 98 Sharp, R.E., 155, 157, 158, 159,182, 183 Shaw, J., 287,315,316 Shaybany, B., 160,183 Sheffield, E., 232, 297,316 Sher, N., 86 Sherwood, R.T., 100 Shih-Ying, H., 161, 183 Shindy, W.W., 157, 164, I83 Shinshi, H., 50, 99 Shiuaev, V.N., 100 Shivji, M.S., 210,228 Showalter, A.M., 29,54,77,99,253,316 Sidler, W., 209,228 Siegrist, J., 73, 99 Silverthorne, J., 181 Simmonds, J., 185 Singh, S., 180 Sisworow, E.J., 172 Sitte, P., 215,218,222,224,228,229 Sitton, D., 126, 140,183 Sivakumaran, S., 160,183 Skare, N., 69, 99 Skene, K.G.M., 108,140,156,170, 183 Skoog, F., 95, 117,176 Slovik, S., 106, I73 Sluiman, H.J., 233,316 Small, E.B., 200,226 Smart, C.M., 131, I83 Smart, M.G., 303,316 Smidsr~d,O., 59, 99 Smit, B., 160,179,183 Smit, B.A., 109, 136, 137, 160, 161, 162,163, 164,171,180,183

AUTHOR INDEX

Smith, A.J.E., 235,243,309,316 Smith, A.R., 124,185 Smith, D.K., 235, 241,309,313,315, 316 Smith, G.L., 245,253,255,316 Smith, H., 120, 121,184 Smith, K.A., 145,183 Smith, O.E., 168,183 Smith, P.G., 108, 112, 119, 164, 165, 183 Smith, R.C., 44, 64, 66,88, 99 Smith, S.M., 92 Snider, J.A., 283,316 Snyder, D., 223 Snyder, F.W., 177 So, H.B., 178 Sogin, M.L., 220,224 Somlyai, G., 77 Sommer, K.J., 178 Somssich, I.E., 54, 55, 99 Sotta, B., 121, 122,179 Southwick, A., 86 Spear-Bernstein, L., 209,229 Speirs, J., 225 Spellman, M.W., 17, 99 Spencer, D.F., 222 Spencer, M.S., 145,168 Spikman, G., 39,60,85 Sponsel, V.M., 139,184 Spray, C.R., 181 Sprinzl, M., 211,229 Staal, M., 116, 132, I77 Stacciarini, E., 171 Stacey, G., 39, 90 Stacey, N.J., 62, 99 Stachowiak, M., 160,183 Stachowiak, M.L., 183 Stanton, D.S., 232,316 Steer, M., 285,311 Steere, W.C., 235,316 Steeves, T.A., 303, 305,314 Stekoll, M., 20, 21, 99 Stelzig, D.A., 96 Stevenson, T.T., 17, 99 Stewart, K.D., 228 Stickel, S.K., 220 Stipanovic, R.D., 78 Stoddart, J.L., 184 Stoddart, R.W., 5,99 Stoecker, D.K., 217,229 Stoffel, S., 228 Stotler, R.E., 235,316 Strand, L.L., 24,68, 95

Strange, R.N., 87 Strangeways, E., 118,177 Street, H.E., 285,309 Strong, F.M., 95 Strout, G.W., 303,307 Stuchbury, T., I73 Stults, J.T., 171 Stutz, E., 17, 18,85 Stypa, M., 165,184 Suda, S., 229 Suire, C., 237,310 Suter, F., 228 Suthar, H.K., 303,313 Svalheim, O., 29, 97 Sweet, G.B., 177 T Takahashi, N., I71 Takaichi, S., 224 Takasaki, S., 92 Takeda, F., 72,77 Takeda, Y., 229 Tal, N., 86 Talmadge, K.W., 80 Tan, H.M., 182 Tang, Z.C., 145,184 Tardieu, F., 110, 151, 152, 153, 155, 166,184 Tashiro, N., 93 Taylor, C., III., 291, 311 Taylor, F.J.R., 190,213,227,229 Taylor, I.B., 149,172,184 Templeton, M.D., 54, 55, 99 Tepper, C.S., 24,40, 99 Termote, F., 58,100 Terry, M.E., 62, I00 Terzi, M., 84, 87 Thain, J.F., 47, 48, 100,101 Thibaud, J.B., 96 Thiessen, W.E., 168 Thomas, J.R., 17,100 Thomas, R.J., 232,316 Thompson, A.G., I85 Thompson, N.S., 96 Thompson, W.F., 210,225,227 Thomson, N., 291,309 Thorne, G.N., 174 Thornley, A.L., 190,224 Thornley, J.H.M., 113,184 Thorpe, S.R., 86 Tietz, A., 156,184 Tiller, P.R., 101 Tilton, V.R., 303,316

