Advances in
BOTANICAL RESEARCH incorporating Advances in Plant Pathology VOLUME 22
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BOTANICAL RESEARCH i...
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Advances in
BOTANICAL RESEARCH incorporating Advances in Plant Pathology VOLUME 22
Advances in
BOTANICAL RESEARCH incorporating Advances in Plant Pathology Editor-in-Chief J.A. CALLOW
School of Biological Sciences, University of Birmingham, Birmingham, UK
Editorial Board J . H . ANDREWS H. G. DICKINSON M. KREIS R.M. LEECH R. A. LEIGH E. LORD D.J. READ I. C. TOMMERUP
University of Wisconsin-Madison, Madison, USA University of Oxford, Oxford, UK Universite' de Paris-Sud, Orsay, France University of York, York, UK Rothamsted Experimental Station, Harpenden, UK University of California, Riverside, USA University of Sheffield, Sheffield, UK CSIRO, Perth, Australia
Advances in
BOTANICAL RESEARCH incorporating Advances in Plant Pathology Series editor
J.A. CALLOW School of Biological Sciences, University of Birmingham, Birmingham, UK
VOLUME 22
1996
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 NWl 7DX
United States Edition published by ACADEMIC PRESS INC. San Diego, CA 92101
Copyright 0 1996 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 catalogue record for this book is available from the British Library ISBN 0-12-005922-3
Typeset by Colset Private Limited, Singapore Printed in Great Britain by Hartnolls Ltd, Bodmin, Cornwall
CONTENTS
................................. SERIES PREFACE ........................................................ PREFACE ................................................................... CONTRIBUTORS TO VOLUME 22
ix xi
...
xiii
Mutualism and Parasitism: Diversity in Function and Structure in the "Arbuscular" (VA) Mycorrhizal Symbiosis F.A. SMITH and S.E. SMITH I . Introduction: Names and Structures ........................................... I1. Transfer of Solutes Between Fungus and Host ............................. A . Mechanism of Nutrient Transfer ........................................... B . Is there Spatial Separation of Transfer of Phosphate and Carbon?
1 3 5 6
...............
9
IV . Variations in Nutritional Efficiency in VA Mycorrhizas ................. A . Cheating by the Fungus ..................................................... B . Cheating by the Host: Linked Plants ...................................
17 17
111. Variations in Structure: Two Classes of VA Mycorrhizas
V . Cheating by Myco-heterotrophic (Achlorophyllous) Plants
..............
VI . Costs and Benefits of the VA Mycorrhizal Symbioses: Fast and Slowgrowing Plants and Related Issues .............................................
VII . Development and Control of the VA Mycorrhizal Symbiosis VIII . Conclusions
............................................................................
Acknowledgements References
..........
..................................................................
.............................................................................
22 25
26 29
33 34 35
vi
CONTENTS
Calcium Ions as Intracellular Second Messengers in Higher Plants ALEX A.R. WEBB. MARTIN R . McAINSH. JANE E . TAYLOR and ALISTAIR M . HETHERINGTON I . Introduction ........................................................................... A . Calcium Ions as Second Messengers - the Animal Cell Paradigm
45 47
I1. Calcium Ions as Second Messengers in Plant Cells ........................ A . Methods for Measurin [Ca2+Iiin Single Cells ...................... B . Measurements of [Ca F+ Ii ...................................................
49 49
Ill . Measurements of Stimulus-induced changes in [Caz+Iiin plants
60
......
68
IV . The Calcium Homeostatic Apparatus .......................................... A . Mechanisms of Generating Increases in [Ca2+Ii ...................... B . .Ca2+.ATPases .................................................................
69
V . The Problem of Specificity ....................................................... A . Other Second Messengers ................................................... B. The Calcium Signature - a Stimulus-specific Calcium Signal C . Calcium Signatures in Plant Cells ........................................
84 84 85
VI
.
Future Prospects Acknowledgements References
69 83
...
86
.....................................................................
87
..................................................................
88
.............................................................................
88
The Effects of Ultraviolet-B Radiation on Plants: A Molecular Perspective BRIAN R . JORDAN I . General Introduction ............................................................... 98 A . Stratospheric Oxone Depletion and the UV Environment ......... 98 B. Potential Effects of Increased UV-B on Plant Growth and Development .................................................................... 101 103 I1. The Perception of UV-B Radiation ............................................ A . Measurement of Plant Responses to UV-B Radiation .............. 103 104 B. Photoreceptors and their Interactions ................................... 107 C . Mechanisms of Signal Transduction ..................................... D . Penetration of UV-B Radiation Through Plant Tissue ............. 112
.
111
Protective Mechanisms Against UV-B Radiation ........................... A . UV-B-absorbing Pigments ..................................................
114 114
CONTENTS
vii
B. DNA Repair .................................................................... C . Antioxidants and Other Protective Mechanisms ......................
117 119
IV . Effects of UV-B Radiation Upon Cellular Processes ...................... A . Photosynthesis .................................................................. B . Carbohydrate, Lipid and Nitrogen Metabolism ...................... C . Reproductive Biology ........................................................ D . Cell-cycle and Cytoskeleton ................................................
121 121 130 131 133
V . The A. B. C.
Effects of UV-B Radiation on Gene Expression ..................... Chloroplast Proteins .......................................................... Defence Genes ................................................................. Factors Affecting Gene Expression .......................................
VI . UV-B Interactions with Other Stresses ........................................ A . Light .............................................................................. B . Water ............................................................................. C . C 0 2 and Temperature ....................................................... D . Pathogens ........................................................................ VII . Conclusion
141 141 143 145 147
.............................................................................
148
..................................................................
149
Acknowledgements References
134 134 135 138
.............................................................................
149
Rapid. Long-distance Signal Transmission in Higher Plants M . MALONE I . Introduction ........................................................................... A . Shoot Responses to Changes in the Root Environment ............ B. Rapid Movements Induced by Localized Mechanical Stimuli ..... C . Remote Responses to Localized Wounding ............................ D . Miscellaneous Related Phenomena .......................................
163 164 164 165 166
I1. Mechanisms of Long-distance Communication Within Plants .......... A . Airborne Signals ............................................................... B. Phloem Translocation ........................................................ C . Hydraulic Pressure Signals ................................................. D . Hydraulic Dispersal Signals ................................................ E . Electrical Signals ..............................................................
167 167 170 171 177 186
I11. Rapid, Long-distance Signalling in Plants: Case Studies ................. 188 A . Systemic Induction of PI by Localized Wounding in Tomato ... 188 196 B. Signal Transmission in Mirnosu ........................................... IV . Implications and Directions for Further Research ......................... 200 A Re-assessment of Electrical Signals in the Higher Plant ........... 201 B. Signalling of Non-wound Stimuli - an Hydraulic Mechanism? . 209
.
viii
CONTENTS
V . Conclusion
.............................................................................
Acknowledgements References
216
..................................................................
216
.............................................................................
217
Keeping in Touch: Responses of the Whole Plant to Deficits in Water and Nitrogen Supply A . J . S. McDONALD and W . J . DAVIES I . Introduction
...........................................................................
228
.............................................
231
Framework for Analysing Limitations to COz Uptake ............. Stomata1 Responses ........................................................... Changes in the Mesophyll .................................................. Acclimation of NO; Uptake ...............................................
................................................
235 235 238 240 243
IV . Acclimation of Extension Growth .............................................. A . Framework for the Analysis of Extension Growth .................. B. Growth of Roots and Shoots when Water Supply is Restricted . C . Growth of Roots and Shoots when N Supply is Restricted .......
246 246 252 258
I1. Manipulating Water and N Supply 111. Acclimation of C and N Uptake
A. B. C. D.
V . Implications ........................................................................... A . Sink Strength ................................................................... B. Regulation of N Balance .................................................... C . Regulation of Water Use Efficiency .....................................
263 263 264 266
VI . Information Transfer ............................................................... A . Respones to Soil Drying ..................................................... B. Responses to N Limitation .................................................
267 261 273
VII . What is in the Xylem Sap and How Can Changes in Water and N Availability Change the Xylem Sap Contents? .............................. A . Collection of Xylem Sap .................................................... B. Soil Drying and N Deprivation and the Effects on Xylem Contents C . Interaction and the Concept of Sensitivity Variation ...............
275 275 276 280
..
286
VIII . Conclusions: An Integrated Stress Response System for the Plant?
............................................................................
289
AUTHOR INDEX
............................................................
301
SUBJECT INDEX
............................................................
319
References
CONTRIBUTORS TO VOLUME 22
W. J. DAVIES, Division of Biological Sciences, IEBS, Lancaster University, Bailrigg, Lancaster LA1 4YQ, UK A. M. HETHERINGTON, Division of Biological Sciences, University of Lancaster, Lancaster LA1 4YQ, UK B. R. JORDAN, New Zealand Institute of Crop and Food Research Limited, Levin Research Centre, Kimberley Road, Private Bag 4005, Levin, New Zealand M. MALONE, Horticultural Research International, Wellesbourne, Warwicks CV35 9EF, UK M. R. MCAINSH, Division of Biological Sciences, University of Lancaster, Lancaster LA1 4YQ, UK A. J. S . McDONALD, Department of Plant and Soil Science, Aberdeen University, Cruick Shank Building, St. Machar Drive, Aberdeen, AB9 2UD,U.K. F . A. SMITH, Department of Botany, The University of Adelaide, SA 5005, Australia S. E. SMITH, Department of Soil Sciences, The University of Adelaide, SA 5005, A ustralia J. E. TAYLOR, Division of Biological Sciences, University of Lancaster, Lancaster LA1 4YQ, UK A. A. R. WEBB, Division of Biological Sciences, University of Lancaster, Lancaster LA1 4YQ, UK
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SERIES PREFACE
Advances in Botanical Research is one of Academic Press’ longest standing serials, and has established an excellent reputation over more than 30 years. Advances in Plant Pathology, although somewhat younger, has also succeeded in attracting a highly respected name for itself over a period of more than a decade. The decision has now been made to bring the two serials together under the title of Advances in Botanical Research incorporating Advances in Plant Pathology. The resulting synergy of the merging of these two serials is intended to greatly benefit the plant science community by providing a more comprehensive resource under one “roof“. John Andrews and Inez Tommerup, the previous editors of Advances in Plant Pathology, are now on the editorial board of the new series. Our joint aim is to continue to include the very best articles, thereby maintaining the status of a high impact factor review series.
This Page Intentionally Left Blank
PREFACE
Harmonious, integrated functioning of the whole plant system requires that its various cells, tissues and organs should be able to communicate with each other, transferring a range of information on environmental conditions, physiological and microbial stresses etc. In this volume of Advances in Botanical Research, no less than three articles are concerned with different aspects of plant signalling. The article by McDonald and Davies considers how shoot systems respond to drying and N-deficient soil, in terms of their stomata1 behaviour and growth, via the transmission of root-derived chemical signals. Abscisic acid has long been suspected as playing a critical role in this signalling system and the authors of this article consider critically the evidence supporting this role, and that of other growth regulators and hydraulic signals. It has been known for some time that plant organs which are wounded or subject to microbial invasion or insect-induced damage, can rapidly transmit information to this effect to other, undamaged or uninfected organs, resulting in modified gene expression in the recipient organ and a variety of physiological effects including an enhanced level of defence. The molecular basis of this systemic response is gradually being unravelled but is still an area of considerable controversy, as is the precise nature of the long-distance signal itself. Malone considers the major hypotheses that have been proposed with particular attention being given to hydraulic pressure signals and the hydraulic dispersal of chemical signals. At a different, intracellular level of communication, a wide variety of second messengers couple extracellular stimuli to a characteristic physiological response. Calcium ions are now well established as playing a central role in intracellular signalling in a wide variety of animal cell types, alongside other signalling molecules. Webb et a/. consider progress made in establishing similar roles for calcium in plant signalling, in the context of the mammalian paradigms. The effects of UV-B radiation on plants have been extensively investigated in recent years. A wide range of responses has been documented and much of the biochemical symptomatology described. However, the underlying primary molecular mechanisms are relatively poorly understood, and in his review Jordan considers progress in understanding the chain of events from perception of UV-B to signal transduction and changes in gene expression, and regulation that underly the diversity of responses.
xiv
PREFACE
The article by Smith and Smith is concerned with vesicular-arbuscular (VA) mycorrhizas, important and widespread mutualistic symbioses of a wide range of most higher, and some lower plants. Despite considerable study of the physiology of these associations a detailed understanding of mechanisms of solute transfer at the endophyte-host interface is still lacking. The authors assess the various hypotheses erected over the years to account for the structure and function of the various interfaces formed in mycorrhizas of this type and relate new ideas on function to their possible ecological significance. JA Callow
Mutualism and Parasitism: Diversity in Function and Structure in the "Arbuscular" (VA) Mycorrhizal Symbiosis
F. A. SMITH and S. E. SMITH'
Department of Botany, 'Department of Soil Science, The University of Adelaide, SA 5005, Australia
I.
Introduction: Names and Structures
......_. ....................................,.
1
3 11. Transfer of Solutes Between Fungus and Host ................................ A. Mechanisms of Nutrient Transfer ........................................... 5 B. Is there Spatial Separation of Transfer of Phosphate and Carbon? ............................................................................. 6 111. Variations in Structure: Two Classes of VA Mycorrhizas IV.
V.
VI. VII. VIII.
..................
9
Variations in Nutritional Efficiency in VA Mycorrhizas .............. ..,.. 17 A. Cheating by the Fungus ....................................................... 17 B. Cheating by the Host: Linked Plants ..................................... 22 Cheating by Myco-heterotrophic (Achlorophyllous) Plants
...............
25
Costs and Benefits of the VA Mycorrhizal Symbiosis: Fast and Slow-growing Plants and Related Issues ........................................ 26 Development and Control of the VA Mycorrhizal Symbiosis
.......-....
29
Conclusions ............................................................................. 33 Acknowledgements ...............................................................- .... 34 References ............................................................................... 35
I.
INTRODUCTION: NAMES AND STRUCTURES
Mycorrhizas are very widespread symbioses both geographically and in terms of the numbers of plant species involved (Brundrett, 1991; Read, 1991; Trappe, 1987). In many cases (but not aI1) the symbiosis is mutualistic in that Advances in Botanical Research Vol. 22 incorporating Advances in Plant Pathology
ISBN 0-12-005922-3
Copyright 0 1996 Academic Press Limited All rights of reproduction in any form reserved
2
F.A. SMITH and S.E. SMITH
it improves nutrition and growth of the plant while supporting growth of the fungus. The most common mycorrhizas involve fungi of the Order Glomales that colonize an enormous range of plants, including herbs, shrubs and trees, and even some lower terrestrial plants (bryophytes and pteridophytes). These mycorrhizas were called vesicular-arbuscular (VA) mycorrhizas on account of the characteristic intraradical structures that are formed, arbuscules being the “little tree”-like structures consisting of branched hyphae (Gallaud, 1905). Recently, there has been a concerted move to drop “vesicular” from the name, since not all of the fungal genera form vesicles (Walker, 1995). Also, emphasis on “arbuscular” fungi is in accord with the prevailing view that the symbiotic function is due to the activity of the arbuscules. However, even the simpler name can be hazardous, since sometimes the major intracellular fungal structures are not arbuscules but hyphal coils. As previously (Smith, 1995), we will retain the old abbreviated name for continuity and to avoid undue structural and functional emphasis on arbuscules alone. In many cases - especially in those that have been commonly studied the colonization process resulting in the formation of a VA mycorrhiza leads to the development of two distinct interfaces. One is intercellular, where the fungal hyphae grow between cortical cells, and the other is intracellular, where the hyphae penetrate cortical cell walls to form arbuscules. In other cases the intercellular interface is absent or insignificant; instead there are extensive intracellular hyphal coils from which arbuscules are formed. Strictly, the term “intracellular” is misleading, since the symbionts are still separated by two plasma membranes and cell-wall material (Smith and Smith, 1990, 1995). Hence they are separated from the host cell by an apoplast, just as the intercellular hyphae are separated from the adjacent cells of the host by a (much larger) apoplast. Vesicles, to which we will pay little further attention, can be intracellular or intercellular. These structural matters are important, as a major purpose of this review is to consider the possible roles of the interfaces between the fungal endophytes and their hosts, particularly in relation to the transfer of solutes on which the symbiosis (whether mutualistic or otherwise) depends. After assessing briefly the validity of traditional perceptions of the nutritional basis of the VA mycorrhizal symbiosis we discuss issues that relate to variations in nutritional efficiency and suggest avenues for future research that might combine a functional and structural approach. This review complements recent reviews of morphological and molecular aspects of VA mycorrhizal fungi (Bonfante-Fasolo and Perotto, 1992; Giovannetti and GianinazziPearson, 1994) and of mycorrhizas in ecosystems (Brundrett, 1991).
3
MUTUALISM AND PARASITISM
EXTERNAL ENVIRONMENT: (RHIZOSPHERE)
nutrients (P etc)
+
EXTERNAL ENVIRONMENT: (AIR)
4
+
I
I
I I I
I I L - - - - - - - - - -
I
signals
*
signals
Fungal f Structure
- - - - - - - - _ -- b INTERFACE b
4
P etc organic C
co2
1 I I
I
7
Light,
+
Plant Structure
b
4
Fig. 1 . Summary of major interactions between VA mycorrhizal plants and their environment. Transfer of material such as nutrients (P, etc.) and organic C, and of energy (light) is shown as solid lines. Transfer of chemical (molecular) signals between the symbionts and their environments is shown as broken lines. For details, see text.
11.
TRANSFER OF SOLUTES BETWEEN FUNGUS AND HOST
Figure 1 summarizes very simply the established physiological interactions in VA mycorrhizas, with emphasis on transfer of phosphate (P) and other nutrients from soil to fungus and then, via the fungal/plant interface(s), to the host. In return, the fungus depends on a supply of photosynthate. This traffic of solutes may be regulated by chemical signals, here undefined. Figure 1 also shows that both fungus and plant can influence the external (soil) environment within the rhizosphere. Again, chemical signals are thought to be involved in events associated with both growth of the fungal propagules and colonization of the host by the fungus. For example, it has been suggested that colonization is influenced by leakage of organic carbon (C) from the host, this being increased at low external concentrations of P (Graham etal., 1981; see also Graham and Eissenstat, 1994). This hypothesis is, however, controversial (e.g. Amijee etal., 1993; Giovannetti eta/., 1994; see also Anderson, 1992; Koske and Gemma, 1992). Figure 2 summarizes in a conventional way the fluxes of solutes across the
4
F.A. SMITH and S.E. SMITH
Interface
Soil
Pi-
NH4+
Zn*+
other ions (e.9. K+)
fPm
Fungus
..
Pi-
1 .
... . I
.. 1.
fpm
. .. .
Ncornpounds
Zn*+
other ions
(e.g. K+)
Root
4B-b
.. 1h
4 B - b
4-
+b 4
rPm
::
A h
- - - - - - - - 4F - - - - - - -4 - -
sugars growth factors?
H+
Fig 2. Membrane transport of inorganic nutrients and organic C in “normal” VA mycorrhizas, showing for simplicity simultaneous bidirectional transfer of nutrients, organic C and growth factors across a generalized interface. The presumed occurrence o f transport ATPases for Hf is also shown. fpm, fungal plasma membrane; rpm, root plasma membrane.
plasma membranes of fungus and host, via a generalized interface. It is generally supposed that bidirectional transfer of solutes in VA mycorrhizas occurs solely and simultaneously across the arbuscular interface. However, as Rhodes and Gerdemann (1980) pointed out, this supposition lacks evidence and arose from two beliefs. One was that, as individual arbuscules have a lifespan that is shorter than that of the root, their senescence and partial breakdown would release P to be taken up by the host. This does not account for uptake of organic C (e.g. sugar) by the fungus, of course. If the sugar were taken up earlier by an “active” arbuscule, the transfer would not be simultaneous in time in the same arbuscule, though it would in the same root, since there will normally be arbuscules of different ages in the one root. In fact, a series of elegant calculations by Cox and Tinker (1976), utilizing measured inflows of P into mycorrhizal roots of Allium cepa (onion) infected by Glomus mosseae and of arbuscular surface areas obtained by image
5
MUTUALISM AND PARASITISM
TABLE I Areas of fungus/host interfaces and estimafed fluxes of P from Glomus sp. ‘City Beach’ (WUM 16) ~~
Harvest time (days)
21 42 63
Infection (0101
64 69 82
Area of interface (m’m[root]-’ x
Flux of P (nmol rn-’s-’)
Arbuscules
Hyphae
Via arbuscules
Via total area
2.33 2.65 0.44
0.31 0.99 0.89
12.8 9.0 5.9
11.3 7. I 3.7
Data are derived from Smith and Dickson (1991) and Smith e f a[. (1994). P fluxes were estimated from inflows of P into mycorrhizal and non-mycorrhizal plants, and are average values over the periods 0-21, 21-42 and 42-63 days.
analysis, showed that in this symbiosis the release of P from senescing arbuscules would be far too slow (by a factor of about 100) to account for P transfer to the host. They emphasized that continuing transfer, presumed to be across only the arbuscular interface, was necessary. Smith and Dickson (1991) and Smith etal. (1994), again using the Allium/ GIomus symbiosis, refined this approach in two ways. One was to stain only living fungal material with nitro blue tetrazolium and the other was to apply computerized image analysis to calculate separately the surface areas of the arbuscular and intercellular interfaces. The fluxes of P across the arbuscular interface were in accord with the single value obtained by Cox and Tinker (1976). More importantly, the work showed that in this symbiosis the contribution of the hyphal interface was too large to be ignored. It increased with time, and after about 50 days its surface area per unit root length was considerably greater than that of the active arbuscules, which was reduced. Representative results are summarized in Table I. The work raised serious doubts about the second (original) reason for focusing solely on the arbuscule as the site of nutrient transfer, namely that it looks to be the right shape: the surface area/volume ratio is large and the wall material is very thin compared with the walls separating the other interfaces. However, the above calculations aside, there is no reason to believe that the walls provide a physical barrier to prevent nutrient transfer at other interfaces (cf. Bonfante-Fasolo, 1984). After all, nutrient transfer in other mycorrhizas is across non-arbuscular interfaces, whether intercellular (ectomycorrhizas) or intracellular (mycorrhizas of the Ericales and orchids). A. MECHANISMS OF NUTRIENT TRANSFER
In the absence of definitive evidence about the nature of individual membrane transport proteins at VA (or other) mycorrhizal interfaces a number of workers have resorted t o speculation, basing their proposals on developments
6
F.A. SMITH and S.E. SMITH
in knowledge of transport mechanisms in free-living fungi and plants. The first detailed analysis of the membrane transport mechanisms that might be involved was done by Woolhouse (1975), who pointed out that transfer of P from fungus to host requires passive efflux across the fungal plasma membrane at the interface (presumed to be the arbuscule - see above), and active influx across the plasma membrane of the host (the periarbuscular membrane). Conversely, transfer of sugar will involve passive efflux from the host and active influx to the fungus. More recently, Clarkson (1985) and Smith and Smith (1986) proposed that activity of H+-ATPases in both fungal and host plasma membranes at an interface (see Fig. 2) would "energize" the membranes by setting up proton motive forces to drive the active influxes of sugar and P by cotransport with H + . Such ATPase activity is a feature of more recent models, such as that of Schwab etal. (1991). Various novel transporters involving direct coupling of P and C transport at one or other of the two membranes have been canvassed (e.g. Woolhouse, 1975; Schwab et ~ l .1991; , Tester etal., 1992) but there is no evidence for any of these on the plasma membranes of free-living plants or fungi. In all cases, overall independence of transport of P and C has been proposed, to allow nonstoichiometric fluxes of these solutes. The flux on which most attention has been focused is efflux of P from the fungus to the interface. As there usually seems to be little efflux of P from free-living fungal or plant material (see Beever and Burns, 1980) it has been suggested that there is a need for a "special" mechanism - that is, enhanced efflux of phosphate from the fungus at the interface, possibly through a gated ion channel (e.g. Smith and Smith, 1990; Tester etal., 1992). It also seems that active transport of P into the fungus must be turned off at the interface, otherwise the efflux would be nullified. It is always necessary to bear in mind that, as P is not the only nutrient that moves from fungus to host, other transport proteins must be present at the interfacial membranes. This also renders unlikely any novel transport protein directly coupling transport of C from the host with influx of P alone (Smith and Smith, 1990). B.
IS THERE SPATIAL SEPARATION OF TRANSFER OF PHOSPHATE AND CARBON?
All of the above mechanisms could exist in plasma membranes at any of the VA mycorrhizal interfaces, whether intracellular or intercellular, As already noted, it is generally assumed that there is simultaneous bidirectional transfer of P and organic C (see Fig. 2) and that this occurs at the arbuscular interface alone. The only direct evidence in support of this traditional model came from cytochemical studies of the activity of membrane-bound ATPases that might be involved, directly or indirectly, in nutrient transfer. Using the onion (Allium)/Glornus and sycamore (Acer)/Glomus symbioses, Marx et al. (1 982) found that the fungal plasma membranes in fine arbuscular branches and
7
MUTUALISM AND PARASITISM
TABLE I1 Occurrence of A TPase activity on plasma membranes of Glomus intraradices colonizing A. cepa Inhibitors
Interface Hyphal
None Molybdate Vanadate or DES CONCLUSIONS: H+-ATPase activity
Arbuscular
Plant
Fungus
Plant
Fungus
(+)
(-1
++ ++
++ ++
(-1
(-)
(-)
(-1
(-)
No
Yes
Yes
No
++
Summarized from Gianinazzi-Pearson el a/. (1991b). ++ , strong activity; ( ), weak activity; ( - ), no or variable activity. DES, diethylstilbestrol.
+
the periarbuscular membrane both showed ATPase activity. The peripheral plasma membrane of uninfected cortical cells also showed ATPase activity, but in infected cells this was weak except in the early stages of formation of arbuscules or where arbuscules were senescent. The membrane-bound ATPase activity was sensitive to the transport inhibitor diethylstilbestrol (DES). In addition there was considerable intracellular (e.g. vacuolar) activity. Other interfaces were not examined. Gianinazzi-Pearson et al. (1991b) carried out a more detailed study, again using Allium/Glomus and applying a wider range of inhibitors to distinguish possible membrane transport ATPases from general phosphatase activity. ATPase activity on membranes of the intercellular interface was also examined. A key finding was that molybdate considerably decreased the cytochemical deposition of lead phosphate, especially that not associated with plasma membranes. The results are summarized in Table 11, which shows that (according to Gianinazzi-Pearson et al., 1991b) the fungal plasma membrane in the arbuscules had little or no membrane ATPase activity, although there was significant activity in the intercellular hyphae. Conversely, the periarbuscular membrane of the plant had ATPase activity but that adjacent to the intercellular hyphae did not. Based on the assumption that a plasma membrane lacking ATPase activity was incapable of active uptake (H+-cotransport) of P or organic C, a new model was developed in which P transfer occurs across the arbuscular interface but transfer of organic C occurs across the intercellular (hyphal) interface. This model is summarized in Table IIIA, along with the traditional model. The term “Arum-type” refers to structural features of interfaces that are discussed below. The radical feature of the new model is that it spatially separates transfer of P from that of organic C and thus provides a possible explanation at the structural level for differences in “nutritional efficiency”. We define this term as the amount of P (for simplicity) transferred by the fungus per C lost from the host, and
8
F . A . SMITH and S.E. SMITH
TABLE 111 Models for transfer of phosphate and carbohydrate across fungdplant interfaces in VA mycorrhizas A. ARUM-TYPE
Interface: HyphaI
Arbuscular
Traditional model Phosphate transfer Carbohydrate transfer
-
+ +
Spatial separation Phosphate transfer Carbohydrate transfer
-
+-
Hybrid model Phosphate transfer Carbohydrate transfer
-
+ +
+
+
B. PARIS-TYPE
Structural basis for different nutritional efficiencies and cheating
No
Yes
Yes
Interface: ~
Coil
Arbuscular
-
+ +
Spatial separation Phosphate transfer Carbohydrate transfer
-
+
+-
Uniform transfer Phosphate transfer Carbohydrate transfer
+ +
+ +
Traditional model Phosphate transfer Carbohydrate transfer
No
Yes
No ~
~~~
The terms Arum- and Paris-types refer to structural features of interfaces defined in the text, as are nutritional efficiency and (later) cheating.
it is reflected by the potential positive growth response of the host compared to an equivalent non-mycorrhizal plant when P is the growth-limiting nutrient. The growth-limiting nutrient for VA mycorrhizal symbioses need not always be P and the same principle could apply to other mineral nutrients, or even to organic nitrogen (N) as amino acids and amides if these too are transferred to the plant by cotransport solely across the arbuscular interface. Put simply, according to the new model, if the intercellular fungal hyphae are particularly numerous or metabolically active compared with the arbuscules, then there is the potential for massive loss of C from the host, with little or no transfer of P , etc. The difference between the findings of Marx et al. (1982) and GianinazziPearson etal. (1991b) with respect to presence or absence of ATPase activity on the arbuscular membrane remains to be resolved. If in fact both plasma membranes at the arbuscular interface retain ATPase activity, even over only part of the lifespan of an arbuscule, then a “hybrid” model is possible which
MUTUALISM AND PARASITISM
9
allows bidirectional transfer of P and organic C across the arbuscular interface but only transfer of organic C across the intercellular interface (Table IIIA). This too would allow variation in nutritional efficiency, depending on arbuscular activity vis-a-vis that of the intercellular interface. Although direct evidence of differential activity of plasma membrane transport ATPases at the two interfaces has been obtained from one investigation with one VA mycorrhizal symbiosis, there is indirect evidence for spatial separation of transfer of P and organic C. Brundrett et al. (1985) found that arbuscules were not formed in leek (Allium porrum) until after external and internal hyphae had reached their maximum extent, which means that carbon requirements for early development that could not be met by the fungal propagules would have to be obtained via intercellular hyphae. More importantly, there is a mutant of Pisum that prevents VA mycorrhizal colonization from proceeding beyond the formation of appressoria and another that allows development of extensive intercellular hyphae but only abortive arbuscules without membrane ATPase activity (Gianinazzi-Pearson et al., 1991a, 1994). In the latter case, the carbohydrate supply must be provided via the intercellular hyphae to the fungus, which presumably grows parasitically. Hall (1977) noted that a strain of Glomusfasciculatus that gave no positive growth response in Cosmospora robusta produced no arbuscules. He suggested that the numbers of arbuscules produced by different endophytes that vary in their efficiency should be compared in a single host and soil combination. A more rigorous approach requires estimation of the surface areas of active arbuscules compared with total fungal biomass (external and internal) per unit root length. This has not been attempted on a comparative basis, as far as we are aware, despite a vast literature on differences in P nutrition, growth response, etc. that are caused by colonization of a single host species by different strains or species of VA mycorrhizal fungi. There is also a vast literature on effects of different environmental conditions, some of which describes changes in formation of arbuscules and suggests that carbon transfer can be decoupled from P transfer. This work is discussed later, as it is relevant to the issue of “cheating” by VA mycorrhizal endophytes. Although present evidence is limited, we believe that the possibility that transfer of P and other inorganic nutrients is spatially separated from that of organic C has important implications with respect to aspects of diversity in both structure and function of VA mycorrhizal symbioses, as follows.
111. VARIATIONS IN STRUCTURE: TWO CLASSES OF VA MYCORRHIZAS Gallaud (1905) described two classes of endomycorrhizas which are now recognized as VA mycorrhizas. These were named from the “type species” in which they were described: the Arum maculatum type and the Paris quadrifolia type:
10
F. A. SMITH and S. E. SMITH
a)Arum type Fig. 3. (1905).
b) Paris type
Interfaces in the two classes of VA mycorrhizas, first described by Gallaud
see Figs 3 and 4. The definitive feature of the Arum-type is that the roots contain extensive cortical intercellular spaces. Having penetrated the epidermis and (where present) the exodermis, fungal hyphae enter the intercellular spaces and can grow rapidly, resulting in the intercellular (hyphal) interface described above. Penetration into cortical cells is then essentially a second colonization stage, after which there occurs formation of intracellular arbuscules and, frequently, vesicles. The mycorrhizas of AIlium spp. (seeTable 11) are classic Arumtypes. Puris-types develop in roots (or other underground organs of lower land plants; see below) which apparently do not have extensive intercelluIar spaces. Once the fungus has penetrated the epidermis and (where present) the exodermis, its growth is intracellular, and coils of hyphae are formed that are similar to, but much more extensive than, those that can form in exodermes of both classes of VA mycorrhizas. The arbuscules are usually intercalary structures rather than, as in Arum-types, terminal on hyphal branches. There are sometimes very few arbuscules and these may be relatively long-lived: see below. Gallaud (1905) stated that in the plants that he examined the Arum-type was more common and gradually the distinctive features of the Puris-types have come to be either ignored or regarded as a curiosity. One complication is that roots can contain a range of fungal endophytes that have some features in common with Glomalean fungi, such as the formation of mainly aseptate hyphal coils, vesicles, but apparently no arbuscules. Examples occur in the
’
’
Intracellular hyphal coils are of course a feature of mycorrhizas of the Ericales and those of orchids, which are taxonomically and physiologically well defined. We will not discuss these here except to note that a cytochemical study by Serrigny and Dexheimer (1985) showed the presence of membrane-bound ATPases at the intracellular interface of an orchid mycorrhiza. There are other endophytes which have coils of highly septate hyphae, are generally considered “non-Glomalean”, and whose taxonomic affinities and physiological features are still unresolved: see Harley (1969).
MUTUALISM AND PARASITISM
11
Fig. 4. Illustrations photocopied from Gallaud (1909, retaining the original numbers of the illustrations, and with translations of the captions. Arum-types: 1 , Arum maculatum, longitudinal section. Penetration at the base of a root-hair; intracellular then intercellular hyphae, with a vesicle and arbuscules. 27, ANium
Caption continued over page
12
F.A. SMITH and S.E. SMITH
Araliaceae and Cornaceae (Boullard, 1953a,b; see also Harley, 1969). ECOlogical surveys of the occurrence of VA mycorrhizas have mostly concentrated, not surprisingly, on the occurrence of arbuscules and vesicles, rather than on other hyphal features which are not solely characteristic of VA mycorrhizal fungi (see, however, Louis, 1990). Furthermore, most of the experimental studies have involved cultivated plants that form Arum-type VA mycorrhizas, rather than wild plants in which Paris-types have been reported. Hence the occurrence of Paris-types may have been underestimated. The studies by Brundrett and Kendrick (1990a,b and references therein) on herbaceous woodland plants in Canada, which include both classes of VA mycorrhiza, have come as a valuable corrective. Brundrett and Kendrick (1990b) suggested that it is not yet clear which class is the most common and, having delved into the literature, we see no reason to disagree. Table IV is a selected list of publications which include illustrations and descriptions of recognizably Arum- and (particularly) Paris-type VA mycorrhizas in pteridophytes, gymnosperms and angiosperms. With only a few exceptions, only examples which show arbuscules are included though, as has been noted above, there are many descriptions of Paris-type structures (intracellular coils and vesicles; aseptate or sparsely septate hyphae) that do not mention arbuscules or which describe arbuscules as sparse or absent. Thus the early extensive survey of tropical plants in Java by Janse (1897) has been excluded from Table IV, although it included examples that are almost certainly Paris-types on the basis of the features that he recorded. These included intracellular coils and vesicles as well as the “sporangioles” that were often senescent arbuscules, as can be seen from his drawings. Nicolson (1959), in surveying VA mycorrhizas in Gramineae (in England), stated: “In grass roots, the main development of hyphae (our emphasis), arbuscules and vesicles is intracellular, with the latter two structures localized in layers.. . . With infection in some species, and particularly in Avena sativa, hyphae and vesicles may be intercellular . . .”. He pointed out that this distinction corresponds to Gallaud’s Paris and Arum classes; the implication is of course that Paris-types were the more common in his survey. Localization of arbuscules near the stele of some Paris-types has been described by others (e.g. Brundrett and Kendrick, 1990b). It applies also to many Arum-types (e.g. Saif, 1977; Abbott, 1982; see Harley and Smith (1983). It must be emphasized that Table IV is far from a complete list and is biased towards Paris-types to illustrate that these are far from unusual. For example, Paris-type VA mycorrhizal structures (not always including arbuscules) are. present in absorbing organs of lower land plants including some bryophytes, and an enormous range of sphaerocephalum, longitudinal section. Paris-types: 16 and 17, Paris quadrifolia, longitudinal and transverse sections. 3 1 , Anemone nemorosa, longitudinal section, ap, epidermis; as, exodermis; pa, root-hair; c, passage cell; end, endodermis; ac, compound arbuscule.
13
MUTUALISM AND PARASITISM
TABLE IV Selected publicafions with descriptions and ilfusfrationsof Arum- and Paris-type VA mycorrhizas Arum-types Paris-types Pteridophytes Psilotaceae Marattiaceae Pteridaceae (etc.)
Shibata (1902)'; Peterson et a/. (1981)' Angiopteris Gallaud (1905) Pteridium (etc.) Cooper ( I 976)
Psiloturn
Gymnosperms Podocarpaceae
Podocarpus
Gingkoacaeae
Gingko Taxus Sequoia
Taxaceae Taxodiaceae Angiosperms a. Monocots: Araceae Burmanniaceae Liliaceae
A risaema Arum
Thismia Allium Ruscus Smilacina
Dioscoraceae Gramineae
Paris Erythroniurn Trillium Tamus Molinia
zea b. Dicots Aristolochiaceae Aceraceae Cucurbitaccae
Asarum Acer
Cucurbito Luff0
Gentianaceae Leguminosae Magnoliacae Meliaceae Ranunculaceae Saxifragaceae Violacacae
Reference
Gentiana Trifolium' Vicia G/ycine Liriodendron Khaya Anemone Ranunculus Parnassia Viola
Shibata (1902)'; Baylis et a/. ( 1963) Bonfante-Fasolo & Fontana (1985) Strullu etal. (1981) Gallaud (1905)
Brundrett & Kendrick (1990b) Gallaud (1905) McLennan (1958) Gallaud (1905); Mosse (1973); Hayman (1974); Brundrett eta/. (1985) Gallaud (1905) Brundrett & Kendrick (1 990b) Brundrett & Kendrick (1 990b) Gallaud (1905) Endrigkeit (1937) Gerdemann (1965) Brundrett & Kendrick (1990b) Kessler (1966); Frankland & Harrison, 1985; Yawney & Schultz (1990) Saif (1977) Saif (1977) Jacquelinet-Jeanmougin & Gianinazzi-Pearson (1983); Kahn & Weber (1986) Abbott & Robson (1978) Holley & Peterson (1979) Carling & Brown (1982) Kinden & Brown (1975) Redhead (1 968) Gallaud (1905) Gallaud (1905) Gallaud (1905) Gallaud (1905)
Generic names do not imply that all species within the genus form the same type, though we have found no examples where this is not the case. No arbuscules recorded. Intracellular hyphae in cortex.
'
14
F.A . SMITH and S. E. SMITH
pteridophytes besides those shown in Table IV (Stahl, 1949; Boullard, 1958; Cooper, 1976; Berch and Kendrick, 1982; Duckett and Ligrone, 1991; see also Harley, 1969 and references therein). The existence of the two distinct types of VA mycorrhizas does not mean that there cannot be structures in the one plant that show features of both, such as development of both extensive intracellular coils and intercellular hyphae in roots or other absorbing structures with restricted intercellular spaces (see Bonfante-Fasolo, 1984). The bryophyte Phaeoceros shows some such combination of features: its gametophyte has intercellular hyphae, with intracellular hyphal “ramifications” and Paris-type intercalary arbuscules (Ligrone, 1988; see also Stahl, 1949). There are mentions of intercellular and intracellular hyphae in Griselinia (Cornaceae) by Greenall (1963), in Vitis (Bonfante-Fasolo, 1984) and in Liquidambar (Ling-Lee et al., 1975). VA mycorrhizas in this last plant have been much studied, but we have seen no illustrations or other reference to it having Paris-type features more recently than the descriptions by Janse (1897). Other examples include Trifofium subterraneum and maize; these are discussed below. Finally in this catalogue, the VA mycorrhizas formed by Gigaspora margarita in Ri T-DNA-transformed roots of carrot (BCcard and Fortin, 1988; Becard and Piche, 1989) appear to have Paris-type features: the light micrographs suggest the presence of hyphal coils within the cortical cells, while limited growth of intercellular hyphae was also recorded. It is important to stress that the definitive fungal structures that distinguish the two types (presence or absence of an extensive intercellular interface) apparently result primarily from the anatomy of the root or other absorbing organ, so that the type of VA mycorrhiza that develops is controlled genetically by the host. Strong evidence for such control was provided by the demonstration (Gerdemann, 1965) that the same Glomalean fungus produced a Paris-type VA mycorrhiza in Liriodendron (tulip tree) and an Arum-type in maize. Similarly, Jacquelinet-Jeanmougin and Gianinazzi-Pearson (1983) showed that the development of Paris-type structures in Gentiana was produced by Glomalean fungi that formed Arum-types in Aflium. Control of major fungal structures by the genome of the host was also emphasized by Daniels-Hetrick et al. (1985). Nevertheless, other work suggests that the fungus has some control over these structures, as well as lesser ones, such as hyphal diameter, etc. A comparison of several Glomalean endophytes in Trifolium subterraneum showed formation of extensive “100ps” of intracellular hyphae in the outer cortex with intercellular hyphae plus (intracellular) arbuscules in the inner cortex (Abbott, 1982). Acaufospora spp. were interesting exceptions in that they produced intracellular hyphae throughout the cortex of T. subterraneum. Descriptions of the hyphal structures in maize also suggest differences in formation of cortical hyphal coils (near sites of initial colonization) and intercellular hyphae (e.g. Winter, 1951; Meloh, 1963; Gerdemann, 1965; Kariya and Toth, 1981; Morton, 1985). However, in
MUTUALISM AND PARASITISM
15
comparing these studies, differences due to the use of different maize cultivars cannot always be ruled out. There is renewed interest in the occurrence of VA mycorrhizas in plants of different ecosystems and hence considerable opportunity to increase our knowledge of the biological diversity in intraradical fungal structures. In particular, the infrequency of arbuscules in some Paris-types and even (apparently) their complete absence in some cases, deserves further study. It is likely that there are seasonal effects, for example in long-lived roots, where P transfer might cease in autumn or winter. Also, there may be effects produced by other environmental factors, for example by shade, similar to those demonstrated experimentally in Arum-types. Infrequency of arbuscules in VA mycorrhizal roots of Acer saccharum (sugar maple) in a hard-wood forest in Quebec was recorded by Cooke etal. (1992) and it was suggested that the trees were stressed and that productivity was declining. In contrast, Yawney and Schultz showed well-developed arbuscules in young A. saccharum, as shown in Fig. V. A further study of the development of these Paris-type mycorrhizas (Cooke etal., 1991) showed that the numbers of vesicles and arbuscules varied with nutritional conditions, decreasing with addition of potassium, calcium and magnesium carbonates. Brundrett and Kendrick (1990a,b) showed that the arbuscules in A. saccharum are much longer-lived than those in Arum-types such as Allium. Very few detailed studies have been done with Paris-types that allow comparison with those of Arum-types whose physiology has been described above. Work with Acer spp. is an exception. As well as that already mentioned, Marx et al. (1982) compared the membrane-bound ATPases of the arbuscular interfaces in sycamore (Acer pseudoplatanus) and onion (Allium cepa). Although this was not noted at the time, A. pseudoplatanus has Paris-type VA mycorrhizas. This is shown by more recent illustrations by Frankland and Harrison (1985). Hence, the work of Marx etal. suggests, perhaps not surprisingly, that there is no functional difference between the arbuscules of the two classes, but there is still the caveat over the presence of ATPase in the fungal membrane as shown by the work of Gianinazzi-Pearson et al. (1991b). The question of the role of the intracellular hyphae (coils) of the Paris-types in nutrient transfer is unresolved and the situation is even riper for speculation than that for Arum-types. The traditional expectation would be that P and organic C are exchanged only across the arbuscular interface (Table IIIB), which would mean that active arbuscules are needed for a nutritionally efficient mycorrhiza; if these were few in number the fluxes of solutes might have to be high. However, it seems totally unrealistic to ignore the other intracellular interface - the coils - as a site for transfer, a point also made by Reinsvold and Brent Reeves (1986). We reiterate that in other, non-arbuscular, endomycorrhizal associations (those of the Ericales and of orchids) transfer of organic C , P or both must involve the coils. Thus there may be Yuniformntwo-way transfer across both interfaces (Table IIIB) or there
16
F.A. SMITH and S.E. SMITH
Fig. 5. Paris-type interfaces in the VA rnycorrhizal symbiosis between sugar maple (Acer sacchurum) and Glomus etunicutum. Scanning electron micrographs of (a) hyphal coils and (b) arbusculate coils. Photographs by W. J. Yawney (see also Yawney and Schultz, 1990).
MUTUALISM AND PARASITISM
17
again may be functional specialization, for example with C uptake only into the coils and loss of P only from arbuscules, where present (Table IIIB). Depending on which case is correct, absence of arbuscules may not impair the mutualistic ability of a Paris-type VA mycorrhiza, or it may allow only transfer of C, and hence a parasitic association. Seasonal effects (lack of arbuscules) might be associated with decreases in P transfer to the host. In other words, the possibilities for solute exchange are analogous to those in Arum-type VA mycorrhizas, with the intracellular coils replacing the intercellular hyphae in the latter as a non-arbuscular interface of significant surface area.
IV. VARIATIONS IN NUTRITIONAL EFFICIENCY IN VA MYCORRHIZAS We have already referred to examples where the nutritional efficiency in VA mycorrhizal symbioses varies from what is considered the normal mutualistic responses associated with two-way transfer of mineral nutrients (P, etc.) for organic C. In extreme cases, far from being symbiotic, either the fungus or the plant becomes parasitic - that is, in ecological terms the partner is then a “cheater” in that it obtains benefit@) while depriving the other partner of its benefit(s) (Soberon and Martinez del Rio, 1985; see also Janos, 1987). A.
CHEATING BY THE FUNGUS
There are many examples in the literature where plants do not show significant positive growth responses when colonized by VA mycorrhizal fungi and others where growth is depressed (responses that are usually considered undesirable by an investigator who is interested in maximizing the benefits of VA mycorrhizas). Sometimes the growth depressions occur only in the early stages of growth and different species or strains of endophyte can have different effects on the one host. These effects have been found often in experiments in glasshouses or growth-cabinets and also occur in the field. Colonization of a single host by species or strains of VA mycorrhizal fungi that do not produce positive growth responses has been reported in many comparative studies (e.g. Mosse, 1972; Abbott and Robson, 1977, 1978; Sanders et al., 1977; Menge et al. , 1978; O’Bannon et al., 1980; Azcdn and Ocampo, 1981; Schenck and Smith, 1982; Van Nuffelen and Schenck, 1984). Apart from these differences, three environmental conditions have been shown to produce negative growth responses. The first two are low light and low temperature, which can result in limitation of photosynthesis and hence in a drain of organic C to the fungus that can become excessive when compared to the gain of P - that is, there is decreased or negative nutritional efficiency. These effects were nicely shown by Hayman (1974) with Allium cepa (onion).
18
F.A. SMITH and S.E. SMITH
Decreased light intensity, temperature or both, inhibited the formation of arbuscules more than the total colonization, and under the most severe conditions the mycorrhizal plants were the smaller. Similar effects on growth, with different light and/or temperature regimes depending on the plants under investigation, have been found in many experiments by others, including ourselves (Son and Smith, 1988; see also Smith, 1980; Miller, 1987). The third condition that can lead to mycorrhizally induced growth depression involves provision of high levels of mineral nutrients, especially P. This results in improved mineral uptake by the non-colonized parts of the root system, and the external fungal hyphae give no additional benefit in terms of plant growth, although they may continue to absorb and transfer P so that the P concentration in mycorrhizal plants is sometimes higher than that of non-mycorrhizal plants of the same or lesser size (Stribley etal., 1980). Sometimes colonization is reduced by the high levels of external nutrients the plant rejects the “unnecessary” symbiont, but the levels of nutrients that produce this effect vary considerably with different fungudhost combinations and growth conditions (e.g. Abbott and Robson, 1978; Thomson et al., 1986). An early demonstration of these effects was by Mosse (1973). Growth of both non-mycorrhizal and mycorrhizal onion plants was depressed at high P levels, with larger decreases in mycorrhizal plants. These latter were attributed to P toxicity caused by the enhanced absorption of P by the mycorrhizal plants. However, the results showed no correlation between internal P concentrations and the mycorrhizal growth depressions, which sometimes occurred when mycorrhizal plants had the same or even lower concentrations of P than the non-mycorrhizal plants. Mosse (1973) noted that as external P was increased the form of the infections changed dramatically. Arbuscules became “smaller and much fewer” but hyphal development was extensive both inside and outside the roots. At high levels of P the growth patterns changed t o the extent that “hyphal growth appeared to be entirely uncontrolled by the root structure and in this respect resembled that of a root pathogen, but there was no discoloration or hypertrophy of the root tissue.. . . Only the connection of these abnormal hyphae with much more normal . . . mycelium on the root surface made it credible that these could be growth forms of the same fungus”. The published illustrations clearly show these “abnormal” structures, which included development of frequent septa in some cells. Many other workers have found similar depressions on growth caused by VA mycorrhizal fungi at high external P and it is often suggested that the depressions are caused by the drain of C to the fungus (e.g. BuwaIda and Goh, 1982; see also Cooper, 1984). However, Amijee etal. (1989) favoured P toxicity as the cause and pointed out that when colonization is decreased by high external P the C demand by the fungus should be small. This issue will of course depend on the density of external hyphae and indirect C costs associated with colonization, including structural effects and root respiration. In some studies, high external P decreased the numbers of arbuscules independently of effects on
19
MUTUALISM AND PARASITISM
‘cheater’: C receiver
P
a) normal symbiosis
b) ‘cheater’
fungus
C)
normal symbiosis?
d) ‘cheated’
fungus
Fig. 6. Possible variations in VA mycorrhizal symbioses. (a) the normal symbiosis, with bidirectional transfer of P and organic C (not necessarily across the same interface); (b) cheating by the fungus (no transfer of P to the plant); (c) the “normal” symbiosis in a donor plant that is linked to a receiver plant (d) via a fungus that transfers both P and organic C to the receiver. The donor and receiver plants may or may not be of the same species. External fungal hyphae are shown by thick broken lines. For details, see text.
overall colonization (e.g. Son and Smith, 1988; Braunberger et a/., 1991; Bruce et al., 1994), but in others this was not the case (e.g. Abbott and Robson, 1979; Amijee et al., 1989). Apart from these examples, little attention has been paid to the details of the changed fungal structures. Often it is not possible to disentangle the various environmental factors that might cause growth depressions in VA mycorrhizal plants whether growing alone or in mixed communities (e.g. Crush, 1973; Hall, 1978; Sparling and Tinker, 1978; Bethlenfalvay ef al., 1982a,b; Abbott and Robson, 1985; Modjo and Hendrix, 1986). Nevertheless, whether or not there is a depression in growth, it is obvious that host plants d o not always obtain the maximum possible nutritional benefits of VA mycorrhizal symbioses, that cheating by fungal partners undoubtedly occurs and that the degree of cheating (nutritional efficiency relative to the theoretical maximum) varies. Figure 6(b) summarizes this situation, compared to a “normal” VA mycorrhizal association (Fig. 6a). Spatial separation of the processes involved in transfer of organic C and P is very relevant to the issue of cheating (see Table 111). If, as suggested by Gianinazzi-Pearson etal. (1991b), the fungus obtains C but does not lose P in an intercellular apoplast, then cheating ought to be relatively easy in Arum-type VA mycorrhizas, where this interface can be extensive, and especially under conditions where development of arbuscules is suppressed.
20
F. A. SMITH and S. E. SMITH
This situation cannot apply to Paris-types because these structures have few if any intercellular hyphae, and the surface area of arbuscules, where present, must be small when compared with that of the coils. It seems to us, therefore, that whether or not the fungi in Paris-types can cheat their host may depend on whether the coils can absorb organic C but not lose P, or whether both of the intracellular interfaces are nutritionally ”tight”, allowing simultaneous transfer of P and C. To work out the structure/function interactions will require detailed comparative studies of ecophysiology and growth responses in the two classes of VA mycorrhizas and, ideally, measurements of transport ATPase activity at the various interfaces. Meanwhile, we can make the obvious and testable prediction that if the model for spatial separation of transfer of P and organic C in the Arum-types is correct, successful fungal cheaters ought to produce extensive intercellular and external hyphae but few or no active arbuscules, while in the Paris-types cheating might also be associated with absence of active arbuscules. (The emphasis on active arbuscules again ought to be obvious.) It is interesting that Atriplex confertifolia, a shrubby member of a family generally regarded as “non-mycorrhizal” can become colonized by VA mycorrhizal fungi when nearby plants of other (non-chenopod) species are themselves VA mycorrhizal (Miller et al., 1983). The main intraradical structures in A. confertifolia are hyphal coils, with very few arbuscules. The authors suggested that in this case there was parasitism by the VA mycorrhizal fungus, presumably via the coils. Further work on plant mutants that allow only restricted VA mycorrhizal colonization will provide a good opportunity to throw light on these issues. Since - despite the tenor of this review so far - the control of transfer of P or other inorganic nutrients across the interface(s) may not be the process that always determines nutritional efficiency of the symbiosis under low nutrient conditions, it will be important to assess how much external mycelium can be supported under conditions where cheating might occur. The reason is that decreases in external mycelium would indirectly reduce transfer of P, etc. to the host by restricting uptake from the soil (e.g. Graham et al. , 1982; Graham and Eissenstat, 1994). The overall effect (little or no positive growth response) would resemble that of a fungal cheater which had extensive colonization both internally and externally. Abbott and Robson (1 985) found that Scutellospora (Gigaspora) calospora infecting Trifolium subterraneum produced many more external hyphae than did Glomus fasciculatum, but did not increase growth of the host whereas G. fasciculatum did. In other work with the same pair of fungi, S. calospora did increase growth of T. subterraneum but was less effective than G. fasciculatum and had a greater demand for organic C (Thomson etal., 1986, 1990b). Thus the former fungus behaved as a cheater, though to different extents in different experiments. This was confirmed in an impressive series of studies which showed that S. calospora translocated much less ”P to the host but accumulated more 32P in its hyphae than other VA mycorrhizal fungi tested, although the proportion
MUTUALISM AND PARASITISM
21
of I4C allocated below-ground was fairly similar in all cases (Pearson and Jakobsen, 1993, and references therein). Thomson et al. (1986) commented that although S . calosporu had the same number of arbuscules within roots relative to its internal hyphae as did G. fasciculatum, the total number of arbuscules per total fungal biomass was lower in S. c a hp o r a due to the larger production of external hyphae, which again fits with the notion that nonarbuscular interfaces are involved in transfer of organic C. (We have found no detailed comparison of numbers of arbuscules in these fungi, but may have missed this.) Since this strain of S. calospora behaved similarly in T. subterraneum and cucumber, it seems a prime candidate for investigation with other hosts to see if it is a constitutive (perpetual) cheater. Not surprisingly there may be multiple effects of the colonization processes on nutrient transfer and growth. Sanders et al. (1977) found that one out of four VA mycorrhizal fungi tested produced little growth increase in the host (onion). This fungus developed little external mycelium and only slowly colonized the roots. However, the authors concluded that as well as the relatively small development of fungal structures, this species also had either low uptake efficiency or transfer capacity for P - that is, there was possibly a cheating component in the effects. Various writers over the years have addressed the general evolutionary issue of why symbioses other than the most beneficial are maintained - that is, in the present context, why plants have not evolved mechanisms to prevent colonization by fungal cheaters. Harley and Smith (1983) extended the viewpoint of Vanderplank (1978) that the selection pressures on mutualistic and parasitic associations are very different, so that “mutations to resistance in mycorrhizal plants are eliminated by specificity because they are disadvantageous; and the elimination also eliminates a major source of specificity” (Vanderplank, 1978). The case that specificity (discrimination) is a defence against cheating in mutualistic associations in general is made by Soberon and Martinez del Rio (1985). Janos (1985, 1987) has explored the issue in some detail in relation to VA mycorrhizas, emphasizing that on evolutionary grounds it might be expected that only the most beneficial (efficient) VA mycorrhizas would be favoured unless low specificity is somehow maintained. As also pointed out by Yallop and Davy (1994), if the plants repelled cheaters (“mimics” in the ecological terminology adopted by Yallop and Davy), they might also repel genuinely beneficial VA mycorrhizal fungi which in most soils would be present as inoculum along with the cheaters and would often colonize the host simultaneously. Even if cheating by some VA mycorrhizal fungi is widespread, as far as we are aware there is no compelling evidence that any of the fungi are always cheaters, with the possible exception of the strain of S. calospora mentioned above. Under some environmental conditions, or with some hosts, or even with later developmental stages of the hosts, they might be true mutualists by increasing transfer of P in return for the C that they gain. This may be another
22
F . A . SMITH and S . E . SMITH
reason why low specificity is maintained. Paraphrasing Janos (1985), environmental fluctuations might change the relative efficiency of different VA mycorrhizal fungi so that none is consistently superior and, further, VA mycorrhizal colonization might be so unpredictable because of limitations in dissemination of propagules and vegetative spread of fungus that neither fungus nor host can afford to reject the symbiosis . . “because encounter of an optimal partner cannot be anticipated” (Janos, 1985). If there are constitutive VA mycorrhizal fungal cheaters, they are essentially long-lived compatible biotrophic root parasites. Lewis (1 974) proposed that mycorrhizas evolved from just such biotrophic parasitic associations. Functionally, there may also be analogies between Arum-type VA mycorrhizal cheaters and the clavicipitaceous endophytes that colonize intercellular apoplasts in the shoots of many grasses (Clay, 1990). We can also note here reports of VA mycorrhizal fungi in unexpected places, such as those of hyphae and vesicles, but no arbuscules, in the xylem of Tradescanria (Taber and Strong, 1982) and in the scale-like leaves on the rhizomes of some members of the Zingiberaceae (Taber and Trappe, 1982, using material bought in grocery stores, and Stasz and Sakai, 1984, using more conventional material!). It is not clear if these colonizations were remote from inoculum in the soil; nevertheless, the suggestion was that they represent a parasitic association that is, the fungi were acting as cheaters in the scale leaves, but not in the roots where arbuscules were found. The functional significance of VA mycorrhizal fungi in the tissues of bryophytes and pteridophytes would also repay further study, as in some cases arbuscules seem to be absent (Peterson eral., 1981; Pocock and Duckett, 1984; Duckett and Ligrone, 1991). Lastly in the context of fungal cheating, there is a danger in assuming, as has been done so far, that selective advantages of the VA mycorrhizal symbiosis lie solely or primarily in transfer of nutrients or even of water, which we do not discuss in this review (see Nelsen, 1987). Fitter (1985, 1991) emphasized that mycorrhizas can confer other benefits on their hosts, including protection against various pathogens, as with some shoot endophytes. Hence, even in the absence of significant transfer of nutrients to the host the cheaters may retain these benefits when they produce a mycorrhiza. This will be more difficult to test experimentally but is not impossible if VA mycorrhizal fungi that act consistently as cheaters can be identified.
.
B. CHEATING BY THE HOST: LINKED PLANTS
Just as one extreme of cheating in a “dysfunctional” VA mycorrhizal symbiosis is transfer of C to the fungus with no gain of P, etc. by the host (zero nutritional efficiency), the other extreme is transfer of P to the host in return for no loss of C (infinite nutritional efficiency). At first sight, this seems impossible since the fungus needs a C supply, but it can occur, at least in theory,
MUTUALISM AND PARASITISM
23
where plants of the same or different species are linked by external mycelium and one plant is a donor of organic C to the other, or at least to the mycelium. The plant that receives (or does not lose) C becomes a cheater, in the sense that it cheats both the fungus and the donor plant (Janos, 1987): see Fig. 6(c and d). Translocation of organic C from donor plants through mycorrhizal fungal hyphae to shaded receiver plants has been repeatedly demonstrated with 14C (see Miller and Allen, 1992). For example Grime eta/. (1987) showed that 14C fed to VA mycorrhizal Festuca ovina, the canopy dominant, was transferred to eight understorey VA mycorrhizal herbs when all were growing in association in experimental microcosms, but not to a non-mycorrhizal species (Rumex acetosa). In sterilized soil there was little transfer of I4C between plants. Of particular interest was Centaurium erythraea, which only survived in the mycorrhizal state, as also found by McGee (1985) with species from the Gentianaceae. McGee showed that Centaurium would grow in the absence of companion plants but could not act as a source of VA mycorrhizal inoculum for other plants. This remains unexplained (cf. Jacquelinet-Jeanmougin and Gianinazzi-Pearson, 1983). Janos (1987, 1995) has suggested that hyphal links are particularly important in humid tropical ecosystems where VA mycorrhizas can cause very large positive growth responses, and where shaded individuals, whether understorey plants or juvenile upper storey plants, might be strong sinks for photosynthate from fully illuminated plants. Of course, such temporary cheating by a receiver plant may occur in any shaded situation. Despite the ecological attractions of these ideas, it is still not clear if transfer of I4C into receiver plants does represent significant net movement of photosynthate, or whether it reflects recycling of organic C as, for example, amino acids or amides, which could involve mixing of pools of C and N derived from both donor and receiver. Such movement was discussed by Smith and Smith (1986, 1990). Transfer of amino-N (and hence organic C) is known to occur from studies with "N (e.g. Haystead etal., 1988; Newman etal., 1992; Frey and Schuepp, 1992). It is possible that experiments involving shading, the favourite experimental treatment to promote movement of C between linked plants, could well affect assimilation of N from soil surrounding the receiver plants. Movement of 14C into mycorrhizal roots of receiver plants does not mean that it leaves the fungus, of course (e.g. Waters and Borowicz, 1994). Some so-called receivers may receive P but with little net gain in organic C. However, transfer of I4C to shoots of receiver plants undoubtedly occurs under some conditions (e.g. Grime ef a/., 1987). Waters and 3orowicz (1994) compared the movement of I4C between pairs of linked Lotus cornicu/atus plants, one of which was clipped to simulate grazing. They concluded that there was net movement away from the clipped (i.e. weaker) plant, contrary to what would be expected by the hypothesis that transfer of C evens-out growth of stressed and non-stressed plants. Obviously, there is a lot to be
24
F.A. SMITH and S.E. SMITH
done to reduce uncertainty about the ecological significance of linkage by VA mycorrhizal fungi. If there is, in fact, net movement of C in large quantities as carbohydrate then, as we have also pointed out previously (Smith and Smith, 1990) the transport processes at the interface(s) between the VA mycorrhizal endophyte and the receiver plant must be very different from the normal (mutualistic) situation. Carbohydrate must efflux passively from the endophyte along with the P and be taken up across the receiver plant's interfacial plasma membranes (Fig.6d), presumably by H + cotransport, as is the P. Which interfaces are involved in this reversed transport of C is unknown. Movement of 32P between plants linked by VA mycorrhizas has also been demonstrated but in this case it is more equivocal in that the "P may be released from senescent roots into the rhizosphere from which it is absorbed by the fungus and translocated to the receiver plant. This issue has been reviewed critically by Newman (1988) and Newman etal. (1992); see also Smith and Smith (1990). Although the amounts and form of C and other compounds that are translocated between linked plants remain unclear, the experiments with 14C, "N, 32P and other tracers h v e given rise to suggestions that host-plant interconnections are very widespread, so that the hosts in effect share a common root system and have a common ability to obtain mineral nutrients (Miller and Allen, 1992). Janos (1987) suggested that the need to share photosynthate might then constrain competition between species, a point also addressed by Grime etal. (1987). However, nobody yet seems to have considered the possibility that although (and despite the experiments with I4C) there may be no or insignificant net gain of organic C to the so-called receiver plant, the latter may still be at a competitive advantage vis-a-visthose of its neighbours which are donors of C to the linking fungus. This last point aside, Janos (1987) pointed out that sharing of photosynthate with another plant will be an additional cost to a mycorrhizal plant in addition to that imposed by loss of C to the fungus per se, and that if this cost were excessive in relation to the benefits (improved mineral nutrition), over evolutionary time there might be selection against the mycorrhizal symbiosis among potential donor plants. However, it is likely that other ways of obtaining nutrients via changes in root growth or architecture would be even more expensive in terms of energy expenditure. In other words, the selective disadvantage to a plant that donates organic C to a receiver plant, while still receiving a share of the P taken up by the fungus, might be very small. The advantage of retaining low specificity to allow colonization by mutualists is again relevant. Janos (1987) suggests that tightly coupled exchange of P and organic C across the membranes at the interface would prevent cheating by the host, but accepts that there is no evidence for such coupling of transport processes in roots, as mentioned above. Janos also discussed the evolutionary constraints on cheating by receiver plants and particularly why fungal colonization is maintained. The issue is
MUTUALISM AND PARASITISM
25
the obverse of that discussed in the previous section: it is now why the fungus colonizes a plant that cheats it (as well as the donor plant to which it is linked) of organic C on which the fungus depends. For canopy dominants this may not be as big a problem as it seems, because growth of an individual plant within the mixed community could mean that in a juvenile or shaded state it was a cheater, but as it grew further into the canopy increased potential for photosynthesis would turn it into a potential donor. The issue is certainly relevant to permanently shaded species, and the possibility of non-nutritional benefits to the fungus again arises, as it does with achlorophyllous (heterotrophic) plants which we discuss next. We leave the evolutionary arguments to Janos (1980, 1985, 1987, 1995) and agree with him that the key benefit to a stable community of linked plants is that improved mineral nutrition to both donors and receivers of organic C must exceed any disadvantages to donors in terms of loss of organic C.
V.
CHEATING BY MYCO-HETEROTROPHIC (ACHLOROPHYLLOUS) PLANTS
There are many types of achlorophyllous plants that are dependent on mycorrhizal associations of one form or another (Leake, 1994) and are an extreme example of cheating by the plant. Not only does the plant not provide the fungal endophyte with a source of organic C, but it always depends on the fungus to provide the organic C, whether from the soil (as in many orchids) or from another plant to which the fungus is linked. The plant is, simply, a total parasite on the fungus. Given that these achlorophyllous plants also obtain at least some of their inorganic nutrients via the fungus, the question (raised in the previous section) of the benefits to the fungus is far from clear, and it has been suggested that the plant provides vitamins, phytohormones, etc. (see Harley and Smith, 1983). Another fall-back position is that the roots provide a safe haven for the fungus in a stressful environment. These possibilities could, of course, apply to receiver plants in general. This evolutionary uncertainty notwithstanding, it seems very relevant that some mycoheterotrophic plants, including members of the Gentianaceae, have symbioses that structurally resemble Paris-type VA mycorrhizas. These structures have been known since the work of Janse (1897) (see also McLennan, 1958), and have received renewed attention in the excellent review by Leake (1994) who, despite lack of identification of the fungal endophytes, accepts that these symbioses are formed by Glomalean fungi. If this is so, then the organic C on which these particular plants depend must come from a donor plant rather than the soil. Thus, it appears that evolution has led to the extreme result of the donor/receiver relationship in achlorophyllous plants which form VA mycorrhizas, just as it has in other myco-heterotrophic plants.
26
F.A. SMITH and S.E. SMITH
VI. COSTS AND BENEFITS OF THE VA MYCORRHIZAL SYMBIOSIS: FAST AND SLOW-GROWING PLANTS AND RELATED ISSUES Variations in nutritional efficiencies of VA mycorrhizal symbioses involve, using other jargon, variations in costs and benefits to the symbionts and this area has been extensively reviewed (e.g. Harris and Paul, 1987; Koide and Elliott, 1989; Fitter, 1991; Tinker et al., 1994). Particular attention has been paid to the cost to the plant of the drain of C that is associated directly or indirectly with the colonization (see also Lambers, 1987). This is highly relevant to the issue of fungal cheaters, as discussed above. Koide (1991) has focused more on the inorganic nutrients, with discussion of nutritionally related traits in plants that might predispose them to high or low growth responses when colonized by VA mycorrhizal fungi. Koide suggested that there might be major differences in growth responses between plants that constitutively have low maximum growth rates and those that have high growth rates. Many wild plants are slow-growers (Brundrett and Kendrick, 1990a), particularly shade plants (Lambers and Poorter, 1992), though ephemerals, some of which do not form mycorrhizas, include many fast-growers. In contrast, most cultivated plants have been bred for fast growth. Following arguments propounded by Chapin (1980), Koide (1991) suggested that most wild plants should show relatively low dependency on VA mycorrhizas (i.e. low growth responses to colonization) because they will have evolved so that their growth rates can cope with relatively low levels of available P. In contrast, according to Koide, the demand for P by cultivated plants with high growth rates might be so high that it outstrips the supply of P; hence there should be a greater dependency on VA mycorrhizas. (Presumably the same argument should apply to other types of mycorrhizas as well, though the limiting nutrient may vary.) This conclusion is not intuitively obvious because the VA mycorrhizal symbiosis is very ancient and is thought to have evolved on nutrient-poor soils in plants of which many might be expected to be slow-growers. There are some obvious complications with this analysis, as acknowledged by Koide. One is that a non-mycorrhizal plant with a low growth rate will have a low rate of extension of roots around which, depending on the level of available P and the uptake capacity of the roots for P, depletion zones of P may develop that are at least as large as those around the (nonmycorrhizal) roots of a fast-grower that explores the same soil more rapidly. Hence we believe that the role of VA mycorrhizas in minimizing localized depletion of P around the roots of slow-growers, and so helping their nutrition and growth, should not be underestimated. This is also the conclusion of Brundrett (1991), who included slow root growth in a list of traits that should be associated with high mycorrhizal dependency. Allsopp and Stock (1993) tested this issue with three slow-growing sclerophyllous woody plant species, all of which showed very large responses to colonization in low-nutrient soil.
MUTUALISM AND PARASITISM
27
Turning to the carbon side of mutualistic VA mycorrhizal symbioses, the needs of the fungi have presumably been “built-in” during the evolution of wild plants as part of the normal sink for photosynthates - they are not an “extra” demand since it is the mycorrhizal condition that is in most cases the usual one. The complication for fast-growers is that while VA mycorrhizas can help to meet a high demand for P, especially in soils low in available P, this demand might sometimes be met only at the expense of organic C that the plants can ill afford if they have evolved (or have been bred) to minimize excess photosynthetic capacity. The complication may be exacerbated if the cultivated plant has a high shoot/root ratio - that is, maximal production of aboveground biomass, whether stems, leaves, fruits or seeds. Lambers and his colleagues (see Lambers and Poorter, 1992; Ryser and Lambers, 1995) and others (e.g. Korner, 1994) have addressed the ecophysiological consequences of fast and slow growth in terms of photosynthetic capacity and especially supply of organic C to roots. It has been suggested that slow-growers, and especially perennials, are often able to lay down comparatively large amounts of organic C as reserves in various forms (Lambers and Poorter, 1992) and that the walls of root cells can be relatively thick, when compared with fastgrowers. If some of this C is excess in the sense that it can be diverted to the mycorrhizal fungi without harm to the plant, then the latter may be better able to support the symbiosis than in fast-growers in which the supply of organic C to the roots is kept to the minimum needed to support growth. Thus, it may be that slow-growers are less liable to growth depressions caused by mycorrhizal fungi or, more generally, that slow-growers can better tolerate fungal cheating. This may be one of the reasons why plants have not evolved ways of repelling the mycorrhizal cheaters - the slow-growers can afford to be cheated while awaiting a nutritionally efficient colonization, as proposed by Janos (1985,1987). Low shoot/root ratio is a trait that is often considered to predispose plants to relatively small positive growth responses to colonization, as emphasized by Koide (1991). However, we should bear in mind Korner’s stricture (1994) that fine roots which are active in nutrient absorption are the important component of total below-ground biomass in this context. Woody roots, often with significant storage capacity, can be a major sink for nutrients additional to the shoot. Setting aside this point, an important factor in terms of costs and benefits is the differences in the ability of plants to adjust their shoot/root ratio. A plant that, when colonized, can decrease the relative allocation of C to root biomass (compared t o that in a non-colonized plant of the same species) is at an additional advantage in supporting the fungus over a plant that does not have this plasticity. As far as the plant is concerned, the important benefit from this trade-off is that, cheating aside, uptake of P (etc.) via the fungus must exceed the loss of uptake via the decrease in root growth. The cause of the “stunt disease” of tobacco that is associated with colonization by Glomus macrocarpurn (Modjo and Hendrix, 1986) appears to be a case
28
F . A . SMITH and S.E. SMITH
where massive reduction in root growth does decrease overall growth of the plant (Jones and Hendrix, 1988). What is particularly unusual is that stunting can occur where colonization is very low indeed, so that transfer of P , etc. by the fungus is minimal. The reason for the effect on root growth is not known. Large growth stimulations caused by VA mycorrhizas in tobacco have been found by others (e.g. Peuss, 1958). As recognized for many years, morphology of roots is an extremely important character in determining mycorrhizal dependency. Plants with roots that are comparatively thick, with little branching and few or no root-hairs (magnolioid-type roots) tend to show greater VA mycorrhizal colonization and positive growth responses than plants with fine, highly branched roots and many long root hairs (graminoid roots) (Baylis, 1975; St John, 1980; see Hetrick, 1991). Menge etal. (1978) found an inverse relation between the dry weights of the roots of several citrus cultivars and their mycorrhizal dependency, and differences in root architecture complicated an attempt by Graham and Syvertsen (1995) to correlate mycorrhizal dependency of citrus root stocks with relative growth rate. Azcon and Ocampo (1981) found an inverse relation between the root lengths of wheat cultivars and their mycorrhizal dependency. The roots of Allium species fall structurally in the magnolioid category and the species that we and many others have used (onion: A. cepa and leek: A. porrum) show large positive growth responses to VA mycorrhizal colonization in soils low in P yet grow relatively slowly compared with many cultivated plants. Thus, these Allium species show that high VA mycorrhizal dependency is not simply a feature of fast-growers. Another anatomical feature of roots which may well have functional significance for VA mycorrhizas is the widespread occurrence of exodermes that is, hypodermes with Casparian bands, which can provide intraradical apoplasts that are relatively isolated from the external environment compared with those in roots that lack exodermes (Peterson, 1988; Perumalla etal., 1990). Such “isolated” intercellular apoplasts may provide levels of organic C that are better regulated than those in roots that lack exodermes. The possible consequences in relation to mycorrhizal efficiency of Arum-type VA mycorrhizas are obvious. The conclusion is that it is unrealistic to analyze the costs and benefits of VA mycorrhizal symbioses in terms of either demand of organic C by the fungus or demand of P or other nutrients by the plant, without considering traits of the roots such as those discussed above and in the reviews by Brundrett (1991), Hetrick (1991) and Koide (1991). Some of these traits are quite subtle, such as rates of turnover of roots, which can also vary and are higher in at least some fast-growers (Schlapfer and Ryser, 1995). Finally, we emphasize again some corresponding fungal traits that are variable, including the rate of growth and extent (i.e. the total biomass) of external hyphae and the distance for which they spread in soil, and even the number of vesicles, which are also part of the C demand of the fungus. It is
29
MUTUALISM AND PARASITISM
TABLE V Possible determinants of VA mycorrhizal responsiveness (adapted and extended from Smith and Gianinazzi-Pearson, 1988) ~~~
Fungus External hyphae: Growth rate Extent Colonization rate Nutrient uptake Nutrient translocation“ Carbon translocation Intraradical hyphae: Growth rate Arbuscule production“.’ Nutrient delivery” Carbon delivery
’
Plant
Symbiosis
Roots: Growth rate Length Branching Thickness Root hairs Dry/fresh weight Nutrient uptake Nutrient concentration Carbon deliverya-’
Interfaces: Area of contact Wall metabolism Carbon transfer from”/to roots Nutrient transfer to roots‘
’
’
” Processes
possibly involved in cheating by fungus.
’ Processes possibly involved in cheating by plant (via linked plants). the total C demand of the fungus, including that used in respiration, that determines the flux of organic C across the interfacial plasma membranes. As yet, there are no estimates of interfacial fluxes of C on the same basis as the few that have been obtained for P, and hence no quantitative basis to analyse the amounts of cheating by VA mycorrhizal fungi under different conditions or by different endophytes. Nevertheless, attempts are now being made to disentangle the many interactions between C economy and P nutritional benefits in VA mycorrhizal plants. A realistic conclusion so far is that this is very difficult (Eissenstat etal., 1993)! Table V (adapted from Smith and Gianinazzi-Pearson, 1988) summarizes the major factors which can determine the extent to which plants respond to colonization by VA mycorrhizal fungi.
VII.
DEVELOPMENT AND CONTROL OF THE VA MYCORRHIZAL SYMBIOSIS
Although cheating by VA mycorrhizal fungi and their hosts was for convenience discussed separately, this separation is to an extent artificial as is apparent when we (and others before us) considered why a cheated partner does not totally reject the other symbiont. In fact, of course, some such rejections are produced physiologically, as when colonization is decreased where external concentrations of P are so high that the plant “does not need” the fungus. Also, there is a relatively small number of plant species that constitutively never form functional mycorrhizas, such as most chenopods, brassicas, etc. (see Tester et al., 1988; Brundrett, 1991; Schreiner and Koide,
30
F.A. SMITH and S.E. SMITH
TABLE VI Regulation of morphological and related characteristics of VA mycorrhizas by plant and fungus (adapted and extended from Brundrett and Kendrick, 1990b) Control by the plant Position of appressoria on epidermis
Control by the fungus Diameter of intraradical hyphae
Penetration of exodermis
Branching pattern of intraradical hyphae
Growth of intercellular or intracellular hyphae (i.e. Arum- or Paris-type)
Arbuscular branching pattern
Amount of intraradical colonization‘” Location of arbuscules within cortex Size and abundance of arbusculesaSb
Diameter of final arbuscular branches Amount of intraradical colonization Amount of external hyphaeusb Presence of intraradical vesicles, spores or hyphal swellingsb Size and abundance of arbusculesusb
Influence by the other symbiont cannot be ruled out as evidence is often limited; Influence by environmental/physiological features; for details see text.
1993a,b). Realistically, to consider whether colonization results in a mutualistic or parasitic (cheating) VA mycorrhiza, or whether there is any successful colonization at all, requires consideration of complex developmental interactions between the partners that are modulated by the environment. Overall, successful colonization obviously includes recognition events involving both the fungus and the host. Morphological and other characteristics that are associated with control by the two symbionts are summarized in Table VI, which has been adapted and extended from Brundrett and Kendrick (1990b). The molecular basis and sequence of events of the initial colonization have been described well by Bonfante-Fasolo and Perotto (1992) and Giovannetti et al. (1994). Inspection of “non-mycorrhizal” chenopods, brassicas, etc. reveals the formation of the appressoria that normally precede successful colonization but only limited penetration of hyphae. Experimental attempts to increase colonization in these plants have had very little success in terms of the length of root infected, formation of intracellular or intercellular structures, etc. (e.g. Hirrel etal. , 1978; Schwab etal., 1982; Glenn et a/., 1988). The mechanisms behind such constitutive lack of colonization still remain unclear and are probably different in different cases (Glenn ef al. , 1988; Schreiner and Koide, 1993a,b; Giovannetti efal., 1994). It is of course a truism that such “non-mycorrhizal” plant species have evoived ways of obtaining their nutrients that are as successful as those provided by mycorrhizas. The interesting question is still why they are so few in number (Trappe, 1987; Tester et a/., 1988) and a simplistic answer is that for most plants the cost in terms of C use by mycorrhizal fungi is apparently less than that necessary for development of root biomass that is equally or more effective in nutrient absorption. It is
MUTUALISM AND PARASITISM
31
the presence of mycorrhizal cheaters that may alter this equation. (Again, it should be borne in mind that mycorrhizas may confer benefits other than nutritional ones: Fitter, 1985, 1991.) The stages that have been mainly discussed in this review are those that follow penetration of the epidermis and (if present) exodermis by the fungus. It is now clear (see Table VI) that much of the further development of a mutualistic symbiosis is under the control of the plant, whose genome determines whether the mycorrhiza is Arum- or Paris-type, whether arbuscules are formed at all (as in the Pisum mutant) and even the size of arbuscules in different types of plants (Alexander et ul., 1988, 1989). In contrast, much less is known about the influence of the fungal genome on VA mycorrhizal structure and function. It is uncertain if the fungus per se has control over the amount of intraradical and external hyphae. Apart from the presence of vesicles and spores, most of the other morphological characteristics in Table VI that are known to be controlled by the endophyte seem to be relatively minor architectural features of the hyphae. The problem of identifying characteristics of the fungus, of course, arises because it is an obligate symbiont. Begging the question of what influences germination of spores or other propagules (see Koske and Gemma, 1992), after this stage the development of the mycorrhiza is always influenced by the host and it becomes extremely difficult to separate causes and effects. For example, there is uncertainty about whether or not uptake of 32Pby VA mycorrhizal germ-tubes prior to colonization requires the presence of organic factors derived from the root. Two studies with Giguspora marguritu gave equally convincing evidence for (Lei et uf., 1991) and against (Thomson et u f . , 1990a) the need for host-derived factors. Uncertainty also surrounds a possible early role for arbuscules in stimulating growth (especially externally) by the fungus. Mosse and Hepper (1975) showed that growth of external hyphae of Gfomus mosseue colonizing root organ cultures of Trifofium spp. was stimulated during formation of appressoria and subsequent intercellular hyphal growth, but before formation of arbuscules. Hepper (1981) came to the same conclusion with intact Trifofium plants. In contrast, Becard and Piche (1989) reported that external hyphal growth of Gigasporu marguritu colonizing transformed roots of carrot required the presence of “intracellular spread with arbuscules”. They suggested that the development of arbuscules enables the fungus to utilize the root as a nutritional source. If “nutrition” in this context included sugar, then this finding is apparently contrary to the model of Gianinazzi-Pearson et uf. (1991b). However, the paper does not mention a phase of intercellular growth of hyphae before formation of arbuscules, although an earlier paper by Becard and Fortin (1988) indicated very limited intercellular spread. Also, the culture medium included a high concentration of sucrose to support growth of the roots (as did that used by Mosse and Hepper, 1975), so it is not clear that this work is a test of the models in Table 111. The possibility that arbuscules transfer nutrients other than carbohydrate that might be needed to support
32
F.A. SMITH and S.E. SMITH
early growth of the hyphae should not be forgotten, nor the need for nonnutritional (molecular) signals from the host that transfer hyphal growth from dependence on the reserves in the spore (or other inoculum) to its biotrophic phase. However, there seems no reason why any such signals that require membrane transport across an interface could not be transferred during the initial penetration of the epidermis, exodermis (where present) and, in Paristypes, via the hyphal coils in the cortex. Irrespective of these developmental uncertainties, once the nutritional reserves ,in the propagule have been exhausted, the production of external hyphae requires prolonged transport of organic C from the host, supplied via the interface(s) between the symbiont. Thus, although continued production of external hyphae undoubtedly varies between VA mycorrhizal fungi, this must also to some extent be partly under the control of the host, modified as well by environmental factors! A key question that is relevant to the occurrence and physiology of fungal cheaters is whether the fungal genome has a role in determining production or otherwise of arbuscules (Table VI). As we have seen, evidence one way or the other is so far conspicuously lacking. Irrespective of whether or not transport of P and organic C across the interface(s) is spatially separated it is advantageous to the plant for it to control formation of arbuscules, since these are universally accepted as the sites of P transfer to the plant. (It might be remembered, however, that the only evidence against P transfer across the intercellular interface is the absence of membrane ATPase in the ANium/ Glomus symbiosis, as found by Gianinazzi-Pearson et al., 1991b.) The relative infrequency of arbuscules in many Paris-type VA mycorrhizas is not easy to explain (assuming that this situation occurs in plants that are growing) unless the intracellular coils have a similar function (the “uniform transfer” model in Table IIIB), so that the arbuscules are simply needed to a lesser extent. If the “spatial separation” or “hybrid” models for function of interfaces (Table 111) are correct, it may be disadvantageous to thefungus to allow continued formation of arbuscules since these are the sites of loss of nutrients which are gained from soil at the expense of a significant drain of energy. What the fungus requires is organic C that might be gained from the intercellular apoplast (Arum-types) or hyphal coils (Paris-types). Here, the issue of control by the symbionts becomes very unclear. Developmental changes in the amount and form of the structural features (external and internal hyphae and arbuscules) inevitably complicate the understanding of genetic control, where observations have been made without regard to the age of the roots or stages of colonization. Also, as discussed above, developmental processes are influenced by environmental factors, such as low temperature, low light intensity or duration, or high external P. In some cases such physiological effects on the rate and/or extent of colonization are clearly greatly influenced by the plant’s genome (Graham and Eissenstat, 1994). In summary, difficulties in sorting out causes and effects arise because of the ways in which one partner may influence the other’s environment. This
33
MUTUALISM AND PARASITISM
ENVIRONMENT:
---1
r--I
0 prop gule
genome
I I
I
- - - - - - - - - - - JL - - - - - - - - 1 I
I I
I
I I
+
Fungal + - - - - Structure INTERFACE
I
I
I
I
* + +
Plant Structure
- - - ’
4
Fig. 7 . Summary of major interactions between VA mycorrhizal plants and their environment, emphasizing developmental interactions and chemical (molecular) signalling. Gene expression is shown by solid lines and signalling by broken lines. For details, see text.
brings us back to the interactions shown in Fig. 1. This has been modified to give Fig. 7 in order to emphasize both the environmental/genetic interactions in the initial stages of colonization and those afterwards. Once the initial colonization (penetration) is complete, it is events within the interface(s), including transmission of recognition signals, that must fine-tune the exchange of nutrients between the partners (Smith and Smith, 1990; Bonfante-Fasolo and Scannerini, 1992).
VIII.
CONCLUSIONS
In this review we have extrapolated from hypotheses about the possible interfacial sites of different transport processes in VA mycorrhizal associations to a wide-ranging discussion about the possible physiological and ecological significance of the various fungal structures. We have tried to encompass relevant aspects of costs and benefits of these symbioses and (briefly) the evolutionary significance of cheating by fungi and their hosts. In thinking about these issues we have been impressed by the limited basis for some of the “perceived wisdom” about VA mycorrhizas. There is a clear need to extend studies of both structure and function to a wider range of plants - especially wild plants - and to more plant/fungus combinations. In other words, more attention should be paid to mycorrhizal biodiversity and particularly to the diversity of intraradical structures in different VA mycorrhizal plants growing
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under different conditions, rather than merely the presence, percentage, etc. of overall colonization. There is certainly a need for less “arbusculocentricity” in thinking about VA mycorrhizal function. Speculation is really only of value if it leads to testable hypotheses something that is very difficult with evolutionary speculation! Some experimental approaches are obvious, especially comparisons of Puris-type VA mycorrhizal plants with Arum-types. Functionally, it will be important to identify the presence of individual transport proteins, including ATPases, at the different interfacial locations using the molecular techniques now widely available and (more difficult) to assess their activity under different conditions. This approach would also be worthwhile in achlorophyllous plants the ultimate cheaters. Molecular taxonomy has an important place in helping to identify the fungal endophytes, whether species or strains, and to provide more confidence in seeking those that provide high nutritional efficiency and hence large agronomic benefits. Molecular taxonomy is also especially needed to identify the symbionts in newly identified or “unusual” mycorrhizas. Those in the Araliaceae and Cornaceae (Boullard, 1953a,b) and the various achlorophyllous plants come immediately to mind. More estimates of fluxes of P across the interfaces are needed and (again more difficult) a start has to be made in measuring fluxes of organic C to give a better quantitative understanding of nutritional efficiency and hence of cheating. Measurement of interfacial surface areas will be facilitated by extension of image-analysis techniques (Smith and Dickson, 1991; Smith etul., 1994), though it will be very difficult in Puris-type associations to resolve arbuscules versus coils. Identification of more mutants lacking development of complete mycorrhizal structures, and investigations of their transport capacities, nutritional efficiencies, etc. will also prove very useful. Even if our ideas about physiological differences between the various interfaces turn out t o be completely wrong, even in the “hybrid” form (Table III), it may prove possible to explain differences in nutritional efficiency and cheating in VA mycorrhizas by identifiable differences in transport mechanisms on a finer scale - that is, within a single interface - at different times, under different conditions, or with different host/fungus combinations. In this case, however, the interesting anatomical differences now becoming apparent (or being rediscovered?) would need explanations different from those which we have suggested here. Finally, we hope that this overview will provide ideas for collaborative studies among VA mycorrhizologists and that these may help to resolve some of the issues that we have raised.
ACKNOWLEDGEMENTS In this wide-ranging review we have tried to keep references within reasonable limits and are very conscious that many relevant original papers and even
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reviews are not included. We hope that references that we have cited will help to rectify these omissions and allow the interested reader to follow the paths that we have ourselves traversed in formulating our ideas. We are grateful to many colleagues for very stimulating discussions, and particularly to Dave Janos for educating us about cheating. We are greatly indebted t o Larry Peterson for his hospitality at Guelph during our Study Leave from the University of Adelaide. Thanks are also due to Roger Koide, Christian Korner, David Read, Peter Ryser and (especially) Adrian Yallop, who set off our whole train of thought about physiological and structural aspects of mimicry/cheating. Sandy Dickson made very helpful comments about the draft document and Marcus Brownlow prepared some of the figures. Last, but not least, we thank the Australian Research Council for financial support.