333

334

AUTHOR INDEX

Tollner, E.W., 169 Tomas, R.N., 215,229 Tomiyama, K., 12,48,86,88, I00 Tommerup, I.C., 94 Tong, C.B., 30,100 Topa, M.A., 118,184 Toppan, A., 87,97 Torgov, V.I., 5,100 Torrez-Ruiz, J., 226 Toubart, P., 95, 100 Townsend, R.R., 7,90 Traas, T.P., 89 Tran Thanh Van, K., 15,27,100 Treharne, K.J., 130, 138,184 Trejo, C.L., 110, 152,184 Trewavas, A.J., 92, 95, 105, 150, 184 Tromp, J., 110, 125,184 Truchet, G., 93 Trudel, J., 80 Truelsen, T.A., 63, 100 Tschaplinski, T.J., 114,115,184 Tsjui, J., 180 Tsukumi, H., 72, 92 Tsurusawa, Y . , 93 Turkova, N.S., 145,185 Turner, N.C., 177 Turner, S., 190, 211, 212,215,222, 223,229

U Usov, A.I., 100 Usui, T., 5,100 V Vaadia, Y., 124, 125, 133, 134, 175, 177,183, 186 Vaizey, J.R., 316 Valent, B., 98 Valent, B.S., 3, 8,14,69, 78,100 Valentin, K., 211,212,226,229 van Andel, O.M., 146,147, 160,185 Van Boom, J., 84 Vance, C.P., 100 Van Cutsem, P., 95 Van Den Bulcke, M., 94 Vanden Driessche, R., 115,185 Vanderhoef, L.N., 18,100 Van Der Plas, L.H.W., 89 Van der Zandt, H., 84 Van Engelen, F., 84 Van Halbeek, H., 101 Van Kammen, A., 84,85,89 Van Montague, M., 94

van Overbeek, J., 124,177 van Staden, J., 115, 123, 124, 125, 126, 131,170,171, 185 Van Toai, T.T., 161,183 Van Went, J.L., 303,317 Varner, J.E., 99 Vaughn, K.C., 279,291,316 Venere, R.J., 90 Verduyn, R., 84 Verma, D.P.S., 63, 100 Vesk, M., 209,222 Vessey, J.C., 73, 96 Vitt, D.H., 243,247,249,316 Vizarova, G., 121, 124,176 Voesenek, L.A.J.C., 145, 146,185 Voetburg, G.S., 182 Vogeli, U., 31,81,83, 98, 100 Vogels, R., 84 Vonlanken, C., 83 Von Saltza, M.H., 95 Voragen, A.G.J., 98

W Wade, M., 83 Wadman-van-Schravendijk, H., 146, 147, 160,185 Wagner, B., 172 Wagner, von H., 107, 132,185 Walker-Simmons, M., 20, 22, 35, 36, 60, 96,100,101 Wallace, T.P., 209,225 Walsh, M.M., 190,229 Walter, C.H.S., 170 Walter, M.R., 190,228 Walton, D.C., 156, I85 Wample, R.L., 150, 181 Wang, M.-C., 101 Wang, T.L., 125, 129, 131, 173, 185 Wang, T.-W., 147,185 Ward, E., 86 Wardlaw, C.W., 232, 297, 306,316 Wareing, P.F., 110, 115, 118, 120, 121, 124, 125, 128, 130, 131, 132, 142,168, 171, 172, 173,174, 181,182,184,185,186 Wartinger, A., 151,173, 185 Waseem, M., 125,185 Watanabe, M.M., 216,224,229 Waters, A.P., 224 Weatherley, P.E., 147, 160, 178 Webb, D.P., 171 Weber, J., 229 Wedemayer, G.J., 291,311

335

AUTHOR INDEX

wedermayer, G.J., 214,230 Weeden, N.F., 190,229 Wehrmeyer, W., 192,208,228,229 Weigel, H., 59, 101 Weil, J.-H., 226 Weiland, J., 183 Weiler, E., 150, 186 Weiss, C., 124, 186 Wellensiek, S.J., 121, 186 Went, F.W., 117, 124,186 Wessels, J.G.H., 34, 101 West, C.A., 3, 8,20,21,22,23,28,68, 82,91, 93, 99, I01 Weste, G.M., 170 Wetherbee, R., 192, 219,220,224 Whatley, F.R., 190, 215,229 Whatley, J.M., 190, 201, 203,215, 219, 229 Wheeler, A.W., 129,186 Whistler, R.L., 4, I01 Whitbread, F.C., 182 Whitford, P.N., 150,186 Whittier, D.P., 297,299,314 Whittington, W.J., 104, 179 Whyte, P., 125, 178 Wickham, K.A., I01 Wiencke, C., 237,245,251,317 Wijesundera, R.L.C., 68,101 Wilcox, L.E., 303,316 Wilcox, L.W., 214,230,291,311 Wildon, D.C., 6, 20,100, I01 Wilkins, H., 146, I86 Wilkins, M.B., 174, 180 Wilkins, S.M., 186 Willemse, M.T.M., 303,317 Williams, D.M., 203,230 Willis, C.L., 174 Willmitzer, L., 91, 96 Wilson, J.B., 113, 186 Wiltshire, G.H., 171 Winter, S., 228 Wise, R.R., 303, 306,317 Woese, C.R., 227 Wolf, O., 106, 164,186

Wollett, G., 224 Wolters, J., 215,222,230 Wong, O.C., 123,180 Wong, Y .-S., 90 Woolhouse, H.W., 107, 128, 168 Woolley, D.J., 130, 186 Wright, S.T.C., 150, 155, 160,173, 186 Wyndaele, R., 63,100 Wynne, M.J., 233,307

Y Yamaguchi, I., 171 Yamashita, K., 92 Yamazaki, N., 3, 25, 78,101 Yan, H., 303, 305,317 Yang, H.-Y., 303,305,317 Yang, S.F., 100, 107, 110, 147, 149, 168,169 Yeung, E.C., 303,317 York, W.S., 4, 14, 56, 75, 92, 101 Yoshikawa, M., 42, 74, 91,101 Young, D.H., 36,48,49, 92, I01 Young, H., 156, 172 Young, P.G., 228 Young, R.E., 72, 79 Young, S.F., I76 Z Zacharius, R.M., 24,40, 93 Zaerr, J.B., 177 Zeevaart, J.A.D., 141, 142, 155, 156, 161,171, 186 Zeidler, R., 229 Zeroni, M., 146,186 Zetsche, K., 211,212,226,229 Zhang, J., 109, 110, 138, 152, 154, 156, 157, 158, 159, 160, 162, 163, 180,184,186,187 Ziegler, E., 46, 94 Zimmerman, P.W., 146,187 Zobel, R.W., I87 Zodda, G., 273,317 Zuber, H., 228


SUBJECT INDEX

A Abiotic elicitors, 21 Abscisic acid (ABA), 105-7, 145, 149-66 chemical identification, 150 evidence against ABA as positive message from roots, 161-3 evidence for drying roots as source of apoplastic ABA, 155 evidence supporting ABA as positive message from roots, 163 in xylem root exudate, 158 mechanisms raising apoplastic ABA in leaves of droughted plants, 155 miscellaneous stress effects, 164-6 physiological significance of in-shoot apoplast and xylem sap, 151-5 physiological studies, 150 reconciliation of findings, 1 6 3 4 soil flooding, 160-4 water deficiency and leaf expansion, 159-60 water deficiency and stomata1 closure, 150-9 Acanthamoeba, 201 Acer, 123 Acer pseudoplatanus, 142 N-Acetylglucosamine, 9 Acrobolbus, 257 S-Adenosylmethionine (SAM), 144 Adiantum, 297 Alcaligenes eutrophus, 212,213 Alder, 114 Alisma, 306 Almond trees; 151 Alnus glutinosa, 114 Amino-oxyacetic acid (AOA), 149 1-Aminocyclopropane-1-carboxylic acid (ACC), 30, 144, 145, 147, 148, 149

Aminoethoxyvinylglycine (AVG), 31, 32, 144, 149 Anacystis nidulans, 206 Andraeobryum, 235 Andreaea, 235, 247, 263,285, 287, 289, 293 Andreaea rothii, 240 Andreaeales, 287, 293 Andreaeidae, 235 Andreaeobryum, 241 Aneura, 265,267,293 Aneura pinguis, 279 Angiosperms, 305 transfer cells in, 302 Anthoceros, 275, 279, 281, 295 Anthocerotaceae, 295 Anthocerotes, 275-83 placental cell walls in, 276 placental cells in, 276 Apricot trees, 151 Arabidopsis, 305, 306 Arabinosyltransferase, 12 Archidium, 235, 251, 283 Aspergillus, 21 Aspergillus niger, 25, 68, 69 Asterella, 255 Asteridae, 305 ATP, 144 Atrichum, 247 Atrichum undulutum, 246 Auxin, 15, 27 AXX, 33 B Barley, 105, 114 Bean cell, 54 Beta vulgaris, 114 Betula pendula, 142 Biotic elicitors, 21 Biotic stresses, 133-5 Blackcurrant, 121