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Schwab, S. M., Johnson, E. L. V. and Menge, J. A. (1982). Influence of simazine on formation of vesicular-arbuscular mycorrhizae in Chenopodium quinona Willd. Plant and Soil 64, 283-287. Schwab, S. M., Menge, J. A. and Tinker, P. B. (1991). Regulation of nutrient transfer between host and fungus in vesicular-arbuscular mycorrhizas. New Phytologist 117, 387-398.
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Taber, R. A. and Strong, M. E. (1982). Vesicular-arbuscular mycorrhiza in roots and xylem of Tradescantia. Mycologia 74, 152-156. Taber, R. A. and Trappe, J. M. (1982). Vesicular-arbuscular mycorrhiza in rhizomes, scale-like leaves, roots and xylem of ginger. Mycologia 74, 156-161. Tester, M.A., Smith, S . E . and Smith, F.A. (1988). The phenomenon of “nonmycorrhizal” plants. Canadian Journal of Botany 65, 419-431. Tester, M. A., Smith, S. E. and Smith, F. A. (1992). The role of ion channels in controlling solute exchanges in mycorrhizal associations. In “Mycorrhizas in Ecosystems” (D. J. Read, D. H. Lewis, A. H. Fitter and I. J. Alexander, eds), pp. 348-351. CAB International, Wallingford. Thomson, B. D., Robson, A. D. and Abbott, L. K. (1986). Effects of phosphorus on the formation of mycorrhizas by Gigaspora calospora and Glomus fasciculatum in relation to root carbohydrates. New Phytologist 103, 75 1-765. Thomson, B. D., Clarkson, D. T. and Brain, P. (1990a). Kinetics of phosphorus uptake by the germ-tubes of the vesicular-arbuscular mycorrhizal fungus Gigaspora margarita. New Phytologist 116, 647-653. Thomson, B. D., Robson, A. D. and Abbott, L. K. (1990b). Mycorrhizas formed by Gigaspora calospora and Glomusfasciculatum on subterranean clover in relation to soluble carbohydrate concentrations in roots. New Phytologist, 114, 217-225. Tinker, P. B., Durall, D. M. and Jones, M. D. (1994). Carbon use efficiency in mycorrhizas: theory and sample calculations. New Phytologist 128, 115-122. Trappe, J. M. (1987). Phylogenetic and ecologic aspects of mycotrophy in the angiosperms from an evolutionary viewpoint. In “Ecophysiology of VA Mycorrhizal Plants” (G. R. Safir, ed.), pp. 5-25. CRC Press, Boca Raton. Vanderplank, J . E. (1978). “Genetic and Molecular Basis of Plant Pathogenesis”. Springer Verlag, Berlin. Van Nuffelen, M. and Schenck, N. C. (1984). Spore germination, penetration and root colonization of six species of vesicular-arbuscular mycorrhizal fungi on soybean. Canadian Journal of Botany 62, 624-628. Walker, C. (1995). AM or VAM: what’s in a word? In “Mycorrhiza” (A. Varma and B. Hock, eds) pp. 25-26. Springer Verlag, Berlin. Waters, J . R. and Borowicz, V. (1994). Effects of clipping, benomyl and genet on I4C transfer between mycorrhizal plants. Oikos 71, 246-252. Winter, A. G. (1951). Untersuchungen iiber die Verbreitung und Bedeutung der Mykorrhizen bei kultivierten Gramineen und einigen anderen landwirtschaftlichen Nutzpflanzen. Phytopathologische Zeitschrift 17, 421-432. Woolhouse, H. W. (1975). Membrane structure and transport problems considered in relation to phosphorus and carbohydrate movements and the regulation of endotrophic mycorrhizal associations. In “Endomycorrhizas” (F. E. Sanders, B. Mosse and P. B. Tinker, eds). pp. 209-239. Academic Press, London. Yallop, A. and Davy, A. (1994). Growth response disparities - a case for mycorrhizal mimicry. Abstracts 4th European Symposium on Mycorrhizas, p. 72. Granada, Spain. Yawney, W. J. and Schultz, R. C. (1990). Anatomy of a vesicular-arbuscular endomycorrhizal symbiosis between sugar maple (Acer saccharum Marsh) and GIomus etunicatum Becker and Gerdemann. New Phytologist 114, 47-57.
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Calcium Ions as Intracellular Second Messengers in Higher Plants
ALEX A . R . WEBB. MARTIN R . McAINSH. JANE E . TAYLOR and ALISTAIR M . HETHERINGTON
Division of Biological Sciences. University of Lancaster. Lancaster LA1 4YQ. UK
I . Introduction ............................................................................... A . Calcium Ions as Second Messengers - the Animal Cell Paradigm .............................................................................. I1 . Calcium Ions as Second Messen ers in Plant Cells ........................... A . Methods for Measuring [Ca B+ Ii in Single Cells ........................... B . Measurement of [Ca2+Ii .......................................................... 111. Measurements of Stimulus-Induced Changes in [Ca2+Ii in Plants
........
IV . The Calcium Homeostatic Apparatus ............................................. A Mechanisms of Generating Increases in [CazfIi ........................... B . Ca2+-ATPases ......................................................................
.
V . The Problem of Specificity ........................................................... A . Other Second Messengers ........................................................ B . The Calcium Signature - a Stimulus-Specific Calcium Signal ........ C . Calcium Signatures in Plant Cells ............................................. VI . Future Prospects ......................................................................... Acknowledgements ...................................................................... References .................................................................................
45 47 49 49 60 68 69 69 83 84 84 85 86 87 88 88
I . INTRODUCTION Over the past decade evidence has been rapidly accumulating that points to the importance of the free calcium ion in the control of a diverse array of plant Advances in Botanical Research Vol . 22 incorporating Advances in Plant Pathology
ISBN 0-12-005922-3
Copyright 0 1996 Academic Press Limited All rights of reproduction in any form reserved
46
A. A. R. WEBB el al.
processes (Bush, 1993; Poovaiah and Reddy, 1993; Bowler and Chua, 1994; Gilroy and Trewavas, 1994). The common role that calcium plays in all these processes is to act as an intracellular second messenger. This implies that alterations (usually increases) in the concentration of free calcium ions in the cytosol ([Ca2'Ii) act as an intermediary in the coupling of an extracellular stimulus to its characteristic intracellular response. An important corollary is that cytosolic calcium ion homeostasis is under strict control. In this review we have chosen to use the animal cell paradigm of calcium-based second messenger systems as a comparator for examining the evidence that plant cells employ a similar mechanism for stimulus-response coupling. Clearly, 'there are inherent dangers in this approach and many pitfalls await the unwary if such a path is followed too closely. However, on the basis of the available evidence it would seem that, provided that one is cautious and is careful not to discard too readily data that do not apparently fit the model, there is much to be learnt from this approach. We start this review with an overview of the workings of calcium-based second messenger systems in animal cells. We then move to plant cells and first consider the methodology that is currently available for studying free calcium in single cells. We believe that an understanding of the technology and its limitations is vital to assessing the subsequent material on the (increasing) range of stimuli which appear to use calcium ions as second messengers in higher plants. As will become apparent the subject of the involvement of calcium ions in signal transduction is a very large field and a detailed discussion of all its facets would be far beyond the scope of this review. Accordingly, we have chosen to concentrate on certain areas that are currently attracting a great deal of interest. One of these areas is concerned with mechanisms of encoding specificity in the calcium messenger system. To examine this in more detail requires a thorough discussion of the calcium homeostatic machinery. Finally, we close with our personal perspective on the direction research in this field may follow for the next few years. It is necessary to mention those areas which, although of great importance to plant cell signalling, will not be covered here. Obviously the lines of demarcation between these various fields are entirely artificial and only serve to delimit the areas for reviewers' convenience. One major area that will not be discussed is other second messengers. In this review we concentrate on the calcium ion but there is evidence that changes in cytosolic pH may also be important on transducing certain signals and it would appear from the literature that the time is ripe for re-evaluation of the role of CAMP and other cyclic nucleotides. Additionally, there is evidence, albeit fragmentary at present, that lipid-derived second messengers such as lyso lipids and diacylglycerol, may also have a signalling role. It seems likely that there will be other second messengers, yet to be discovered, that may play a critical role in stimulus-response coupling. Within calcium signalling one area that we will not be discussing in any great detail is the field of downstream processing of
CALCIUM IONS AS INTRACELLULAR SECOND MESSENGERS
47
the calcium signal. Obviously this is of great importance to the generation of the final response and many of the primary and secondary effectors involved in these downstream events are now being identified and characterized. This subject is covered in detail in other reviews (Bush, 1993; Poovaiah and Reddy, 1993; Bowler and Chua, 1994; Gilroy and Trewavas, 1994). Finally, it is important to note that the area of calcium signalling in plants has been the subject of number of recent useful reviews (Johannes et al., 1991; Bush, 1993; Poovaiah and Reddy, 1993; Bowler and Chua, 1994; Gilroy and Trewavas, 1994) and it is recommended that the reader should consult these to complete the picture of calcium-based stimulus-response coupling pathways in plants.
A.
CALCIUM IONS AS SECOND MESSENGERS - THE ANIMAL CELL PARADIGM
Intracellular second messengers are used to couple extracellular stimuli such as hormones to their characteristic intracellular responses. The interaction of the extracellular stimulus with its receptor sets in motion a train of events which results in an alteration (most frequently an increase) in the concentration of another molecule in the cytosol. This alteration then triggers the cell's internal machinery to produce the response (such as secretion, contraction or alterations to gene expression) that is typical of the primary stimulus. As the field of stimulus-response coupling had its origins in mammalian physiology where the primary stimulus is itself frequently secreted from an endocrine gland and is known as a first messenger, then it follows that the cytosolic molecules that link the first messenger with the final response should be called intracellular second messengers. In terms of second-messenger based signal transduction pathways, mammalian cells are the best understood. cAMP was the first second messenger to be described (Robinson etal., 1971) and since then the list has expanded to include GMP, cytosolic free calcium ions ( [Ca2'Ii), diacylglycerol, inositol( 1,4,5)trisphosphate (Ins(1,4,5)P,) and cADP ribose (cADPR) and it seems likely that others will be identified as our understanding of signalling pathways increases. Next t o cAMP the best studied of these second messengers is [Ca2+Ii.In mammalian cells an array of first messengers make use of calcium-based signal transduction pathways in their mechanism of action. Interestingly, these range from simple molecules such as neurotransmitters to the relatively complex peptide growth factors. Perhaps equally striking is the breadth of the responses that are elicited by these first messengers. These include physiological examples such as muscle contraction, through calcium-mediated regulation of metabolic pathways such as in the control of glycogen metabolism in hepatocytes, to responses that must ultimately reflect alterations to gene expression as in the control of cell proliferation (Berridge, 1993a; Tsunoda, 1993). Finally, the fact that the calcium messenger
48
A. A.
R. WEBB
ef at.
system has been employed and is retained in such a diverse array of cells would suggest that it is both effective and flexible. This latter point is important as it implies that there is an inherent capacity for encoding specificity in the system which allows it to couple faithfully with the appropriate intracellular responses. Having introduced the concept of calcium ions as second messengers it is now time to consider the events that lead from first messenger reception to the generation of the increase in [Ca2+Ii- the calcium signal. Although we deal with these steps in greater depth later as the detailed comparison of plant and animal cell proceeds, it is worth looking at the process in overview. The calcium signal may be fuelled by two possible sources. In animal cells the concentration of calcium outside the cell is at least 1000-fold greater than the cytosolic concentration. Clearly, one source of calcium is from the cell’s exterior, and animal cells are indeed equipped with a range of calcium channels the operation of which allows the selective and controllable entry of calcium down its electrochemical gradient into the cytosol (Tsien and Tsien, 1990; Hofmann et al., 1994). Voltage operated calcium channels (VOC) are regulated by the membrane potential, which in turn can be influenced by a number of first messengers. Other calcium channels are linked to receptors (receptor-operated channels [ROC]) and, as they are regulated by receptor occupancy, provide a direct link between first messengers and the influx of calcium. A third class of calcium channel in the plasma membrane is regulated by intracellular second messengers (second messenger-operated channels [SMOC]) and gives the cell the potential to utilize feedback to either amplify or abolish the calcium signal. Clearly, the machinery exists at the plasma membrane to allow calcium entry and through the regulation of these channels to exert a high level of control over the process. When these factors are coupled to regulation of the spatial distribution of these channels across the surface of the cell the possibilities for generating controlled, localized calcium entry and generating a high degree of specificity in the calcium messenger system are obvious. The other possible sources of free calcium used to generate the calcium signal are the internal stores. Release of calcium from these sites is indirect and usually relies on the participation of other second messengers. Of these mechanisms, perhaps the best understood is the coupling process which proceeds through the operation of the phosphoinositide cycle. In this system, the binding of an extracelldar first messenger to a receptor belonging to the seven membrane pass or serpentine superfamily of membrane receptors generates a signal, which results in the activation of the membrane-bound enzyme phospholipase C (PLC). Transmission of the signal to the PLC from the receptor proceeds through the involvement of heterotrimeric G proteins. Once the PLC is activated it hydrolyses the membrane lipid phosphatidyl inositol(4,5)bisphosphate [PtdIns(4,5)P2] to yield diacylglycerol (DAG) and Ins(1 ,4,5)P3. The DAG produced is involved in the activation of
CALCIUM IONS AS INTRACELLULAR SECOND MESSENGERS
49
the pleiotropic enzyme protein kinase C while the Ins( 1 ,4,5)P3,being water soluble, diffuses into the cytosol. Once in the cytosol it is available for binding to its receptor located on the surface of intracellular membranes. Binding of the ligand to its receptor results in opening of outwardly directed calcium channels and causes calcium to be released into the cytosol. Importantly, these membranes also contain receptors for cADP-ribose, which is another second messenger involved in the generation of the calcium signal (Galione, 1993). Once generated, the increase in calcium can become autocatalytic and from a local site can generate global increases in calcium which can take a variety of forms such as oscillations, spikes and waves depending on the agonist and cell type. Importantly, it has been suggested that the calcium signal contains information that may contribute to the specificity of the response (Berridge, 1990). Finally, calcium 'is a somewhat paradoxical messenger in that if its concentration is allowed to increase unchecked it can act as a cytotoxin. To prevent this, under physiological conditions Ca2+-ATPasesare responsible for dissipating the calcium signal and returning the concentration of free calcium to its resting level by either pumping the calcium out of the cell or back into intracellular stores.
11.
CALCIUM IONS AS SECOND MESSENGERS IN PLANT CELLS
In the following sections we review the evidence that indicates that calcium ions behave as second messengers in the cells of higher plants. Central to the study of calcium ions as second messengers is the ability to measure accurately the concentration of free calcium ions in the cytosol of single cells and to detect changes that occur after the application of an appropriate stimulus. For this reason, we begin this section with an account of the different techniques that can be used to address this question and indicate what information each of the protocols reveals about cytosolic calcium dynamics. A.
METHODS FOR MEASURING [Ca2+]i IN SINGLE CELLS
Several techniques are available for measuring [Ca2+liin single cells, most of which were originally developed for use with animal cells. These include the use of ion-selective electrodes, luminescent proteins and Ca2+-sensitive dyes. Over the past 10 years the number of reports of stimulus-induced changes in [Ca2+Iiin plants has increased markedly (Table I). In the majority of these studies fluorescent Ca2+-sensitive dyes have been used for the measurement of [Ca"], . Recombinant aequorin techniques have also been developed for monitoring [Ca2+Iiin plants (Knight eta/., 1991, 1992, 1993).
TABLE I Measurements of stimulus-induced changes in plant cell [Ca2+Ji using fluorescent Ca2+-sensitivedyes and aeguorin-based techniques Stimuli
Cell type
Ca2+ response
Reference
ABA
Guard cells
Increase
Coleoptiles Hypocot yls Roots Aleurone protoplasts Guard cell protoplasts Guard cells Coleoptiles Hypocotyls Roots Root hairs
McAinsh eta/. (1990, 1992); Gilroy et a/. (1991); Irving et a/. (1992) Gehring et al. (1990a) Gehring et a/. ( 1990a) Gehring et al. (1990a) Wang eta/. (1991) Schroeder & Hagiwara (1990) Allan et al. (1994) Gehring et al. (1990a) Gehring et al. (1990a) Gehring et a/. (1990a)
(3‘43
Aleurone protoplasts
Increase Increase Increase Decrease Increase Increase Decrease Decrease Decrease Variable (increases & decreases) Increase
Cytokinin H2 0 2
Protonema Guard cells Aequorin-transformed seedlings Guard cells Leaf protoplasts
Increase Increase Increase Increase Increase
Leaf protoplasts Leaf protoplasts Alga Root protoplasts Mesophyll protoplasts Aequorin-transformed seedlings
Increase Decrease Increase Increase Increase Increase
Aequorin-transformed seedlings
Increase
Injected “caged” ABA Auxin
Methyl viologen Red light Far-red light UVA (pulses) Salt stress Heat stress Mechanical stimuli (touch & wind) Cold shock
Ayling et at. (1994) Bush & Jones (1988); Gilroy & Jones (1992) Hahm & Saunders (1991) McAinsh (1994) Price et al. (1 994) McAinsh ( I 994) Chae eta/. (1990); Shacklock eta/. (1992); Volotovski e t a / . (1993a,b) Volotovski e t a / . (1993a,b) Chae et al. (1990) Russ eta/. (1991) Lynch et al. (1989) Biyaseheva et al. (1 993) Knight eta/. (1991, 1992, 1993) Knight e t a / . (1991, 1992, 1993)
Fungal elicitors
Aequorin-transformed seedlings Suspension culture cell protoplasts
Increase Increase
IP, Injected “caged” Ins( 1,4,5)P3 Injected GTPyS Mitosis (anaphase) a-Factor Incompatible stigmatic S-glycoprotein Electric fields Fertilizationlegg activation Cell polarization [Ca2 I,
Guard cells Leaf protoplasts Stamen hairs Endosperm Yeast Pollen tubes
Increase Increase Increase Increase Increase Increase
Pollen tubes
Increase Increase
Injected “caged” Ca2+
Injected Ca2+ A23187 Ca2+ Br-A23 187
+
Fucus
Fucus Guard cells
Increase Increase
Leaf protoplasts Aleurone protoplasts Rhizoid Pollen tubes
Increase Increase Increase Increase
Moss protonema Spores Alga Isolated mitochondria Guard cells
Increase Increase Increase Increase Increase
Leaf protoplasts Pollen tubes
Increase Increase
Stamen hairs Rhizoid Pollen tubes Suspension culture cell protoplasts
Increase Increase Increase Increase
Knight ef a/. (1991) Messiaen et a/., (1993); Messiaen & Van Cutsem ( 1994) Gilroy et a/. (1990) Shacklock eta/. (1992) Zhang eta/. (1990) Keith eta/. (1985) Iida eta/. (1990) Franklin-Tong et a/. (1993) Malho eta/. (1994) Robinson (1990); Roberts et a/. (1994) Berger & Brownlee (1993) Gilroy et a/. (1991); McAinsh et a/. (1995) Volotovski eta/. (1993a,b) Bush & Jones (1987) Hodick eta/. (1991) Obermeyer & Weisenseel (1991) Hahm & Saunders (1991) Scheuerlein eta/. (1991) Russ eta/. (1991) Zottini & Zannoni (1993) Gilroy et a/. (1990); McAinsh etaf. (1995) Shacklock eta/. (1992) Franklin-Tong eta/. (1993); Malho et a/. (1994) Zhang eta/. (1990) Brownlee & Wood (1986) Franklin-Tong et a/. (1993) Messiaen & Van Cutsem (1994)
TABLE I continued
+
Ionomycin CaZ+ Injected Caz+-saturated Br,-BAPTA Verapamil
Isolated mitochondria Stamen hairs
Increase Increase
Zottini & Zannoni (1993) Zhang et al. (1990)
Rhizoid Root hairs Pollen tubes
Decrease Decrease Decrease
Brownlee & Pulsford (1988) Clarkson et al. (1988) Obermeyer & Weisenseel
Calmodulin antagonists (C4/80, W-7) Vanadate
Pollen tubes
Decrease
Obermeyer & Weisenseel
Pollen tubes
Decrease
Obermeyer & Weisenseel
EGTA
“Seedling” (cotyledon & hypocot yl) protoplasts Guard cells Root protoplasts Spores Alga Stamen hairs Embryogenic suspension culture cell Pollen tubes Stamen hairs
Decrease
Elliot & Petkoff (1990)
Decrease Decrease Decrease Decrease Decrease Decrease
Gilroy et al. (1991) Gilroy et a/. (1 986) Scheuerlein et ul. (199 1) Russ eta/. (1991) Zhang et at. (1990) Timmers et al. (1991)
Decrease Decrease
Miller eta/. (1992) Zhang et al. (1990)
Stamen hairs Rhizoids Suspension culture cell protoplasts
Increase Increase Increase
Zhang eta/. (1990) Hodick etal. (1991) Messiaen & Van Cutsem
Guard cells Suspension culture cell protoplasts Suspension culture cell protoplasts Root hairs Pollen tubes
Increase Increase Increase Increase Increase
Gilroy eta/. (1991) Gilroy et al. (1 989) Gilroy et a/. (1989) Clarkson eta/. (1988) Malho et a/. (1994)
~
a
~
’
(1991) (1991) (1991)
Injected EGTA A23187 EGTA
+
Br2-BAPTA2+ Injected Ca -free Br,-BAPTA Injected C1- K +
(1994)
Decrease in K f &NO, NaN, M-N-ethyl maleimide Iontophoretic microinjection
ns(1,4,5) ABA, abscisic acid; GA3, gibberellic acid; UVA, ultra-violet A; Ins(1,4,5)P3,inositol trisphosphate; GTPyS, guanosine S‘-O-(3’thiotrisphosphate); BAPTA, bis-(O-aminophenoxy)-ethane-N,N,N’,N’-tetraacetic acid; EGTA, ethyleneglycol-bir-(~-aminoethyl)-N,N,N’,N‘tetraacetic acid.
CALCIUM IONS AS INTRACELLULAR SECOND MESSENGERS
53
TABLE I1 Non-ratiometric and ratiometric (dual-excitation and dual-emission) fluorescenl Ca -sensitive dyes +
Excitation wavelengths (nm) Non-ratiornetric dyes F~uo-3 Rhod-2 Calcium Green ( - I , -2 and - 5N) Calcium Orange ( - 5N) Calcium Crimson Ratiometric dyes Dual-excitation dyes Fura-2 Quin-2 Fura Red BTC Dual-emission dyes Indo- 1 Structurally linked dyes Calcium Green-1 and Texas Red 70000 Dextran
Emission wavelengths (nrn)
490 550
530 580
490 550 590
530 590 610
340 330 420 400
and and and and
380 350 480 480
510 490 660 540
360
405 and 480
490 and 570
530
I . Fluorescent Ca2+-sensitivedyes Several fluorescent Ca 2+-sensitive dyes, and their conjugates, are commercially available (Table 11). These can be broadly grouped into two categories: non-ratiometric or single-wavelength dyes, and ratiometric or dual-wavelength dyes. Non-ratiometric dyes. The most commonly used non-ratiometric dyes are fluo-3 and Calcium Green. These exhibit an increase in fluorescence across the whole of the emission spectra on binding Ca2+ (Fig. 1A). Therefore, at a given wavelength [Ca2+Iiis proportional to the intensity of fluorescence. Typically, the emission maximum, approx. 530 nm for fluo-3 and Calcium Green (490 nm excitation), is used. However, the amount of fluorescence measured in a cell is not simply dependent on [Ca2+Iibut also on the amount of dye present, which will vary from cell to cell, the distribution of the dye within the cell, and the degree of dye loss and photobleaching that occurs during measurements of [Ca2+Ii.These factors complicate the quantification of [Ca2+Iiusing non-ratiometric dyes. Ratiometric dyes. Ratiometric dyes exhibit a shift in either their excitation or emission spectra when they bind Ca2+. Consequently, they are subdivided
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.-z
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Fig. 1. Emission spectra for (A) fluo-3 (a non-ratiometric dye); (B) fura-2 (a dualexcitation ratiometric dye); (C) indo-1 (a dual-emission ratiometric dye); and (D) Calcium Green-1 and Texas Red dextran (a 70 000 MW dextran conjugate of both Calcium Green and Texas Red which can be used as a dual-excitation ratiometric dye). Redrawn from Haugland (1992) with permission.
into: (i) dual-excitation dyes, the most widely used of which is fura-2 (Fig. 1B); and (ii) dual-emission dyes, the most commonly used of which is indo-1 (Fig. 1C). This shift is utilized in the measurement of [Ca2+Ii.For example, fura-2 (a dual-excitation dye) exhibits a shift in its excitation maximum (510nm emission) on binding CaZ+, such that the fluorescence at shorter wavelengths, typically 340 nm, increases with increasing [Ca2+Jiwhereas the fluorescence at longer wavelengths, typically 380 nm, decreases with increasing [Ca2+li(Fig. 1C). By calculating the ratio of the fluorescence intensities at
CALCIUM IONS AS INTRACELLULAR SECOND MESSENGERS
55
these two wavelengths a measurement is obtained that is proportional to [Ca2+Iibut independent of the amount of dye present in the cell. The same principle applies to dual-emission dyes except that it is the shift in the emission maximum that is utilized for calculating the Ca2+-dependent ratio. This technique eliminates potential artefacts that may be associated with cell-to-cell variations in dye loading, dye distribution and dye loss (through leakage or photobleaching). In addition, it allows accurate calibration of the fluorescence ratio (see Tsien and Rink, 1980; Grynkiewicz e t a / . , 1985). The majority of ratiometric measurements of [Ca2+Ii have been made using dyes that are excited by short wavelength ultraviolet light (UV), for example fura-2, 340/380 nm excitation maxima; indo-1, 360 nm excitation maximum. However, in general, UV-excitable dyes cannot be employed in studies requiring the release of active compounds from their inactive "caged" forms by UV-photolysis (McCray and Trentham, 1989) or in imaging studies using confocal scanning laser microscopy (CSLM) (White et al., 1987; Shotton, 1989). The latter would require a laser capable of emitting lines in the UV range. Advanced confocal microscopes equipped with this facility have only recently become commercially available. Therefore, these studies have relied heavily upon non-ratiometric dyes with excitation maxima at longer wavelengths. Recently, long wavelength ratiometric dyes have become available. These include Fura Red, which may act as a ratiometric dye, using excitation of 490nm for the Ca2+-sensitive signal and of 440nm for the Ca'+-insensitive signal (535 nm emission), and BTC, a coumarin benzothiazole-based indicator, which exhibits an excitation maximum shift from 480 nrn to 400 nm on binding Ca2+ (540 nm emission). In addition, cells can be coloaded with two separate non-ratiometric dyes, the spectral properties of which allow a ratio to be calculated that is independent of dye concentration, for example with fluo-3 and Fura Red. This technique assumes that both dyes adopt equivalent distribution patterns within the cell, which may not always occur. A 70000MW dextran conjugate of both Calcium Green and Texas Red (Calcium Green-1 and Texas Red dextran) has been developed to avoid this problem. This uses excitations of 490 nm (Calcium Green-1) for the Ca2+-sensitive signal and 570 nm (Texas Red) for the Ca2+-insensitive signal (530nm emission) (Fig. 1D). Since both dyes are structurally linked an identical distribution is assured. However, many of these dye combinations have still to be applied successfully to the measurement of [Ca2+liin plant cells. The majority of fluorescent Ca*+-sensitive dyes are produced in both the free acid and the acetoxymethyl esterified (AM) form. Many of them are also available conjugated t o specific biomolecules. The most common conjugates are those in which the dye is attached to a high molecular weight (MW) water-soluble dextran (typically with molecular weights of 10 000, 70 000 or 500 000). However, Molecular Probes, Inc. (Eugene, Oregon, USA) have developed an amine-reactive fura that can be covalently attached t o any
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suitable biomolecule. Fura-BSA is one example of such a conjugate. In addition, site-selective conjugates of certain Ca2+-sensitive dyes are available. These include NuCaGreen, a 70 000 MW dextran conjugate of Calcium Green and a nuclear localization peptide, which can be used specifically to monitor changes in nuclear [Ca2+Ii. Other site-selective dyes include rhod-2 AM, which is the only cell-permeant dye that has a net positive charge causing its sequestration into mitochondria in some cells, and fura-C,, and fura-indoline -CIS,which are lipophilic Ca2+ indicators that preferentially associate with cell membranes allowing changes in [Ca2+Iioccurring close to membranes to be studied (Etter et al., 1994). With the exception of fura-indoline-CI8, which exhibits a shift in its excitation maximum from approx. 575nm to approx. 510 nm upon binding Ca2+, making it a potential longer-wavelength ratiometric dye, the site-selective forms of the dyes and their conjugates possess similar fluorescence characteristics to the parent dyes. Ca2+-sensitive dyes have also been developed which possess different affinities for Caz+ making them suitable for determining [Ca2+Iiacross a wide range of concentrations. For example, there are three types of Calcium Green commercially available (see Table 11); Calcium Green-1 has the highest affinity for Ca2+ (Kd 189nM), making it best suited for monitoring small changes in [Ca2+Ii(Haugland, 1992), while Calcium Green-5N (and Calcium ) it suitable for Orange-5N) has a low affinity for Ca2+ (Kd 3.3 p ~ making measuring unusually large pulses of [Ca2+Ii(e.g. during Ca2+-induced Ca2+ release) (Kuba and Takeshita, 1981; Goldbeter et al., 1990; Berridge, 1993b). Calcium Green-2 has an intermediate affinity for Ca2+.The magnesium indicators (mag-fura-2 and -5, mag-fura red, mag-indo-1, mag-quin-1 and -2, Magnesium Green), also have a moderate affinity for Ca2+ (e.g. Magnesium Green: Kd for Ca2+ 4.8 pM) and may therefore also be useful in detecting large "spikes" in [Ca2+Iiup to 1 p~ (Collins etal., 1991; Konishi et al., 1991; Hurley et al., 1992).
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2. Dye loading techniques A range of techniques have been developed for introducing Ca2+-sensitive dyes into single cells. Several of these have been used to load plant cells, including: microinjection, low pH loading, ester loading, electroporation and digitonin permeabilization. These have met with varying degrees of success. The reasons for this remain unclear.
Microinjection. This technique has been widely applied to the study of [Ca2+Iiin plant cells, and has met with a high degree of success (Brownlee and Pulsford, 1988; Clarkson etal., 1988; Gilroy etal., 1990, 1991; McAinsh etal., 1990, 1992, 1995; Zhang etal., 1990; Hodick etal., 1991; Miller etal., 1992; Thiel et al., 1993; Allan et al., 1994; Ayling et al., 1994; Franklin-Tong etal., 1993; Malh6 etal., 1994; Roberts etal., 1994). There are both advantages and drawbacks associated with microinjection. The major advantage is
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that it allows a wide range of both charged and uncharged compounds to be rapidly introduced directly into the cytoplasm of a cell enabling the intracellular environment t o be manipulated and the effects monitored. The major drawback is that it is an invasive, and potentially disruptive technique requiring the impalement of a cell with a microelectrode or micropipette with the associated structural damage this may cause. Therefore, it is essential to establish the viability of a cell following microinjection. A range of parameters have been used to assess cell viability following microinjection (see Blatt et al., 1990; McAinsh et al., 1990,1992,1995; Gilroyet al., 1991).Microinjection techniques fall into two groups: iontophoretic injection and pressure injection (Callaham and Hepler, 1991). In addition, patch clamp techniques have also been used to introduce compounds into protoplasts held in the whole-cell configuration in a manner analogous to microinjection (Schroeder and Hagiwara, 1990). Iontophoretic microinjection is a technique that relies on the movement of charged molecules, for example dyes with a net negative or positive charge, driven by an electrical current. Under such conditions currents are carried by the ions in the solution rather than by the transfer of electrons resulting in ionic movements without any accompanying change in cell pressure or cell volume (Callaham and Hepler, 1991). This makes the iontophoretic microinjection ideally suited for use in plants, particular in studies on turgor regulating cells, for example stomatal guard cells (Gilroy etal., 1990, 1991; McAinsh et al., 1990, 1992, 1995; Allan et al., 1994). Both continuous current and current pulses have been used for the iontophoretic microinjection of plant cells. The latter avoids the protracted hyperpolarization of the plasma membrane which occurs when passing negative currents (depolarization when using positive currents) and reduces the effect of charging cells by allowing time for the cell to discharge in between successive current pulses. Although cell loading by iontophoretic microinjection can be controlled by regulating the iontophoretic current, additional factors also influence the delivery of a dye from the electrode. These include competing ions, differences in characteristics between individual electrodes (e.g. tip resistance) and cell type. However, in practice, the same quantity of a dye can be repeatably injected into cells although the absolute amount present may be difficult to calculate (see Purves, 1981 for additional details regarding microelectrode techniques). At Lancaster we use microelectrodes (<0.25 pm tip diameter), pulled from 0.68-mm filamented electrode glass, to impale stomatal guard cells in detached epidermis. The tip diameter of the electrode is dictated by the size of cell to be impaled and the molecular weight of the compound to be injected. External diameters which have been used for studies in plants range from 0.1 to 1 .O pm (see Read et al., 1992). Negatively charged compounds, for example fura-2, are iontophoretically injected into the cytosol of the cells using negative current pulses (1 .O nA, 2 Hz, 200 ms duration) for 1 min (McAinsh et al., 1990, 1992, 1995) using an isolated stimulator and an intracellular amplifier (World
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Precision Instruments [WPI], Sarasota, Florida, USA). Positively charged compounds are injected by positive current, while uncharged compounds may be co-injected into cells together with a high K + buffer. Currents used for the iontophoretic microinjection of plant cells range from <0.1 to 10nA passed for 10s to 10min (see Read etal., 1992). The tips of the electrodes are filled with the Ca’+-sensitive dye at a concentration of 10mM and backfilled with 3 M KCI for use with WPI electrode holders containing a sintered Ag-AgC1 pellet to make the connection with the external circuit. Concentrations of dyes used to fill electrodes range from 5 0 p to ~ 10mM (see Read etal., 1992). Using apparatus described above it is also possible to monitor membrane potential as an additional indicator of cell viability (Brownlee and Pulsford, 1988; Clarkson et al., 1988; Callaham and Hepler, 1991; McAinsh et a/., 1992). Pressure microinjection uses pressure to inject uncharged compounds or proteins that are not sufficiently mobile in an electrical current to allow successful iontophoretic microinjection. In addition, it can be used to inject mixtures of either charged and/or uncharged compounds. Iontophoretic injection of mixtures will tend to preferentially inject individual components on the basis of the different charges they carry. Pressure microinjection, however, will maintain the integrity of the mixture during injection, ensuring that the composition of the mixture entering the cell is the same as that in the pipette. Injection of compounds into the cytosol of cells using pressure is, in general, harder to achieve than iontophoretic microinjection due to the large pipette tip diameter required. This is a particular problem with walled cells (rather than protoplasts) when the requirement for a sharp pipette to penetrate the wall conflicts with the need to have a large, open tip. Bevelling of the micropipettes, giving them a profile similar to that of a hypodermic needle, may help to overcome this probIem by increasing the internal tip diameter while ensuring that the pipette remains sharp (Kaila and Voipio, 1985). In addition, pressure microinjection is complicated by the high pressure that must be applied inside the pipette in order to overcome the back pressure exerted by the cell’s turgor. For example, the turgor inside the guard cells of open stomata is approximately 60 bars (Weyers and Meidner, 1990). This is at the working limit for most commercial injection systems. Pressure microinjection has been successfully used in such diverse cell types as fungal hyphae (Money, 1990) and the eggs and rhizoids of Fucusserratus (Berger and Brownlee, 1993; Roberts et al., 1994).
Acid loading. The free acid form of the Ca2+-sensitive dyes dissociate at physiological pHs into the anionic state rendering them cell impermeant. However, at mildly acid pHs this is prevented, allowing them to enter cells in the uncharged, undissociated form. Once inside the cell the dyes encounter the higher pH of the cytosol and dissociate into the cell impermeant anionic state and become trapped inside the cell. This phenomenon, referred to as
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“ion trapping”, can be used to load cells using the acid-loading technique (Callaham and Hepler, 1991). This technique was first described by Gilroy etal. (1986) as a method for loading root protoplasts with quin-2 and has subsequently been used to load protoplasts from a range of different tissues (see Table I); for example protoplasts from barley aleurone layers (Bush and Jones, 1987, 1988), oat (Volotovski el al., 1993a), maize root tips (Lynch et al., 1989; Kiss et al., 1991) and pea mesophyll (Biyaseheva etal., 1993). There are few reports of this method working with walled cells (e.g. Halachmi and Eilam, 1989; Callaham and Hepler, 1991; Hahm and Saunders, 1991; Russ etal., 1991). In addition, the extreme conditions employed during acid loading protocols can have a marked affect on cell viability following loading. Typically, cells are maintained at pH 4.5 for > 2 h at dye concentrations > 5 O p M (Callaham and Hepler, 1991). These factors, therefore, severely limit the potential usefulness of this technique for studying [Ca2+Iiin plant cells.
Ester loading. The acetoxymethyl (AM) esters of the Ca’+-sensitive dyes are highly lipophilic and therefore are supposed to pass freely through the plasma membrane into the cell where they are hydrolysed by intracellular esterases. This traps the dye inside the cell, within the cytoplasm (Tsien, 1981). The AM esters of the dyes are insensitive to Ca2+ and some, for example flU0-3 AM, are also non-fluorescent. Therefore, hydrolysis releases the active form of the dyes. Ester loading is commonly used to introduce dyes into animal cells. Inclusion of compounds such as Pluronic F-127 (Molecular Probes, Inc., Eugene, Oregon, USA), a low-toxicity detergent, and foetal calf serum in the loading solution have also been reported to increase the success of loading (Haugland, 1992; Graziana et al., 1993). A wide range of conditions (20min to 24 h incubations in 1-50 pM of the AM esters at 4-30’ C) (see Read et al., 1992) have been used in an attempt to load plant cells with the AM esters of the CaZ+-sensitivedyes with varying degrees of success. These include studies of [Ca2+Iiin both individual plant cells (Keith etal., 1985; Nobiling and Reiss, 1987; Chae et al., 1990; Elliot and Petkoff, 1990) and tissue sections (Gehring eta/., 1990a,b; Williams et al., 1990) (see Table I). The success of ester loading varies markedly from cell type t o cell type (ester loading appears to be most successful in non-green tissues) and with the dye used. This variability may at least be partially the result of cell wall associated esterases which cleave the dye before it enters the cell (Callaham and Hepler, 1991; Cork, 1986). In addition, dyes may become rapidly compartmentalized on entering the cell, rather than remaining in the cytosol, through the same mechanism as ester loading due to the presence of extracytoplasmic esterases in subcellular compartments such as the endoplasmic reticulum and vacuole (Callaham and Hepler, 1991). Electroporation (electroperrneabilization). This technique is a general
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method that allows nearly any compound to be directly introduced into cells. It is best suited for use with wall-less cell suspensions - that is, protoplasts (Callaham and Hepler, 1991) - but can also be applied to walled cells growing on suitable support media (e.g. Jackson and Heath, 1990). High voltage (1.1-7.5 kV cm-') electrical pulses (10-6-10-3 s duration) are used to cause a transient permeabilization of the plasma membrane (Tsong, 1991). The conditions required to achieve this vary with each type of cell and the compound to be loaded (see Read etal., 1992 for a review of conditions used). A range of protoplasts have been loaded with Ca2+-sensitive dyes using this technique, including barley (Gilroy et al., 1986), wheat (Shacklock et af., 1992), yeast (Iida et af., 1990) and fern (Scheuerlein etal., 1991) (see Table I). There are two major drawbacks with electroporation: (i) the enzymatic isolation of protoplasts may alter the physiology of the cell or destroy vital surface proteins; and (ii) the transient permeabilization of the plasma membrane may allow the leakage of vital compounds and metabolites out of the cell as well as facilitating the loading of dyes. Therefore, the usefulness of this technique as a tool for studying [Ca2+Iiin plants is questionable.
Digitonin permeabilization. Permeabilization of plant cells, allowing the entry of the free acid forms of the Ca2+-sensitive dyes, may also be achieved using digitonin. A concentration of 0.1070 digitonin has been shown to enable the loading of fluo-3 into the cytosol of embryonic cells of Daucus carota (Timmers et al., 1991). Although fluo-3-loaded Daucus embryos have been reported to continue their development, this technique still remains to be shown to be a viable method of dye loading in plants. B.
MEASUREMENT OF [Ca2+]i
Quantification of the fluorescence signal from cells loaded with Ca2+-sensitive dyes can be achieved using either photometric or imaging with both conventional microscopy and confocal scanning laser microscopy techniques. Each of these provides a different level of information regarding the quantitative, temporal and spatial aspects of changes in [Ca2+Iiwithin cells. The apparatus required for each of these techniques differs markedly although they all have four components in common. These are: (i) an excitation light source (allowing the selection of excitation wavelengths); (ii) specimen holder (for the immobilization and maintenance of cells in order to facilitate dye loading, often by microinjection, and repeated measurements of fluorescence signals); (iii) detector (enabling the emission wavelengths t o be specified); and (iv) signal processing (for amplification, recording and analysis of data). Many of the practical aspects of the measurement of [Ca2+],have been reviewed previously (e.g. see Cobbold and Rink, 1987; Tsien and Hartoonian, 1990; Callaham and Hepler, 1991; Read et al., 1992).
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I . Fluorescence ratio photometry This technique provides information about both quantitative and temporal aspects of the [Ca”], dynamics of cells, and is characterized by a very high degree of temporal resolution (but no spatial resolution: measurements of [Ca2+],obtained are an average value for the whole of the cell). Consequently, ratio photometry has a low dependence on complicated hardware and computer software. Figure 2 shows a schematic representation of the apparatus required for ratio photometry. Typically, excitation light is provided by a xenon light source. This provides a more uniform spectral output than a mercury lamp. The excitation wavelength is selected using narrow band-pass filters. For dualexcitation photometry, the excitation wavelength is alternated using a rapid filter changer or spinning rotor (a monochromator can also be used in combination with a “Chopping” device). Cells are viewed using an inverted epifluorescence microscope and quartz optics. This allows a high degree of flexibility with respect to the perfusion and microinjection of cells. A dichroic mirror is used to reflect short wavelength excitation light onto cells while passing the longer wavelength emission light to the detector. Fluorescence emissions are detected using a photomultiplier tube (PMT). When a dualemission dye is used two PMTs are required. These are mounted at 90” to each other and a second dichroic mirror is used t o split the two emission wavelengths; the shorter are reflected into one PMT and the longer are passed to the other. Specific wavelengths are selected using narrow band-pass filters. Commonly, shutters are used to protect both cells and the detector from stray illumination. Excitation of cells and data acquisition are synchronized by computer. The data, in the form of the analogue output from the PMTs are subsequently digitized and fed into the host computer for storage and ratio analysis.
2 , Fluorescence ratio imaging Imaging techniques provide information about the spatial distribution of [Ca”], , in addition to quantitative and temporal changes, generating a “Ca2+-map”of the cell. This adds an additional layer of complexity t o both the hardware and software required for these measurements. Excitation of cells is achieved as with ratio photometry (Fig. 2). However, fluorescence emissions are recorded using a video camera. Typically, a low light level CCD (charge coupled device) camera is employed for imaging [Caz+Iiin plant cells. At Lancaster we use a cooled Extended ISIS-M intensified CCD camera (Photonic Science, Kent, UK). This contains two intensifier screens in series and is typically operated at -5°C to reduce the dark current (or “thermal noise”) generated by the detector in the absence of any light input. A rapid filter changer or spinning rotor is required for use in conjunction with dual-emission dyes to enable images at the two emission wavelengths to be captured. Excitation of cells and image acquisition are
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Fig. 2. A schematic representation of the apparatus used at Lancaster for the measurement of [Caz+Iiusing fluorescent Ca*+-sensitive dyes. Microscope system: Systems are based around a Nikon (Telford, UK) Diaphot TMD inverted epifluorescence microscope. Excitation light is provided by a Nikon 75/100 W xenon light source. Excitation wavelengths and intensities are selected by a spinning rotor holding six narrow band-pass filters, in combination with metal gauze neutral density filters, and transmitted to the microscope via a liquid light guide (Cairn Research Ltd, UK). A Nikon C F Fluor DL 40x, oil immersion lens (aperture 1.30) and non-fluorescent immersion oil (Sigma, UK) are used for all measurements. Excitation and emission wavelengths are split using an appropriate combination of filters and dichroic mirror (Nikon). Photometry: Photometric measurements are made using a Cairn Spectrophotometer system (Cairn Research Ltd, Kent, UK). The system consists of: two 9924B photomultiplier tubes (Thorn EMI, UK) and amplifiers, together with an analogue-digital converter located in the host computer (Cairn Research Ltd). Emission wavelengths are specified using narrow band-pass filters (NikonICairn Research Ltd) and split into the two photomultiplier tubes for dual emission measurements using an appropriate dichroic mirror (Cairn Research Ltd). Data are acquired and processed using Cairn C32 software (Cairn Research Ltd). Imaging: A cooled Extended ISIS-M intensified CCD camera (Photonic Science, Kent, UK) is used to record images. Images are acquired using an ARGUS-50 image analysis system (Hamamatsu Photonic UK Ltd, Middlesex, UK), consisting of an ARGUS-50 image processor and video monitor, together with a frame store located in an 80486 host computer (Viglen, USA) running ARGUS-50 Ca2+ concentration analysis software (Hamamatsu Photonic UK Ltd). Emission wavelengths are specified using narrow band-pass filters (Nikon). Images are subsequently processed using either ARGUS-50 control software or ARGUS-50 Ca2+ concentration analysis software (Hamamatsu Photonic UK Ltd). SimuItaneous photometry and imaging: The two photomultiplier tubes and imaging camera are mounted on a purpose built detector changer enabling rapid switching between C a 2 + measurement systems. This is connected to the side camera port of the microscope via a variable aperture PFX shutter system (Nikon).
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synchronized by computer. The output of the camera is digitized, typically up to a maximum resolution of 512 x 512 pixels, sent to the host computer for storage and analysis. Fluorescent images are normally displayed in 256 grey level intensities. Ratio images, calculated on a pixel by pixel basis, are coded as different pseudocolours on the basis of [Ca2+Ii. There are two important factors that must be taken into account when using fluorescent dyes to measure [Ca2+Iiin plants: (i) cell autofluorescence; and (ii) the signal-to-noise ratio. Plant cells are highly autofluorescent at the excitation wavelengths of many of the Ca2+-sensitive dyes. It is essential to correct for the contribution this makes to the total fluorescence signal at both excitation/emission wavelengths. In photometric studies autofluorescence is normally subtracted on-line using a mean value determined for each cell prior to loading with dye (e.g. see McAinsh etal., 1990, 1992, 1995). However, in imaging studies cell movements complicate this processes, making it impossible to subtract the initial image of the unloaded cell from all subsequent images. Therefore, a calculated mean autofluorescence is subtracted from images, pixel by pixel, at the end of each experiment (e.g. see Gilroy etal., 1990). Following autofluorescence correction the signal from dye-loaded cells is often very low making measurements extremely noisy. The noise can be reduced by integrating successive fluorescence measurements, increasing the signal-to-noise ratio. At Lancaster we have systems for making both photometric and imaging measurements of [Ca’’], using either non-ratiometric or ratiometric Caz+sensitive dyes. We have also developed a system that enables us to make simultaneous photometric and imaging measurements (see Fig. 2). The major advantage of this is that it combines the high level of temporal resolution obtained with photometry with the spatial resolution of imaging. In addition, photometry provides a Ca2+-dependent ratio “on-line” so that changes in [Ca2+Iican be monitored throughout the course of the experiment. This allows image capture to be optimized, images being obtained at the crucial points during changes in [Ca2’], . Sophisticated imaging systems (using either conventional microscopy or CSLM) have improved temporal resolution. However, this is still significantly less than that obtainable by photometry. They are also capable of calculating ratio images on-line, although they do this at the expense of temporal resolution. The large amount of computer memory required for storing the images obtained during rapid, on-line ratio imaging is also prohibitive, limiting the time course of experiments substantially. 3. Confocal scanning laser microscopy (CSLM) Images obtained by conventional microscopy are prone to out of focus blur due to light from both above and below the plain of focus. This may distort the image of the part of the cell which is in the focal plain, reducing the spatial resolution of the technique. CSLM allows optically thin sections of cells to be examined producing images that do not suffer from blur due to out of focus
CALCIUM IONS AS INTRACELLULAR SECOND MESSENGERS
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light (White et al., 1987; Shotton, 1989). Sophisticated hardware and software are both required to capture these images. However, the high resolution images produced should increase the level of spatial information obtained. Confocal microscopes illuminate a very small, precisely defined spot on the cell with the image of a pin hole. The fluorescence is viewed through a second pin hole at a n identical image plane. Fluorescent light from out of focus regions of the cell is out of focus when it reaches the observation pin hole, and as such is almost totally excluded from entering the detector. Pin point illumination is achieved using a laser while fluorescence emissions are detected using a single PMT. An image is built up by scanning the confocal pin holes in two dimensions over the cell. In addition, the scan can be moved vertically through the thickness of the cell allowing a series of sequential, optically pure images to be obtained. These can be reconstructed to produce a threedimensional representation of the distribution of [Ca2+Iithroughout the cell. There are several disadvantages associated with CSLM, the major one of which is the restricted number of lines emitted by lasers which can be used for exciting Ca2+-sensitivedyes. Until recently only the non-ratiometric dyes with long wavelength excitation maxima were suitable for use with confocal microscopes. In addition, although most of the commercial confocal microscopes are capable of achieving a high degree of spatial resolution they have a very low level of temporal resolution. Photobleaching of dyes and cell damage, resulting from the intensity of laser illumination and localized heating effects, may also limit the number of applications for which CSLM is suited. 4. Aequorin Aequorin is a photoprotein from the coelenterate Aequorin victoria consisting of apoaequorin, a 22 000 MW polypeptide, and coelenterazine, a hydrophobic luminophore. The photoprotein has three Ca2+-bindingsites. Upon binding Ca2+ the luminophore is discharged and emits a finite amount of blue light (470nm). Consequently, aequorin has been used to measure [Ca2+Iiin both animal (see Cobbold and Lee, 1991) and plant cells (Williamson and Ashley, 1982; Gilroy etal., 1989). There are both advantages and disadvantages associated with the use of aequorin for measuring [Ca2+Iiin single cells as compared to fluorescent Caz+-sensitive dyes (see Cobbold and Lee, 1991). Advantages include: 1. Measurements of [Ca2+Iiusing aequorin require less complicated hard-
ware and software. 2. The technique involves the quantification of blue light emissions, therefore, there is no autofluorescence since no excitation light is used. 3. There is no intracellular compartmentalization or leakage of the photoprotein due to its high molecular weight and negative charge. 4. There is n o buffering of [Ca2+liby the photoprotein.
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5 . Aequorin has no toxic effects within cells.
Disadvantages included: 1 . In general, photoproteins have to be introduced into cells by pressure microinjection due to their high molecular weight. This requires the use of micropipettes with a large tip diameter increasing the potential for cell damage (similar problems may also be encountered with microinjection of the high molecular weight dextran conjugates of the fluorescent Ca2+-sensitive dyes into certain smaller cell types). 2. The signal recorded from aequorin-loaded cells is normally too low to allow imaging of changes in [Ca2+Iiusing this technique.
Therefore aequorin and fluorescent Ca2+-sensitive dyes can be viewed as complementary techniques for measuring [Ca2+Ii,providing different approaches to study similar questions. 5 . Recombinant aequorin Recently, a new technique has been developed for the measurement of [Ca2+Ii in plants using aequorin. This employs recombinant DNA technology. Tobacco plants have been transformed with a chimeric gene construct consisting of the 35s cauliflower mosaic virus promoter fused to the apoaequorin-coding region from complementary DNA (cDNA), so that they constitutively express apoaequorin (Knight et a f . , 1991). Treatment of transformed seedlings with coelenterazine results in the reconstitution of functional aequorin inside cells. Consequently, the luminescence of these plants should provide a direct measure of [Ca2+Iiwithin cells. Using this technique a wide range of stimuli have been shown to stimulate an increase in [Ca2+jiin intact plants. These include wind, touch, cold and fungal elicitors (Knight e t a f . , 1991, 1992, 1993; also see Fig. 3). In addition to reporting “whole-plant” [Ca”], , it is now possible to measure [Ca2+Iiin specific subcellular organelles using recombinant aequorin techniques. This is achieved by fusing the apoaequorin cDNA to an organelle specific target sequence. In animals, a chimeric mitochondria1 apoaequorin cDNA has been used to transform bovine endothelial cells, specifically targeting the apoaequorin to mitochondria (Rizzuto et a f . ,1992). These cells have been used to demonstrate ATP-induced increases in mitochondrial [Ca2+Ii (Rizzuto etaf., 1992). Work is currently in progress to specifically target the apoaequorin to different cell types, subcellular organelles and membranebound proteins in plants. As with the use of aequorin microinjected into cells there are both advantages and disadvantages associated with the recombinant aequorin technique for measuring [Ca2+Ii.The major advantage is that it provides a non-invasive method for introducing a Ca2+-sensitive indicator directly into the cytosol of cells. This eliminates potential cell damage (although it prevents manipulations
67
CALCIUM IONS AS INTRACELLULAR SECOND MESSENGERS
30
1
-I I I I ?O0
2
3
4
5
C)
0.5
0 !
3
3
0
sL
L
%i
I 1.5
1.5
0
'0 Time
Fig. 3. Changes in the cytosolic Ca 2+-dependent luminescence of aequorintransformed tobacco seedlings, recorded using a chemilurninometer. Traces show the effects of (a) touch (seedlings were touched once every minute [arrows] for four successive minutes with a fine wire, after which water at 0°C was added); (b) temperature shock (seedlings were rapidly transferred from 20°C to between 0 and 50°C by the addition of water at the appropriate temperature); (c) fungal elicitors (seedlings were treated with [ 11 untreated yeast elicitor; [2] trifluoroacetic acidhydrolysed yeast elicitor; [3] void volume from a Sephadex G-25 column of yeast elicitor preparation; [4] proteinase K digested yeast elicitor; and [ 5 ] untreated Gliocfudiurn deliquescens elicitor); (d) wind (seedlings were subjected to wind forces of 1-12N). Data redrawn from Knight era/. (1991, 1992) with permission.
of the intracellular environment during the course of experiments). In addition, measurements can also be made on intact seedlings rather than isolated tissues or cells. The major disadvantage is that it is not clear whether all cells or only certain cell types exhibit a change in [Ca2+li(Poovaiah and Reddy, 1993). This problem is compounded by the lack of data regarding the uniformity of expression of apoaequorin throughout transformed plants. The first
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of these problems may be answered using a sensitive photon counting camera to image aequorin luminescence at the single cell level (Read etal., 1992). Recently, Knight el al. (1993) have imaged changes in the Ca2+-dependent luminescence of aequorin-transformed tobacco seedlings; in the cotyledons, hypocotyls and roots in response to cold shock, and in the cotyledons in response to mechanical stimuli (wounding and touch). However, the second problem may prove a little harder to address. Other disadvantages include: 1 . Only a finite amount of functional aequorin is reconstituted within cells. Once this has been discharged the transformed plants are no longer capable of reporting changes in [Ca2+Ii. This problem is compounded by the relatively low levels of expression which have so far been achieved in these plants. This may be overcome by the use of a stronger promoter in the apoaequorin chimeric gene construct or the use of coelentrazine analogues, with different biological activities, to reconstitute functional aequorin within plants (Knight el al., 1993). 2. At present, the lack of both tissue and cell specificity severely limits the number of applications for recombinant aequorin techniques. Single cell studies, for example stomata1 guard cells, will not be possible until the advent of suitable cell-specific promoters for the apoaequorin chimeric gene constructs used in plant transformations. 3. The kinetics (i.e. rapid, transient increases) of all the changes in [Ca2+Ii determined by recombinant aequorin techniques vary markedly from those measured using fluorescent Ca2+-sensitive dyes (see Fig. 3). This makes comparisons between data difficult.
111. MEASUREMENT OF STIMULUS-INDUCED CHANGES IN
[Ca"], IN PLANTS There are an increasing number of reports of stimulus-induced changes in plant cell [Ca2+Iidetermined using fluorescent Ca2+-sensitive dyes or aequorinbased techniques. Studies have been conducted in a variety of different species on a range of different cell types. These have revealed both increases and decreases in [Ca2+liin response to a diverse array of treatments. Table I provides a list of the studies conducted to date, including the type of cell under investigation, the stimuli used and the change in [Ca2+Ii reported. It is apparent that calcium ions act as second messengers in coupling a diverse range of important stimuli to an equally broad array of physiological responses and that this occurs in a number of different cell types. What is even more striking is that if we look at the most intensely investigated cell (the guard cell) it is apparent that stimuli which bring about opposite effects on guard cell turgor, namely auxins and abscisic acid, both stimulate increases in [Ca2+Ii.We discuss the implications of this apparently paradoxical result later. However,
CALCIUM IONS AS INTRACELLULAR SECOND MESSENGERS
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before addressing this point it is necessary to consider the processes that contribute to calcium homeostasis in plant cells. As we have discussed already, a great deal more is known about the processes that regulate calcium influx and efflux in animal cells and for this reason we relate what is known of calcium homeostasis in the plant cell to the picture that has emerged from studies with animal cells.
THE CALCIUM HOMEOSTATIC APPARATUS
IV. A.
MECHANISMS OF GENERATING INCREASES IN [Ca2+Ii
Agonist-induced elevations in [CaZ+Iiare brought about via a number of complex and interconnecting pathways. In plant cells these pathways are beginning to be elucidated. Studies of vertebrate and invertebrate cells have described elegant systems that are capable of generating waves or agonistspecific oscillations and spikes in [Ca2+Ii.In order to describe what has been learnt about the mechanisms by which plant cells elevate [Ca2+Ii,it will be necessary to summarize the pertinent components of animal cell signalling pathways (Fig. 4). For more detailed information regarding animal signalling pathways the reader is referred to excellent reviews of the subject by Berridge and Dupont (1994), Fasolato e t a / . (1994), Berridge (1993a,b), Fewtrell(1993), Galione (1993) and Tsien and Tsie (1990). 1. Direct pathways for elevating [Ca2+], Ca2+ may enter the cytosol either from the extracellular space via an influx across the plasma membrane or via release from intracellular compartments which act as Ca” stores. Release of Ca2+ from intracellular stores requires the involvement of diffusible messengers in indirect pathways. These are described later. Influx of calcium across the plasma membrane is due to the movement of the ion, down the electrochemical gradient, through membranespanning pore-forming proteins. Agonist-sensitive opening or closing of these Ca’+-permeable channels can be via an indirect pathway involving diffusible second messengers. Such channels are known as second messenger-operated channels (SMOC). Alternatively, channels such as receptor-operated channels (ROC) and voltage-operated channels (VOC) are regulated directly. The most extensively studied calcium influx pathways are those mediated by the VOC (Tsien and Tsien, 1990). The Ca2+-permeable VOC have been characterized on the basis of their pharmacology, electrophysiology and cellular distribution into four main types, L, T, N and P. These definitions may require adjustment in the light of more recent molecular investigations (Tsien and Tsien, 1990). The characteristic features of these VOC are that they are activated by membrane depolarization, exhibit high selectivity for
Fig. 4. A schematic representation of the mechanisms for increasing [Ca2+Iiin mammalian cells. Details and abbreviations can be found in the text. Solid lines indicate pathways resulting in an increase in [Ca”];. Dashed lines represent feedback and regulatory mechanisms. Ins(1,4,5)P3 is represented by 4 and Ins(1,3,4,5)P4 by
s.
CALCIUM IONS AS INTRACELLULAR SECOND MESSENGERS
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Ca2+ over other ions as a result of high affinity Ca2+-bindingsites on the protein, are selectively modulated by a number of agonists and their subcellular distribution is heterogeneous. The L,-type VOC are found in both excitable and non-excitable cells. The L-type VOC are unique among the Ca'+-permeable VOC in that they are blocked by the 1,4-dihydropyridines (Tsien and Tsien, 1990). Channel activity can be studied using patch-clamp analysis, in which changes in electrical current across the plasma membrane are monitored by sealing a very small diameter glass electrode to the membrane (Satter and Moran, 1988). Such analysis has demonstrated that the L-type VOC are activated by high voltages. This is also the case with N and P but not T-type VOC (Tsien and Tsien, 1990). A number of cDNAs encoding subunits of the mammalian L-type channels have been cloned and sequenced providing structural data. Microinjection of mRNA encoding sense and antisense for subunits of the L-type VOC into Xenopus oocytes, followed by expression and subsequent patch-clamp analysis, has provided information regarding the role of the individual subunits of the channel protein in pore formation and gating (Tsien and Tsien, 1990). Higher plant channels which allow the flux of CaZ+into the cytosol across the plasma membrane were first identified in guard cells (Schroeder and Hagiwara, 1990; Cosgrove and Hedrich, 1991). Schroeder and Hagiwara (1990), using the whole cell patch clamp configuration, identified a relatively non-selective plasma membrane channel which was permeable to C a 2 + . This channel showed repetitive and transient activation by abscisic acid (ABA), which was accompanied by concomitant increases in [Caz+li.The process by which ABA gates this channel is unknown but Schroeder and Hagiwara (1990) suggested that the repetitive nature of the activation of the channel makes it unlikely that it is an ROC, and is probably an SMOC. Given the apparent importance of this channel in contributing to ABA-induced increases of [Ca2+Ii, it is clear that further study of the regulation of this channel is required. The stretch activated channel reported by Cosgrove and Hedrich (1991) is discussed later. Ca2+-influx into carrot protoplasts can be inhibited by pharmacological agents such as the phenylalkylamines (verapamil, D600, D800), the diphenylbutylpiperidines (fluspirilene, R66204) and bepridil which are all believed to bind and block L-type channels in mammals (Graziana etal., 1988; Hetherington etal., 1992). Furthermore, binding sites for these agents have been located on the plasma membrane of carrot protoplasts (Graziana et al., 1988). In the study of Graziana el al. (1988), binding of 1,4 dihydropyridines (nitrendipine, nifedipine, (+)PN200-110) was not detected and these compounds had little effect on 4sCa2+ uptake, even though the 1,4,-dihydropyridines are thought also to antagonize L-type VOC. Further analysis of Ca2+-channel antagonist binding in carrot protoplasts resulted in the purification of a
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75 kDa plasma membrane protein which bound an azido derivative of phenyl-
alkylamines ([3H]LU49888) (Thuleau et al., 1990, 1993; Hetherington et al., 1992). Verapamil-binding proteins also have been purified from maize plasma membranes (Harvey et al., 1989). Reconstitution of the [3H]LU49888-binding protein in liposomes resulted in the formation of a Ca2+-permeablechannel (Thuleau etal., 1993). Patchclamp studies of this channel identified that it was short-lived. Following its disappearance the channel was replaced by one that was permeable to both CaZ+ and C1-. This second channel could be blocked by the addition of bepridil and verapamil providing the first direct measurement of block of a plant Caz+-channel (Thuleau et al., 1993). The above findings suggested the presence of Ca2+-channelsin the plasma membranes of plant cells which on the basis of pharmacology at least showed some similarity to those present in mammalian cells. Recent major advances suggest that Ca2+-permeableVOC present in the plasma membrane of plant cells differ significantly from those in mammalian cells. A Ca2+-permeableVOC in the plasma membrane of carrot cell protoplasts has been reported by Thuleau etal. (1994). This channel was activated by depolarization, with a permeability sequence as follows Ba2+ > Ca2+ > Mg2+. The channel was selective for Ca2+ over K + but a permeability to K+ was not entirely excluded. Uptake of 45Ca2+into plasma membrane vesicles and whole protoplasts was used to identify a voltage-dependent calcium transporter in roots of Zea mays (Marshall etal., 1994). Maximum 4sCa2+ uptake was activated at - 80 mV suggesting that in vivo, this Ca2+current is activated by depolarization of the membrane from resting (estimated to be - 150mV). A comprehensive study of the pharmacology of the 45Ca2+uptake was undertaken. This described differences between plant plasma membrane Ca2+-permeableVOC and their mammalian equivalents. The maize Caz+-flux was insensitive to verapamil, nifedipine and diltiazem, cadmium and Cu2+. Ruthenium red (which is usually considered an inhibitor of endomembrane Ca2+ channel activity) was inhibitory. 45Ca2+uptake was inhibited by 20-30070 by the following ions: Ba2+,Cs+, Ni2+, Zn*+,MgZ+,Gd2+.Whereas, La3+,Nd3+, Mn2+ inhibited the influx by approximately 70%. It was demonstrated that Z . mays root cells possess two Ca2+ uptake pathways, the first is lanthanumsensitive and dominates, accounting for approximately 70% of uptake with maximal uptake occurring at - 80 mV, the second is Gd3+-sensitive, accounts for 30% of uptake and peaks at -60mV. 45Ca2+uptake was almost completely inhibited by the presence of 1 mol m-3 of each La3+ and Gd2+ (Marshall et al., 1994). Pineros and Tester (1993) reported a Ca2+-permeablechannel in plasma membrane vesicles of root cells of wheat. This VOC also was activated by depolarization from the resting potential. Verapamil inhibited the channel ~) blocked the channel. These open time whereas AIC13 ( 7 0 ~ completely
CALCIUM IONS AS INTRACELLULAR SECOND MESSENGERS
73
authors suggested a role for the root plasma membrane Ca2+-permeable VOC in aluminium toxicity. The wheat Ca2+-permeable VOC and the voltage-dependent Ca2+-fluxes across maize root cell plasma membranes differed in their sensitivity to verapamil. This suggests that the activities of different Ca2+channels were being observed during the studies of Pineros and Tester (1993) and Marshall etal. (1994). Whether these channels were activated by dissimilar conditions or reside in different root cells or species remains to be established. It is not known whether any of the voltage-dependent Ca2+-influx activities described in carrot protoplasts, maize root protoplasts or plasma membrane vesicles of wheat root are involved in signal transduction processes. A number of investigations have attempted to identify the role of plasma membrane Ca2+-channelsin physiological responses by testing the sensitivity of these responses to agents known to antagonize mammalian C a 2 + permeable VOC (e.g. McAinsh e f al., 1991; Knight et al., 1992). However, the Ca2+-permeable VOC in plant plasma membranes show very different sensitivity to inhibitory agents compared to that shown by mammalian channels (Marshall et al., 1994). Therefore, some caution should be employed before agents whose pharmacology has been determined from studies of mammalian cells are used in plant-based studies. Additionally it is clear that plant plasma membrane Ca2+-permeableVOC show altered sensitivity to certain agents in different cells and conditions (Pineros and Tester, 1993; Thuleau et al., 1993, 1994; Marshall et al., 1994). Verapamil, bepridil, G d 3 + ,La3+ and certain 1-4 dihydropyridines have all been demonstrated to inhibit directly the flux through outward rectifying K channels, further complicating the use of these compounds in assessing the importance of Ca 2+-channels in contributing to agonist-induced increases in plant [Ca2+Ii(Terry et al., 1992). ROCs open in response to the binding of an external ligand. The activation is direct and occurs without the intervention of a diffusable messenger, though such messengers may be involved in modulation of the channel activity. ROC may [e.g. the N-methyl-D-aspartate (NMDA) receptor] or may not (e.g. the ATP receptor) be additionally gated by voltage (Tsien and Tsien, 1990). As far as we are aware there are no reports of Ca2+-permeableROC activity in the plasma membrane of plant cells. A considerable inward Ca2+ conductance in smooth and skeletal muscle cells is carried by stretch activated channels (SAC) (Tsien and Tsien, 1990). Cosgrove and Hedrich (1991) have identified Ca2+-permeable SAC in the plasma membrane of guard cell protoplasts of V. faba, which when activated allowed Ca2+-influx. The role of the SAC Ca2+-permeable channel in the signal transduction network of guard cells remains to be determined. In addition, SAC permeable to K + and anions were also located in the guard cell plasma membrane, these may also be important in regulating stomata1 movements. Another Ca2+-permeable SAC has been identified by patch-clamp analysis of the plasma membrane of epidermal cells of onion bulbs (Ding and +
14
A. A .
R. WEBB et al.
Pickard, 1993a,b). The onion Ca2+-permeable SAC exhibited a voltagedependent component to its gating by mechanostimulation. This channel showed a biphasic response to Gd3+ and L a 3 + .At low concentrations G d 3 + (< 1 p ~ and ) La3+ (< 25 pM) stimulated channel activity in response to mechanoactivation, whereas at higher concentrations both G d 3 + (> 1 p ~ ) and La3+ (> 4 0 ~ were ~ ) inhibitory. The effects of G d 3 + and La3+ could have been a result of blockage and/or interference with the mechanosensor mechanism. The tension-dependent activity of the onion Ca2+-selectiveSAC increased as temperature was dropped from 25°C to 6°C (Ding and Pickard, 1993b). Ding and Pickard (1993b) proposed that temperature-sensitive plasma membrane Ca2+-selective SAC may be involved in thermonastic responses, such as the closure and reopening of petals and thermotropic responses. Communication between some animal cells can involve the spread of waves of Ca2+ (or another diffusable messenger such as Ins(1,4,5)P3) via gap junctions (Tsien and Tsien, 1990; Berridge, 1993a). Whether plant cells communicate with each other in a similar manner via plasmodesmata requires investigation. It is worthy of note that the most well understood plant signalling pathways are found in the guard cell, which lack such connections (Willmer, 1983).
2. Indirect pathways for generating an increase in [Ca2+li In many animal cells binding of an agonist to a cell surface G-protein linked receptor or tyrosine kinase receptor results in the accumulation of a diffusible messenger such as Ins(1,4,5)P3 (or possible cADPR) which in turn elevates cytosolic free calcium via release from internal stores or via influx through SMOC (Fig. 4).
Ins(l,4,5)P3-sensitive Ca2+ release. Hydrophilic Ins(1 ,4,5)P3 (as well as another messenger, DAG) is generated by the action of PLC upon PtdIns(4,5)P2 which is found in the inner leaflet of the plasma membrane. PtdIns(4,5)P2 is a phospholipid formed via the successive phosphoryiations of phosphatidylinositol (PtdIns) to phosphatidylinositol 4-phosphate (Ptd(4)InsP) and to PtdIns(4,5)P2 (Berridge and Irvine, 1989). Activation of a G-protein linked receptor results in a conformational change in loops I1 and 111 of the receptor which causes the G protein to dissociate. The cy subunit exchanges guanosine diphosphate (GDP) for guanosine triphosphate (GTP) and activates PLC-01 and the GPy subunits activate PLCp, both of which generate Ins(1 ,4,5)P3. Agonists such as platelet-derived growth factor cause the dimerization of tyrosine kinase receptors. Dimerization allows the kinase domains of these receptors to autophosphorylate specific tyrosine residues, thereby exposing docking sites for SH2 domains on PLC-yl. Phosphorylation of this enzyme results in the generation of Ins( 1,4,5)P3 (Berridge, 1993a). Ins(1 ,4,5)P3 diffuses into the cytosol where it binds the Ins(1,4,5)P3-
CALCIUM IONS AS INTRACELLULAR SECOND MESSENGERS
75
receptor (IP3R) resulting in release of Ca 2+ from Ins( 1,4,5)P3-sensitive stores, thereby elevating [Ca”], (Berridge, 1993a; Mikoshiba et al., 1993). IP3R is a tetrameric transmembrane protein which contains the Ins( 1,4,5)P, binding site at the N-terminal end located in the cytoplasm. The C-terminal contains a membrane-spanning region which forms a Ca2+ channel. Binding of Ins( 1,4,S)P3 results in a conformational change which is probably associated with channel opening. IP3R are found on the membranes of rough and smooth ER, therefore these are believed to be the Ins(1 ,4,5)P3-sensitive stores (Berridge, 1993a). Ins( 1,4,5)P3-induced Ca2+-releasevia the IP3R has been demonstrated via the challenge of permeabilized cells with Ins(1,4,5)P3 (Hill et al., 1988), microinjection of Ins( 1,4,5)P, into single cells (Payne et al., 1988), photolysis of caged-Ins( 1,4,5)P3 in Xenopus oocytes (Parker and Ivorra, 1990), challenge of intact membrane vesicles (Galione et al., 1993b), patch-clamp recordings of purified IP3R reconstituted in planar lipids (Ehrlich and Watras, 1988) and transfection of L-fibroblasts with IP3R-encoding cDNA (Mikoshiba etal., 1993). Such studies have demonstrated that the IP3R has a bell shape response to C a Z + ,with this ion being stimulatory in the 100-300nM region (Berridge and Dupont, 1994). The role of C a 2 + in regulating the IP3R will be discussed later. IP3R is inhibited by heparin, caffeine and ethanol. The use of heparin in whole cell studies is complicated by the inhibition of Ins( 1,4,5)P3 generation by this agent (Berridge, 1993a). Cloning of the IP3R has identified at least four families, all of which share significant homology (Berridge, 1993a; Mikoshiba et al., 1993). The primary sequence of the IP3R shows no homology with those of plasma membrane VOC C a 2 + channels (Mikoshiba etal., 1993). A highly conserved region in the free C-terminal domain appears to be involved in channel opening because antibodies directed against this region can either enhance or inhibit Ins( 1 ,4,5)P3-induced Ca2+-release (Berridge, 1993a,b). Deletion of any fraction of another highly conserved 650 amino-acid sequence at the N-terminal prevents Ins( 1,4,5)P3 binding (Mikoshiba et al., 1993). Each of the four Ins(l,4,5)P3-binding sites of the tetrameric channel are available, though there is some debate as to whether the binding of the four molecules of Ins( 1 ,4,5)P3 act co-operatively or not, to open the channel (Berridge, 1993a). Ins(1,4,5)P3 can release only a proportion of the Ca2+ present in Ins(1 ,4,5)P3-sensitive stores. This “quanta1 calcium release” is probably a result of variations in receptor sensitivity. These variations in sensitivity may be due to receptor modulation by a sensor which increases IP3R sensitivity as the store fills and/or due t o receptor heterogeneity as a result of transcription from different genes or post-translational modification (Berridge, 1993a). In addition to its role of releasing intracellular C a 2 + , Ins(1,4,5)P3 can generate an influx of Ca 2+ across the plasma membrane via activation of an SMOC which is an inositol phosphate receptor (IPR). The IPR is more sensitive than the IP3R t o inositol 1,3,4,54etrakisphosphate,a phosphorylated
76
A. A.
R. WEBB et al.
15000
3500 GroPl
Gr"P'P
-E
3000
-
n U
=-
2500
c
-2000
UJ
n 10000
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0.
= 0
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Q)
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5000
c ;'
7
-1000 m=
I
c.
P
L
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0
10
20
30
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fraction Fig. 5. High performance liquid chromatography of deacylated phospholipids from [32P]-labelled guard cell protoplasts of Commelina communis $0 ), chromatographed with mammalian [3H]-glycerophosphatidylinositol(GroPI), [ HIglycerophosphatidylinositol phosphate (GroPIP) and [3H]-glycerophosphatidylinositol bisphosphate (GroPIP,). Tritiated compounds (0).Webb, A. A. R., Drobak, B. K. and Hetherington, A. M. (unpublished observations).
derivative of Ins(1 ,4,5)P3. Therefore it appears that both these inositol phosphates can mobilize an influx of calcium (Berridge, 1993a,b; Fasolato et al., 1994). Plant cells possess PtdIns, PtdIns(4)P and PtdIns(4,5)P2, along with other inositol-containing phospholipids and the appropriate enzymes for their interconversion (Drobak, 1992, 1993; Hetherington and Drobak, 1992; Cote and Crain, 1993; Parmar and Brearly, 1993). However, the relative abundance of PtdIns(4,5)P2 is very low in plant ceIls compared to that in animaI cells (Fig. 5 ; and see Hetherington and Drobak, 1992).
CALCIUM IONS AS INTRACELLULAR SECOND MESSENGERS
77
Despite the presence of the inositol-containing phospholipids and PLC activity in plant plasma membranes, a clear identification of Ins( 1,4,5)P3 in a higher plant cell has yet to be achieved (Hetherington and Drobak, 1992; Cote and Crain, 1993; Drobak, 1993; Drobak e t a f . , 1994). However, plant cells are competent to respond to Ins( 1,4,5)P3. Release of Ins( 1,4,5)P3 in the cytosol of guard cells resulted in an increase in [Ca2+Ii,inactivation of the plasma membrane K+-influx channel, activation of the depolarizing "leak" current and subsequent stomata1 closure (Blatt et a f., 1990; Gilroy et a f . , 1990). The Ins( 1,4,5)P3-sensitive Ca2+-store in guard cells remains to be determined. We describe the progress that has been made in identifying Ins( 1,4,5)P3-sensitive stores and receptors in other higher plant cells. On the basis of the current evidence plant cells possess an IP3R that is similar to that identified in mammalian cells. However, the plant IP3R is on the tonoplast and does not appear to be located on the endoplasmic reticulum (ER) (Canut e t a f . , 1993). Ins(1,4,5)P3 has been reported to release Ca2+ from isolated vacuoles (Drobak and Ferguson, 1985; Ranjeva et al., 1988; Alexandre et a f . , 1990) and tonoplast vesicles or microsomes (Schumaker and Sze, 1987; Brosnan and Sanders, 1990; Canut e ta f., 1993). Patch-clamp analysis of whole vacuoles isolated from red beet has demonstrated that Ins(] ,4,5)P3 releases C a t + from the vacuole via a Ca2+-permeable channel (Alexandre et a f . ,1990). Ins( 1,4,5)P3-induced Ca2+-release measured either by 4sCa2+ flux from tonoplast vesicles and whole vacuoles or by patch-clamp analysis, has a number of features in common with Ins(1 ,4,5)P3-induced calcium release via the IP3R in mammalian cells. Ca2+-release by the vacuole is specific for Ins( 1,4,5)P3 over other inositol phosphates, this specificity is similar to that demonstrated by the mammalian IP3R (Johannes et a f . , 1992b). The vacuolar Ins( 1,4,5)P3-sensitive Ca2+-releaseis inhibited by heparin and this inhibition appears to be due to competition with Ins(1,4,5)P3 for binding sites, rather than due to blockage of the channel (Johannes e t a f . , 1992b; Brosnan and Sanders, 1990, 1993). The estimated Kd for Ins( 1,4,5)P3-induced Cat+release from the higher plant vacuole is in the range 0.2-1 pM which compares well with the Kd for release via the mammalian IP3R (0.1-1 p ~ (Alexandre ) and Lassalles, 1992). Ins(1,4,5)P3 is unable to release all the C a z + taken up by tonoplast vesicles (Brosnan and Sanders, 1990; Canut et a f . , 1993). Whether this quanta1 release by the vacuole is a result of modification of receptor sensitivity as appears to be the case with the IP3R in animals, or is a result of the presence of both Ins( 1,4,5)P3-sensitive and -insensitive vacuolar stores is not known. However, an Ins( 1,4,5)P3-independent release pathway is present at the tonoplast (see below). A striking difference between the vacuolar 1ns(l,4,5)P3-sensitive Ca2+-release and the mammalian IP3R is the lack of inhibition of the vacuolar system by extravacuolar C a t + . Alexandre e ta f. (1990) were able to detect activity of the vacuolar Ins(1 ,4,5)P3-sensitive Ca2+-channel in the
78
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presence of millimolar Ca2+ at the cytoplasmic face of the tonoplast. “Cytoplasmic” Ca2+ concentrations of this order result in the inhibition of the mammalian IP3R (Berridge, 1993a; Berridge and Dupont, 1994). The mammalian IP3R consists of both a receptor and channel. It is not known whether the Ins(1 ,4,5)P3-receptor in plants and the tonoplast Ins( 1,4,5)P3-sensitive Caz+-channel reside on the same protein. However, Brosnan and Sanders (1993) describe the identification of high affinity binding sites for Ins(1,4,5)P3 on the tonoplasts of red beet. Ca2+-releaseby red beet vacuoles has previously been shown to be Ins( 1,4,5)P3-sensitive (Alexandre et al., 1990; Brosnan and Sanders, 1990). The Ins(1 ,4,5)P3-specific binding site appears to be proteinaceous. Furthermore, the binding of Ins(1,4,5)P3 is , inhibited by competition with heparin, has an estimated Kd of 0.1 p ~ and is specific for Ins(l,4,5)P3 over other inositol phosphates. The above data suggest that the red beet tonoplast Ins( 1,4,5)P3-specific binding site may be involved in Ins( 1,4,5)P3-induced Ca2+-releasefrom the vacuole (Brosnan and Sanders, 1993). Binding of Ins( 1,4,5)P3 to the red beet tonoplast Ins( 1,4,5)P3-binding site was inhibited by sulphydryl reagents (Brosnan and Sanders, 1993). Preincubation of red beet microsomes with Ins(1,4,5)P3 protected the binding site from sulphydryl reagents. These results are very similar to those obtained with the mammalian IP3R. It is believed in the case of the mammalian IP3R that sulphydryl reagents act upon cysteine residues in the Ins( 1,4,5)P3-binding site. It has yet to be confirmed that these reagents act in a similar manner on the plant Ins( 1,4,5)P3 receptor.
Ryanodine receptors and cADPR-sensitive release. Another principle intracellular Ca2+-release channel is the ryanodine receptor (RYR) (Tsien and Tsien, 1990; Berridge, 1993a,b; Fewtrell, 1993; Berridge and Dupont, 1994). The RYR shows many structural and functional homologies with the IP3R suggesting common evolutionary origins, though the RYR is larger. There are three families of RYR. RYRl is found in skeletal muscle, RYR2 in cardiac muscle and RYR3 in non-muscle cells. The RYR are tetrameric and show considerable sequence homology between families and a certain amount with the IP3R, especially in the C-terminal membrane-spanning region (Berridge, 1993a,b). At nanomolar concentrations the plant alkaloid ryanodine results in channel opening and release of Ca2+ from stores, whereas at concentrations greater than micromolar, ryanodine inhibits RYR. Caffeine activates RYR, unlike the IP3R (Berridge, 1993a). RYRl can also be activated by the cell surface dihydropyridine receptor which appears to be in physical contact with the channel protein (Berridge, 1993a; Fewtrell, 1993). Recently, advances have been made in understanding the in vivo regulation of RYR. Caz+ can be released from ryanodine-sensitive stores by the messenger cADPR (Galione, 1993; Galione et d . ,1993a,b; Thorn et al., 1994). In sea urchin eggs cADPR synthesis from P-NAD+ (P-nicotinamide adenine
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dinucleotide) is activated by the action of 3 ' ,5'-cyclic guanosine monophosphate (cGMP) on the synthetic enzyme ADP-ribosyl cyclase and it has been proposed that cADPR may transduce signals generated by cell surface receptors linked to cGMP production (Galione etal., 1993b). However, it is unlikely that cGMP will regulate cADPR production in all cell types (Thorn et al. , 1994). Recently, Allen et al. (1995) have demonstrated that cADPR can release Ca2+ from plant vacuoles via a voltage-dependent pathway.
IP3R and R YR and calcium-induced calcium release. Both ryanodine- and Ins(l,4,5)P3-sensitive stores can reside in the same cells. Not all animal cells possess both types of stores. In sea urchin eggs the presence of both types of stores appears to represent redundant calcium-release pathways (Galione et al., 1993a), whereas in pancreatic acinar cells the operation of both release pathways appears to be required for the generation of the full Ca2+ response (Thorn et al., 1994). The common sensitivity of RYR and IP3R to Ca2+ has led to the suggestion that both these stores are involved in calcium-induced calcium release (CICR) (Berridge, 1993a,b; Berridge and Dupont, 1994). CICR is a feedforward process by which small elevations in [Ca2+],stimulate large releases of Ca2+ from both ryanodine and Ins( 1,4,5)P3-sensitive stores. The CICR properties of the IP3R result in an all-or-none response in which a sudden and near maximal release occurs as Ins(1,4,5)P3 concentrations rise. This is a consequence of released Ca2+ inducing further C a 2 + release (Berridge, 1993a). Berridge and his co-authors believe that an important role of cADPR and Ins(1,4,5)P3 is to sensitize RYR and IP3R respectively to C a 2 + in order to induce CICR (Berridge 1993a,b; Berridge and Dupont, 1994). Other researchers place greater emphasis on the ability of Ins(1 ,4,5)P3 to evoke release, while acknowledging the role of Ca2+ in sensitizing the IP3R (Berridge, 1993a; Fewtrell, 1993 and references therein). Agonist-induced oscillations and waves of increase of [Ca2+Iiacross the cell have been observed in many animal cells types (Berridge, 1993b; Fewtrell, 1993; Miyazaki and Shirakawa, 1993). Release of C a 2 + from intracellular stores can form an important component in the generation and maintenance of waves and oscillations. A number of models have been proposed which describe how the above mechanisms interact to generate complex [Ca2+Ii signals. Fewtrell (1993) classified these models into three main classes; those where the abundance of Ins(1 ,4,5)P3 oscillates, those where Ins(1 ,4,5)P3 increases but does not oscillate and those where no increase of Ins(1 ,4,5)P3 is required to generate the C a 2 + signal. Whether such models will require modification in the light of recent discoveries concerning the role of cADPR as a messenger remains to be determined. The reader is referred to the following reviews where models of spatial and temporal variations in [Ca2+Iiare discussed in detail: Berridge and Dupont (1994), Fewtrell (1993), Berridge
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(1993a,b), Tsien and Tsien (1990). An interesting recent finding suggests that the accumulation of Ca2+-storesin restricted regions of the cell may also be important in defining subcellular [Ca2+Iigradients (Stendahl et al., 1994). Ca2+-release pathways have been described in plant cells which appear to be distinct from the Ins (1 ,4,5)P3 ryanodine/cADPR-sensitive systems described above (Johannes et al., 1992a,b; Gelli and Blumwald, 1993; Allen and Sanders, 1994; Ward and Schroeder, 1994). The Ins( 1,4,5)P3-insensitive channels are located in the tonoplast of the vacuole, though it is not known yet whether this represents the same Ca2+-store as the Ins(l,4,5)P3-sensitive store. One of the Ins(l,4,5)P3-insensitive influx pathways at the tonoplast may be involved in CICR (Ward and Schroeder, 1994). In describing fluxes across the tonoplast we shall adopt the conventions of Bertl etal. (1992) in which the voltage across the tonoplast (V,) is defined as follows: Vrn =
Vcytosol
-
Vvacuole
Positive or outward currents, therefore, represent the flux of cations out of the cytosol (i.e. into the vacuolar lumen). Therefore, under resting conditions the cytoplasm is negative relative to the vacuole due to the action of H+-translocating ATPases and pyrophosphatases in the tonoplast (Rea and Poole, 1993). Not all the articles referred to here have used the convention proposed by Bertl etal. (1992). For clarity such data have been redefined accordingly. Ward and Schroeder (1994) proposed that in plant cells CICR requires the co-ordinated action of at least two distinct classes of tonoplastic channel, a voltage-independent Ca*'-sensitive K+-influx channel (VK) and a very high conductance, voltage- and Ca2+-gatedcationic channel which has previously been designated the slow vacuolar (SV) channel. The VK channels identified by Ward and Schroeder (1994) in the tonoplast of guard cells of V. faba allowed K+-influx from the vacuole and were activated by increases in [Ca2+li;however, they were not permeable to C a z + . Alkalinization of the cytosol resulted in a reduction of VK activity. The SV channel, unlike the VK channel, was demonstrated to be capable of permitting Ca2+-influxfrom the vacuole to the cytosol (Ward and Schroeder, 1994). SV-type channels have been described in the tonoplasts of a number of plant cells; they are characterized by high conductances, activation at positive membrane potentials and permeability to cations, though so far only in the guard cell have they been demonstrated to be Ca2+-permeable(Ward and Schroeder, 1994 and references therein). The SV channel is open at positive membrane potentials, which are unlikely to occur in resting cells. This has meant that it has proved difficult to ascribe a physiological role for the SV channel, but Ward and Schroeder (1994) propose that it is via this channel that CICR may occur in plant cells.