337

338

SUBJECT INDEX

Blasia, 265, 267, 293 Blasia pusilla, 275 Blasticidin S, 24 Blindia, 251 Blindia acuta, 264 Brachythecium, 251 Brachythecium velutinum, 251 Brown algae, 210 Bryales, 249-53 Bryidae, 243-5 Bryophytes, 233, 234-83 placenta in, 283-95 Bryopteris, 257 Bryum, 245, 251, 257, 287 Bryum capillare, 263 Buxbaumia, 251 Buxbaumia piperi, 249 Buxbaumiales, 287 C Ca2+concentration, 36, 53-4 Callose, 36 Calobryales, 255, 269, 289, 293 Calobryum, 269 Calobryum blumei, 253,269, 285, 286 Calobryum indictum Udar et Chandra, 253-5 Campylomonas, 208 Capsella, 305, 306 Carbohydrates, 46 Carrot cells, 23 Carrot protoplasts, 15 Carrpos, 273, 291 Carrpos monocarpos, 291 Casbene synthase, 21,22 Castor bean cell wall, 22 Cavicularia, 265 Cell wall composition, oligosaccharininduced changes in, 28-9 Cellulysin, 32 Cephalozia, 263, 265 Cephalozia bicuspidata, 271 Chaetomium globosum, 35 Chalcone synthase, 31, 54 Chenopodiurn polyspermum, 121 Chitin, 73 oligosaccharides of, 34-5 Chitin-derived oligomers, 28 Chitinases, 12, 32, 73 Chitosan, 28, 73 Chitosan oligosaccharides, 35-7 Chitosan oligosaccharins, 44 Chlorarachnion, 214-16

Chlorarachnion reptans, 217 Chloroplast, 208-13 chromosome, 210-1 1 gene sequences, 211 genome, 210-13 membranes, 208 photosynthetic pigments, 208-10 rRNA, 212-13 second-hand, 189-230 storage product, 208 Chloroplast endoplasmic reticulum (CER), 196,200,203,208,218 Chromophyte algae, 201-3 Cichorium intybus, 121, 122 Citrus “polygalacturonic acid”, 17 Cladophascum, 251,283 Cladophascum gymnomitrioides, 264 Cladosporium cucumerinum, 28, 29,39 Cladosporium fulvum, 24, 39,49, 68,

74 Cocos nucifera, 124 Coleochaete, 29 1 Colletotrichum lagenarium, 29, 46, 73 Colletotrichum lindemuthianum, 24, 29, 32,40, 52, 54,55,61, 68, 70 Commelina communis, 152, 157 Conocephalum, 255, 271, 273 Corsinia, 255, 271 Coscindiscus, 211 Cowpea pods, 25 Crithidia, 2 15 Cryptomonad algae evolution of, 189-230 see also Chloroplast; Nucleomorph Cryptomonads ancestors of chromophyte algae, 201-3 as endosymbionts, 213-14 cell layout, 192-3 overview of, 192 parasites of, 213-14 rRNA genes, 20&1 taxonomy, 219-20 Cryptomonas, 208, 214 Cryptothallus, 265,267, 283,285, 293 Cryptothallus mirabilis, 279, 28 1 Cucumber cell walls, 28, 29 Cucurbitapepo, 132 Cyanidium caldarium, 212 Cycloheximide, 24 Cytokinins, 123-38 development in unstressed plants, 125-8