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The guard cell VK is activated by elevations in [Ca2+],.Therefore, Ward and Schroeder (1994) proposed that this channel is activated by an initial rise in [Ca2+Iiin response to a stimulus which promotes stomata1 closure, such as ABA (McAinsh etal., 1990, 1992; Schroeder and Hagiwara, 1990). Opening of the VK results in an influx of K + which in turn makes the tonoplast potential more positive. At positive potentials SV is activated by increases in [Ca2'Ii and therefore in the model of Ward and Schroeder (1994) this channel will allow influx of C a 2 + ,which will serve to both further elevate [Ca2+Ii and make the membrane potential even more positive. The activation of SV by increased [Ca2+Ii,in barley aleurone cells, appears to involve the action of calmodulin (Bethke and Jones, 1994). K+-influx across the tonoplast cannot be maintained at potentials positive of about 100 mV. Ward and Schroeder (1994) proposed that the activity of H+-translocating pyrophosphatases and ATPases in the tonoplast maintains a potential negative enough to allow continued influx of K + into the cytosol from the vacuole. The Ward and Schroeder model is very intriguing and it proposes an elegant mechanism for CICR which is dissimilar to those evolved by animal cells. It is perhaps surprising that plant cells possess an Ins( 1,4,S)P3-sensitive Ca2+-channelwhich on the basis of current data appears to be similar to the IP3R, but have evolved an alternative pathway for CICR. At least three other Ins( 1,4,5)P3-insensitive Ca2+-permeable influx channels have been identified in the tonoplast (Johannes eta/., 1992a,b; Gelli and Blumwald, 1993; Allen and Sanders, 1994). The first of these was described by Johannes etal. (1992a,b) in sugar beet tap roots. This voltage-gated Ca2+-influxchannel is very different from the SV described above. It is open at negative potentials and has a much lower conductance (12pS), which saturates at -40mV. The channel is inhibited by Zn2+ and G d 3 + and appears to be insensitive to Ins(1 ,4,5)P3, heparin, ryanodine and [Ca2+Ii.A very similar channel has been identified in the tonoplast of V.faba guard cells alongside another Ins( 1,4,5)P3-insensitive Ca2+-permeable channel (Allen and Sanders, 1994). This second channel shows some similarities with the one described above. It is active at negative membrane potentials and is inhibited by G d 3 + . They differ in that the second channel has a higher conductance (27pS), is inhibited by nifedipine and is gated open by high vacuolar Ca2+ concentrations (Allen and Sanders, 1994). The physiological significance of the presence of two similar tonoplastic Ca2+-influx channels is not known. Allen and Sanders proposed that the two channels are involved in transducing different signals or may contribute to different types of elevations in [Ca2+Ii. Gelli and Blumwald (1993) also identified an Ins(1 ,4,5)P3-insensitive Ca2+-influx channel in the tonoplast which is active at negative membrane potentials. Unlike those channels identified by Sanders and his coworkers, this channel is regulated by increasing [Ca2+Ii.The Ca2+ channels in sugar beet
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tonoplasts described by Gelli and Blumwald (1993) were inhibited by nifedipine, verapamil and La3+. It has proved difficult to identify physiological roles for the tonoplast Ins( 1 ,4,5)P3-insensitive Ca2+-influx channels which are open at negative membrane potentials (i.e. all those described above except the SV channel). This is because their voltage dependencies are such that at physiological membrane potentials the channels would always be active, resulting in uncontrolled Ca2+-influx. However, Allen and Sanders (1994) report that the two Ca2+-influx channels, active at negative membrane potentials in the guard cell tonoplast, are also regulated by vacuolar pH. Assuming a vacuolar pH of 5 .O, these channels would not normally be open. Therefore, it is possible that tonoplastic CaZ+channels which are open at negative membrane potentials may be gated by other unidentified agents. If these putative regulators are identified, it may prove possible to define a role for these channels in regulating increases in [ c a 2 + l i . It is clear that a number of distinct pathways exist in the tonoplast to regulate Ca2+-influx into the cytosol. Whether these pathways contribute to the elevations in [Ca2+Iiin response to different stimuli or whether they are involved in generating a variety of patterns of increase in [Ca2+Iiremains to be determined. Another Ca2+-permeablechannel has been identified in the tonoplast of sugar beet cells. The channel described by Pantoja e t a f. (1992)was outwardrectifying and active at positive membrane potentials. Pantoja et af. (1992) proposed that this channel is involved in the uptake of Ca2+from the cytosol following CICR. However, it is unlikely that under normal conditions Ca2+], would reach the millimolar concentration required for this channel's activity. It is interesting that the model proposed by Ward and Schroeder (1994) for CICR would result in the positive membrane potentials required for this Ca2+-efflux channel to open. Other Ca2+-uptake mechanisms, i.e. the Ca2+-ATPases, are discussed later.
Capacitative Ca2+entry. Depletion of Ca2+-containingstores by a number of agents stimulates an influx of Ca2+ across the plasma membrane (Putney and Bird, 1993;Fasolato eta f . , 1994). The process has been terr-ied kapacitative entry". Recently evidence has accumulated that depletion of intracellular stores results in the release of a diffusible messenger, Ca2+-influx factor (CIF) (Parekh e t a f . , 1993; Randriamampita and Tsien, 1993). CIF has not been fully characterized but it is known to be a small (< 500 Da), lipophilic, phosphate-containing anion that can induce Ca2+-influx in a number of cells. It is found in the cytosol of resting cells and the organelles of active cells (Randriamampita and Tsien, 1993; Fasolato e t a f . , 1994). A small G protein may also be involved in capacitative Ca2+ entry (Putney and Bird, 1993; Fasolato et af.,1994).
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CIF probably acts upon a Ca2+-release activated Ca2+-channel (CRAC) present in the plasma membrane. This influx channel is activated by agents which deplete intracellular stores and is inhibited by high [Ca2+Ii(Fasolato et al. , 1994). The presence of a Ca2+ chelator outside the guard cell can deplete intracellular [Ca2+Ii(Gilroy etal., 1991; Webb etal., In Press). It has not been investigated whether such a depletion results in an influx of Ca2+ across the guard cell plasma membrane. Furthermore, it has not been demonstrated that an extracellular Ca2+-chelator depletes intracellular Ca2+-stores stores in addition to cytosolic Ca2+ in guard cells.
B.
Ca2+-ATPases
In mammalian tissues, [Ca2+Iiis returned to and maintained at resting levels by the action of Ca'+-translocating ATPases in the plasma membrane (PM Ca2+-ATPase) and the membranes of the ER and sarcoplasmic reticulum (SR). These Ca2+-ATPasesremove C a 2 + from the cytosol, and in the case of the ER/SR Ca2+-ATPases consequently bring about loading of intracellular Ca2+-stores(Tsien and Tsien, 1990; Berridge, 1993a). The PM Ca2+ATPases are directly activated by calmodulin and show little sequence similarity with ER/SR Ca2+-ATPases which are indirectly activated by calmodulin. The current literature concerning Ca2+-ATPaseactivity in plant cells has recently been reviewed by Evans (1994). T o avoid repetition we shall summarize the major conclusions of that excellent article, to which the reader is referred. In plant cells, calmodulin-stimulated Ca2+-ATPase activity is associated with the plasma membrane and intracellular membranes. Membrane purification, protein purification and reconstituted and immunological studies have demonstrated that the vast majority of calmodulin-stimulated Ca2+-ATPase activity is associated with the intracellular membranes, most likely the ER. This is totally unlike the situation in mammalian cells. Plants also possess a Ca2+-ATPaseactivity which is not stimulated by calmodulin and molecular studies have identified plant homologues of a mammalian SR Ca2+-ATPase. The complexity of plant Ca2+-ATPases means that further work is required before the role of each type of pump in signalling can be assessed. However, in the case of gibberellic acid (GA)-induced secretion of a-amylase by barley aleurone, a physiological role for the calmodulin-stimulated ER Ca2+-ATPase has been described. Calmodulin-stimulated calcium transport into the ER is associated with secretion of a-amylase. It is believed that Ca2+-binding stabilises a-amylase in vivo. Furthermore GA also stimulates increased calmodulin levels in this tissue (Evans, 1994).
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V.
THE PROBLEM OF SPECIFICITY
It is clear from the data presented in Table I that calcium ions are used as second messengers to couple a diverse array of stimuli to their characteristic responses. On the basis of this evidence it would seem likely that this list will expand in the next few years as the techniques for measuring [Ca2+Iiare applied to other cell types. Given the apparent ubiquity of the calcium messenger system the question of specificity arises. Clearly, there is every reason to believe that specificity can be built into signal transduction systems at the level of the receptor and indeed there has been progress on the isolation of molecules with properties which would suggest that they may well serve this purpose (Chang etal., 1993; Jones, 1994). Based on work on the animal heterotrimeric G proteins it also seems possible that there is the capacity for building in specificity in terms of receptor43 protein interactions where specific G-protein subunits can interact with specific effector molecules. However, difficulties begin to manifest themselves at the level of the calcium signal. One example, which has been alluded to already, should illustrate the problem. This is the action of auxins and ABA on stomata1 guard cells. Although these compounds bring about opposite physiological effects they both cause an increase in [Ca*'], . The question, then, is how can an increase in calcium control the fluxes of ions responsible for both an increase or decrease in turgor?
A.
OTHER SECOND MESSENGERS
One possible solution to this problem relies on the participation of other second messengers in the generation of the final response. Is there any evidence to suggest that this may be the case in plant cells? Again guard cells provide some of the most relevant evidence. It has now been shown convincingly that the elevation of free calcium induced by ABA is accompanied by an increase in cytoplasmic alkalinization while in the case of auxins the pH of the cytoplasm drops (Irving etal., 1992; Blatt and Thiel, 1993). Although it is not clear whether protons are behaving as second messengers and their source has yet to be accounted for, the ability to modulate pH concurrently with calcium begins to present an attractive mechanism for the differential control (in this specific example) of the ion channel activity responsible for mediating the changes in guard cell turgor. Again, space prevents a full discussion of the possibilities for fine control using this approach but for a very useful and detailed account the reader should consult Blatt and Thiel (1993). In addition to protons/hydroxyl ions there is evidence for the involvement of other putative second messengers in plant cells. Again much of the most recent evidence has come from guard cells where it is possible to use patch clamp analysis to study the effect of putative second messengers on the ion channels
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responsible for bringing about turgor changes. Such studies have generated data which suggest that guard cells are capable of responding to CAMP, DAG and Ins(1,4,5)P3 (Assmann, 1993; Li e t a / . , 1994). In addition to these data are the results from the Chua laboratory which strongly support the involvement of cGMP in the phytochrome stimulus-response coupling pathway (Bowler etal. , 1994).
B. THE CALCIUM SIGNATURE - A STIMULUS-SPECIFIC CALCIUM SIGNAL
Another possibility for generating specificity which would not necessarily rely on other second messengers would be t o encrypt information into the calcium signal itself. Such an idea has generated much interest in animal cell biology. Whittaker and colleagues (Galione et a/., 1993b) have coined the term “calcium signature” for a stimulus-specific calcium signal. There are two obvious (but not necessarily separate) ways of encoding information in the calcium signal. The first is to generate increases in calcium which occur in localized subregions of the cell that are close to the primary and secondary effector molecules required to generate a particular response. Such a mechanism would require the localization of either plasma membrane calcium channels or subcellular release sites to an area overlying the effector molecules. In the case of the plasma membrane channels this might be fairly simple and depend on receptor clustering. However, the situation with the subcellular sites is much more complex as it would also require the participation of other second messengers. In animal cells there is evidence these strategies are utilized. In the case of pancreatic acinar cell it has been shown that application of acetylcholine or cholecystokinin at physiological concentrations results in a localized increase of free calcium and this correlates in spatial terms with the cellular distribution of the a-amylase-containing zymogen granules (Kasai et al. , 1993; Thorn etal., 1993). Temporal aspects of the calcium signature may also be very important, especially in situations where an agonist induces intracellular calcium to oscillate. This phenomenon has been recorded in many cells in response to a variety of agonists. In overview, it has been found that the pattern of oscillations depends on both the type and strength of the agonist. These observations led to the suggestion that the frequency and amplitude of the oscillations may encode signalling information. The frequency of the oscillations varies from approximately 10 s up to several minutes (Tsunoda, 1993). Superimposed on these temporal elements are spatial considerations. It is clear that in certain cells the oscillations can propagate as a wave which traverses the cell. At the moment the precise physiological significance of waves and standing oscillations is a matter of some debate (Tsien and Tsien, 1990; Amundson and Clapham, 1993). However, their characterization and in particular the mechanism of their generation is much better understood (Berridge, 1993a,b;
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Berridge and Dupont, 1994; Bootman, 1994). Here there is good evidence that the delicate interplay of influx across the plasma membrane in combination with the release from intracellular stores is capable of generating the subtle but highly reproducible pattern observed in a number of cell types. When oscillations and localized increases are superimposed on an asymmetric distribution of effector molecules then it is obvious that there is great scope for generating specificity.
C . CALCIUM SIGNATURES IN PLANT CELLS
Is there any evidence for a calcium signature in plant cells? Again the guard cell is probably the model in which to address this question. Imaging studies (Gilroy etal., 1991; McAinsh etal., 1992; Shacklock etal., 1992) have certainly revealed that there is a markedly heterogeneous distribution of calcium in single guard cells and other cell types. Interestingly, it is apparent that after the addition of ABA certain areas in the guard cell remain calcium quiescent while others demonstrate marked increases in calcium (McAinsh et al., 1992). However, we do not yet know whether it is possible to correlate these localized “hot spots” with the distribution of ion channels. Asymmetric distribution of calcium is also observed in tip growth, where there is a localized calcium gradient towards the tip (and growing region) of both Fucus rhizoids (Brownlee and Wood, 1986) and pollen tubes (Miller etal., 1992). In these cases it is possible that the elevated calcium is associated with the control of tip growth (Battey and Blackbourn, 1993). Oscillations in cytosolic free calcium have also attracted attention in higher plants, although many of the earlier reports have been discounted on the basis of imaging artefacts (Read etal., 1993). Recently, however, we have found good evidence that [Ca”], in the guard cell oscillates in response to elevated external free calcium and that the pattern of oscillations is absolutely dependent on the strength of the external stimulus (McAinsh et al., 1995). This result has added significance as it is also possible to correlate both the pattern of the oscillations and the strength of the external stimulus with the extent of the physiological response. We have investigated the origin of the calcium used to generate the oscillations and have found evidence to suggest that there is both calcium influx across the plasma membrane and calcium release from internal stores. The next key question to address is whether this pattern of oscillations can be altered by the addition of another external stimulus. The second feature that these results highlight is the importance of measuring the concentration of free calcium in the apoplast as we have shown that external calcium above and including 100pm induced oscillations in guard cell [Ca2+Ii.The possibility that cytosolic free calcium in these cells may be in a natural state of oscillation requires further investigation. In summary, although the evidence is as yet fragmentary it seems likely that,
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as in animals, plant cells will rely on the interaction of different signalling pathways and different forms of agonist-induced signal to define specificity. When this is combined with differential localization of receptors and effectors there would seem to be ample opportunity for encoding specificity of the calcium signal.
VI. FUTURE PROSPECTS Given the large number of physiological stimuli which are already documented to use calcium ions as second messengers in plants it seems likely that this list will continue to expand in the near future. This process will certainly be aided by the new technological approaches such as recombinant aequorin and long wavelength indicators which will make the quantification of cytosolic free calcium much easier. However, there is still the wider question of the overall control of calcium homeostasis to consider. For example, there is still much work to be done in the characterization of the intracellular stores. In particular, although research has tended to focus on the vacuole, is there a major role for the ER and do other organelles contribute to the maintenance of cytosolic calcium levels? In this review we have not discussed the role of the family of calcium-binding proteins including calmodulin. These proteins could have a major role to play in the control of calcium homeostasis and this will require detailed investigation. Recently, there have been some exciting developments in terms of defining plant calcium-permeable channels (Thuleau etal., 1993, 1994). It can be anticipated that the next few years will result in the opening up of this field through expression of the cloned genes in heterologous systems combined with patch clamp analysis. This type of experiment is likely to reveal some of the complexities and subtleties associated with the regulation of these channels, Similarly, isolation of the genes for signal transduction components will be important for a number of reasons. Over-expression studies in heterologous systems will be important for defining in biochemical terms the activity of the encoded protein. The access to purified proteins will certainly help to shed some light on specificity through the accurate documentation of substrates and activation optima. Over-expression is also the first step in the series of steps leading to a full molecular characterization of the protein either by NMR or X-ray crystallography. Here solution structures may be of great value in working out the molecular basis of interactions with other components of signal transduction components. Access to signalling genes and their promoters will also be of use in studies of the physiological function of the gene product through transgenic strategies. Already this work is bearing fruit (reviewed by Taylor et al., 1994). However, one of the greatest challenges will be to disentangle the complexities of interacting signal transduction pathways which combine to generate
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a single response. Here the picture is getting more complex with the appearance of additional putative second messengers such as cADPR, protons, DAG, CAMP and cGMP. Teasing apart the fundamentals of how these individual messengers are orchestrated to produce the final response will be one of the greatest challenges.
ACKNOWLEDGEMENTS AARW, MRM, JET and AMH are all grateful to the BBSRC for research grant support (Biological Adaptation to Global Environment Change Programme and Intracellular Signalling Programme), the results of which are described in this review. In addition MRM is grateful to the Royal Society for the award of a University Research Fellowship.
REFERENCES Allan, A. C., Fricker, M. D., Ward, J. L., Beale, M. H. and Trewavas, A. J. (1994). Two transduction pathways mediate rapid effects of abscisic acid in Commelina guard cells. The Plant Cell 6, 1319-1328. Allen, G. J. and Sanders, D. (1994). Two voltage-gated, calcium release channels coreside in the vacuolar membrane of broad bean guard cells. The Plant Cell 6, 685-694. Allen, G . J., Muir, S. R. and Sanders D. (1995). Release of CaZ+ from individual plant vacuoles by both InsP, and cyclic ADP-ribose. Science 268, 735-737. Alexandre, J. and Lassalles, J. P. (1992). Intracellular CaZ+release by InsP3 in plants and effect of buffers on Ca2+ diffusion. Philosophical Transactions of the Royal Society of London B, 338, 53-61. Alexandre, J., Lassalles, J. P. and Kado, R. T. (1990). Opening of Ca2+ channels in isolated red beet root vacuole membrane by inositol 1,4,5-triphosphate. Nature 343, 567-570. Amundson, J. and Clapham, D. (1993). Calcium waves. Current Opinion in Neurobiology 3, 375-382. Assmann, S. M. (1993). Signal tranduction in guard cells. Annual Review of Cell Biology 9, 345-375. Ayling, S. M., Brownlee, C. and Clarkson, D. T. (1994). The cytoplasmic streaming response of tomato root hairs to auxin; observations of cytosolic calcium levels. Journal of Plant Physiology 143, 184-188. Battey, N. H. and Blackbourn, H. D. (1993). The control of endocyclosis in plant cells. New Phytologist 128, 307-338. Berger, F. and Brownlee, C. (1993). Ratio confocal imaging of free cytoplasmic calcium, gradients in polarising and polarised Fucus zygotes. Zygote 1, 9-15. Berridge, M. J. (1990). Calcium oscillations. Journal of Biological Chemistry 265, 9583-9586. Berridge, M. J. (1993a). Inositol trisphosphate and calcium signalling. Nature 361, 315-325. Berridge, M. J. (1993b). Ca2+-induced and Ca2+ release and inositol trisphosphate, intracellular channels and calcium spiking. Biomedical Research 14, 21-29. Berridge, M. J. and Dupont, G. (1994). Spatial and temporal signalling by calcium. Current Opinion in Cell Biology 6, 261-274.
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Tsien, R. Y.and Hartoonian A. T. (1990). Practical design criteria for a dynamic ratio imaging system. Cell Calcium 11, 93-109. Tsien, R. Y. and Rink, T. J . (1980). Neutral carrier ion selective microelectrodes for measurement of intracellular free calcium. Biochimica et Biophysica Acta 599, 623-638. Tsien, R. Y. and Tsien, R. W. (1990). Calcium channels, stores and oscillations. Annual Review of Cell Biology 6 , 715-760. Tsong, T. Y. (1991). Electroporation of cell membranes. Biophysical Journal 60, 296-334. Tsunoda, Y. (1993). Receptor operated C a 2 + signalling and cross-talk in stimulus secretion coupling. Biochemica et Biophysica Act! 1154, 105-156. Volotovski, I. D., Sokolovski, S. G.,Nikiforov, E. L. and Zinchenko, V. P. (1993a). Influence of CaZ+ transport inhibitors on light-induced Ca2+ oscillations in plant cell suspension. Photosynthetica 29, 169- 176. Volotovski, I. D., Sokolovsky, S. G.,Nikiforov, E. L. and Zinchenko, V. P. (1993b). Calcium oscillations in plant cell cytoplasm induced by red and far-red light irradiation. Journal of Photochemistry and Photobiology B-Biology 20, 95-100. Wang, M., Van Duijn, B. and Schram, A. W . (1991). Abscisic acid induces a cytosolic calcium decrease in barley aleurone protoplasts. FEBS Letters 278, 69-74. Ward, J. M. and Schroeder, J . I. (1994). Calcium-activated K + channels and calciuminduced calcium release by slow vacuolar ion channels in guard cell vacuoles implicated in the control of stomatal closure. The Plant Cell 6 , 669-683. Weyers, J . and Meidner, H. (1990). “Methods in stornatal physiology”. Longman, London. White, J . G.,Amos, W. B. and Fordham, M. (1987). An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy. Journal of Cell Biology 105, 41-48. Williams, D. A., Cody, S. H., Gehring, C. A., Parish, R. W. and Harris, P. J . (1990). Confocal imaging of ionised calcium in living plant cells. Cell Calcium 11, 291-297. Williamson, R. E. and Ashley, C. C. (1982). Free CaZ+ and cytoplasmic streaming in the alga, Chara. Nature 296, 647-651. Willmer, C. M. (1983). “Stomata”. Longman, London. Zhang, D. H., Callaham, D. A. and Hepler, P. K . (1990). Regulation of anaphase chromosome motion in Tradescantia stamen hair cells by calcium and related signaling agents. The Journal of Cell Biology 111, 171-182. Zottini, M. and Zannoni, D. (1993). The use of Fura-2 fluorescence to monitor the movement of free calcium ions into the matrix of plant mitochondria (Pisum sativum and Helianthus tuberosus). Plant Physiology 102, 573-578.
The Effects of Ultraviolet-B Radiation on Plants: A Molecular Perspective
BRIAN R . JORDAN
New Zealand Institute for Crop and Food Research Limited. Levin Research Centre. Kimberley Road. Private Bag 4005. Levin. New Zealand
I . General Introduction .................................................................. 98 A . Stratospheric Ozone Depletion and the UV Environment ............ 98 B . Potential Effects of Increased UV-B on Plant Growth 101 and Development ............................................................... I1. The A. B. C. D.
Perception of UV-B Radiation ............................................. Measurement of Plant Responses to UV-B Radiation ............... Photoreceptors and their Interactions .................................... Mechanisms of Signal Transduction ...................................... Penetration of UV-B Radiation through Plant Tissue ...............
103 103 104 107 112
I11 . Protective Mechanisms Against UV-B Radiation ............................ A . UV-B-absorbing Pigments .................................................... B . DNA Repair ..................................................................... C . Antioxidants and Other Protective Mechanisms .......................
114 114 117 119
IV . Effects of UV-B Radiation Upon Cellular Processes ....................... A . Photosynthesis ................................................................... B . Carbohydrate. Lipid and Nitrogen Metabolism ........................ C . Reproductive Biology .......................................................... D . Cell-cycle and Cytoskeleton .................................................
121 121 130 131 133
V . The A. B. C.
Effects of UV-B Radiation on Gene Expression ...................... Chloroplast Proteins ........................................................... Defence Genes ................................................................... Factors Affecting Gene Expression ........................................
134 134 135 138
Advances in Botanical Research Vol . 22 incorporating Advances in Plant Pathology
ISBN 0-12-005922-3
Copyright 0 1996 Academic Press Limited All rights of reproduction in any form reserved
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VI.
UV-B Interactions with Other Stresses ......................................... A. Light ............................................................................... 9. Water .............................................................................. C. CO, and Temperature ......................................................... D. Pathogens .........................................................................
141 141 143 145 147
VII.
Conclusion .............................................................................. Acknowledgements ................................................................... References ...............................................................................
148 149 149
I. GENERAL INTRODUCTION A.
STRATOSPHERIC OZONE DEPLETION AND THE UV ENVIRONMENT
Life on earth is protected from ultraviolet radiation (UV) by an ozone layer present within the stratosphere (20-50 km above the earth’s surface). Ozone is produced when radiation from the sun breaks down oxygen into oxygen atoms. These oxygen atoms then combine with oxygen to form ozone. The production of ozone is greatest over the tropics where the sun’s radiation is strongest. Ozone can then be redistributed around the globe and towards the poles by winds in the upper atmosphere. The concentration of ozone within the stratosphere is greatest over the polar regions (c. 350 Dobson units) and is reduced at the equator (c. 245 Dobson units). The UV composition of the equatorial solar environment is subsequently greater than in the upper latitudes. In 1985 it was reported that the Antarctic ozone layer was depleted during the Austral spring (Farham et al., 1985). This observation, made using ground-based Dobson instruments, caused concern and the immediate re-evaluation of data from the total ozone mapping spectrometer (TOMS) aboard the Nimbus-7 satellite. From these studies it was apparent that during the spring a yearly decrease in the ozone layer was taking place (Kerr, 1988). The level of destruction of ozone is now so extensive that overall losses of 50% have been recorded over Antarctica, representing about 5% of global ozone. At lower altitudes within the stratosphere, temperature conditions are optimum for ozone destruction ( - 80°C) and can result in > 90% depletion. The depletion of ozone over the Arctic has been less dramatic, due to atmospheric conditions not being as suitable for the ozone destruction. However, recent data suggest substantial losses of ozone over the Arctic and the ozone layer may realistically be described as being “poised for destruction” (Brune er al., 1991; Austin et al., 1992). Global ozone has also decreased over the period 1979-1991 by 3-5% in mid-latitudes and 6-8% at higher latitudes, with no losses near the equator (Gleason etal., 1993). The data for 1992 have shown a lower global level than any other previous measurements and preliminary data for 1993 suggest even further reductions are taking place (Fig. 1). The destruction of the ozone layer is thought to be primarily caused
99
UV-B RADIATION: A MOLECULAR PERSPECTIVE
.g t
320
1979-90 average
3
.--
L
0
2
290
1992
-
--.
1993 Preliminary data
/--*--
I
I
I
J
F
I
M
I
A
I
M
I
I
I
J
J
A
I
S
I
O
I
I
N
D
Month Fig. I . Stratospheric ozone depletion. Daily global average ozone amount (areaweighted 65"s to 65"N).Adapted from Gleason etaf. (1993). by man-made chlorofluorocarbon (CFC) and other halons. A complex chemical reaction that involves low temperature, C1, nitrogen and their oxides, is taking place to break down the ozone within polar stratospheric clouds (Fig. 2). The CFCs have been widespread in their use as refrigerants, aerosol propellants and in the manufacture of foam and are particularly long-lived molecules. For instance, C F l l and CF12 have an estimated atmospheric lifetime of 75 and 120 years respectively. As a result of their increased usage and longevity the concentration of CFCs in the stratosphere has approximately tripled between 1960 and 1985 (Kerr, 1988). Although the damaging CFCs are being phased out, they will remain a significant problem as they have accumulated in the troposphere to very high levels. These CFCs will continue to permeate slowly into the stratosphere well into the next century. Although ozone is only a small percentage of the atmospheric gaseous constituents, it is the only component that absorbs appreciably radiation below 300nm. The extinction coefficient of ozone increases by orders of magnitude with decreasing wavelength (five orders of magnitude between 350 and 280nm) and completely excludes UV-C radiation of < 280nm which would otherwise destroy the biosphere. UV-A radiation between 320 and 380nm is not absorbed by ozone and passes unaffected through the stratospheric ozone layer. It is therefore only UV-B radiation (280-320 nm) that could be influenced by small changes in the level of global stratospheric ozone. The official definition of the UV-B range was 280-315 nm, established
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+ C10 + M --> C1202 + M C1202 + sunlight --> C1 + ClOO ClOO + M --> C1 + 0 2 + M
C10
2(C1
+ 0, --> c10 + 02)
Fig. 2. Mechanism of ozone depletion. For further details see Brune etal. (1991). M is molecular nitrogen and oxygen.
by the commission International de I'Eclairage in 1935. As no photons in the 280-290 nm region are detectable in the solar spectrum reaching the earth, the lower limit of UV-B could be either wavelength 280 or 290 nm. However, the upper limit is now generally considered to be 320 nm (Peak and van der Leun, 1993). Any change in UV-B radiation resulting from ozone depletion will only be a few per cent of the total solar spectrum. The impact of this change, however, is likely to be significant as these wavelengths are particularly biologically active. Thus, the major concern of a global decline in stratospheric ozone is that there will be an increase in the shorter, more biologically damaging, wavelengths within the UV-B range. The general value of the increase in UV-B is 1.5- to 2-fold increase for every I To loss of ozone. There have, however, been relatively few long-term studies from which an accurate evaluation can be made. Blumthaler and Ambach (1990) made measurements at an altitude of 3000 m in the mountains above Innsbruck. At this altitude they could alleviate absorption of incident UV-B radiation from pollution of urban populations. From 1979 to 1990 they recorded a 0.7-1.0% increase per annum in UV-B radiation. This is approximately consistent with changes in the ozone layer over the northern hemisphere. A number of studies have recently been carried out to compare UV-B levels in the mid-latitudes of the northern and southern hemispheres (Roy et al., 1990; Seckmeyer and McKenzie, 1992). Seckmeyer and McKenzie (1992) compared the UV-B levels in summer between latitudes 45"s in New Zealand and 48"N in Germany. The results showed UV-B irradiances were almost a factor of two greater in New Zealand than in Germany. This was explained by the decrease in stratospheric ozone in New Zealand (266 Dobson units compared t o 352 Dobson units) and increased levels of tropospheric ozone over Germany. Although the precise nature of the changes in the UV environment are not certain, it is apparent that there is likely to be increased amounts of UV-B radiation reaching the earth's surface. This is particularly so for regions in the southern hemisphere were ozone depletion is most severe
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and the atmosphere is relatively unpolluted. Because plants are constantly exposed to solar radiation, the consequences of any increase in UV-B radiation are likely to be most obvious in their effect upon plant growth and development.
B.
POTENTIAL EFFECTS OF INCREASED UV-B ON PLANT GROWTH AND DEVELOPMENT
There have been many studies on the effects of UV-B radiation on plants and a number of excellent reviews are available (Teramura, 1983; Caldwell et al., 1989; Tevini and Teramura, 1989; Tevini et al., 1989; Teramura and Sullivan, 1991; Bornman and Teramura, 1993; Caldwell and Flint, 1993; Tevini, 1993, 1994). Most of the data on plant responses to UV-B radiation have been obtained using controlled environment facilities. The conditions in these facilities are not typical of the natural environment and therefore may not truly reflect the actual response (Caldwell and Flint, 1990, and section IIA). However, it is clear from these studies and the limited field research, that the UV-B environment can be a major factor determining plant growth and development. The research has shown a diverse range of responses depending upon the species, the nature of the UV-B treatment and the interaction of other environmental factors (Table I). Over 300 species have been studied and of these, two-thirds are sensitive to UV-B radiation (Caldwell el al., 1989). Some species are particularly sensitive and show signs of damage at moderate increases in UV-B irradiance, while other species show no response even when exposed to relatively high levels of UV-B radiation. The specific effects upon plants include inhibition of photosynthesis, changes in leaf area and morphology, loss of fresh and dry weight, changes in assimilate partitioning, alterations in pigment biosynthesis and effects upon flowering and reproduction. These responses resulting from an increase in UV-B radiation would clearly lead to a loss of crop productivity and to substantial changes in the nature of plant communities (Barnes etal., 1988, 1990). Among the more susceptible species are agricultural crops from temperate latitudes, such as pea and soya. Differences in response to UV-B radiation are also found between varieties of the same species and this can lead to significant variation in yield. For instance, of 40 cultivars of soya 59% were sensitive to UV-B radiation (Teramura, 1986), the most sensitive being ”James Bay” and “Essex” which gave loss of vegetative growth of 26-38070 in controlled environments and 11-22070 in field trials. Other varieties, such as “Williams”, remained tolerant to the UV-B exposure. Similar results have been found for a number of other dicotyledonous species, notably cucumber (Murali and Teramura, 1986a). Tropical crops, including rice, can also suffer damage from increased UV-B radiation (Teramura eta/., 1990b, 1991; He eta/., 1993; Huang etal., 1993;
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TABLE I
Effects of ultraviolet-B radiation on plants ~
~
~~
~
Effects vary between species and within varieties of the same species. Temperate species are generally more susceptible than tropical species. The developmental stage of the tissue also influences the response
Loss of photosynthetic activity, changes in leaf area and morphology, alterations in fresh weight and dry weight, changes in assimilate partitioning: yield loss in economic crop species Changes in protective pigments caused by the rapid and coordinated induction of enzymes of the phenylpropanoid pathway: potential loss of crop quality Alterations in the surface layers, such as increased amounts of wax, could alter the interactions with pathogens, etc. Effects upon plant development, for instance changes in the timing of the switch from vegetative to reproductive growth Complex interaction with other environmental parameters such as photosynthetically active radiation (PAR), CO, and water stress Influence on the competitive ecology between plant species and with other organisms
Ziska etal., 1993). Recently, 22 cultivars of rice from diverse origins were tested for UV-B sensitivity (Barnes etal., 1993). For most cultivars, the effects on shoot morphology was greater than on biomass production. However, there were significant differences among cultivars in the extent of the changes; with modern, high yielding cultivars among the most sensitive. Studies of UVB radiation effects on trees remain relatively limited in the range of species examined and have usually been carried out over short time periods (Teramura and Sullivan, 1991). Gymnosperms such as the important forest conifer species of Pinus have been studied in some detail and can suffer UV-B damage (Sullivan and Teramura, 1988, 1989, 1992). Of 15 species of conifer tested, seven were damaged, five were resistant and three favoured by UV-B radiation (Teramura and Sullivan, 1991). More recently, Sullivan and Teramura (1992) carried out experiments on loblolly pine over a number of growing seasons and concluded that the effects of the UV-B radiation could be accumulative and significantly reduce the growth over the lifetime of the trees. A major factor determining the UV-B response is the interaction of other environmental parameters (Teramura, 1986; Jordan, 1993, and section VI). Studies of UV-B responses, in combination with other stresses, suggest that any prediction as to the consequences of increased UV-B can only be realistic in the context of interactions with other stresses. The ability of plants to adapt to UV-B radiation is also likely to be an important factor in evaluating the
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overall response. In general, plants growing naturally at higher altitudes or in an equatorial region are less sensitive to UV-B radiation (Barnes et al., 1987). Thus, many plant species have the potential to grow successfully in a higher ambient UV-B environment. In addition, the biological implications of changes in UV-B radiation is not just in the absolute levels of the increase, but the relative increase as a percentage of the biologically active radiation. Thus, many plant responses depend upon the quality of light at different waveIengths and this may be changed by modification of the light environment (shading, solar angle, etc.). Consequently, any alteration in the quality of the light environment could have a substantial effect upon the plant’s response to UV-B radiation (Caldwell et al., 1994). Although the effects of UV-B radiation on terrestrial plants have been extensively investigated and reviewed in some detail, few reviews have focused on the molecular events taking place in the cells and tissues of the plants. Recently, a number of reviews have addressed these aspects of UV-B effects (Bornman, 1989; Stapleton, 1992; Jordan, 1993; Strid et al., 1994). Most, however, have concentrated on photosynthesis and protective pigment biosynthesis. In this review, the effects of UV-B radiation on plants are addressed from a molecular perspective. The chain of events from the perception of UVB radiation to the diversity of responses at the cellular level are described.
11.
THE PERCEPTION OF UV-B RADIATION
A. MEASUREMENT OF PLANT RESPONSES TO UV-B RADIATION
It is not a trivial matter to determine accurately the UV-B radiation to which plants are exposed, and many important problems have been previously considered in detail (Caldwell et al., 1986; Coohill, 1989, 1991, 1993; Caldwell and Flint, 1993). Consequently, the major aspects will only be covered briefly in this section. To determine the biological effectiveness of the UV-B radiation, the following functions are used: UV-BBE= IxEhdX, where UVBEE is the integrated biologically effective radiation, I, is the spectral irradiance and Eh is the biological weighting function which is usually determined from a biological action spectrum (Caldwell and Flint, 1993). It is therefore apparent that the index of biological effectiveness will depend significantly upon the nature of the action spectrum used. Thus, if the action of UV-B radiation on DNA is used a very serious prognosis is obtained. However, the effects may be moderated by using the generalized plant action spectrum (Caldwell, 1971). Furthermore, if the biological effectiveness is derived from a polychromatic action spectrum, the UV-B radiation is even less damaging (Tevini, 1994, and references therein). The advantage of polychromatic action spectra is that they include the ameliorating effects of other wavelengths of light. For instance, these wavelengths may stimulate DNA
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repair or protective pigment biosynthesis. A recent absolute action spectrum for pyrimidine dimer formation (Quaite etal., 1992) also suggests that the effect of increased UV-B radiation may have less impact than has previously been predicted. This is also supported by the work of Caldwell etal. (1994), showing the ameliorating influence of both UV-A radiation and photon flux density. Plant responses to UV-B have been determined in a wide variety of conditions, including controlled environment cabinets, glasshouse experiments, long-term field trials and experiments where natural UV-B radiation is filtered out. These approaches and the problems involved are discussed in Caldwell and Flint (1993, and references therein). In most studies, UV-B emitting lamps are used as a supplement to controlled environment lighting or natural irradiance. These must be used with filters (such as cellulose acetate) to exclude shorter wavelength UV-B ( c 295 nm) which is never part of the solar flux. Although controlled environment experiments are convenient, they do not represent a realistic simulation of UV-B radiation in a natural solar environment. Consequently, these studies routinely give exaggerated effects which are usually related to the relatively low PAR and high UV-B radiation in the cabinets. Controlled environment experiments may, however, be indicative of a plant’s UV-B sensitivity and are certainly useful for mechanistic studies. Field trials are more realistic as, to a greater extent, they take into account the natural irradiance. However, the solar irradiation fluctuates during the day and the UV-B component fluctuates more than other wavelengths. This, and other aspects such as cloud cover, need to be considered in these type of experiments. In addition, a major problem is accounting for the variations in other environmental parameters that are known to influence the UV-B response, such as rainfall. Thus, long-term field studies should be carried out to establish the pattern of the response and the variation in the climate taken into account throughout the experimental period.
B.
PHOTORECEPTORS AND THEIR INTERACTIONS
To enable the UV-B radiation to have an effect it must be absorbed by the plant. The plant may perceive the UV-B radiation by a specific mechanism involving photoreceptor molecules or by non-specific absorption by other cellular constituents. One of the reasons that UV-B radiation is potentially so damaging is that it can be absorbed by a wide range of biologically important molecules. These include proteins, quinones, lipids and most significantly, nucleic acids. Such non-specific absorption would almost certainly have deleterious consequences. In addition, this would not provide the regulatory potential to allow the plant to adjust to the UV-B environment. Plants may, however, “sense” their UV-B environment by either UV-B photoreceptors alone or in conjunction with other photoreceptors detecting various regions
UV-B RADIATION: A MOLECULAR PERSPECTIVE
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of the solar spectrum. As plants readily monitor their light environment by photoreceptors, it is certainly possible that UV-B radiation could be detected in a similar manner. This would give the plants the potential to acclimate to the UV-B radiation and regulate its influence. Plants have a number of photoperception mechanisms that monitor the light environment and regulate their growth and development. These can be divided into mechanisms that involve specific photoreceptors, including phytochrome, a blue/UV-A photoreceptor and possibly a specific UV-B receptor. Alternatively, the perception of the light environment may be part of a complex biological system, such as photosynthesis (Chow etal., 1990) or photoperiodism (involving phytochrome and a circadian timer; Lumsden, 1993). Of the photoreceptors, phytochrome has been most extensively characterized and indeed is one of the most studied plant molecules (see reviews by Pratt, 1982; Jordan etal., 1986; Partis and Thomas, 1991; Quail, 1991; Furuya, 1993). Phytochrome acts both to perceive the light environment and to transmit this information within the plant cell. Consequently, it has a major role in determining plant photomorphogenesis and is known to control many aspects of plant development, such as flowering, germination and morphology. Phytochrome is a chromoprotein with a characteristic photoreversibility between an ostensibly inactive red absorbing form (Pr: X max. 666 nm) and an active farred absorbing form (Pfr: h max. 730nm). Although it does have absorption in the blue part of the spectrum it is thought to function largely through the perception of red and far-red light. Phytochrome also exists in several forms, notably light-labile type I phytochrome that is present in dark-grown tissue and type I1 that is present in relatively small amounts but predominant in green tissue. These types of phytochrome are thought to regulate different aspects of plant growth and development. Far less detailed information is available about the molecular character of the blue-light/UV-A photoreceptor system, although it is likely to be a flavin or flavoprotein (see Short and Briggs, 1994, for a comprehensive review). Although the biochemical properties are still not characterized, the bluelight/UV-A photoreceptor(s) is known to regulate many specific responses. These responses may even be controlled by different blue-light/UV-A photoreceptors. For instance, phototropism and inhibition of elongation by blue light display different kinetics and sensitivity. In addition, other photoreceptors may be involved in blue-light/UV-A light responses. Thus, phytochrome may absorb in the blue region of the spectrum and elicit photomorphogenetic responses directly or interact with the blue-light/UV-A photoreceptor system. Recently, the gene for a blue-light photoreceptor has been isolated from the Arabidopsis hy4 mutant by T-DNA tagging (Ahmad and Cashmore, 1993). The gene for the photoreceptor has substantial amino acid sequence similarity to microbial photolyase. There are, however, significant differences in its structure, suggesting that it is functionally different. It is expressed in all tissues of the plant and is likely to be a soluble protein. The
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identification of the gene for this putative photoreceptor could considerably increase the present understanding of blue-light/UV-A photoregulation. Detailed information on a specific UV-B photoreceptor is still relatively poor. A number of studies have indicated the involvement of such a photoreceptor, but alternative mechanisms could explain these findings, such as non-specific damage and the action of other photoreceptors (discussed in Wellmann, 1983). More recently, Ballark etal. (1991) have provided good evidence for the existence of a specific UV-B photoreceptor. They used wildtype and long-hypocotyl mutants ( f h mutants are deficient in stable phytochrome) of cucumber to study the effect of UV-B radiation on hypocotyl elongation. These studies demonstrated that the signal resulting in inhibition of growth was perceived in the cotyledons and therefore not due to direct UVB damage to the hypocotyl tissue. The effect was not caused by changes in area or dry matter, and is therefore unlikely to involve assimilate limitation. Furthermore, inhibition was the same for the f h mutant as for wild-type, suggesting that phytochrome was not the photoreceptor in the cotyledons. Photomorphogenesis routinely requires the precise regulation of gene expression. It is not surprising therefore to find that all the photoreceptors are known to regulate gene expression. Thus, phytochrome regulates its own gene expression and that of many other genes, notably those for chloroplast proteins (Jordan et al., 1986). Blue-light/UV-A has also been found to regulate gene expression (see details in the reviews of Thompson and White, 1991; Short and Briggs, 1994) as has UV-B radiation (see section V). The changes in gene expression can involve either up- or down-regulation and the effectiveness of a particular photoreceptor may vary with the stage of development. For example, phyA may control gene expression and chloroplast development during greening of etiolated tissue. In more mature tissue other phytochrome types may be more influential in regulating development. Consequently, the perception of UV-B radiation may vary with the changing nature of other photoreceptors and this could lead to different responses during development. Another important aspect of photoreceptor control is in their regulation of different members of multigene families. Thus, in a number of studies, the activity of a particular member of a gene family is controlled by different photoreceptors and this may vary with the stage of development (Karlin-Neumann et al., 1988; DeRocher and Bohnert, 1993). The complexity of the response to UV-B within the overall light environment is clearly reflected in a number of studies on flavonoid biosynthesis (Bruns ef af., 1986; Batschauer etaf., 1991; Frohnmeyer etaf., 1992). In parsley tissue culture ceIls, chalcone synthase (chs) is strongly induced by UV light. The increase takes place after a lag of 2 h. If the cells are pretreated with blue light the lag phase can be removed. In addition, red/far-red light treatments which control the phytochrome system will alter both the magnitude and extent of the chs induction. Thus, three separate light qualities can be involved in this one induction process. The complex role of different
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photoperception mechanisms is further illustrated during the development of petunia (Koes e t a f . , 1989) and mustard (Batschauer e t a f . , 1991). In petunia seedlings and cell suspension cultures, chs expression is UV dependent, while in petals it is controlled by red light. In mustard, red light regulates chs in cotyledons, but blue and UV control the expression in primary leaves. The influence of UV-B radiation on chs gene expression is therefore dependent upon the nature of the tissue and the stage of development. Similarly, the response to UV-B radiation of cab gene expression changes during development (Jordan etal., 1994, and section VC).
C.
MECHANISMS OF SIGNAL TRANSDUCTION
Once the UV-B radiation is perceived by the plant, the information must be transmitted through cells or tissue to target sites were it may elicit a response. This transmission is frequently referred to as the signal transduction pathway and is comprised of the secondary messenger@), an amplification mechanism and the responsive component within the cells. It is important to stress that although the message may arrive at the correct site within the cell or tissue, that system must be appropriately primed to respond. Most of our knowledge of plant signal transduction systems is based upon information obtained in animals. There is now, however, considerable information on plant systems and a range of molecular mechanisms have been demonstrated (see the review by Lumsden, 1993). Components of these pathways may include G proteins, Ca2+, the production of CAMP and the inositol phosphate pathway. Lightinduced responses are thought to involve many of these components (Lumsden, 1993; ROUX,1994). As yet, little is known about the specific mechanisms stimulated by UV-B radiation. However, they are likely to involve some or all of the known pathways. The most information available on lightinduced transduction pathways is on those mediated through phytochrome (Roux, 1994). It is therefore appropriate to consider them as they may reflect similar UV-B induced pathways. One response that has been used to investigate light-induced signal transduction pathways, is the phytochrome-mediated swelling of protoplasts. This was first reported by Blakeley et af. (1983) who showed a red-light-induced swelling of wheat protoplasts that was far-red photoreversible over two cycles. The protoplast swelling was considered to be caused by changes in plasma membrane permeability as indicated by a K + stimulation of the response and inhibition by vanadate (Blakeley et al., 1987). Bossen e ta f . (1988) reported that swelling of wheat protoplasts was dependent on exogenous Ca2+ and this requirement could not be replaced by MgZ+,Ba2+ or K + . In the presence of the calcium ionophore A23187, protoplast swelling takes place in the dark to the same extent as after red irradiation. Ca2+-channel blocking agents, verapamil and La3+, inhibited the phytochrome-induced swelling. The work
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was extended to examine the role of GTP-binding proteins (G proteins) and the phosphatidyl inositol (PI) transduction pathway in protoplast swelling (Bossen et al., 1990). Activation of G proteins is thought to be a primary event in signal transduction and has a role in regulating Ca2+-channels(Gilman, 1987; Kaziro et al., 1991). Red-light-induced swelling of protoplasts was inhibited by the G-protein inhibitor GDP-0-S. In darkness or control far-red irradiation, the G-protein activator GTP-y-S gave equivalent swelling to red irradiation. These results suggested the involvement of G proteins in the phytochrome response. A range of compounds known to inhibit enzymes of the PI transduction mechanism were also tested. These included neomycin and Li+ that inhibit phospholipase C and phorbol 12-myristate, which is responsible for stimulating protein kinase C. The results with these compounds were consistent with the predicted response based on animal studies of the PI transduction pathway. Further support for a role of PI in plant signal transduction has come from the unambiguous identification of PI cycle intermediates (Parmar and Brearley, 1993). Recently, the role of Ca2+ in mediating phytochrome-induced swelling of protoplasts was examined by Shacklock et al. (1992), using caged Ca2+ and inositol 1,4,5-triphosphate (IP3). Wheat leaf protoplasts were loaded with the caged Ca2+ and photoactivated with ultraviolet light. The [Ca"] was raised to 1 PM in localized areas of the cytosol within 1 min and protoplast swelling increased by > 20% within 10min. Thus a transient rise in cytosolic [Ca2+]can induce a longterm physiological change in the cell. These studies also showed that IP3 could increase [Ca2+]and a transient increase in [Ca*+]could be induced by phytochrome action. From the studies of this model system, the results suggest the involvement of Ca2+, G proteins and the PI pathway in phytochromeregulated signal transduction. The phytochrome signal transduction pathway has also been investigated in the aurea mutant of tomato (Neuhaus etal., 1993). In this elegant study, a more analytical procedure was employed to define the components of the pathway. Thus, etiolated aurea mutant seedlings contain less than 5% of the phytochrome present in wild-type plants. They also have reduced anthocyanin levels (20%) and under-developed chloroplast ultrastructure. These features allow the signal transduction pathway to be dissected by microinjection of putative cell-signalling intermediates. The results suggest that the phytochrome transduction pathway initially involves one or more heterotrimeric G proteins. These G proteins are activated by the phytochrome system at an early stage and control anthocyanin biosynthesis, chloroplast development and the expression of a cab-GUS reporter gene. These G proteins are coupled to two or more different pathways. One of these pathways involves calcium and activated calmodulin as essential components. This pathway leads to the expression of the cab-GUS reporter and to partial development of the chloroplast. The calcium/activated calmodulin pathway is not, however, able to stimulate either anthocyanin biosynthesis or complete chloroplast develop-
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ment. The incomplete development of the chloroplast was due to limited biosynthesis of chloroplast proteins. Thus, the calcium/activated calmodulin pathway induces the synthesis of a number of chloroplast components, such as Rubisco and subunits of photosystem I1 and the ATP synthase complexes, but not photosystem I or the cytochrome b,f complex. It was also significant that the calcium-dependent pathway could stimulate the synthesis of chloroplast encoded proteins. As these proteins are regulated at a number of levels (transcription, translation and post-translation), the calcium/activated calmodulin pathway shows a wide network of interactions with the regulation of gene expression. Recently it has been shown that cGMP is able to stimulate anthocyanin biosynthesis and in combination with calcium can induce the development of fully mature chloroplasts containing all the photosynthetic components (Bowler et al., 1994). To date, mainly biochemical approaches have been used to study UV-B and blue/UV-A signal transduction pathways. The plasma membrane of plant cells undergoes a number of changes in response to UV-B exposure (see Murphy et al., 1993,for further details and references). These changes include an efflux of K + , depolarization of the cells electrical potential, synthesis of H z 0 2 and oxidation of reduced glutathione (GSH) to oxidized glutathione (GSSG).The changes to the plasma membrane are considered to be induced in response to the UV-B and not a result of direct photochemical damage and subsequent loss of membrane integrity. Using Ca2+-channelblockers such as La3+ and nifedipine, evidence has been obtained for a role of CaZ+in UV-B-induced signal transduction. Chlorotetracycline, monoclonal anticalmodulin antibodies, fluphenazine and 4’-6-diamidino-2-phenylindole-dihydrochloride monohydrate have also been used to visualize UV-B radiation-induced changes in calcium, calmodulin and nuclear DNA in Euglena gracilis (Tirlapur et al., 1992). The results showed a significant influx of Ca2+ into the cytoplasm of the irradiated cells which is followed by a massive accumulation of these ions in and around the nuclei. While initially the cytosolic calmodulin distribution was high, the level decreased to a very low level in the cytosol after 6 h of irradiation and a weak signal was present in the degenerating nucleus. Recently a light-responsive in vitro transcription system from evacuolated parsley protoplasts has been described (Frohnmeyer et al., 1994). Using different light treatments, including UV, to study chs transcription, it was concluded that this system contained a largely intact signal transduction chain from the photoreceptor(s) to the chs promoter. Such systems are likely to prove invaluable in defining the UV-B signal transduction mechanism. The role of G proteins has been investigated in blue-light/UV-A signal transduction (see Short and Briggs, 1994, for further details and references). Warpeha et al. (1991)reported a blue-light dependent rapid transient increase in GTPase activity in plasma membranes isolated from etiolated pea apical buds. They also used immunodetection and photoaffinity labelling to identify G proteins in the plasma membrane. From these studies they concluded that
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a heterotrimeric G protein was involved in the blue-light signal transduction pathway These signal transduction pathways must accept information from a range of photoperception mechanisms and accurately direct the response. The molecular mechanisms involved in transmitting the signal from the photoreceptor to the components of the pathway(s) is not known. One obvious inconsistency is the soluble nature of the known photoreceptors (e.g. phytochrome and the recently isolated blue-light photoreceptor) and the membrane location of components of the transduction pathway. It is likely from the experimental data that components of the transduction mechanism (notably G proteins) are associated with the plasma membrane. Intuitively, such a location would make most sense. However, real progress will only be achieved when the link between the photoreceptor(s) and transduction pathway(s) is fully understood. An additional complexity is that many cellular responses can be induced by a number of factors. For instance, the anthocyanin biosynthetic pathway can be induced by light and or pathogens (see section VID for further details). However, UV light and pathogens elicit separate components of the pathway, so that UV irradiation induces flavone glycosides, and fungal elicitors induce the furanocoumarin phytoalexin pathway. Furthermore, the air pollutant ozone can stimulate both of these pathways simultaneously (Eckey-Kaltenbach et al., 1994). There must therefore be sophisticated regulation of the “cross-talk” between pathways. In addition, the target sites for these pathways must be able to respond appropriately. This is illustrated by the expression of chs in parsley. The expression of this gene is influenced by a different quality of light and by the stage of development; this regulation taking place through different photoreceptors (see section IIB). However, in parsley, there is only a single gene and therefore the promoter of this gene must have the potential to respond to a variety of light environments, developmental stage and tissue (cell)-specific regulation (Frohnmeyer et al., 1992). It is clear from the details above that a degree of consensus is emerging as to the likely components of the light-induced signal transduction pathways. There are still, however, many aspects of the system that need to be clarified and this is particularly true for UV-B-induced pathways. Using a combination of biochemical, molecular and genetic approaches, it should soon be possible to define the signal transduction pathway from UV-B and the interactions between one or more separate photoreceptor systems. In addition to specific photoreceptors initiating a transduction pathway, other possible systems exist. For instance, down-regulation of genes for photosynthetic proteins, especially cab, is frequently cited as evidence for a chloroplast signal transduction pathway (reviewed by Taylor, 1989; Susek and Chory, 1992). This signal is associated with a lack of chloroplast development and/or oxidative damage to the chloroplast. The gene expression for other cytoplasmic proteins remains unaltered by the oxidative damage. As an early consequence of UV-B exposure is chloroplast membrane disruption, probably
.
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resulting from oxidative damage (Chow eta/., 1992), and there is also a rapid down-regulation of genes for chloroplast proteins (Jordan et al., 1991, 1992, 1994 and further discussion below). It is possible that the chloroplast signal could be involved in UV-B-induced repression of gene expression. Recently, a number of Arabidopsis mutants (gun mutants: genome uncoupled) have been produced in which the chloroplast signal has been uncoupled (Susek eta/., 1993). In the presence of herbicides that allow photo-oxidative damage to the chloroplasts, these mutants continue to express genes for photosynthetic proteins. Using gun mutants the role of the chloroplast signal in UV-B-induced down-regulation of gene expression has been investigated (Jordan, Susek and Chory; unpublished observations). From these studies gun mutants that have been photo-bleached with herbicide continue to express genes such as cab and rbcS. However, plants that are also exposed to UV-B radiation continue to show down-regulation. Thus, in plants in which the chloroplast signal is not present, UV-B radiation still affects gene expression (Fig. 3). This strongly suggests that the chloroplast signal is not involved in these UV-B-induced changes. An additional piece of evidence against the role of oxidative damage in the down-regulation of genes for chloroplast proteins, is that higher irradiance should increase oxidative damage, However, increased irradiance acts to protect mRNA levels (Jordan eta/., 1991, 1992). In contrast, oxidative damage may play a role in inducing the genes of the flavonoid pathway (see section IIIC below).
Fig. 3. UV-B effects on Arubidopsis gun mutant gene expression. Arubidopsis gun (genome uncoupled) or pOCA (control line) transgenic mutants were exposed to UV-B radiation for 3 days and the cub RNA transcripts determined. S, sandoz; U, supplementary UV-B treated; C, white light control plants.
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PENETRATION OF UV-B RADIATION THROUGH PLANT TISSUE
Plants can intercept a large percentage of solar radiation and assimilate it into organic material. This requires that visible radiation penetrates through the epidermal layers of leaves to the underlying palisade and mesophyll tissues where the majority of photosynthesis takes place. A similar penetration of UVB radiation into these cells would, however, be harmful to the plant. In most plants that have been studied, reflectance from the leaf surface is relatively low ( < 10%) and attenuation by UV-B-absorbing pigments is the method of screening out this harmful radiation (Caldwell etal., 1983). Phenolic compounds, such as flavonoids, strongly absorb in the UV-B region but also transmit photosynthetically active radiation. They rapidly increase in concentration on exposure to UV-B radiation and are therefore a primary means of attenuating UV-B. The role of epicuticular waxes has also been examined (Day et al., 1992). After removal of these waxes little difference in UV-B penetration was observed and therefore they are not considered to play a major role in UV-B attenuation. Similarly, little affect on UV-A (360 nm) penetration has been found (Bornman and Vogelmann, 1988). There are technical problems with the evaluation of the spectral radiation within the tissue as a result of factors such as the internal architecture of the tissue. The radiation may be absorbed, scattered or reflected and the relative extent to which these processes take place will vary depending upon the tissue (Vogelmann, 1993). Growth conditions have a profound influence upon plant morphology. For instance, leaf ultrastructure, biochemical composition and morphology are substantially modified by increases in irradiance (see references in section VIA). UV-B radiation itself will modify pigment composition and lead to attenuation of the UV-B levels. An alteration in UV-B can also lead to changes in the distribution of photosynthetically active radiation (PAR) within a leaf (Bornman and Vogelmann, 1991). Detailed studies of UV-B penetration have been carried out on a variety of species (Robberecht and Caldwell, 1986; Day et al., 1992, 1993). These studies suggest that there is a wide range of epidermal transmittance between species. In a study of 22 species, conifer needles were particularly effective in reducing UV-B penetration into underlying tissue while UV-B penetration was greatest in herbaceous species with woody dicotyledonous plants and cereals being between the two extremes (Day et al., 1992). Using a fibre-optic microprobe, penetration of UV-B radiation (300 nm) was shown to be approximately 150 pm in leaves of herbaceous species such as Chenopodiurn album, but only 17 pm in Picea pungens (Day et al., 1993). Epidermal transmittance in these two species was 39% and 0% respectively. The spatial pattern of penetration was also investigated. By placing epidermal peels over a thin polymer that fluoresced blue when irradiated with UV-B, the distribution of UV-B penetration could be examined microscopically. The investigation showed that in herbaceous species, UV-B radiation penetrated through the anticlinal cell walls
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of the leaf tissue and not through the protoplasts of the epidermal cells. In the herbaceous plant, Srnilucinu stellata, epidermal transmittance was 90% through anticlinal cell walls, but less than 10% through protoplast regions of the epidermal cells. Transmittance was also high through stoma1 pores, but not through the guard cells. Penetration through the epidermal layer was not detected in conifer and thus it provides a more uniform protective layer in these species, the lack of UV-B transmittance through the anticlinal cell walls being caused by the presence of more phenolic compounds. One important factor that will determine the incident radiation on the leaf surface is the angle of the lamina to the radiation (Bornman and Teramura, 1993). For instance, the relative lack of sensitivity of cereal species to UV-B has been suggested to be due to their upright position. In studies on rice, where the leaf laminae were placed perpendicular to the incident UV-B radiation (He etal., 1993), it was found that some increase in UV-B effects took place as a result of the leaf position. However, it was noted that pea leaves which were naturally perpendicular to the UV-B radiation suffered much more extensive damage than did the rice leaves. A wide range of plants track the passage of the sun and can move their leaves to avoid photodamage. The ability to adjust leaf orientation to the sun may even account to some extent for UV-B sensitivity between cultivars (Bornman and Teramura, 1993, and references therein). Another interesting aspect of these studies is the relationship of the UV-B penetration to the inhibition of biological function. As UV-B penetrates into the mesophyll cell layers in herbaceous plants, they would be expected t o suffer some form of inhibition to the photosynthetic apparatus. However, in many of the herbaceous species examined no inhibition of intact-leaf photosynthesis has been found (Caldwell etal., 1989). There are a number of possible explanations for this. For example, there could be substantial attenuation of UV-B radiation by protective pigments at or within cells. Similarly the internal architecture of the tissue may limit damage. Protective mechanisms may also be very effective in the repair of DNA damage or in suppressing oxidative damage. Furthermore, it has also been observed that the effect of UV-B radiation may vary depending upon the developmental stage of the tissue (Jordan etul., 1994; Lois, 1994). Thus, in etiolated pea seedlings cub gene expression is not affected by UV-B exposure, even after 7 days of greening under supplementary UV-B radiation (Jordan eta/., 1994; Fig. 4). This contrasts with the severe inhibition of cab expression in more mature tissue. A number of different reasons may account for this differential response. For instance, the difference may be caused by variation in the signal transduction pathways between etiolated and green leaf tissue. In addition, different members of a multigene family may be expressed at the different stages of deveIopment and be more or less sensitive to UV-B exposure. It may also be due to more physical properties, such as a greater concentration of protective pigments
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Fig. 4. UV-B effects on cub gene expression during greening. Autoradiographs of northern-blot analysis of total RNA (20 pg) from pea tissue undergoing greening for 7 days. The RNA was isolated from dark-grown tissue exposed to white-light irradiance (L), dark-grown tissue exposed to UV-B radiation alone (U), dark-grown tissue exposed to white-light irradiance and UV-B radiation (LU) and dark-grown bud tissue (D). The RNA was then hybridized to 32P-labelled cub (data from Jordan et ul., 1994).
(flavonoids, carotenoids, etc.) per unit of tissue. In addition, there may be more of these compounds in the cell walls which will reduce the penetration of the UV-B radiation.
111.
PROTECTIVE MECHANISMS AGAINST UV-B RADIATION A.
UV-B-ABSORBING PIGMENTS
Plants have a range of mechanisms to protect themselves against UV-B radiation damage (Fig. 5 ) . A well-characterized response of plants to UV-B radiation is the synthesis of protective pigments that absorb short-wavelength radiation. These pigments are mainly phenylpropanoids and absorb light between 230 and 380 nm (Markham, 1982; Hahlbrock and Scheel, 1989; Strid and Porra, 1992). The major protective pigments are water-soluble, colourless flavonoids including flavones, flavonols and isoflavonoids. Other related phenolic compounds, such as sinapate esters, also accumulate in epidermaI cell vacuoles after exposure to UV-B radiation (Li etal., 1993, and references therein). In addition, anthocyanins also readily accumulate under UV-B irradiation, although they absorb maximally around 530 nm. Anthocyanins esterified to cinnamic acid can, however, provide UV-B protection. Exposure to UV-B induces a rapid and coordinated increase in the enzyme activities of the phenylpropanoid biosynthetic pathway, the flavonoid pigments being deposited mainly in the cell vacuoles and walls. Flavonoids are found in most
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Protective mechanisms against UV-B increase in surface wax reflectance) flavonoid biosynthesis (UV E absorption)
anti oxidants
UV B penetration through anticlinal
DNA repaii
upper epidermis changes i n cell metabolism
changes in gene expression
changes in stomata1 opening
Fig. 5. Protective mechanisms against UV-B radiation. Representation of a leaf cross-section exposed to UV-B radiation and an enlarged mesophyll cell illustrating potential protective mechanisms.
plant tissue including leaves, petals, pollen and stems. In parsley leaves the biosynthesis has been specifically localized in the epidermal cells (Schmelzer eta/., 1988). Thus, both the chs mRNA and protein increase within the epidermal cells as flavonoids accumulate. The accumulation of flavonoids can increase to high concentrations of between 1 and 10 mM in the epidermal layer (Vierstra eta/., 1982), providing the major source of attenuation of UV-B radiation. This increase in flavonoids is a consistent and widespread plant response to UV-B radiation, both under controlled environments and in natural plant communities. The accumulation of these pigments is therefore considered to provide protection to normal plant functions. Until recently, however, there have been relatively few experimental examples confirming this inferred protection. In rye seedlings increased flavonoids in the epidermal layer provide protection to the photosynthetic apparatus (Tevini eta/., 1991). Plants were pre-irradiated with long-wavelength UV-B, causing the accumulation of flavonoids. These plants were less susceptible to photosynthetic damage than controls, as monitored by chlorophyll fluorescence, when exposed to short-wavelength UV-B radiation. In addition, in Brassica napus the degradation of the photosystem 11, D1 protein was reduced in UV-B adapted plants. The reduction in the rate of degradation was comparable to the flavonoid concentration (Wilson and Greenberg, 1993). In suspension cells
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of Centuureu cyunus the effect of UV-B on cell survival and pyrimidine dimer formation has been determined (Takahashi et ul., 1991). In these suspensions, cell sensitivity to UV-B decreased with increased levels of protective anthocyanins. Furthermore, pyrimidine dimer formation resulting from UV-B exposure was decreased in cell suspensions that accumulated anthocyanins. More direct evidence for the protective function of flavonoids has come from the use of transparent testa mutants of Arubidopsis (Li et ul. , 1993). The tt-4 mutants, which have reduced flavonoids and normal levels of sinapate esters, were more sensitive to UV-B irradiation than were wild-type plants. However, the tt-4 and tt-6 mutants which have both reduced flavonoid levels and sinapate esters, are highly sensitive to UV-B radiation treatments. Thus both types of phenolic compound are involved in UV-B protection. These mutants have also been used to study the protective role of flavonoids on gene expression (Jordan et ul., unpublished observations). In these studies, gene expression for cub was more severely inhibited by UV-B exposure than it was in wild-type Arubidopsis. Another UV-B sensitive Arubidopsis mutant has been generated by chemical mutagenesis (Lois and Buchanan, 1994). This mutant is specifically sensitive to UV-B compared to other forms of stress and the mutation appears to influence only the genes involved in the biosynthesis of kaempferol. These results suggest that a few specific flavonoid compounds may be particularly important in UV-B protection. In similar experiments on maize it was found that DNA damage was reduced in plants that contain flavonoids compared with isogenic lines lacking regulatory genes for anthocyanin biosynthesis (Stapleton and Walbot, 1994). Both cyclobutane pyrimidine dimer (CPD) and 6,4 photoproducts were increased in the plants lacking flavonoid pigments. Interestingly, experiments on field-grown plants showed no significant differences in CPD levels between maize plants with or without flavonoids. UV-B radiation causes both quantitative and qualitative changes in epicuticular waxes (Steinmiiller and Tevini, 1985; Barnes etul., 1994). These waxes are a complex mixture of hydrophobic lipids that do not significantly absorb UV-B radiation. The change in their composition with UV-B may, however, have considerable indirect effects. Thus, in addition to changing the reflectance properties of the leaves, the changed wax composition may reduce transpiration, change the splash-dispersal of pathogens, alter the uptake of aqueous chemicals and even affect the response of herbivores to the plants. The effect of UV-B on cuticular wax was examined in three species, cucumber, bean and barley (Steinmiiller and Tevini, 1985). The cuticular wax of both cucumber and bean was a homogeneous thin layer over the leaf surface. In contrast, in barley, crystal-like structures could be seen on the leaves. In addition, the amount of wax was five times greater in barley than in the two dicotyledonous species and increased with increasing age along the leaf lamina, The wax content of barley was comparable to that found in other monocotyledonous plants, such as wheat (Steinmiiller and Tevini, 1985). On
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irradiation with UV-B, the wax content of all of the plants tested increased by approximately 25%. As leaf area was also decreased by the UV-B treatment, the apparent increase in wax may only be a consequence of the change in morphology and not a change in biosynthesis of these lipids. The results, however, are likely to reflect changes in wax biosynthesis because the increase was mainly associated on the adaxial surface of the leaf. Also, in addition to the increase in wax content, there was a qualitative change in composition with more shorter-chain waxes in UV-B irradiated tissue. Barnes eta/. (1994) studied the wax composition of UV-B irradiated tobacco leaves. Tobacco wax consists of three major components: straight-chain hydrocarbons (C27-C33 comprising 59%), branched-chain hydrocarbons (C2s-C32comprising 38%) and fatty acids (C14-CIs comprising 3%). After 28 days exposure to enhanced UV-B (5.66 kJ m-2 d-': approximately 25% enhancement) the quantity of straight-chain lipids decreased and the branched-chain lipids and fatty acids increased. The overall change was towards a shorter chain composition of the leaf surface wax. This is likely to be caused by inhibition of membrane-localized elongases responsible for the biosynthesis of the longchain hydrocarbons (see Tevini and Steinmuller, 1987, for further discussion and references). These changes in the surface wax were associated with an increased wettability of the leaf surfaces.
B.
DNA REPAIR
DNA is a highly reactive molecule that is sensitive to damage from a wide range of both physical and chemical agents. It is, however, remarkably stable, even in comparison with other nucleic acids such as RNA. This stability is partly due to its double helix configuration, allowing duplication of information and also to a variety of repair mechanisms (Sancer and Sancer, 1988). UV radiation is strongly absorbed by DNA with a maximum absorption around 260 nm (UV-C radiation), decreasing substantially in absorption through the UV-B region (260 nm/280 nm ratio of 1.65 to 1.85) to low levels at 320 nm. Any damage caused by UV-B to the genetic machinery of the cell is potentially a significant problem for plant growth and development. It is therefore imperative that plants have an effective system of DNA repair capable of coping with increasing levels of UV-B radiation. The damage to DNA and its repair mechanisms have been investigated extensively (Jackson, 1987; Sancer and Sancer, 1988; Stapleton, 1992). The major damage caused by UV radiation is the formation of CPDs and pyrimidine-(6-4 ')-pyrimidone photoproducts. A number of types of mutations also occur, including transitions (G:C- > A:T and A:T- > G:C) and transversions (G:C- > T:A, A:T- > T:A, A:T- > C:G and G:C- > C:G). The transitions frequently cluster around pyrimidine dimer sites creating hotspots for mutation (Hutchinson, 1987). Other DNA photoproducts can be formed, such as
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DNA strand breaks and cross-linking of DNA to proteins (Smith, 1977). This DNA damage leads to a multitude of potential problems including incorrect incorporation of bases during replication and frame shifts of the coding regions (Smith, 1977; Jiang and Taylor, 1993). DNA damage has been observed in plants (Stapleton, 1992) and an in vitro action spectra produced (Setlow, 1974, and see section IIA). More recently, an absolute action spectra for CPDs has been produced from irradiated alfalfa seedlings (Quaite et al., 1992). The action spectra differed from those produced on in vitro DNA by having a maximum sensitivity at 280 nm and not 260 nm. This shift probably resulted from attenuation by pigmented epidermal layers and i n t e r h t r a cellular tissue organization. Direct irradiation of DNA also resulted in greater damage at 280 nm by a factor of 100 than found in the intact tissue. In addition, the DNA damage in alfalfa was found to extend to wavelengths up to 365 nm, although at a longer wavelength (405 nm) no dimer formation was found. Thus, the UV-A region of the spectra may be important for both DNA damage and for the mechanisms of repair (see below). Taken together, these results suggest that the impact of increased UV-B, based on previous predictions, may have been over-stated (Quaite et al., 1992 and section IIA). Until recently, few investigations have focused upon the repair mechanisms of higher plants and it has been assumed that similar mechanisms were operable to those in other organisms. These include direct repair mechanisms in which the covalent modification of the DNA is reversed. Of this type of repair, photoreactivation by photolyase is the best characterized (Sancer and Sancer, 1988; Stapleton, 1992). The DNA photolyase uses light energy (between 300 and 5 0 0 nm) to reverse the covalent dimerisation of cis,syn cyclobutane dipyrimidines. Thus, in Arabidopsis, photolyase activation showed a broad wavelength dependency of 375-400 nm which is also similar to maize pollen (Pang and Hays, 1991). A particularly interesting finding was that the Arabidopsis photolyase activity, both in vitro and in vivo was very temperature dependent. This could be important in a global scenario in which the temperature and the UV-B levels increase (see section VIC). Recently, a photolyase gene has been isolated from mustard (Batschauer, 1993). This gene is functional in a photolyase-deficient Escherichia coli, illustrating the functional conservation of these genes to produce active enzymes. In addition, the expression of the photolyase has been shown to be light-dependent. The wavelength of light that induced the expression, however, did not include UVB radiation. Thus, although the UV-B radiation will cause CPD formation, their repair is dependent upon longer wavelengths of light. Another DNA repair mechanism is nucleotide excision repair, which is widespread from bacteria to mammals, and involves a five-step pathway that can eliminate a variety of structurally unrelated helix-distorting lesions, including CPDs, pyrimidine-(6-4 ‘)-pyrimidone photoproducts and DNA cross-linkages (Hoeijmakers, 1993a,b). In Arabidopsis, Pang and Hays (1991) have shown that UV-induced CPD formation can be rapidly repaired in the light (to,sof 1 h)
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and much slower by excision repair in the dark. This rapid rate of CPD photoreactivation and inefficient (if present at all) dark repair has also been confirmed by Britt efal. (1993), who also noted that the plants continued to grow normally despite the persistence of CPDs at a frequency of one dimer per 42000 bases. This relatively high level of CPDs suggests a degree of tolerance to these dimers. This may involve some selectivity in the sites of repair, so that priority would be given, for example, to actively transcribing genes (see Tanaka and Wood, 1994 for discussion). Britt etal. (1993) also studied DNA repair mechanisms in a UV-sensitive Arabidopsis mutant. They found that repair of CPDs was equivalent in the wild-type and the mutant. However, dark repair of pyrimidine-(6-4 ’)-pyrimidone was defective in the UV-sensitive plants. The sensitivity of these mutant plants to low levels of UVB suggests that repair of this lesion in the DNA is essential for normal growth. Recently, it has been shown that pyrimidine-(6-4’)-pyrimidonephotoproducts can be effectively removed in a light-dependent manner and that this process is independent of the dark repair pathway (Chen et af., 1994). The repair of both CPDs and pyrimidine-(6-4 ’)-pyrimidone photoproducts is dependent upon blue light (435nm), but not light of longer wavelengths. It was also observed that pyrimidine-(6-4’)-pyrimidone photoproduct damage was constitutively photo-repaired whereas CPDs required both photoinduction and exposure during the repair. These results suggest that there are two distinct photolyases, working on either CPDs or pyrimidine-(6-4 ’)-pyrimidone photoproducts. A number of DNA repair mechanism are also effective in pea seedlings (Taylor et a f . , 1995). Using an ELISA and monoclonal antibodies against either a cyclobutane pyrimidone dimer or a pyrimidine (6-4) pyrimidone, the formation of both of these lesions was demonstrated in UV-B irradiated wheat leaf tissue. The thymidine dimers were rapidly repaired in the light, but repair was significantly reduced in the dark, indicating the action of a lightdependent repair mechanism. In contrast, the repair of the 6-4 photoproduct appears to be almost independent of light.
C. ANTIOXIDANTS AND OTHER PRODUCTIVE MECHANISMS
Environmental stress on plants is thought, in many instances, to induce oxidative damage to cellular constituents caused by increased concentrations of peroxides and free oxygen radicals (Winston, 1990). Many essential compounds in plant tissue are susceptible to oxidative damage, particularly the polyunsaturated lipids and chlorophyll within the chloroplast thylakoids. To prevent this damage, plant cells have a wide range of low molecular weight antioxidants, such as glutathione and ascorbate, these compounds being synthesized by stress-induced protective enzymes including superoxide dismutase, ascorbate peroxidase and glutathione reductase (GR). Because short-wavelength radiation such as UV-B is known to produce superoxide
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radicals, it is likely that an early consequence of UV-B damage is oxidative damage. To examine the role of antioxidants, a number of research groups have produced transgenic plants that over-express specific antioxidants. Using superoxide dismutase, these approaches have shown protection (Van Camp et al., 1994) or not (Tepperman and Dunsmuir, 1990). Tobacco plants have also been transformed with GR and tested for enhanced protection (Aono etal., 1991, 1993; Barnes etal., 1994). GR is present both inside and outside of the chloroplast and transgenic plants have been produced that over express Escherichia coli GR in the cytosol (Aono etal., 1991). These plants show enhanced protection against the herbicide paraquat indicating increased antioxidant activity. In addition, using a chloroplast transit-peptide E. coli GR was introduced specifically into tobacco chloroplasts. Immunological detection suggested up to three-fold increased levels of GR in these tobacco leaves (Aono etal., 1993). These plants were less susceptible to sulphur dioxide induced oxidative damage. Both cytosolic and chloroplast GR transgenic plants, however, showed no enhanced protection against increased levels of ozone. These plants have recently been used to study their response to UV-B radiation (Jordan et al., unpublished results). In these experiments, pigment composition, gene expression and growth characteristics were determined for control and transgenic plants (both cytosolic and chloroplast GR transgenic plants). Although tobacco leaves do show UV-B-induced damage, no protection was afforded by the increase in GR. Similar results have been obtained with tobacco plants that over-express a pea GR (Nigel Paul, pers. comm.). Changes in antioxidants such as GR can also lead to rapid induction of the flavonoid pathway (Wingate et al., 1988). These results suggest a strong link between oxidative damage and UV-B-induced changes in gene expression. One component that could be involved in the signal transduction from the oxidative damage is methyl jasmonate (Farmer and Ryan, 1992; Sembder and Parthier, 1993). This compound is produced as a result of lipoxygenase activity and has recently been shown to induce phenylalanine ammonia lyase (PAL) expression (Gundlach et al., 1992). Polyamines are simple aliphatic bases involved in intermediary nitrogen metabolism. These molecules can act as protectants and are increased in concentration on exposure to stress, including UV-B radiation (Kramer et al., 1991). Polyamines also increase in response to high PAR and this may provide an additional explanation for the ability of high irradiance to ameliorate against UV-B.damage (Kramer et al., 1992, and see section V1.A). One suggestion for the action of polyamines in limiting UV-B damage is that they combine with hydroxycinnamic acids to form conjugates that can protect against free radicals. As both polyamines and cinnamic acid derivatives are rapidly produced in UV-B, this is a possible protective mechanism. Recently, polyamines have been localized in the photosynthetic complexes of the thylakoid membrane (Kotzabasis et al., 1993). Furthermore, the stability of thylakoid
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membrane complexes has been increased by polyamines in oat leaves subjected to osmotic stress (Besford et af., 1993). This may be caused by altering membrane rigidity (Roberts etaf., 1986) or by inhibition of phospholipase activity (Sechi et al., 1978). Lipase activity can have substantial effects upon chloroplast function (Jordan et al., 1983) and would undoubtedly play a role in UV-B induced damage to the thylakoid. Thus, increased amounts of polyamines could restrict the damage caused by lipolytic activity.
IV. EFFECTS OF UV-B RADIATION UPON CELLULAR PROCESSES A.
PHOTOSYNTHESIS
Photosynthesis is one of the most important physiological plant processes and is essential for crop productivity. Many studies have examined the effects of UV-B radiation, both on net photosynthesis and on the partial reactions (reviewed in Bornman, 1989; Jordan, 1993). These effects are summarized in Table 11. Inhibition of photosynthesis has usually been found under relatively high UV-B levels and low PAR, higher PAR ameliorating any deleterious effects of the UV-B radiation (see section VIA). In contrast, however, a number of studies using natural environmental conditions of PAR and realistically increased levels of UV-B have also demonstrated inhibition of photosynthesis (Bornman, 1989). Plant species that have either C3 or C4
TABLE I1 Effects of ultraviolet-B radiation on photosynthesis ~
Decreased photosynthetic efficiency with C3 plants generally being more susceptible than C4 plants Inhibition of chloroplast functions associated with C 0 2 fixation, electron transport, photophosphorylation, etc. Reduction in chloroplast protein concentration, usually reflecting a loss of Rubisco Rapid reduction in mRNA transcripts and protein biosynthesis Changes in carbohydrate metabolism Changes in the structural and physical properties of chloroplast membranes Recovery usually takes place with time after removal from the UV-B exposure Other environmental parameters, such as high photosynthetically active radiation (PAR), strongly influence the response
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photosynthetic pathways have been studied and in general C3 species are more sensitive than C4 plants (Basiouny eta /. , 1978; Vu eta/., 1982). Important C4 crop species, such as Zea mays, are, however, susceptible to inhibition by UVB radiation under certain conditions (Vu et al., 1982). The resistance of C4 compared to C3 plants may be due to the different internal cellular organization (Kranz anatomy) and biochemistry. There is also, however, a large range of responsivity to UV-B in C3 species and it is likely that the variation in UV-B inhibition of photosynthesis is largely dependent on leaf morphology and the development of protective pigments by the plant. T o address this relationship, arctic and alpine plants collected from different locations along a latitudinal gradient were used to investigate the response of photosynthesis to UV-B radiation (Barnes et al., 1987). The plants collected from equatorial, alpine sites were not inhibited in either light-saturated or light-limited photosynthetic, as determined by leaf gas exchange measurements. In most ecotypes that were collected from higher latitudes photosynthesis was inhibited, although significantly some were tolerant. In addition, protective flavonoids increased substantially in these ecotypes, but not in those from the equatorial region. This increase in protective pigments did not necessarily relate to an increase in the protection of photosynthetic efficiency. Damage to the photosynthesis apparatus was also not related to plant leaf weight or area. From these results, UV-B sensitivity cannot be ascribed simply to the amount of protective pigments or to leaf structure (see previous discussion: section IIIA). UV-B radiation effects on the partial reactions of photosynthesis have been extensively studied and clearly demonstrate multiple sites of inhibition (Bornman, 1989; Strid eta /. , 1990). UV-B induced changes within the chloroplast include damage to the photosynthetic reaction centres, impairment of electron transport, inhibition of photophosphorylation, loss of enzyme activity and changes in the composition of chloroplast pigments (Fig. 6 ) . The diverse processes that are affected reflect both the range of molecules that absorb UV-B radiation and the energetically unstable nature of the photosynthetic apparatus. These changes subsequently leading to inhibition of photosynthetic function and decreased efficiency of photosynthesis. In a comprehensive study, Strid el a/. (1990) showed a differential sensitivity of photosynthetic function to UV-B radiation. Thus, on a chlorophyll basis, after 8 days of UVB exposure photosystem I1 activity, ATP hydrolysis and Rubisco activity declined by 55, 47 and 80% respectively, whereas photosystem I and cytochrome b/f were stable. These changes took place progressively over the time period and appear to be a response to the cumulative UV-B dosage. This is consistent with the studies of Sisson and Caldwell (1977) that showed short periods of high UV-B irradiance were equivalent to long periods of low irradiance in the suppression of leaf photosynthesis. In a further study, Chow et al. (1992) investigated the effects on pea seedlings of exposure to short-term UV-B radiation and the ability of photosynthetic functions to recover. After 8 h of UV-B treatment, chlorophyll per unit leaf area and chlorophyll a/b ratio
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Potential sites of UV-B damage to the Chloroplast lightharvesting and electron transport photophosphorylation soluble enzymes membrane lipids and pigments gene expression
Fig. 6. Potential sites of UV-B damage. Representation of a chloroplast illustrating the components of the photosynthetic apparatus that have been shown to be effected by UV-B radiation.