SUBJECT INDEX

early research, 123-5 miscellaneous stresses applied to roots, 133-8 responses to mineral nutrient shortage, 131-3 root excision studies, 128-31 D 2,4-D, 14, 15, 63 Dendroceros, 275,279, 281, 295 Dendroceros tubercularis, 298 Dendrocerotaceae, 295 Dicranum, 249, 251 Dicranum majus, 263, 287 Dictyota dichotoma, 211 Diphyscium, 249,251,253, 283, 287 Diphyscium foliosum, 264 Diphyscum, 257 Diploid nucleomorph, 207 Diplophyllum, 263, 265 Diplophyllum albicuns, 273 Dodeca-a-( 1+4)D-galacturonide, 22 Douglas fir, 135 “Driselase”, 26 Drought, 133-5 Dumortiera, 271 E Elicitors abiotic, 21 biotic, 21 formation of pectic oligosaccharides as, 68-72 of phytoalexin synthesis, 2C-3 transport of, 76-7 Embryo, formation of, 233 Embryonic phase, 232 Embryophytes, 233 Encalypta, 245 Endosymbionts, cryptomonads as, 21314 Enzymes acting on chitin and chitosan, 73 direct effects of oligosaccharides, 568 Ephemerum, 235 Equisetum, 297 Erwinia carotovoru, 22, 68, 69 Erwinia chrysanthemi, 49 Erwinia rubrifaciens, 24 Escherichia coli, 206 Eschscholtziu, 31 Ethylene, 144-9

339

effect of flooding, 145-9 synthesis, 12 induction by pectic oligosaccharides, 30-2 “Ethylene-forming enzyme” (EFE), 144 Ethylene-inducing xylanase (EIX), 32, 33 Eucalyptus marginata, 135 Eukaryotic ribosomes around nucleomorph, 196-200

F FAXX, 33 Ferns, 297, 299 Ferulate, 33 Fissidens, 251 Fissidens crassipes, 25 1 Flax hypocotyls, 15 Flooding, 145-9, 1 6 M Flowering, 12@1 Folioceros, 275, 279, 281, 295 Folioceros fuciformis, 305 Fossombronia, 265, 267, 293 Fossombronia echinata, 273,283 Frullania, 257 L-Fucose-containing oligosaccharide, 15 2’-Fucosyl-lactose, 15 Funaria, 245, 251 Fungal cell walls, 12 components of, 8 Fungal infection, 20 Fungal oligo-p-glucans, 7-12 Fusarium, 21 Fusurium oxysporum, 61 Fusurium solani, 36 G GalAI2, 44 D-Galacturonic acid, 21 a-(1+4)-D-galacturonic acid, 22 Galacturonic acid residues, 20 a-D -galacturonidase, 31 Gametophyte-sporophyte junction in land plants, 231-317 Gas chromatographylmass spectometry (GC-MS), 123, 125 Gel-permeation chromatography, 19, 25, 31 Gibberellic acid, 130

340

SUBJECT INDEX

Gibberellins, 138-44 effects of root excision and environmental stresses applied to roots, 142-3 studies of unstressed plants, 139-42 a-(1+3),(1+4)-D-glucan, 20 p-(1+3),(1-+6)-glucan, 8-9, 12, 20,73 p-( 1+3)-linked D-glucan, 42 p-glucanase , 32 p-( 1+3)-D-glucanase, 22 P-D-glucanase, 7p-D-glucopyranose, 9 Glucosamine, 9 p-( 1+3)-linked D-glucose residues, 36 Glutathione, 50 Glutathione peroxidase, 50 Glyceollin, 50 Glycine, 305 Glycine rnax, 126, 142 Glycoprotein-derived oligosaccharins, 61-2 Glycoproteins, 20, 38 synthesis, 12 Goebelobryurn, 257 Goniornonas, 192 Gorse, 43 Gossypiurn hirsuturn, 107, 133 Grape vine, 115 Growth regulators, 12-17

H Haplomitrium, 269, 289 Haplornitriurn gibbsiae Steph., 253 Haplornitrium hookeri, 285 Haustorium, 253 Hedera helix, 142 Helianthus, 305 Helianthus annuus, 131, 132, 135, 140, 155, 158, 160 Heptasaccharide, 9 Herberta, 263, 265,269,271, 291 Hexadecadienoic acid, 37 Hexasaccharide, 9 Hordeurn vulgare, 114, 137 Hordeurn vufgare L. cv. Midas, 105 Hormones, 74-7 assessing developmental impact of messages, 111-12 criteria for implicating regulation of naturally occurring developmental phenomenon, 111

evidence for regulation of root:shoot ratio by roots, 112-16 hormone-like action of roots on shoots, 117-23 in root to shoot communication, 103-87 message concept, 106-12 quantifying messages in transpiration stream, 107-11 Hornworts, 275 HPLC, 125 HRGP, 29-31 Hydroxycinnamates, 29 6-Hydroxymellein, 21 Hydroxyproline-rich glycoproteins. See HRGP I Impatiens glandulifera, 140 Indoleacetic acid (IAA), 27-8, 50-1, 57-8,72, 130 In shoot apoplast, 151-5