were unaltered. However, after removal from the UV-B radiation, a decline in the concentration and change in composition of chlorophyll took place for up to 40 h. Other parameters showed a decline during UV-B treatment that continued after removal from the UV-B irradiation and then began to recover. For instance, functional photosystem I1 reaction centres declined by 23% during the 8 h of UV-B irradiation. This decrease continued by a further 29% after 40 h and then began to recover. In contrast, some parameters were found to change very rapidly on exposure to UV-B. Thus, flash-induced charge separation in functional reaction centres generates an electric field that can be measured as an electrochromic shift at 5 15 nm. The subsequent relaxation of the electric field occurs due to ion movement across the membrane and this is dependent upon the membrane permeability. The maximal electrochromic shift decreased by approximately 30% after 8 h of UV-B exposure and continued t o decline for 80 h. The relaxation time decreased from I .2 to 0.2 s after 8 h and recovered only slightly after 80 h. These changes could be detected after short time periods (15-30 min), indicating that the structural integrity of the thylakoid is rapidly damaged by the UV-B radiation. This is also supported by the research on pea by Brandle etal. (1977). They showed that using half the UV-B irradiance used by Chow etal. (1992), some chloroplast thylakoid membranes became dilated within 15 min of UV-B exposure. These are some of the earliest changes that take place in response to UV-B (see Strid eta/., 1994, for comparisons of response rates to UV-B radiation). Early studies suggested that photosystem I1 was sensitive to UV-B radiation
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O.*t
;b) 17
,
I
,
19
21
23
Days after sowing Fig. 7. UV-B inhibition of photosystem I1 as determined by chlorophyll fluorescence. Typical traces from assaying of the chlorophyll fluorescence of UV-B-exposed and control pea leaves (attached to the plants) on the 4th day of UV-B treatment (a). F, denotes the maximal fluorescence, and F, the fluorescence when the photosystem reaction centres are open. F, is defined as F, - F,. F,/F, (a measure of the photochemical efficiency of photosystem 11) is shown as a function of the time of exposure to UV-B-supplemented light, commencing on the 17th day after sowing (b); ( 0 ) controls and (0) for UV-B-treated leaves. The standard errors of the data are shown with bars (data from Strid et al., 1990). (Erixon and Butler 1971; Brandle etaf., 1977). Figure 7 illustrates the effect of UV-B radiation on efficiency of photosystem I1 photochemistry as determined by chlorophyll fluorescence. Chlorophyll fluorescence is an effective approach to determine the efficiency of photosystem I1 and can be expressed as the ratio of the rate of the photochemical activity and the total rate of absorbed energy dissipation (F,/F,). Thus, F, is the variable fluorescence, F, -F, , where F, is maximum fluorescence and F, is the fluorescence of the open photosystem I1 reaction centres (Davies et af., 1986b). From these and similar studies, photosystem I1 efficiency has been shown to be severely impaired by exposure to UV-B radiation (Strid etaf., 1990). Photosystem I1 is a multifunctional complex of protein-pigments that is comprised of watersplitting components, light-harvesting complexes and a reaction centre. The core of the reaction centre is comprised of the D2 and D1 proteins which act as apoproteins for the electron transport intermediates plastoquinone QA and Qe respectively (Melis eta/., 1992, and references therein). Many studies have attempted to locate the precise site of inhibition within the photosystem I1 complex (Greenberg el af., 1989a; Renger et af., 1989; Strid et a/., 1990; Melis et al., 1992; Jansen et al., 1993). These studies have indicated a number of different primary sites of inhibition, for instance, water splitting (Renger et af.,
UV-B RADIATION: A MOLECULAR PERSPECTIVE
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1989) or the primary quinone acceptor QA (Melis etaf., 1992). In addition, these studies have revealed multiple sites of inhibition that may be directly damaged by UV-B or a consequence of some other inhibition. The reaction centre polypeptide D1 is rapidly turned over and the efficiency of this lightdependent process is essential to the correct function of photosystem I1 (Barber and Anderson, 1992). Studies by Greenberg et af. (1989a) showed that the quantum efficiency of degradation of the D1 polypeptide was greatest in the UV-B region and that plastoquinone (in one or more of its redox states) was the photoperception mechanism for the degradation. This explanation relies on a specific excitation of the quinone bound at the Q B site and alternative interpretations have been suggested (Prfiil et af., 1992). Recently, this aspect of UV-B-induced D 1 degradation has been investigated further (Jansen etaf., 1993). Inhibitors of electron flow that displace plastoquinone in the molecular niche of D1 were used to investigate the effect upon D1 degradation. All inhibitors that displaced plastoquinone were found to inhibit UV-B induced D 1 degradation, but only some inhibited visible-light driven degradation. Therefore, for UV-B radiation to inhibit D1 degradation plastoquinone must be present in the D1 niche. In contrast, D1 degradation in the light will take place as long as specific areas within the niche are not engaged. Thus, normally substrate stabilization by Q B takes place to regulate the degradation of D1. Despite the different opinions on the primary mechanism of inhibition, these studies consistently indicate the sensitivity of photosystem I1 to UV-B radiation and that multiple sites within the complex become inhibited. In contrast to the sensitivity of photosystem I1 activity to UV-B radiation, photosystem I remains largely uninhibited (Renger el af., 1989; Strid etaf., 1990; Prfiil et af., 1993). This is consistent with other studies on different environmental stresses where photosystem I remains fully functional while photosystem I1 is damaged. Photoinhibition of photosystem I1 is also caused by high levels of visible radiation. However, significant differences exist between UV-B damage and photoinhibition, such as the recovery kinetics (Chow et af., 1992). The degradation rate of 5-7 h) induced by UV-B is also much slower than found during photoinhibition (to,sof 60 min), although it may occur through a common mechanism (Greenberg et af., 1989b) and involve the PEST region on the Dl peptide (this region shows a preponderance of proline-P, glutamateE, serine-S and threonine-T; see PrGil et af., 1992, for discussion). There is, however, some controversy over the precise protease cleavage sites under normal irradiance and during photoinhibition (Barber and Anderson, 1992). Consequently, the mechanism of UV-B-induced damage to the D1 polypeptide and its relationship to other degradation processes is still uncertain. The degradation rate of D1 protein is 30% faster in full spectrum sunlight than when all detectable UV radiation is removed with selective filters. As UV radiation contributes only a few per cent of the photon flux
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between 300nm and 700nm, the relative contribution to the degradation of D1 is significant (Greenberg eta/., 1989a). This becomes particularly important with the predicted increase in the proportion of UV-B as a percentage of the total spectral radiation. Furthermore, UV-B and photoinhibition are likely to be synergistic and this may account for photoinhibition in the field being higher than expected under the prevailing light environment ( P r N et al., 1992). Another important component of the thylakoid membranes is the chlorophyll integrated into the chlorophyll-protein complexes. In many studies, both qualitative and quantitative changes in chlorophyll have been seen in response to UV-B exposure (Takeuchi et at., 1989; Strid eta/., 1990; Deckmyn etal., 1994). In pea seedlings, the total chlorophyll decreased by 40% over 8 days of UV-B treatment compared to control plants (Strid et al., 1990), chlorophyll a decreasing to a greater extent than chlorophyll b. The decreased reduction in chlorophyll b on exposure to UV-B may be due to conversion from chlorophyll a or by the greater stability of chlorophyll b (Brown et al., 1991). To extend these findings, Strid and Porra (1992) analysed tetrapyrrolic pigments to examine the changes in biosynthesis and degradation of chlorophylls. Using the same UV-B treatments of pea seedlings, they found increased levels of chlorophyllides a and b, resulting from chlorophyll degradation. In contrast, they found no increase in intermediates of chlorophyll biosynthesis, such as uro- or copro-porphyrins 111, protoporphyrin IX, or phytol-free Mg-tetrapyrollic intermediates. They concluded that the biosynthetic apparatus for chlorophyll was relatively stable on exposure to UV-B radiation and that the overall decrease in chlorophyll resulted from increased degradation. This increased degradation takes place at a time when the gene expression for the chlorophyll a/b binding protein is also severely inhibited by UV-B radiation (Jordan et af., 1991). Because chlorophyll is normally stably-associated with proteins in the thylakoid membrane, the reduction in chlorophyll a/b binding proteins (resulting from the reduction in gene expression) may allow increased degradation of the chlorophyll molecules. This is, however, unlikely to be the entire explanation. For instance, in etiolated pea exposed to UV-B radiation alone, chlorophyll accumulation is severely reduced, although cab gene expression remains high (Jordan et af., 1994). Furthermore, in 6-week-old Arabidopsis, cab gene expression is reduced with UVB exposure, but chlorophyll and the chlorophyll-binding-protein remain constant (Jordan eta/., unpublished observations). Other research has also found increased chlorophyll a/b ratios on exposure to UV-B (Deckmyn et al., 1994 and references therein), suggesting a complex response depending upon the species and the conditions of the experiments. It is clear that further investigation are necessary to establish the precise mechanisms involved in these chlorophyll changes. Ribulose 1,5-bisphosphate carboxylase (Rubisco, EC 4.1.1.39) is the primary enzyme of Cot fixation in C3 plants and is also the most abundant
UV-B RADIATION: A MOLECULAR PERSPECTIVE
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protein in leaves, representing 40-80% of total soluble leaf protein (Huffaker, 1982; Miziorko and Lorimer, 1983; Spreitzer, 1993). Consequently, in addition t o its role in carbon fixation, Rubisco is a major resource of carbon and nitrogen within the plant. Changes in Rubisco enzymatic activity or protein level should therefore have a significant influence on photosynthetic activity and on leaf protein composition. Rubisco enzyme activity is inhibited by UV-B exposure (Fig. 8a; Vu etal., 1982, 1984; Strid et al., 1990; Jordan etal., 1992) and this inhibition is likely to be caused by protein degradation and or inactivation of the enzyme. Vu etal. (1982) showed that Rubisco activity declined in pea, soybean and tomato when exposed to UV-B radiation. In pea and soybean a parallel decrease in soluble protein took place. In contrast, tomato leaves increased their content of soluble protein by as much as 30% at the highest UV-B irradiance tested. Similar increases in soluble protein have been found in barley, corn, bean and radish (Tevini etal., 1981). These results indicate that a tight correlation cannot be made between a UV-B-induced decline in soluble protein and Rubisco content. Studies by Vu etal. (1984) on pea and soybean suggested that a specific loss of Rubisco protein did take place in response to UV-B irradiation and could be the main reason for the inhibition of enzyme activity. However, in a more recent study on pea (Jordan et al., 1992), the Rubisco activity declined more rapidly than did the Rubisco protein at any time point following UV-B exposure. For instance, the Rubisco polypeptides (LSU and SSU) both decrease by 10-15% after 1 day and 56% after 3 days of UV-B exposure. The enzyme activity, however, declined by 38 and 71 070 over the same period. Thus, although Rubisco protein does decline the enzymic activity itself is being lost more rapidly. Interestingly, the percentage activation of Rubisco in vivo (measured as the ratio of the initial rate of carbon assimilation to the maximum rate after incubation with Mg2+ and bicarbonate for 7 min) was increased when plants were exposed to UV-B radiation (Fig. 8b). The activation increased as the maximum rate decreased during 3 days of exposure. A similar increase in activation of Rubisco has been reported during senescence of tomato (Yelle et al., 1989). The rapid loss of Rubisco enzyme activity is likely to be caused by UV-B damage to the Rubisco holoenzyme. This could be as a direct result of UV-B absorption by aromatic amino acids (phenylalanine, tryptophan and tyrosine) causing ring opening or free radical production (tyrosine). Alternatively, damage could result as a consequence of free radicals created within the chloroplast by UV-B exposure. Free radicals are potent electrophiles and would react with nucleophilic amino acid side chains such as cysteine-SH, lysine and arginine-NH, and the aromatic amino acids. The modification of proteins by these radicals leads to an increase in hydrophobicity, partial denaturation and loss of catalytic activity (Cadenas, 1989). A partial disruption of the Rubisco enzyme could decrease the maximum rate of carbon assimilation by the enzyme under conditions where it would normally be fully activated. This lower ceiling imposed on the maximum Rubisco activity by the UV-B treatment would not be
128
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a
50
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40
0" 0
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10
0
UV-B RADIATION: A MOLECULAR PERSPECTIVE
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expected to affect to the same extent the lower initial rate of carbon assimilation measured before the enzyme is fully activated with Mg2+ and bicarbonate. Thus, this would account for the increased activation of Rubisco as the increase in the ratio of initial rate to maximum rate (Jordan et al., 1994). The data on pea leaf mRNA transcripts (see below) for both the large and small subunits of Rubisco, suggest that synthesis of the protein is rapidly switched off on exposure to UV-B radiation. Furthermore, "S-methionine labelling of pea leaf proteins shows that incorporation into Rubisco and other proteins is severely inhibited within hours (Jordan et al., 1994). As Rubisco polypeptides are relatively stable in mature leaves (Petersen et al., 1973), it seems likely that after UV-B exposure the biosynthesis of Rubisco stops and the polypeptide levels decline slowly as the Rubisco pool gradually depletes (Jordan et al., 1992). Similarly, in Arabidopsis, Rubisco mRNA levels decline while the Rubisco protein remains constant after exposure to UV-B radiation. However, degradation of Rubisco is known to occur under certain conditions involving oxidative stress (Mehta et al., 1992; Garcia-Ferris and Moreno, 1994). For instance, Rubisco activity and stability was found to be highly susceptible to oxidative stress, resulting in intermolecular cross-linking of large subunits by disulphide bonds, translocation of the soluble enzyme complex to chloroplast membranes and protein degradation (Mehta et al., 1992). In experiments in which UV-B-induced degradation of Rubisco protein is observed, such a mechanism could be involved. Another important photosynthetic function that is effected by UV-B is photophosphorylation and CF,F, -ATPase activity (Strid et al., 1990; Zhang etal., 1994). In pea leaves, ATPase activity was sensitive to UV-B radiation and decreased by 47% over 8 days of exposure (Strid etal., 1990). However, in further research it was shown that the CF,-ATPase protein content decreased substantially more (Zhang et al., 1994). Thus after 4 days of UV-B irradiance, the activity declined by 25% in comparison to a 60% decrease in the protein. Therefore this implies that an activation of the remaining protein takes place. Such an activation could take place via a reduction in the disulphide bridges of the cystine residues within the y-subunit of the CF, -ATPase (Zhang etal., 1994, for further details). These results for the CF,-ATPase contrast with those for Rubisco in that the enzyme activity of Rubisco declines much faster than the protein level; although Rubisco activation is increased.
Fig. 8. Effect of UV-B radiation on Rubisco activity. Maximum Rubisco carboxylase activity (a) and the in vivo activation of Rubisco carboxylase activity (b) in pea leaves exposed to supplementary UV-B radiation. The data are from a representative experiment: (0)control leaves; ( ) UV-B-treated leaves (data from Jordan et al., 1992).
130
B.R. JORDAN B. CARBOHYDRATE, LIPID AND NITROGEN METABOLISM
In a number of studies, changes in carbohydrate composition have been detected in response to UV-B exposure (Takeuchi et al., 1989; Santos et al., 1993; He etal., 1994). In cucumber cotyledons the levels of soluble sugars (sucrose, glucose, fructose and inositol) increase by 2-10 fold over 8 days growth under normal irradiance. In contrast, apart from inositol, the individual soluble sugars did not increase to the same extent in seedlings exposed to UV-B for 8 days (Takeuchi et al., 1989). Overall, total soluble sugars were only 44% of control levels after 8 days of UV-B exposure. In the leaves of pea seedlings, UV-B exposure caused an increase in starch, leading to large starch granules accumulating within the chloroplast (He et al., 1994). The diurnal increase and decrease in starch and soluble sugars was also disrupted. Significantly, these studies were carried out under the same conditions as the experiments showing decreased levels of Rubisco activity, Rubisco protein and the corresponding mRNA transcripts (Jordan et al., 1992 and section VA). There have been many studies that show changes in leaf carbohydrate status can cause a feedback inhibition leading to the inhibition of photosynthesis. More specifically, these studies show a decrease in Rubisco activity caused by a decline in its biosynthesis (Krapp etal., 1991). Thus the rapid decrease in mRNA levels for photosynthetic proteins in response to increased UV-B may be caused by some form of metabolic feedback (see section VC for further discussion). Very little is known about the impact that increased UV-B radiation would have on nitrogen metabolism (see also section IIIC). However, as nitrogen from Rubisco is a major component in the nitrogen regime of plants (Huffaker, 1982), any degradation by UV-B radiation could have a substantial effect. Rubisco can act as a readily available nitrogen store for the plant which can then be remobilized to other organs of the plant (Millard and Catt, 1988). Rubisco can also be used to supply nitrogen as a long-term store in woody perennials (Millard and Thomson, 1989). For instance, in apple trees, degradation of Rubisco in the autumn can account for 32-48'3'0 of the nitrogen remobilized from woody tissue (stems and roots) for leaf growth the following spring. Furthermore, Rubisco can be rapidly turned over during senescence, under nitrogen deficiency and when the plant is exposed to oxidative stress (Garcia-Ferris and Moreno, 1994 and references therein). Any UV-B-induced change in Rubisco is therefore likely to have a wide range of consequences for the plant's nitrogen balance. The effect of UV-B radiation on enzymes of nitrogen assimilation has been investigated in the turions of Spirodela polyrhiza (Appenroth et al., 1993). The activities of all the enzymes examined were decreased by the UV-B treatments, with nitrate reductase being the most severely inhibited. As neither the uptake of nitrate nor of ammonium was affected by the UV-B irradiation, the inhibition of enzyme activity was not thought to be caused by a reduced level of substrate. It was concluded that
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UV-B-induced changes in gene expression for these enzymes was the most likely explanation for the decreased activities. However, because of the very strong inhibition of nitrate reductase, inactivation of previously synthesized enzyme could also take place. Furthermore, a number of important enzymes of nitrogen assimilation (glutamine synthetase and glutamate synthase) are present in the chloroplasts and require ATP provided through photosynthesis (Jordan and Givan, 1979, and references therein). It is therefore likely that inhibition of photosynthesis will, as a consequence, influence the efficiency of nitrogen metabolism. A major component of plant cell membranes are the acyl-lipids. These lipids are comprised of fatty acid chains (mainly C16 and C18) attached to glycerol backbones. The major lipids of most cell membranes are phospholipids with phosphatidylcholine as the major component. In contrast, the lipid composition of the chloroplast is comprised largely of the galactolipids, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), which account for approximately 70% of the acyl-lipid composition. The thylakoid membranes also contain the sulpholipid, sulphoquinovosyldiacylglyceroland unlike other membranes, the major phospholipid is phosphatidylglycerol containing the unique fatty acid, trans-A-3 hexadecanoic acid. The major fatty acids of these membranes are polyunsaturated C18 chains, such as linolenic acid (C18:3). These molecules are prime targets for UV-B-induced oxidative damage. Studies on barley, corn, bean and radish showed a decrease in the galactolipids with the ratio of MGDG/DGDG decreasing as has been observed in senescing tissue (Tevini etal., 1981). The effect upon the phospholipids varied according to species with some even showing increased amounts. In a study on cucumber cultivars, a decrease in unsaturated fatty acids was correlated with increased peroxidation (Kramer et al., 1991). The level of peroxidation caused by UV-B was also related t o the UV-B sensitivity of the cultivars. The changes in lipid composition may have a profound influence on cellular regulation. Within the chloroplast perturbation of the membrane lipids can dramatically effect photosynthetic function (Jordan eta/. , 1983; Jordan, 1984; and references therein). This could be effected, for instance, by altering the lipid environment of the photosynthetic complexes or by altering the permeability properties of the membrane. Oxidative damage can also cause changes in gene expression for chloroplast proteins (see sections 1I.C and VC). In addition, increased saturation of membranes has recently been found to switch on desaturase genes (Vigh el a[., 1993). Thus, UV-B-induced effects on membrane lipids can cause a cascade of widely different responses.
C.
REPRODUCTIVE BIOLOGY
One of the most profound influences of increased UV-B radiation is likely to be upon the reproductive biology of plants. Considering the importance of this
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aspect of plant development, it is remarkable how little research has been carried out. The ability of plants to flower is controlled by an endogenous mechanism that is dependent upon the developmental stage of the plant (Thomas, 1993). It is therefore apparent that any changes in the UV-B radiation level could have a profound effect upon flowering due to its influence upon plant growth and development. For example, plants cannot flower while they remain juvenile and increased UV-B radiation could potentially interfere with such a stage of development. Furthermore, flowering is influenced by the carbohydrate status of the plants and this may be modified during development by UV-B effects upon photosynthesis and or assimilate partitioning. In addition to the developmental control of flowering, a number of environmental parameters that can determine floral development are overlaid on the endogenous regulation (Thomas, 1993). Thus, light is a major environmental determinant of flowering and a relative increase in UV-B radiation may modify the signal to flower. This may take place by interfering in the perception of the light environment through the coaction of different photoreceptors or by damage to important molecules involved in the signal to flower. For instance, photoperiodic induction is perceived in the leaves and leads to evocation at the apex. A change in the light environment could interfere with the photoperiodic timing of flowering. Studies on the long-day plant, Hyoscyarnus niger have shown that the photoperiodic floral induction was dependent upon the UV-B fluence rate. These changes have been correlated to changes in gibberellic acid content (Tevini, 1993). UV-B exposure also caused earlier flowering in Petunia hybrid@(Staxen and Bornman, 1994). An increase in UV-B radiation may also cause direct damage to molecules, such as DNA, at important stages in the reproductive cycle. Thus, the transition from vegetative to reproductive growth takes place at the apex and involves complex regulation of cellular events. This includes switching on of controlling homeotic genes, changes in cell-cycle and alterations in protein biosynthesis (Jordan and Anthony, 1993; O’Neill, 1993). These important modifications all involve molecules that are particularly sensitive to UV-B radiation damage and therefore evocation may be a particularly vulnerable stage of the reproductive cycle. Other stages of reproduction are known to be protected from damage by UV-B-absorbing pigments. The anthers filter out greater than 98% of UV-B radiation and the pollen itself is also protected by UV-B absorbing compounds (Flint and Caldwell, 1983; Jackson, 1987). In addition, pollen contains repair enzymes that can protect against DNA damage (Jackson, 1987). The ovules within the ovaries are also likely to be well protected from UV-B radiation damage. However, UV-B damage may be caused during germination of the pollen on the stigmatic surface of the style. This is especially so for binucleate germ cells which are generally slower to germinate and penetrate into the tissue (Flint and Caldwell, 1986). In many species, even moderately low fluence of UV-B (50-70 mWm -2) can cause over 50% inhibition of germination within a period of hours (Tevini, 1993). A combination
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of UV-B radiation and other stresses may also exaggerate the inhibition of pollen tube growth. For instance, in vitro pollen tube growth was reduced by ozone and also by UV-B radiation. However, in combination, ozone and UVB radiation gave increased inhibition of the pollen growth, compared to either stress alone. The effects appeared to be additive, implying that different aspects of the growth process were being affected (Feder and Shrier, 1990).
D.
CELL-CYCLE AND CYTOSKELETON
UV radiation has been known for a long time to effect the cell-cycle. These studies have shown that irradiation in the S phase of the cycle is most effective in delaying mitosis. As the S phase of the cycle is involved with DNA replication, it is assumed that the delay is caused by a requirement for DNA repair due to UV-induced damage. The G1 and G2 parts of the cycle, however, also can delay mitotic division to some extent, suggesting other mechanisms may be involved. These studies have predominantly used germicidal UV (< 280 nm) and therefore the results do not necessarily reflect the response to UV-B irradiation. The microtubules are another important component for cell division and growth. These have been shown to be disrupted by UV radiation and their synthesis from tubulin dimers inhibited. Tubulin itself may be particularly sensitive to UV radiation as it contains a high proportion of aromatic amino acids. Unfortunately, most of our information on the effects of UV do not come from studies on plants. This is a remarkable omission considering the inhibition of plant growth by UV-B and the likely influence on the cellcycle. Recently, the effect of UV-B irradiation has been studied on Petunia hybrida protoplasts (Staxen et al., 1993). In this study, progress through the cell-cycle was delayed with G1, G2 and the S phase being affected. The cortical microtubules were also shortened by dose-dependent UV-B-induced breakage. Under the UV-B conditions, the protoplasts remained viable and could recover from the treatment. The ability to recommence cell division was also dependent on the microtubules attaining a similar length and this supports previous findings on Nicotiana and Hibiscus (Hahne and Hoffman, 1984). Studies on intact tissue of Petunia hybrida show UV-B effects upon morphology rather than the cytoskeleton (Staxen and Bornman, 1994) with cell division stimulated and cell number increased. The UV-B-exposed plants showed increased branching and earlier flowering compared to controls. Clearly these findings and those of the previous studies on other organisms illustrate a potentially severe problem for plant growth and development. In monocotyledonous plants, cell division takes place in the basal meristem and the surrounding tissue may afford substantial protection from UV-B. In many other tissues, however, cell division will be exposed to UV-B radiation. This could be particularly important for reproductive biology as one of the earliest changes that take place during evocation at the vegetative meristem is a synchronization of the cell-cycle.
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Changes induced by increased UV-B levels could influence this process and consequently have a profound effect upon floral development.
V.
THE EFFECTS OF UV-B RADIATION O N GENE EXPRESSION A. CHLOROPLAST PROTEINS
Recently, the effects of supplementary UV-B radiation on gene expression for chloroplast proteins has been reported (Jordan et a f . , 1991, 1992, 1994; Zhang et al., 1994). In these studies mRNA transcripts for important chloroplast proteins declined in response to UV-B exposure. Subsequently, there was a loss of enzyme activity and protein content over a period of days. These losses also correlated to a reduction in photosynthetic function (Strid etaf., 1990; Chow etaf., 1992). This research was the first such study and provided a molecular mechanism that could account to some degree for the overall inhibition of photosynthesis by UV-B radiation (see section IVA). In addition, a number of important points have emerged from these studies. Thus, the changes in gene expression for a particular gene are dependent upon the developmental stage of the tissue (Jordan etaf., 1994, and section VC). Another feature of the response is that higher PAR ameliorates the reduction in RNA transcripts to some extent (Jordan etal., 1991, 1992, and see section VIA below for further discussion). These studies also show that the plants can recover the efficiency of gene expression. For example, following a 4-h exposure of UV-B radiation, the mRNA transcripts continue to decline for approximately 24 h to a low level and then recover to 60% after 3 days. The reduction in gene expression and recovery correlate with a similar pattern in quantum yield of photosynthesis (Chow et af., 1992). Repetitive exposure to high UV-B levels is therefore likely to maintain an inhibited and inefficient photosynthetic apparatus in susceptible species. Both nuclear (cab, rbcS and the y-CF,A TPase) and chloroplast encoded (rbcL, psbA, CF,-A TPase B-E, cytochrome b and subunit IV of the cytochrome b/f complex) mRNA transcripts are severely reduced. Thus, both genomes are affected by exposure to UV-B radiation. The reduction in RNA transcripts can be very rapid (80% reduction after only 4 h for cab and rbcS) or take place more slowly @sb A mRNA transcripts remain 35% of the control level after 3 days of UV-B exposure). In general, the chloroplast RNA levels are maintained for longer periods than nuclear-encoded genes. The decline in the mRNA levels is not necessarily reflected in a loss of a particular photosynthetic function. For instance, the cytochrome b6f complex is not inhibited by UV-B irradiation to any extent (Strid etal., 1990), whereas the mRNA for this complex is rapidly downregulated (Zhang etal., 1994). This suggests that the protein of the complex is more stable than the mRNA. However, as the activity was used to determine
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the level of the protein, it is possible that enzyme activation takes place (as seen in Rubisco and the CF,F,-ATPase). Other studies do clearly demonstrate that the protein component is substantially more stable than the mRNA moiety. For instance, in Arabidopsis exposed to UV-B radiation, the cab mRNA level declines substantially over a 7-day period, whereas immunologically quantified chlorophyll a/b-binding protein remained the same (Jordan et al., unpublished observations). Furthermore, Rubisco protein is much more stable than its corresponding mRNA (Jordan etal., 1992). Unfortunately, there are at present no specific data available to define at what level the inhibition of gene expression is taking place. It is possible from similar studies on the photoregulation of gene expression, that the control of the nuclear encoded genes is largely at the level of transcription (Thompson and White, 1991; Kuhlemeier, 1992). Thus, after transcription is inhibited there is a decline in the RNA transcripts that depends upon their relative stabilities (Klaff and Gruissem, 1991). The UV-B radiation also causes defence genes, such as chs, to be simultaneously switched on as the photosynthetic genes are repressed (Jordan et al., 1994). Figure 9 illustrates the rapid up- and down-regulation of chs and cub respectively in Arabidopsis exposed t o short periods of UV-B radiation. As the defence genes are regulated at the level of transcription there must be a carefully regulated gene-specific response to the UV-B stimulus. The regulation of the UV-B-induced response may, however, be still more complex. For instance, in "S-methionine labelling of pea leaf discs, the results indicated that the methionine may have been incorporated into the initiation complex (80 S. Met-tRNA.mRNA) without further elongation of the peptide chain (Jordan et al., 1994). This suggests that there is also some post-transcriptional inhibition. Furthermore, the levels of chloroplastencoded mRNA are thought to be primarily controlled through translational or post-translational regulation (Mullet, 1988). Thus, it is likely that UV-B radiation is affecting gene expression for chloroplast proteins at a number of control levels. In addition, the response to UV-B is determined by the development of the tissue (Jordan etal., 1994, and unpublished observations). This control may be at the level of gene regulation (i.e. working via different members of a multi-gene family) or simply that the tissues vary in the extent of UV-B penetration due to protective pigments or tissue morphology (see section 1I.D). It is certainly clear from these studies that this is an area that should be addressed more extensively, as it will give significant insights into the mechanisms involved in the inhibition of gene expression.
B.
DEFENCE GENES
The formation of protective flavonoid pigments is a widespread response of plants, particularly to changes in the light environment. This increase is due to dramatic changes in gene expression for enzymes of the phenylpropanoid
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Fig. 9. Changes in gene expression in response to UV-B radiation. Autoradiographs of northern-blot analysis of total RNA (20pg) from Arubidopsis tissue after short periods of supplementary UV-B exposure. The RNA was then hybridized to 32Plabelled cub and chs.
pathway. Thus, mRNA transcript levels for enzymes such as PAL, chalcone synthase (CHS), chalcone isomerase (CHI) and dihydroflavonol reductase (DFR) increase to maximum levels within hours and then decline to low levels. The genes for these proteins are sequentially induced in the order of the enzymes of the flavonoid biosynthetic pathway (Kubasek et al., 1992). The regulation of the pathway is controlled by plant development and the developmental stage then determines the response to the light environment (see section IIB). Although a number of photoreceptors are involved in the regulation, UV-B radiation is particularly effective in the induction of this pathway. For instance, in Arabidopsis, UV-B irradiation was much more efficient at inducingpal, chs, chi and dfr gene expression than blue light (Kubasek er al., 1992). In pea, UV-B irradiation alone was more effective in inducing chs than either white light or white light plus a supplement of UV-B (Jordan etal., 1994). Figure 9 shows the rapid increase in mRNA transcripts for chs after exposure of Arabidopsis to supplementary UV-B radiation.
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These increases have been shown, by run-off transcription assays on isolated nuclei, to be caused by changes in transcription (Chappell and Hahlbrock, 1984; Feinbaum and Ausubel, 1988). The light-dependent regulatory transcription elements within the parsley chalcone synthase gene have now been extensively characterized (Schulze-Lefert et af., 1989; Weisshaar et af., 1991a,b). Although the wavelengths used in these studies ranged between 300 and 400 nm, the results are likely to be indicative of the molecular mechanisms involved. Four light-dependent regions of the parsley chalcone synthase promoter have been identified by in vivo DNA footprinting (Weisshaar etaf., 1991b). They are arranged as two cis-acting units each containing two separate footprints and separated by 60 bp. These sequences contain guanosine residues that show differential reactivity to dimethyl sulphate (DMS) methylation in control and UV-treated tissue. In vivo methylation protection takes place in UV-treated tissue and reflects the interaction of trans-acting protein factors with these cis-acting elements on exposure to UV irradiation. Furthermore, the altered reactivity to DMS has a time course that correlates to the known transcription kinetics of chs. Recently, in vivo footprinting and transient expression assays have been used to study the presence of separate promoter regions responsible for UV-B and blue light activation of chs gene expression (Merkle el af., 1994). Pre-irradiation with blue light is known to alter the timing of UV-B induced gene expression of chs (see section IIB). This response was also manifest in the in vivo UV-B induced footprints as they were detected earlier in the pre-irradiated blue light treated cells than in those only treated with UV-B irradiation. Although there was a clear shift in the timing of the footprint, the pattern of the footprint did not alter. Using light-induced transient expression assays, the shortest promoter tested was sufficient to retain the UV-B induction and also the blue light kinetic shift. These results argue strongly against a spatial separation of UV-€3 and blue light responsive ciselements on the chs promoter. Thus, the signal transduction pathways from the photoperception of UV-B and blue light must merge at or before the chs promoter. Using transient expression assays in isolated parsley protoplasts, it has been shown that boxes 1 and 2 represent the minimal promoter sequence for lightresponsive chs transcription. The distance between these boxes is important for their function and replacement of box 2 with an additional copy of box 1 will provide full light regulation. One of the regions, box 2, contains a heptameric “core” sequence that is strongly conserved to the G box in a number of light regulated Rubisco genes, hex in histone promoters and the ABA responsive rab gene promoters. Its presence in developmental (patatin), anaerobic (adh)and pathogenic (rofbc)genes illustrates that these cis-elements do not have a specific light/UV responsivity function and that common regulatory cis-elements are shared by genes that show regulation by a range of different factors. These studies also indicate that the regulation is dependent on the nature of the transacting factors binding to the promoters.
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Recently, a number of cDNA clones have been isolated that encode proteins that bind to box 2 and specifically t o the ACGT motif common to many ciselements (CPRF, common plant regulatory factors: Weisshaar et al., 1991a). The deduced amino-acid sequence of these proteins contain conserved basic and leucine-zipper domain characteristics of transcription factors of the bZip class. The CPRF’s bZip domains must be intact for DNA binding and although they will bind to other ACGT-containing promoter elements, they have markedly different affinities (Armstrong et al., 1992). One of these, CPRF-1, showed mRNA accumulation that corresponded to chs expression and supports the suggestion that this transacting factor is involved in UV light regulation. Other regulation of the flavonoid pathway may involve components of the pathway acting through feedback control mechanisms. For example, trans-cinnamic and trans-p-coumaric acid regulate a chs promoter in alfalfa (Loake etal., 1991). One important aspect of the UV-B regulation of the flavonoid pathway is whether it operates through regulatory genes that control the normal pattern of pigmentation within the plant or if it involves an independent mechanism. The regulatory genes that control anthocyanin biosynthesis have been extensively documented (see Martin and Gerats, 1993ab, for detailed reviews). However, relatively little is known of the interaction with light. Taylor and Briggs (1990) investigated the role in maize of the regulatory loci R, B, PI on anthocyanin accumulation and genes of the biosynthetic pathway during an inductive light treatment. The results demonstrated that the anthocyanin pathway is regulated solely by R; the contribution of the other loci being negligible in young seedlings. Expression of R occurs in dark-grown tissue but is stimulated by high fluence rate white light. R tightly regulates dfr gene expression, but has less control of chs and ufgt expression. In addition to genes that code for enzymes of the phenylpropanoid pathway, the gene expression for other defence-related enzymes is also altered (Strid, 1993). Thus, enzymes involved in protection against oxidative damage, such as glutathione reductase, are increased. In contrast, the gene expression of superoxide dismutase is reduced in response to UV-B irradiation. Enzymes that apparently will function in a similar manner are therefore regulated in a different way.
C. FACTORS AFFECTING GENE EXPRESSION
There is now substantial evidence that UV-B radiation has a dramatic influence on plant gene expression. This has been most extensively investigated during the biosynthesis of protective pigments, such as the flavonoids. Recently, other defence genes have been shown to be switched on and the activity of many genes (mostly for photosynthetic proteins) inhibited. These changes clearly suggest a complex regulation of gene activity in response to
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UV-B radiation. As similar patterns of gene activation and repression take place in response to other stresses, we can assume that this is not an unusual strategy for the plant to adopt. Thus, a pathogen infection can induce chs gene activation and rbcS repression (Kombrink and Hahlbrock, 1990) in a similar manner to UV-B radiation. These changes in gene expression can also be induced by many other environmental stresses, suggesting the cellular resources of the plant are switched from the normal biosynthetic processes to those in which protective molecules are made. Thus, the UV-B induced changes in gene expression are a further example of what is a normal plant strategy to stress. A significant difference, however, is that UV-B radiation can act directly on molecules that absorb it (notably DNA) to alter gene expression or it may mediate its effects through more sophisticated mechanisms involving photoreceptors and signal transduction chains. The factors that are likely t o influence UV-B-induced gene expression are illustrated in Table 111. One of the most important aspects that will determine
TABLE Ill Factors affecting UV-B induced changes in gene expression
Perception of the light environment Perception could involve a specific photoreceptor or a wide range of biologically active molecules. The nature of the perception may involve the interaction between different regions of the light environment Penetration of UV-B light into the tissue Penetration o f UV-B will largely depend upon the attenuation by protective pigments in the epidermis. Penetration of UV-B is also dependent on the species: (conifer < monocot < herbaceous). UV-B exposure may itself alter the plant morphology and pigment composition, thus affecting the penetration Damage to DNA and efficiency of repair mechanisms DNA is vulnerable to UV-B induced damage and this would impair gene expression. The effective repair of these lesions is therefore necessary to maintain a functional genome Indirect regulation of gene expression This could be caused by a number of UV-B induced cellular perturbations, such as oxidative damage or metabolic feedback Developmental stage of the tissue The changes in gene expression are dependent on the stage of development. For instance, etiolated tissue is less sensitive than green tissue and older tissue is more sensitive than younger tissue Interaction of other environmental parameters Both quantitative and qualitative changes in gene expression occur as a result of the interaction o f other environmental factors. Thus, high photosynthetically active radiation (PAR) will reduce the extent of UV-B induced damage and pathogens or ozone may alter the nature of the expression
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the response at the level of the genome, is the nature of the UV-B perception. For instance, if the UV-B radiation acts as a stimulus to activate a signal transduction chain that leads to the genome, we would expect regulated and gene-specific responses. Particular wavelengths of the light environment frequently mediate responses through changes in gene expression. These are environmental cues to which the plant will respond. It is therefore likely that changes in the UV-B part of the light environment will act in a similar manner either independently or in combination with other photoreceptor molecules. Thus, through a signal transduction pathway, the changing UV-B radiation transmits information to the genome and induces changes in transcription. The activation or repression of the genes depends upon cis-elements and or trans-acting factors associated with particular genes. This may also involve the responsivity of different members of multi-gene families, particularly at different stages of development. Alternatively, a direct and non-specific effect of UV-B radiation on nuclear or chloropIast DNA, such as pyrimidine dimer formation, couId induce inhibition of gene activity. A number of lines of evidence, however, suggest that the down-regulation of gene expression for chloroplast proteins is not primarily due to non-specific damage to DNA. First, although gene expression for chloroplast proteins is repressed, there is an increase in the expression of enzymes of the phenylpropanoid pathway (Jordan et al., 1994; see Fig. 9). Thus, the response to UV-B is gene specific, which is unlikely to be the case if UV-B was being generally absorbed by DNA. Second, during de-etiolation of dark-grown seedlings, it is well established that the cab gene expression increases substantially from very low levels. Greening pea seedlings continue to express cab mRNA transcripts in a similar manner when exposed to UV-B radiation for a period of 7 days (see Fig.4; Jordan etal., 1994). These results suggest that a specific mechanism regulates the changes in gene expression in response to UV-B radiation. However, another possibility is that there is a spatial component involved in the regulation of gene expression. Thus, the activation of the phenylpropanoid pathway takes place in the epidermal layers of the tissue through well-documented photoreceptor mechanisms. In contrast, penetration of UV-B radiation into the underlying palisade and mesophyll layers may allow non-specific DNA damage, the penetration of the UV-B being dependent on the species or developmental stage of the tissue. This suggestion is supported by experiments with the Arabidopsis tt mutants, showing that reduced protective pigments allow much more severe reductions in cab gene expression on exposure to UVB irradiation (Jordan et al., unpubIished observations). The precise mechanism remains complex however, as the chloroplast encoded psbA gene transcripts do not seem to be reduced in UV-B irradiated tt mutants compared to controls. UV-B radiation absorbed by other cellular constituents could also impinge on gene regulation through, for instance, metabolic feedback control or the chloroplast signal transduction pathway. Thus, UV-B absorption by chloro-
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phyll or polyunsaturated acyl-lipids can cause oxidative damage and this is known to be involved in a signal transduction pathway that changes gene expression (Taylor, 1989; Susek and Chory, 1992). From the studies of Jordan etal. (unpublished data; Fig. 3 and discussion in section IIC), this pathway is unlikely to be involved. Effects of UV-B radiation may also alter cellular metabolism and Iead t o feedback control that could change gene expression. The carbohydrate products of photosynthesis are important regulators of chloroplast function. Thus, it has been well established that increase in the amounts of sugars, such as glucose, will cause profound changes in photosynthetic efficiency. Increase in these sugars leads to inhibition of Calvin cycle enzymes, particularly Rubisco (Krapp et al., 1991). Recent studies have also shown that an increase in carbohydrate can cause a concomitant decrease in the mRNA levels for nuclear-encoded proteins such as Rubisco (Van Oosten and Besford, 1994; Van Oosten etal., 1994). Using maize protoplasts and a transient expression system, Sheen (1990) has shown that promoters for photosynthetic genes are specifically and co-ordinately repressed by photosynthetic end products, such as glucose and sucrose. The metabolic repression of photosynthetic genes overrides other forms of regulation, including light and the developmental stage. How then could UV-B exposure to leaves cause this repression? The first studies of down-regulation of genes for photosynthetic proteins was carried out on pea leaf tissue (Jordan et al., 1991 , 1992). Under the identical conditions, experiments were carried out to look at chloroplast ultrastructure (He et al. , 1994). This work showed the accumulation of starch granules within the chloroplast which caused membrane disruption. In addition, soluble sugars increased in the leaves. These increases took place over a number of days and it was suggested that the physical disruption could be involved in reducing photosynthetic function in UV-B irradiated leaves. However, an increase in these soluble carbohydrates could undoubtedly cause the observed repression in gene expression. It is also possible that this may account for the differential effect of UV-B at different stages of development as the relationship between photosynthesis and carbohydrate status will vary substantially during chloroplast development.
VI.
UV-B INTERACTIONS WITH OTHER STRESSES A.
LIGHT
The different quantity and quality of light has a marked affect on UV-Binduced changes in plant growth and development. Usually low PAR (400700nm) and relatively high UV-B irradiation give a range of detrimental responses that include inhibition of photosynthesis, changes in assimilate partitioning, reduction in biomass, changes in pigment composition and stomata1 resistance. Increased irradiance, both before and during exposure to UV-B
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irradiation, has been shown to ameliorate the inhibitory effects on photosynthesis of the UV-B treatment (Warner and Caldwell, 1983; Mirecki and Teramura, 1984; Cen and Bornman, 1990; Caldwell et al., 1994). Caldwell et al. (1994) investigated the sensitivity of soybean to UV-B radiation under different combinations of UV-A (320-400 nm) and PAR. They found that significant UV-B effects on aerial biomass and growth in soybean were only found when PAR and UV-A were reduced to less than half their flux in sunlight. When the PAR was low, UV-A was especially effective in reducing UV-B effects. However, when PAR was high, UV-A did not have a significant role in ameliorating the UV-B response. These studies suggested that there was a threshold PAR, above which deleterious effects of UV-B were ameliorated. Under all conditions, UV-B caused an increase in UV-absorbing pigments and decreased whole leaf transmittance. There are, however, some observations that do not follow the consistent pattern of UV-B protection under high PAR. For instance, wheat dry matter content is reduced by high PAR and UV-B radiation (Teramura, 1980). Furthermore, pretreatment of soybean with high PAR provided protection against UV-B radiation, suggesting an indirect affect, while simultaneous high PAR and UV-B had an inhibitory effect on photosynthesis (Warner and Caldwell, 1983). The influence of high PAR prior to UV-B could be a result of acclimation to high PAR leading to changes in leaf morphology, increased levels of protective pigments or the alteration of the internal cellular environment (Bornman, 1989; Bornman and Vogelmann, 1991). Thus increased PAR could cause thicker leaves and thereby extend the pathlength for UV-B and increase the protective pigmentation within the epidermal cells. However, these mechanisms do not explain all of the observations relating to high PAR protection. For example, from the work of Jordan et al. (1992), the extent of a UV-B-induced decrease in mRNA transcripts can be reduced by increased PAR. Thus in some way, the higher PAR protects gene expression. Although these data are consistent with the physiological studies on photosynthesis which show that higher irradiance can afford protection, the protection of mRNA can be demonstrated after only a few hours and so is unlikely to be caused by changes in leaf morphology or pigment content. The protection by higher PAR may be caused by increased photorepair and photoprotection mechanisms (Pang and Hays, 1991, and references therein). However, because normally these mechanisms are saturated at relatively low irradiance they are unlikely to be the explanation. Alternatively, the protection may be afforded through photosynthesis. Thus, high irradiance is known to alter the thylakoid membrane appression (Davies et al., 1986a), photosynthetic function (Davies et al., 1986b) and biochemical composition of chloroplasts (Davies et al., 1987, 1991, and references therein). These changes could influence the response to UV-B by, for instance, increasing the provision of more light-dependent biochemical energy through photosynthesis. This has been directly tested by Adamse and Britz (1992). They used different concentrations of C 0 2 to modify the photosynthetic potential and demonstrated that increased C 0 2 could counteract the effect of UV-B on plant growth,
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indicating an important role for photosynthesis in ameliorating UV-B effects. Interestingly, the increased CO, had no influence on the accumulation of UV-B-induced flavonoid pigments. The influence of high PAR on gene expression has recently been investigated further (Mackerness et al., 1996). Using a combination of approaches that included, high CO, , inhibitors of photosynthesis and low pressure sodium lamps, it was concluded that the protection of gene expression was related to photosynthetic activity and not photorepair mechanisms. The results also suggest that there is a requirement for ATP provided by photophosphorylation. Increased PAR may also afford protection by causing the synthesis of antioxidants and other protective molecules. For instance, high PAR can increase the formation of polyamines (Kramer et at., 1992). Another important protective mechanism against photoinhibitory light is the xanthophyll cycle (Demmig-Adams and Adams, 1992). This involves the light-dependent conversion of violaxanthin via antheraxanthin to zeaxanthin and the reversal by epoxidation. The reactions of the xanthophyll cycle are thought to dissipate excess excitation energy generated within the chloroplast thylakoid. In a recent study (Pfiindel etal., 1992), the violaxanthin de-epoxidase was inhibited in isolated chloroplasts and leaves. The UV-B dose required for the inhibition in vivo was, however, 2 orders of magnitude greater than for chloroplasts. In agreement with other workers, they found UV-B inhibited photosystem I1 electron transport and suggested that a decrease in violaxanthin availability is controlled by the redox state of plastoquinone. From these and many other studies it is apparent that natural PAR will afford substantial protection against UV-B radiation. This protection by high PAR may be very important in moderating the influence of UV-B under natural daylight conditions. It is, however, important to consider plants that are generally in the shade and will not have developed UV-B protection. These plants will be particularly vulnerable under sudden exposure caused by sunflecks, etc., to high levels of UV-B. In addition, plants growing under canopy shade are exposed t o high levels of far-red irradiation due to red-light absorption by chlorophyll. Under these conditions of relatively high far-red, morphological, biochemical and ultrastructural changes take place and these may predispose some species to be particularly susceptible to UV-B damage.
B. WATER
Water stress is an important environmental constraint on crop productivity, both quantitative and qualitative (Boyer, 1982). Water stress can also strongly influence UV-B-induced responses (Teramura, 1986; Tevini and Teramura, 1989; BaIakumar et al., 1993). As in many UV-B-induced effects, the response of plants to the interaction of water and UV-B radiation depends upon the species. For instance, in cucumber, the adaxial leaf diffusion resistance to water loss increased by three-fold in the first few days of UV-B treatment
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and then stomatal function was lost as the leaf resistance rapidly declined. Thus, an increase in UV-B irradiance prevented stomatal closure and exacerbated water loss. Therefore an increase in UV-B could potentially enhance the susceptibility of cucumber to water stress. In contrast, radish showed no additional response to UV-B (Teramura, 1986). This resistance of radish was accounted for by UV-B and water stress promoting a synergistic increase in flavonoid synthesis and hence UV-B protection. The flavonoid levels decreased with increased UV-B in cucumber and therefore this could account for its susceptibility. Recently, Balakumar et al. (1993) also noted a number of beneficial responses to the combination of UV-B and water stress rather than either of them separately. Most noticeably, the combination of UV-B and water stress alleviated morphological changes induced by drought alone. Thus plant height, fresh weight and dry weight was reduced much less by the combination of UV-B and water stress than either stress alone. A number of changes were also observed at the biochemical level. For instance, under water stress molecules such as proline increase and this increase is reduced by the addition of UV-B radiation. This may suggest that the UV-B is reducing the stress induced by water alone. However, if proline is necessary to act as a drought protectant, this reduced proline level may not be of benefit. The increased synthesis of protective compounds and enzymes such as catalase, superoxide dismutase and anthocyanins was also likely to provide the protective synergism. Sullivan and Teramura (1990) studied the combination of UV-B and water stress on photosynthesis in soybean. Under well-watered conditions, increased UV-B radiation reduced photosynthetic capacity, plant dry weight, leaf area and number of pods. The combination of UV-B and water stress did not increase the responses compared to either individual stress. Both UV-B and water stress affected plant growth and photosynthetic gas exchange. Wholeleaf gas exchange analysis indicated that stornatal limitations were only significant under the combined stresses of water and supplemental UV-B; UV-B alone had only a minimal influence on stornatal conductance as has been previously shown (Negash and Bjorn, 1986). A number of factors may be involved in masking the UV-B response in water-stressed plants, in addition to morphology and protective pigmentation. For instance, drought may reduce the plant’s content of phosphorus and phosphorus deficiency reduces UV-B sensitivity in plants. In addition, water stress may delay cell division and therefore reduce growth. As UV-B radiation is particularly damaging during cell division, reduced growth is recognized as a means of UV-B protection. Consequently, reduced growth or phosphorus content resulting from water stress may negate UV-B damage (Sullivan and Teramura, 1990). These results suggest that the influence of increased UV-B radiation on crop productivity may be most inhibitory when there is no shortage of water. The considerable influence of water status has also been demonstrated by a 6-year field study (Teramura et al., 1990a). Two cultivars of soybean, Essex and Williams, were grown under field conditions with either ambient or supplements of UV-B radiation. Overall, Essex was found to be sensitive to UV-B radiation with
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yield losses of 20%, while Williams was tolerant. The yield loss was strongly influenced by the prevailing climate throughout the seasons. Thus the loss of yield in Essex was most severe during seasons when water was readily available. This supports previous findings that water deficit can mask the UV-B response (Murali and Teramura, 1986b; Sullivan and Teramura, 1990). However, the cultivar Williams was only affected by UV-B radiation in a year in which drought and high PAR was most prevalent. Another factor that has been suggested to be involved in cultivar differences is the ability of some cultivars to orientate their leaves to avoid direct incident solar radiation. Thus the extent of incident radiation on the leaves will have wide-ranging effects including changes in leaf temperature, transpiration, photoinhibition and carbon fixation. In experiments on soybean cultivars with different UV-B sensitivities (UV-B sensitivity: Forrest > Cumberland > Essex) the least sensitive cultivars changed their leaf angle in reduced water levels. The most sensitive cultivar to UV-B radiation did not reorientate its leaves. This work clearly illustrates the complex interactions between UV-B radiation, water, temperature and light, with water being a major interactive influence.