J

Jackiella, 257 Jubula, 257 Jubulaceae, 291 Jungermaniidae, 289 Jungermanniales, 253,257-65,265, 267,273,285,289,293,299 K Kornrna caudata, 194, 197, 199 Kurzia, 263, 265 Kurzia trichoclados, 265, 271 L Land plants gametophyte-sporophyte junction in, 231-317 life-cycle of, 232 Leaf expansion and water deficiency, 159 Leaf senescence, 117-19, 126 Lectins, 43 Lejeunaceae, 291 Lejeunea, 257 Lepidodiniurn viridae, 215, 216 Lignification, 20, 28, 34 Lignin, 29 Lipid peroxidation, 52 Lipoxygenase, 52

34 1

SUBJECT INDEX

Liverworts, 233,253-75 anacrogynous, 265 placenta in, 258 placental cells in, 260 Lolium perenne, 131 Lophocolea, 263,265 Lophocolea heterophylla, 269 Lunularia, 271 Lupinus albus, 140, 157 Lycopersicon esculentum, 123, 137, 139, 161 Lycopodium, 297 Lycopodium appressum, 299 Lycopodium cernuum L., 299 Lysozyme, 12

M Macerase, 30 Magnaporthe grisea, 25, 26,33 Maize, 26, 114, 153, 154 Mannia, 271 Marchantia, 255, 271 Marchantiales, 255,271,273, 289,291 Marchantiidae, 271-5, 285, 291, 293, 305 Marchesta, 255 Marsupella, 263,265 Marsupella funckii, 267 Megaceros, 275,279,281, 295 Melons, HRGP biosynthesis in, 30 Membrane depolarization, 46 Messenger RNA (mRNA), 21, 29, 41, 54,55 6-Methoxymellein, 21 4-0-Methyl ether, 32 5’-Methylthioadenosine, 144 Metzgeriales, 257,265-7,267, 273, 289,291,293 Mineral nutrient shortage, 131-3 Mniurn, 251 Mnium hornurn, 251,263 Monoclea, 255, 289 Monoclea forsteri Hook, 257 Monocleales, 253, 271, 291 Mosses, 233,235-53 acrocarpous , 245 arthrodontous, 243 nematodontous, 243 placenta in, 236 placental cells in, 238 Myrionecta rubra, 213

N

Nicotiana , 119 Nicotiana rustica, 117, 124, 129, 131 Nicotiana tabacum, 50 Notothyladaceae, 295 Notothylas, 275,279, 281, 295 Notothylas orbicularis, 305 Nuclear DNA, separation of, 203-6 Nucleomorph, 192-207 derived from red algal nucleus, 201 DNA content, 195-6 electrophoretic karyotype of, 207 eukaryotic ribosomes around, 196200 isolation of, 203-7 nucleus-like characteristics, 192-5 origin of, 200-3 role of, 216-18 structure, 192 Nucleomorph DNA, 216 separation of, 203-6 Nutrient control theory, 112-16 shortcomings of, 113-16 0 Oak, 131 Ochromonas dancia, 211 Odontella, 211 Oligogalacturonides, 19, 22, 23, 27, 28, 44, 50, 51, 70 Oligo-P-glucans, 7-12, 22,42, 43 receptors for, 41-3 Oligo-P-(1+3),(1+6)-glucans, 58 Oligo-P-glucosides, 9, 10 Oligosaccharides, 2 direct effects on enzymes, 56-8 evidence for receptors, 41-6 fucose-free, 14 of chitin, 34-5 of chitosan, 35-7 of pectin, 17-32 purification and chemical characterization, 6-7 sequencing, 7 structure-activity relationships, 9 xyloglucan-derived, 12-17 Oligosaccharin-induced changes in cell wall composition, 28-9 Oligosaccharins, 1-101 artificial, 3 bioassays, 5-6 diversity of, 38 from N-linked glycoproteins, 37-8

342

SUBJECT INDEX

Oligosaccharins (cont.) Pectinase-inhibiting proteins (PGIPs), glycoprotein-derived, 61-2 61, 70-1 mechanism of formation and Pectinases, 17, 20, 25, 33 degradation, 62-74 Pectinmethylesterase, 26 membrane depolarization, 46 “Pectolyase”, 23 mode of action, 41-58 Pellia, 265, 267, 283, 293 movement within plant, 74-7 Pellia epiphylla, 273, 279 natural occurrence, 58-62 Pentasaccharide, 36 oligo-a-xylans as, 32-4 Perilla frutescens , 128 origin of concept, 2-3 Perilla ocimoides, 121 oxidative metabolism, 49-52 Petunia, 12 physiology of effects, 7 4 1 Phaeoceros, 275, 279, 281,283, 295 preparation, 3-5 Phaeoceros carolinianus, 279 method l:, 3 Phaeoceros laevis, 298, 305 method 2:, 3-4 Phascum, 283 method 3:, 4 Phaseolus, 61, 306 method 4:, 4-5 Phaseolus coccineus, 142 protein phosphorylation, 52-3 Phaseolus vulgaris, 54, 70, 117, 123, rapid effects of, 4 6 5 6 130, 134, 136, 137, 139, 146, receptors for, 43-6 147,161, 162, 164, 165 second messengers, 5 3 4 Phenylalanine ammonia lyase (PAL), successful host or successful 23, 31,32, 48, 54, 70 pathogen?, 39 Phloem, 106 synergism between, 4&1 Phoenix dactylifera L, 305 xylan-derived, 34 Photosynthesis, 120-1 Oligotrichurn, 247 Photosynthetic pigments, 208-10 Oligotrichurn hercynicum, 249 Phycobiliprotein, 209 Oligo-P-xylans as oligosaccharins, 32-4 P-Phycoerythrin, 209-10 Olisthodiscus luteus, 211 Physcomitriurn, 251, 297 Phytoalexins, 8-10, 2&3,26, 36, 40, 42,48,50, 53 P Phytophthora, 35,39 Pallavicinia, 265, 267, 293 Phytophthora cinnamomi, 135 Pallavicinia indica, 275, 283 Phytophthora infestans, 24, 40, 48, 51 Pea stem segments, 16, 27 Phytophthora megasperma, 40,42,50, Peach, 130 52,74 Pectic oligosaccharides, 17-32 Phytophthora megasperma f.sp. as elicitors of phytoalexin synthesis, glycinea, 46, 54 2&3 Phytophthora parasitica, 32 as growth regulators, 18 Phytophthoraparasitica var. nicotianae, 46 degradation in plant tissue, 72 Pinus, 306 formation as elicitors, 68-72 Pinus serotina, 118 formation as wound signals, 6 6 8 Pinus taeda, 118 formation in ripening fruit, 72 Pisum, 306 induction of ethylene synthesis, 30-2 Pisurn sativum, 138, 140, 161, 163, 164, morphogenesis-regulating activity, 210 27-8 Placenta, 233 natural occurrence, 59-61 in bryophytes, 283-95 Pectic oligosaccharins in liverworts, 258 hypersensitive response by, 23-6 in mosses, 236 transcription of protease inhibitor Placental cell walls in anthocerotes, 276 genes, 48 Placental cells Pectin lyase, 26 in anthocerotes, 276

SUBJECT INDEX

in liverworts, 260 in mosses, 238 Plagiochasrna, 271 Plagioselmis palustris, 195 Plant hormones. See Hormones Plantaginales, 305 Plantago, 116 Plantago major, 132 Pogonaturn ,247 Pogonatum neesii, 246 Polygalacturonic acid, 28 Polypodium, 297 Polytrichales, 245-7, 263, 285, 287, 293 Polytrichum, 245,247, 285 Polytrichum formosum, 249 Populus, 143, 162 Populus deltoides, 136 Populus trichocarpa, 136 Porellaceae, 291 Porphyridiurn aerugineum, 212 Potato protoplasts, 12 Potato tuber disks, 51 Preissia , 255, 271 Prolyl hydroxylase, 12 Pronase E, 23 “Protease inhibitor inducing factor” (PIIF), 19,48,75 Protease inhibitors, 19, 20 Protein gene phylogeny, 212-13 Protein phosphorylation, 52-3 Proterornonas steinii, 214 Prunus armenica, 151 Prunus dulcis, 151 Prunus persica, 130 Pseudomonas syringae, 49,69 Pseudotsuga menziesii, 135 Pteridium, 297 Pteridophytes, 233, 295-301 Puccinia, 61 Puccinia graminis, 38, 45 Pylaiella littoralis, 210 Pyrenoid-nucleomorph complexes, 206

Q

Quercus rubra, 131

R Radula, 257, 263, 265, 291 Radula complanata, 273 Radulaceae, 291 Reboulia, 255, 271,273 Reboulia hemisphaerica, 291, 293

343

Reboulia hemisphaerica var. macrocarpa Zodda, 273 Red algae, 201,202,217,219,233 Rhamnogalacturonans I and I1 (RG-I and RG-11), 17, 20, 26 L-Rhamnose, 21 Rhizobium, 43 Rhizobium meliloti, 37 Rhizophora, 306 Rhizopus stolonifer, 68 Rhodella , 217 Rhodomonas salina, 204-7 Ribes nigrum, 121 Ribonuclease A, 21 Ribosomal RNA (rRNA), 196, 200, 212-13,216,219 Ribulose-1 ,5-bisphosphate carboxylaset oxygenase, 211 Riccardia, 265,267,285, 293, 295 Riccardia multijida, 275,279,281, 283 Riccia, 255, 271, 273, 291 Riccia sorocarpa, 293 Rice blast pathogen, 25 RNA, 54, 196 Root excision studies, 128-31 Root:shoot ratio of plants, 112-16 Rubisco phylogeny, 211-12 Rubus, 15 Rudbeckia tricolour, 121 Rumex palustris, 146 S Saccharum spp. hybrid, 128 Salinity, 133-5 Salix viminalis, 120 Scapania, 263,265 Scapania gracilis, 271 Scrofularia arguta, 121 Scrophulariales, 305 Second-hand chloroplasts, 189-230 Second messengers, 53-4 Seed plants, 301-6 Selaginella, 301 Shoot extension, 120-1 Silene, 61, 121 Soil flooding, 145-9, 160-4 Soil waterlogging, 135-8 Solanum andigena, 130, 131 Solanum pennelli, 134 Solanum tuberosum, 132 Sorghum saccharum, 121 Sorghum vulgare, 135 Soybean, 21,36,44, 126, 128

344

SUBJECT INDEX

Sphaerocarpales, 255,271, 289, 291 Sphaerocarpos, 271 Sphagnidae, 241-3 Sphagnum, 235,241, 263,283,285, 287,295,299 Sphagnum cuspidatum, 241, 243,245 Sphagnum fallax, 241 Sphagnum fimbriatum, 241,243 Sphagnum subnitens, 241 Sporophyte. See Gametophytesporophyte junction Spring sap in woody plants, 125-6 Stellaria, 306 Stomata1 closure and water deficiency, 15&9 Striga hermonthica, 106, 135 Sugar beet, 114 Sugar cane, 128 Sycamore cells, 25 T Takakia, 235, 241,263,269, 285, 289 Takakia ceratophylla, 243 Takakiales, 293 Targionia, 255, 271 Targionia hypophylla, 271 Tetraphidales, 247-9 Tetraphis, 287 Tetraphis pellucida, 247, 249, 251 Thalictrum rugosum, 12 Timmiella, 25 1 Tmesipteris, 301 Tobacco leaf, 27 Tomato, 19, 36, 109 Transfer cells in angiosperms, 302 Trichoderma viride, 33 Trichomonas, 218 Triticum aestivum, 142, 152, 155 Tropaeolum , 306 U

Ulex europaeus, 43 Urtica dioica, 132 V Vaucheria, 211 Verticillium dahliae, 44, 50 Viciafaba, 210

Vigna, 25 Vitis vinifera, 115, 130, 140, 151 W Water deficiency and leaf expansion, 159-60 and stomata1 closure, 150-9 Willow, 120 Wound hormones, 18-20 Wound signals, 18-20, 6 6 8 non-transport of, 75-7

X Xanthium stromarium, 128, 156 Xanthomonas malevacearum, 24 XET, 65,66 XGS, 15 XG7, 16 XG8, 14, 16 XG9, 14-16, 33, 44, 45, 63, 64, 66, 73, 75 XG9n, 16 Xylan-derived oligosaccharins, 34 Xylanase, 26, 32, 33 p-(1--+4)-D-xylanases,32 Xylem, 107, 119 Xylem sap, 106-12, 123-6, 128, 133, 135, 148, 151-5, 158, 162 Xyloglucan, 41 sugar residues in, 13-14 Xyloglucan-derived oligosaccharides, 12-17 Xyloglucan endotransglycosylase, 56-7, 64 Xyloglucan oligosaccharides degradation of, 66 natural occurrence, 58-9 synthesis of, 62-6 transport of, 75 D-xylose, 21 Z Zeamays, 114, 121, 124, 153, 154, 159, 160 Zeatin, 131 Zeatin riboside, 126, 127, 131, 136 Zoopsis, 263,265,291

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