C. C02 AND TEMPERATURE
Another environmental parameter that is likely to increase in the future is the global concentration of C 0 2 (Bowes, 1993). Most predictions suggesting a rise in concentration from the ambient level of 340pl 1-' t o 6 8 0 ~ 11-' by about the year 2050. It is thought that such an increase in C 0 2 could be beneficial to crop productivity in a wide range of C3 plant species, as photorespiration would be suppressed. Long-term studies, however, suggest that plants can acclimate to the increased C 0 2 and therefore this increased productivity may not take place (Sage et al., 1989). Furthermore, C4 plants will probably show little benefit from a rise in ambient C 0 2 level. In addition, any predicted increase in crop productivity is based on the consequences of increasing only the C 0 2 levels and few studies have been made on the interactions between increased C02 and increased UV-B - a likely future situation. From the studies that have been made, it is clear that any beneficial effects of increased C 0 2 may be reversed by an increase in UV-B radiation (Tevini, 1993 and references therein). For example, when wheat, rice and soybean were subjected to increased C 0 2 (ambient to 650 pl I-'), there was a significant increase in total biomass and seed yield in all three species (Teramura et al., 1990b). However, the addition of increased UV-B radiation (8.8-15.7 kJ m-2 UV-B,,) prevented any increase in seed yield (wheat and rice) or total biomass (rice). In contrast, C0,-induced increase in seed yield and total biomass in soybean was maintained or increased in the elevated C 0 2 and UV-B environment. Two different cultivars of rice have also been investigated (Ziska and Teramura, 1992). In elevated CO,, both cultivars exhibited a decrease in respiration and an increase in photosynthesis, total biomass and yield.
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Simultaneous exposure to C 0 2 and UV-B radiation eliminated the C 0 2 effects on respiration, photosynthesis and total biomass completely for one cultivar and significantly reduced them in the other. Analysis of the site of inhibition suggested that it differed for the two cultivars. Thus, in one cultivar the inhibition was due to a limitation on the capacity to regenerate ribulose bisphosphate and was consistent with damage to the photochemical efficiency of photosystem 11. In the other cultivar, Rubisco carboxylase activity was inhibited, but no damage was apparent to photosystem 11. The lack of damage to photosystem I1 was also correlated to the production of flavonoids by the less-sensitive cultivar. Another prediction for the future is that the greenhouse effect will lead to an increase in global temperature of several degrees centigrade. Such an increase would increase crop productivity for many species assuming that their temperature optima was not exceeded. Seedlings of four species (sunflower, maize, rye and oat) were grown at 28°C and 32°C in growth chambers using the ozone filter technique to represent a 20% UV-B radiation difference between growth chambers (Tevini, 1993). Except for oat, growth of the seedlings, measured as plant height, leaf area and dry weight, was greater at 32°C. UV-B radiation significantly reduced the growth of sunflower at both temperatures. Maize and rye, however, could compensate for UV-B-induced growth reduction if the plants were grown at the higher temperature. These results suggest that higher temperatures may compensate for the loss of productivity that may take place under increased UV-B radiation. Further studies have also shown that growth of cucumber under enhanced UV-B radiation increased their ability to withstand elevated temperatures (Caldwell, 1994). However, the overall response is likely to be very species-dependent. In a recent study, the effect on sunflower and maize of a combination of increased C 0 2 , temperature and UV-B was tested (Tevini, 1993). The plants were subjected to high solar UV-B (equivalent to a 12% ozone depletion), double ambient CO, concentration and 4°C higher daily temperature (32°C instead of 28°C). Sunflower seedlings had a 10% increase in net photosynthetic rate in the higher CO, , temperature and UV-B radiation environment, compared to those plants receiving only higher temperature and UV-B radiation. In contrast, maize seedlings had slightly lower photosynthetic rates, indicating that C4 plants may not generally benefit from any future increase in CO, levels compared to C3 plants. Other gaseous pollutants, such as ozone, in combination with UV-B, can cause a more inhibitory effect than either ozone or UV-B alone (Krupa and Kickert, 1989; Feder and Shrier, 1990). Similarly, studies on the combination of UV-B and toxic metals, such as cadmium, may reveal interactions that are more damaging then the influence of either stress given alone (Bornman and Dub& 1991). Another important aspect of the effect of increased temperature is its influence upon enzyme activities that are involved in UV-B defence. Thus, Arabidopsis photolyase has been shown to be inhibited by increased temperature, both in vivo and in vitro (Pang and Hays,
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1991). The combination of increased UV-B radiation, causing DNA damage, and the inhibition by temperature of repair enzymes could potentially create a very serious future situation.
D.
PATHOGENS
An important aspect of the UV-B effect upon plants is the combined influence of UV-B and pathogens (Orth etal., 1990; Panagopoulos etal., 1992, and references therein). As with many UV-B effects, its influence upon pathogenicity depends upon the species and prevailing conditions. Viral pathogens exposed to increased UV-B radiation are likely to be affected as they are primarily composed of high UV-B absorbing compounds; nucleic acids coated with proteins. Thus, in Chenopodium quinoa, potato virus infection was severely reduced by UV-B treatment and could be prevented completely by high levels of UV-B irradiation (Semeniuk and Goth, 1980). However, these types of results will depend upon the pathogen being exposed to the UV-B radiation and this will depend upon the site of infection. Thus the infection potential of a pathogen on the leaf surface may be inhibited by UV-B, but not if the site of infection is through, for instance, the root. Fungal infections have been increased when exposed to UV-B radiation in cucumber (Cucumis sativus). Three cultivars were tested; one susceptible and two resistant to the fungal pathogens (Orth et al., 1990). If the plants were exposed to UV-B prior to the application of the pathogen, the severity of the disease increased in the susceptible cultivar and became apparent in the resistant cultivar. However, no effect on the disease took place when the cultivars were exposed t o the pathogens and UV-B together. This result strongly suggests that the influence of the UV-B treatment has been to modify the leaf surface so as to allow fungal penetration and consequent infection. However, in sugar beet there is an accumulative interaction of Cercospora beticola and UV-B to give an increased reduction in chlorophyll content, dry weight and lipid peroxidation (Panagopoulos et at., 1992). The nature of the fungal spore may also influence its resistance to enhanced levels of UV-B, although the results at present are equivocal (Bornman and Teramura, 1993, and references therein). Studies on pigment biosynthesis have revealed a complex interaction of UV and fungal elicitors. Both stimuli can activate enzymes of the phenylpropanoid pathway, although UV irradiation results in vacuolar accumulation of flavonoids, whereas the fungal elicitors cause the secretion of furanocoumarins. Together the UV irradiation and fungal elicitor result in quantitative changes in each of the individual responses. Thus, elicitor-induced furanocoumarins are reduced and the light-induced accumulation of flavonoids is completely inhibited. These changes are a result of differential responses of enzymes within the general phenylpropanoid pathway. For instance, an early observation showed that treatment of parsley cells with both fungal elicitor and
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UV-light prevented the UV stimulation of chs, whereas PAL biosynthesis was increased by UV, elicitor or both (Hahlbrock eta/., 1981). Similarly, a number of other enzymes are differentially regulated. Using transient expression analysis of regions of the chs promoter (Lozoya eta/., 1991) it has been demonstrated that elicitor-sensitive regions exist in the promoter and that they are identical to those required for light-inducibility (see section V.B). It is also important that other stress factors rapidly down-regulate gene expression for some proteins and activate the expression of other genes. Thus it is well established that the phenylpropanoid pathway is switched on by pathogens and that the expression of genes such as rbcS are switched off (Kombrink and Hahlbrock, 1990). This seems very analogous to the UV-B response at the level of changes in gene regulation. UV radiation may also stimulate pathogenrelated gene expression (Brederode et al., 1991). Furthermore, during heat shock, there is also a rapid reduction in the expression of some genes and the biosynthesis of the heat shock proteins (HSP). The expression of proteins similar to HSP has recently been reported in Vigna sinensis seedlings exposed to UV-B radiation (Nedunchezhian et al., 1992). It is clear from these few examples that a combination of stress factors can bring about a variety of responses that cannot be predicted on the basis of studying a single environmental parameter. The present predictions and scenarios can only touch upon the true complexity of the possible future situation. Consequently, unless multiple simultaneous stress factors are studied, it is likely that the environmental impact of UV-B will not be correctly anticipated.
VII.
CONCLUSION
A substantial amount of research has been carried out to investigate the effects of UV-B radiation on plant growth and development. These studies describe a wide range of responses caused by increased levels of UV-B radiation. The diverse nature and severity of these responses may, in part, be a result of experimental procedures. However, the results are clearly indicative that plants and plant communities may suffer in response to any future increase in UV-B radiation. In addition, many of these studies demonstrate the complex interaction between UV-B radiation and other environmental parameters. It is also clear from these studies that the underlying molecular mechanisms of UV-B responses are poorly understood. A large number of biochemical changes have been described, but many appear as indirect consequences of exposure to UV-B radiation rather than the primary response. It is therefore particularly important to determine the mechanism of UV-B perception and the signal transduction pathways involved in these responses. This will lead to a greater understanding of the regulatory mechanisms controlling the UV-B responses. Approaches are now becoming available that will provide insights
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into these molecular mechanisms. This will give the potential t o develop longterm strategies to protect susceptible species, either by conventional breeding methods or by genetic engineering.
ACKNOWLEDGEMENTS This review was largely written at Horticulture Research International, Littlehampton, West Sussex, UK and I would like to acknowledge the financial support of the AFRC. I am also grateful for the scientific collaboration of my colleagues at HRI, particularly Drs Brian Thomas and Mick Partis who have provided many helpful comments during the preparation of this manuscript. Also Dr Soheila A.-H. Mackerness, Pat James and Richard Anthony for their experimental efforts that have provided much of the insight in this review. 1 would particularly like to acknowledge the collaboration of Drs W. S. Chow and Jan Anderson, CSIRO, Canberra, Dr like Strid, Goteborg University and Dr Alyson Tobin and colleagues at Manchester University. In addition, 1 would like to thank all those scientists who provided information for this review and Geraldine Linfield for her assistance in the preparation of the manuscript.
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Rapid. Long-distance Signal Transmission in Higher Plants
M . MALONE
Horticulture Research International. Wellesbourne. Warwicks CV35 9EF. UK
I.
Introduction ............................................................................. 163 A . Shoot Responses to Change in the Root Environment ............... 164 B . Rapid Movements Induced by Localized Mechanical Stimuli ....... 164 C . Remote Responses to Localized Wounding .............................. 165 D . Miscellaneous Related Phenomena ......................................... 166
I1.
Mechanisms of Long-distance Communication Within Plants ............ A . Airborne Signals ................................................................. B . Phloem Translocation .......................................................... C . Hydraulic Pressure Signals .................................................... D . Hydraulic Dispersal Signals ................................................... E . Electrical Signals .................................................................
167 167 170 171 177 186
I11. Rapid, Long-distance Signalling in Plants: Case Studies ................... 188 A . Systemic Induction of P I by Localized Wounding in Tomato ..... 188 B . Signal Transmission in Mimosa ............................................. 196
IV . Implications and Directions for Further Research ............................ 200 A . Re-assessment of Electrical Signals in the Higher Plant ............. 201 B . Signalling of Non-wound Stimuli - an Hydraulic Mechanism? ... 209 V.
Conclusion ............................................................................... Acknowledgements .................................................................... References ................................................................................
216 216 217
I . INTRODUCTION Irritability. the capacity to sense and respond to the environment. is one of the characteristics of the living organism . It is particularly important in land Advances in Botanical Research Vol . 22 incorporating Advances in Plan1 Pathology
ISBN 0-12-005922-3
Copyright 0 1996 Academic Press Limited All rights of reproduction in any form reserved
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plants because they must cope in situ with environmental stresses, which may be long- or short-term, predictable or unpredictable. The entire sequence of events that link stimulus perception to response could be described as “signal transduction”. However, this term is usually restricted to the intracellular cascade of events which generate a response (reviews in Poovaiah and Reddy, 1993; Verhey and Lomax, 1993; Gilroy and Trewavas, 1994). Intracellular transduction alone will suffice for some local responses and for simple or unicellular organisms. Co-ordination of activity in the higher organism, however, will require long-distance signal transmission as well as intracellular signal transduction. For example, environmental stresses may not impinge uniformly on the higher plant, and appropriate responses will often involve tissues at considerable distance from those immediately affected by a stimulus. In this review, “long distance” refers to stretches that cannot be spanned by a couple of hours of diffusion in the aqueous phase: several millimetres or more, and sometimes up to metres. “Rapid” refers to rates of perhaps 0.2 mm s-’ and over. A broad view is taken of the concept of signalling, and almost any event that could connect a remote stimulus with a physiological response is considered. Discussion on the fundamental nature of signalling and signal transmission can be found in Bentrup (1979), Canny (1985), Firn (1985), Due (1989) and Wayne (1994). In higher animals, very rapid (40 m s-’) co-ordination within the organism is enabled by a specialized nervous system. Three hundred years of histology has not revealed nerves in plants. Nevertheless, plants can exhibit long-distance communication at rapid rates. According to some reports, rates of up to 300mm s-’ are possible (Sibaoka, 1966; Oda and Linstead, 1975; Umrath and Kasterberger, 1983). The major examples of rapid long-distance signalling within plants can be grouped into the following categories: A. SHOOT RESPONSES TO CHANGE IN THE ROOT ENVIRONMENT
Treatments applied to the root may cause dramatic changes in leaf growth rate beginning within a couple of minutes. This has been demonstrated with root cooling (Dale et af., 1990; Malone, 1993a; Pardossi etal., 1994), osmotica (Chazen and Neumann, 1994), salt (Cramer, 1992; Alarcon and Malone, 1995), and anaerobiosis (Nakahori etal., 1991). Over both short and longer time courses, stomata1 aperture in leaves can also respond sensitively to changes in conditions in the root environment (Davies and Zhang, 1991). B. RAPID MOVEMENTS INDUCED BY LOCALIZED MECHANICAL STIMULI
Rapid movements can be induced by touch in leaves of most species of the insectivorous genera Drosera, Dionaea, Aldrovanda and Utricularia (Darwin,
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1875; Williams, 1976) as well as in sensitive plants exemplified by Mimosa spp. Similar responses are found in a range of organs in Stylidium (Findlay, 1978), Berberis, Sparmania, Mahonia, Mimulus, Bignonia (Hill and Findlay, 198 l), Zncarvilka (Sinyukhin and Britikov, 1967), Oxalis, Carambofa (BurdonSanderson, 1882) and others (see Darwin, 1881). For centuries, these rapid visible responses have fascinated botanists (Jones, 1923, on Dionaea: “The most wonderful plant in the world”) and laymen (e.g. King Charles I1 of England, 1661, cited in Houwink, 1935) and they have attracted close study. By contrast, relatively few invisible responses to localized mechanical stimuli have been characterized (Bose, 1928; Jaffe, 1980; Braam and Davis, 1990; Galaud et al., 1993) and it seems likely that many more such responses, both local and remote, await discovery.
C.
REMOTE RESPONSES TO LOCALIZED WOUNDING
These include “defence reactions” in which damage at one leaf leads to accumulation of toxic compounds throughout the shoot. These responses have attracted particular interest because of their potential in crop protection (Ryan, 1989, 1992). Systemic accumulation of inhibitors of insect digestive enzymes is found after localized wounding in cultivated tomato (Green and Ryan, 1972), wild tomato (Wingate and Ryan, 1991), potato (Peiia-Cortes et a/., 1988), tobacco (Pearce‘ et al., 1993), maize (Eckelkamp et a/., 1993; Corder0 et al., 1994), poplar (Parsons er al,, 1989), alfalfa and Datura strumonium (Shukle and Murdock, 1983). Endogenous elicitors from tomato leaves can also induce proteinase inhibitors in leaves of squash, cucumber, strawberry, grape and clover (Walker-Simmons and Ryan, 1977). In tomato and potato, induction of proteinase inhibitors (PI) is detectable in remote leaves, within 20 min to 2 h from wounding (Peiia-Cortes et af., 1988; Graham et al., 1986; Malone er al., 1994b). The mechanism of wound signalling in this system is considered in more detail in section IIIA. Another defence reaction induced throughout the shoot by localized treatment is known as “systemic acquired resistance“ (SAR or ISR). It occurs after limited pathogen infection in a restricted (De Wit, 1985), or wide (Lawton et al., 1994) range of plants. When one leaf becomes infected by any of a diverse array of pathogens, uninfected leaves on the same plant may develop increased resistance to a similarly diverse range of pathogens (KuC, 1987). Normally, development of SAR requires many days, but after treatment of one leaf with certain chemicals, such as oxalic acid, SAR may develop within 20 h (Doubrava el al., 1988) and the signalling system could be much faster than this. Systemic emission of volatile pheromones and other semiochemicals or alIelochemicaIs may occur soon after localized insect feeding in certain plants. These compounds may attract predators or parasitoids to the attacking
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herbivore (Turlings and Tumlinson, 1992; Dicke et a / . , 1993; Dicke, 1994) and they are thus thought to be involved in defence. A variety of other remote or systemic responses, mostly with no known defence function, are reportedly induced soon after localized wounding. These include stomata1 closure (Van Sambeek and Pickard, 1976; Wildon etal., 1992), reduction in the rate of isoprene emission (Loreto and Sharkey, 1993), increased polysome formation (Davies and Schuster, 1981), changes in the pattern of bud break in Bidens (Desbiez et al., 1991), surface electrical phenomena (Houwink, 1935; Van Sambeek and Pickard, 1976; Malone and StankoviC, 1991; Stahlberg and Cosgrove, 1995), and changes in the activity of certain ions in the xylem (Ries etal., 1994). In many species, localized wounding also triggers a general increase in cell turgor pressure (Malone and StankoviC, 1991) and a swelling of the shoot (see Figs 3, 4 and 7; Boari and Malone, 1993). These effects are described in section IIC and IID. They are systemic except that tissues very close to the wound site may show the opposite effect: a contraction (Malone, 1993b, 1994; see section IID and Figs 10, 13, and the leaf marked * in Fig. 7). Swelling and/or contraction of the stem was also reported by Bose (1928) after application of small electric and mechanical stimuli to stems of Antirrhinum, Tradescantia, tomato, and “woody rose”.
D. MISCELLANEOUS RELATED PHENOMENA
Many further examples of long-distance signalling in plants can be identified but the transmission rates are often relatively slow. Selected examples are considered briefly: a classic paradigm of signal transmission in plants was formerly provided by the Cholodny-Went theory of phototropism and geotropism. However, that theory has proved to be mostly incorrect (Firn and Digby, 1980). Even so, there is clear evidence that mobile signals from the root cap play some role in facilitating geotropism (Moore and Maimon, 1993). The polar transport of auxin (Goldsmith, 1977) provides a potential system for long-distance (basipetal) signalling at perhaps 10 mm h-’, for example, in apical dominance (Sachs and Thimann, 1967). Unidentified signals of daylength are generated in leaves and travel to the apical meristem to control the transition to flowering in various species (Penel et a/., 1985). Signals probably involving cytokinins, and which are generated mainly in the seed pod, trigger active senescence of soybean leaves (Nooden and Murray, 1982; Nooden and Letham, 1993). Plants may also transmit chemical or physical signals to neighbouring organisms. These include attractants (colour, scent, heat) produced by insectpollinated flowers, “warning” signals of heavy chemical defences (Augner, 1994), and phytotoxic root exudates to reduce competition from neighbours (Woods, 1960; Krebs, 1972, p.41). Some signals are released that have no obvious benefit to the transmitter and may even be deleterious. Many of these are probably unavoidable by-products of the plant’s biochemical or biophysical
SIGNAL TRANSMISSION IN HIGHER PLANTS
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presence; they are hardly signals in a physiological sense but they can be ecologically important. Examples include the far-red light reflected from photosynthetic pigments of leaves in sunlight. This may induce competitive reactions in neighbouring plants (Ballare et a/., 1994). Also, volatile substances released from plants can be used by herbivores to locate a food source (Finch, 1986; Could, 1994). Water vapour is the major volatile compound released from land plants and, depending on the degree of physical coupling between canopy and atmosphere, water vapour released from one plant may have a significant influence on the microenvironment of neighbouring plants (Jones, 1992). Some reports discuss changes induced in one plant following wounding of a different plant (Baldwin and Schultz, 1984; Fowler and Lawton, 1985). Such interaction wouId seem to have little benefit to the transmitter unless the neighbouring plants are its close relatives. There are also occasional reports of more mysterious communication between plants. These tend to be on the metaphysical fringe (Backster, 1968; Wagner, 1989; and reinvestigation by Horowitz et al., 1975). In the following section, various mechanisms by which rapid, long-distance signalling within the plant might occur are considered.
11.
MECHANISMS OF LONG-DISTANCE COMMUNICATION WITHIN PLANTS
At least five potential mechanisms for rapid long-distance signalling within the plant can be identified: 1. airborne flow of volatile chemical messengers; 2. phloem transmission of chemicals;
3. self-propagating changes in hydraulic pressure; 4. xylem transmission of chemicals (=“hydraulic dispersal”); 5 . self-propagating changes in electrical potential.
In some cases the stimulus itself can propagate through the plant from a site of application: in Mimosa pudica, for example, vigorous shaking of one leaf can cause distant leaves to react, but the mechanical vibrations of shaking are themselves transmitted through the plant. The response is merely local (Sibaoka, 1991). Light may also be “piped” for considerable distances through the fabric of the plant (Mandoli and Briggs, 1984). This is probably best considered as stimulus transmission rather than true signalling. A.
AIRBORNE SIGNALS
Volatile messengers can move rapidly by diffusion through the plant’s air-space network. In some plants such movement may be facilitated by “internal winds”, which are mass flows of air within the plant (Armstrong and
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Armstrong, 1990). The flow velocity can reach 2 mm s-' in petioles of Nuphar lutea (Dacey, 1981). Few endogenous plant growth regulators are significantly volatile under biological conditions. Exceptions include ethylene (boiling point -103°C) and methyl jasmonate (MeJ). Ethylene is released by most plant tissues. It has a wide spectrum of effects and has been recognized as an important plant hormone for a century (Neljubow, 1911; Osborne, 1975). MeJ is also released by many plant tissues, especially after damage (Albrecht et al., 1993). It may arise during membrane breakdown (Farmer and Ryan, 1992; Peiia-Cortes etal., 1993). MeJ has recently been shown to be effective at extremely low levels in some systems (Farmer and Ryan, 1990; Falkenstein etal., 1991) and it should probably be considered as a true plant hormone. Under most circumstances, volatile compounds will quickly disperse from their sites of production into the surrounding atmosphere. They will therefore play little role in long-distance signalling within the plant. However, if plants are closely sheltered or enclosed in some way, volatiles released in one tissue may accumulate to significant levels and may reach and affect distant tissues on the same or neighbouring plants. This can be demonstrated by experimental enclosure of plants (Farmer and Ryan, 1990). In the natural environment, effective enclosure of plants occurs during submergence. This enables adjustment to changing water depth in a number of semi-aquatic plants (Ku et al., 1970). Nymphoides peltata, for example, has leaves that float on the surface of slow-moving water, but which are anchored by long petioles to roots in the substratum. The leaves produce ethylene continuously. Normally, the gas diffuses quickly away into the surrounding atmosphere, and there is no significant accumulation within the tissue. However, if the leaves become submerged by a rise in the water level, ethylene can no longer escape because its diffusion rate in water is slower by a factor of lo4 than that in air (Burg and Burg, 1965). Ethylene therefore accumulates rapidly in air spaces within the flooded plant; it may increase 20-fold to over 1 pI I-' within an hour of submergence (Fig. 1; Malone, 1983). At such elevated levels ethylene stimulates rapid petiole elongation in Nymphoides beginning within about 30 min (Fig. 2; Malone, 1983), as in many other water plants. This elongation returns the leaf lamina to the surface, whereupon the ethylene dissipates to the atmosphere, and petiole elongation ceases. In this system, therefore, elevation of ethylene levels within the petiole is both a signal of leaf submergence and a trigger of the depth-accommodation response. In the sundews (Drosera spp.) insects are captured by globules of viscous fluid secreted at the apex of glandular leaf hairs. Once an insect becomes entangled at one hair or group of hairs, these hairs quickly bend towards the leaf centre. Later, more distant hairs on the same leaf may bend towards the insect to enclose it further and to aid with its digestion (Darwin, 1875). In some species, the leaf lamina also bends markedly to enclose the insect (Darwin, 1875, p. 12). The signals that initiate bending in neighbouring leaf hairs and in the leaf lamina could be hormonal (Williams and Pickard, 1980) but there
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SIGNAL TRANSMISSION IN HIGHER PLANTS
0 I
I
1
0
2
4
1
1
6
/
I
1
8
I
10
time from submergence (h) Fig. 1 . Kinetics of ethylene accumulation in leaves of a water plant after submergence. Air was vacuum-extracted from batches of nine submerged, excised leaves of Regnellidium diphyllum (the petiole of each leaf was first trimmed to 30mm). Ethylene content was determined by gas chromatograph (G C). From Malone (1983).
is anecdotal evidence that airborne factors released by the insect might also play a part. For example, the following observations on Drosera filiyormis, by one “Mrs Mary Trent” are recounted in Knight and Step (1905, p. 64): “At ten o’clock I pinned some living flies half an inch from the leaves, near the apex. In forty minutes the leaves had bent perceptibly toward the flies. At twelve o’clock the leaves had reached the flies and their legs were entangled among the bristles and held fast”. A “Mrs Treat” is mentioned and cited several times in connection with D. filiyormis in Darwin’s Insectivorous Plants (1875, pp. 278, 281). I presume that “Mrs Trent” is identical with “Mrs Treat”, and that she was a considerable authority on Drosera. Her observations here may seem unlikely, but airborne signalling is not inconceivable in Drosera. For example, the tentacles of D. rotundifolia are incredibly sensitive to ammonium ions, and can react to quantities “. . . comparable to those in rain water” (Darwin, 1875, p. 167); the level of ammonium in Darwin’s rainwater was probably considerably less than in present-day Kentish rainwater). Wounded or decaying insects might emit sufficient ammonia to trigger tropic reactions in nearby leaf tissue.
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4 h
E E m 3 c
u
C
a,
ka, m
5 2
f 0 C
a, C .-
c & m
'
5
!
-4
-2
,
,
/
I
0
,
,
,
I
,
2
I
,
I
,
4
time from ethylene addition (h)
Fig. 2. Kinetics of ethylene-promoted growth in the petiole of Nymphoidespeltata. The height of a vertical column of 4 x 10-mm apical petiole segments was monitored using a displacement transducer. Air containing a saturating level of ethylene (0.1% v/v) was applied directly to the segments at time zero. From Malone (1983).
B. PHLOEM TRANSLOCATION
The phloem is the major pathway for long-distance transport of solutes in the plant (Baker and Milburn, 1989). Various chemical messengers are believed to move through the phloem, including abscisic acid (ABA) (Wolf et a/., 1990; Jackson, 1993), MeJ (Anderson, 1985), the tomato proteinase-inhibitor elicitor "systemin" (Pearce eta/., 1991), salicylic acid (Rasmussen eta/., 1991) and others (Hall and Baker, 1972). The phloem is the most obvious potential route for long-distance transmission of chemical signals in plants (Ishiwatari etal., 1995). In addition, the movement of many signals displays an association with the vascular system (Ryan, 1974; Van Sambeek and Pickard, 1976; Roblin and Bonnemain, 1985; Keil etal., 1989; Stanford eta/., 1990; Davis etal., 1991), and extends in both basipetal and acropetal directions. This again suggests movement in the phloem (Peiia-Cortes eta/., 1988; Davis eta/., 1991; Weiss and Bevan, 1991). Some workers perceive the phloem as being the only system capable of conveying solutes rapidly in both basipetal and acropetal directions in plants (e.g. Beraud eta/., 1992; Rigby etal., 1994); a view which is certainly incorrect (see section 1I.D).
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Estimates of the flow velocity of phloem sap range around 0.1-1 mm s - ' (MacRobbie, 1971; Nelson etal., 1983; Nobel, 1991, p. 515) with values up to 4 m m s-' (Baker and Milburn, cited in Wildon etal., 1992). Many longdistance signals in plants are slower than this, but a significant number are substantially faster. It is difficult to measure accurately the movement of signal molecules in the phloem. The most common method of extraction involves incubating the base of a cut petiole in a solution containing ethylenediaminetetraacetic acid (EDTA) as chelating agent (Anderson, 1985; Pearce etal., 1991). It is questionable whether extracts obtained in this way represent pure phloem sap (Jackson, 1993). Techniques based on severed aphid stylets (Ishiwatari et al., 1995) or on histochemical approaches (Narvaez-Vasquesz et al., 1995) should facilitate more precise analysis of the identity and flow rate of signal molecules in the phloem. Even here, however, caution must be excercised because various substances can pass between the xylem and the phloem (Van Bel, 1984; Minchin and McNaughton, 1987). Thus the presence of a molecule at any point in the phloem does not necessarily mean that it has arrived there entirely via the phloem. Some putative signal molecules, such as oligosaccharides, appear to have very limited phloem mobility (Baydoun and Fry, 1985).
C. HYDRAULIC PRESSURE SIGNALS
Hydraulic pressure signals are propagating changes in water pressure. They will pass throughout the hydraulic continuum of the plant (Boyer, 1969, 1989; Malone, 1993b) and they offer "a potentially ubiquitous system of integration within plants" (Palta et al., 1987). Hydraulic theories of communication in plants date back to 1661 (Clarke, cited in Houwink, 1935); the fact that vessels were not then known was an inconvenience. Hydraulic theories thus predate electrical (Burdon-Sanderson, 1873) and hormonal (Neljubow, 1911) theories by a couple of centuries. A minority of tissues do not have good hydraulic connections to the main stem of the plant. These may include cortical regions of the mature root (Sanderson, 1983), and some unusual structures (Morse, 1990), as well as material destined shortly to part company with the mother plant, such as maturing fruit and seed (Darlington and Dixon, 1991; Welbaum etal., 1992), abscising leaves, etc. Most tissues, however, have plentiful hydraulic connections furnished by the xylem. This is particularly important in leaves because, except in some crassulacean acid metabolism (CAM) systems and plants of aquatic or exceptionally humid environments, the process of harvesting C 0 2 inevitably involves major loss of water (Cowan, 1977). Extensive xylem connections ramifying to all parts of the leaf (Canny, 1988) are required to replenish this water efficiently. These connections also provide a route for long-distance transmission of hydraulic signals.
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The front of a pressure wave will travel through the plant at up to the speed of sound (1500m s-’ in water). However, to be physiologically relevant, an hydraulic signal must cause a significant change in turgor pressure in the live cells receiving it. Most plant tissues are appreciably elastic; their turgor will change only if net influx (or efflux) of water occurs, that is significant relative to the hydraulic capacity of the tissue. Thus, all physiologically important hydraulic signals must involve significant mass flow of water. As a rough example, net water influx equivalent to 1-5% of the total volume of water in a leaf might be required to raise the turgor pressure of its cells through I bar (Malone, 1993a). The kinetics of pressure change in tissue receiving a hydraulic signal will depend on the magnitude and distribution of hydraulic resistance along the flow path, and of hydraulic capacitance of the receiver tissue. Despite several advances (Zimmermann, 1983; Canny, 1990; Tyree and Ewers, 1991 ; Peterson et al., 1993), the detailed information on plant hydraulic architecture required for prediction of the systemic pattern of pressure propagation is not available. The mass flow associated with hydraulic signals between organs will usually involve a relatively long axial pathway, through the xylem, and a short radial pathway through cells (or cell walls) of the tissue at each end. The volumetric rate of fluid flow through the open tubes of the xylem can be approximated from the Hagen-Poiseuille “law”:
where J, is volumetric flow rate through the tube, r is tube radius, AP is pressure gradient, q is kinematic viscosity of fluid, and I is tube length. This equation states that the flow rate (per tube) will depend on the prevailing gradient of hydrostatic pressure, on the 4th power of the tube radius, and on the viscosity of the fluid (Nobel, 1991; Niklas, 1992). The xylem usually contains only dilute solutes. These have negligible effect on flow rate. More concentrated solutes, such as sugars, can increase the viscosity of water considerably (Zimmermann, 1983). The viscosity of water can also change significantly with temperature over the biological range (Boyer, 1993). The Hagen-Poiseuille law often yields good approximations for water flow through xylem (Zimmermann and Brown, 1971, p. 199; Frensch and Steudle, 1989). Discrepancies can be ascribed to the various features by which xylem conduits differ from ideal tubes. These include occasional end walls, uneven internal walls (see Fig. 6 ) , non-circular members, and non-straight vascular bundles (Zimmermann, 1983; Tyree and Ewers, 1991). The radial pathway from the xylem into the tissue is comparatively short, but it appears to present more hydraulic resistance than the axial pathway, at least in herbaceous plants (Boyer, 1969; Malone, 1992). For example, Fig. 3 shows the kinetics of swelling at various points along an individual wheat leaf, in response to a remote wound. The half-time of swelling can be seen to vary
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1
-4
1
1
0
1
1
4
~
8
I
,
12
I
16
,
I
,
20
Time from wound (min) Fig. 3. Kinetics of change in wheat-leaf thickness after wounding a neighbouring leaf. Five displacement transducers were distributed along a single wheat leaf, at the positions indicated in the diagram (inset). This leaf was about 120 mm in length. At time zero the neighbouring leaf was scorched with a flame for 3 s , at the position indicated by the vertical arrow. The curves show changing thickness a t each position, normalized so that the total change by 16min from wounding is the same for all. Symbols alternate between filled and open for successive curves. From Malone (1992).
inversely with the distance from the treatment site - that is, swelling appears faster further from the wound site. In fact, the kinetics depend on leaf thickness at the point of measurement, rather than on distance from the treatment site; the leaf becomes thinner towards its distal end, so the kinetics are faster there. This will occur only if the radial component of the flow path offers a larger resistance than the axial. In a range of herbaceous plants, change in xylem pressure at one location causes leaves over the entire shoot to swell with a half-time of 2-4 min (Fig. 4; Boyer, 1969; Boari and Malone, 1993). An abrupt change in xylem pressure can be imposed by cutting one leaf under water, or by damaging cells at one location and releasing their cell sap to the xylem (see Fig.9, section IIDl). Each of these treatments make water available to the xylem at the treatment
~
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Fig. 4. Remote wound-induced swelling in a range of plants. Each curve shows the mean change in leaf thickness from four similar plants treated simultaneously. A displacement transducer was placed on one leaf of each plant then, at the time indicated by the vertical line, one remote leaf on each plant was scorched for 3 s with a flame. Most of the plants were seedlings of about 15 cm in shoot height and having at least four expanded leaves. Time and vertical scales are indicated on the figure, as are the generic names. Wound-induced swelling of remote leaves was found in all plants tested. From Boari and Malone (1993).
SIGNAL TRANSMISSION IN HIGHER PLANTS
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site. In young wheat leaves, a radial file of only three to four cells separates the xylem from the epidermis. Since the half-time for water-exchange across individual wheat-leaf cells is only 1 s or so (Malone and Tomos, 1990), it is surprising that the whole leaf displays a half-time of several minutes (Fig. 3). Even allowing for the concentric arrangement of cell layers around the xylem, the half-time for the whole leaf should be faster. This suggests the presence of an additional barrier to radial water efflux from xylem. The approximately exponential kinetics observed for leaf swelling (Fig. 3) also suggest the presence of a single major barrier to radial water flux, rather than a distributed series of resistances. This barrier may be similar to that identified by Canny (1990) in the bundle sheath that surrounds the vascular tissue in leaves of many monocotyledonous plants. Analogous barriers are present in the root endodermis and are postulated in some other tissues (Welbaum etal., 1992; Zimmermann et al., 1993). In a transpiring plant, the water status of all tissues will approach a dynamic equilibrium with their local xylem and thus with the entire plant. A change in the flux of water at any one site will alter local xylem pressure and will thus be transmitted throughout the xylem and throughout the plant. This means that a change in flux anywhere in the plant will be buffered by the hydraulic capacity of the entire plant, and it will affect the turgor pressure of live cells throughout the entire plant. Local changes in flux will occur, for example, when some roots encounter drying soil, or on a change in lighting or microenvironment at one leaf, even with a gust of wind or passing cloud. These events must cause small pressure transients to pass through the entire plant. Plants growing in the natural environment probably experience such pressure ripples throughout most of the day, but they may not be very large. 1. Transduction of pressure signals There is evidence that plant cells can be extremely sensitive to pressure (Staves et al., 1992). Various ways in which pressure changes might be transduced into physiological effects have been considered (Zimmermann, 1977). Two that deserve particular attention, from the whole-plant perspective, are:
Hydropassive effects on stomata. Raschke (1970) showed that small changes in the xylem pressure of maize leaves have rapid effects on stomatal aperture throughout the leaf. A decrease in xylem pressure causes opening of stomata, beginning almost immediately, and vice versa. The half-time here (a few seconds) is markedly faster than that for leaf swelling. This may indicate amplification of stomatal movement by mechanical interactions between guard cells and their neighbours, or the existence of an hydraulic by-pass direct from xylem to stomatal complex (Cowan, 1977; Boyer, 1985). In any case, the hydropassive opening of stomata by decreasing leaf water status produces a positive feedback loop: under transpiring conditions this could cause any
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small local decrease in water status to be amplified by acceleration of transpiration over the entire plant. Compensation for such effects occurs partly through change in leaf temperature, which automatically follows change in transpiration rate (Cowan, 1977), but it may also require active regulation of stomatal aperture. This suggests that ion-pumps and channels at the guard cell membrane could be very sensitive to change in leaf water status or guard cell turgor pressure. Many (but by no means all) transient or oscillating stomatal responses t o environmental change can be explained in terms of interactions between hydropassive and hydroactive stomatal movement (Cowan, 1977). Effects of turgor on cell growth rate. The commonly used model of Lockhart envisages a simple relationship between the extension rate of a cell and its turgor pressure (Lockhart, 1965; Cosgrove, 1986, 1993): dV Vdt
-=
0 ( P - Y)
where dV/Vdt is relative growth rate, 0 is cell wall extensibility, P is turgor pressure, and Y is wall yield threshold. Some recent models postulate lesser dependence on turgor (Zhu and Boyer, 1992), but all incorporate at least a threshold requirement. A decrease in the availability of water in the soil would therefore be expected rapidly to inhibit shoot growth rate, by reducing xylem pressure and cell turgor throughout the plant. Such effects are observed; treatments that cause a decline in root (or xylem) water status usually inhibit growth rate. The kinetics of growth inhibition in these cases are often very fast (Serpe and Matthews, 1992; Malone, 1993a; Chazen and Neumann, 1994; Stahlberg and Cosgrove, 1995); much faster than those that occur for transverse leaf shrinkage under the same treatment (Malone, 1993a). The faster effect on growth rate, compared to leaf swelling, may arise because: 1 . Cells that determine elongation rate (in grass leaves at least) may be immediately adjacent to the xylem and should thus respond more quickly to changing xylem pressure than the bulk of cells in the leaf (which determine transverse shrinkage). 2. Elastic changes in cell length will occur during change in turgor pressure, causing large but transient changes in apparent growth rate. The turgor change will act on the inherent elasticity in each cell (Cosgrove, 1981) and it may also be amplified through elastic interactions between the different tissues of the growing organ. Similar elastic interactions are manifested as “tissue tensions” in growing organs (Kutschera, 1989). 3. Only a small decline in turgor may be necessary to lower turgor to the yield threshold, and thus to stop growth (growth rate will be zero when P 5 Y , according to Lockhart’s equation, above; Boyer, 1985).
An increasing number of workers now argue that root-shoot hydraulic
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signals are not sufficient to account for shoot responses to soil conditions (Cowan, 1977; Passioura, 1988; Davies and Zhang, 1991; Munns, 1993). Many of these workers favour additional control by root-sourced chemical signals (section IID). However, while various additional factors will undoubtedly play a role in the longer term, the limited number of high-resolution measurements currently available often indicate a close correlation between purely hydraulic effects, and shoot growth responses in the short and medium term (Malone, 1993a; Pardossi etal., 1994; Alarcon and Malone, 1995). This indicates that chemical signals may not be necessary for such communication. Maturing fruit often show relatively long half-times for equilibration of water status with the rest of the plant. In these, hydraulic capacity may be large and xylem connections limited, and the majority of water influx may arrive via the phloem rather than the xylem (Ho etal., 1987). Since phloem translocation is thought to involve pressurized flow, it is possible that phloemborne hydraulic signals might become significant in large fruits. However, because of the higher sap viscosity, lower tube diameters, and internal membranes and constrictions, phloem-borne hydraulic signals are likely to be much weaker than those in xylem. Some plants have extensive tubular systems in addition to the phloem and xylem. Species such as Hevea have a systemic laticifer network. Mimosa pudica has a near-systemic network of large tube cells ("Schlauchzellen", Haberlandt, 1914) also termed "secretory cells" (Esau, 1970). Similar structures are present in many non-sensitive Leguminoseae. In Mimosa pudica there is also a large perivascular sheath of sclerenchyma that appears to consist of open tubes. In principle, any of these open tubular systems could also conduct rapid hydraulic signals. D.
HYDRAULIC DISPERSAL SIGNALS
Hydraulic dispersal refers to transmission of chemical signals by mass flow in the xylem. The xylem seems largely free of transverse membranes, and it offers potentially rapid distribution of solutes over long distances. Many inorganic nutrients, notably calcium, are believed to be delivered to leaves primarily in this way (Canny, 1993). However, for rapid signalling, chemical transmission in the xylem has a number of limitations: 1. Mass flow in xylem is generally unidirectional. Unlike hydraulic pressure signals, therefore, hydraulic disperal signals can usually contribute only to signalling from root to shoot. 2 . If hydrophobic barriers surround the xylem in the leaf (see section IIC), solutes may be trapped within the xylem by ultrafiltration (Canny, 1990) and may not reach the living tissue. Unless these peripheral ultrafiltering membranes are patchy or leaky, specific receptors may be necessary for transmission of chemical signals beyond the xylem.
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3. Xylem flow rate is unpredictable and will vary enormously with weather
conditions. Signal chemicals synthesized at a particular rate by some basal transmitting tissue will thus arrive in the xylem at a concentration that varies with the prevailing flow rate. The response of receiving tissues in the shoot could not, therefore, be based on the immediate concentration of a signal in the incoming stream. Receiving tissues would have to interpret the signal further, perhaps by integrating it over an extended period, or by somehow distinguishing its true delivery rate from its concentration (Gowing et al., 1993; Jackson, 1993). In principle, the system could operate if the transmitting tissue produced a chemically similar, but nonmodulated, reference signal in addition to the modulated signal. The ratio of signal: reference would be conserved regardless of the flow rate, and could be read off in the receiver tissue. A related problem is the “residence time” of solutes in the xylem. During periods of reduced transpiration, such as at night, chemical signals produced in the roots might not be conveyed to the leaves for many hours. Conversely, at other times the velocity of flow in the xylem might greatly exceed 1 mm s-I (Zimmermann and Brown, 1971; Nobel, 1991). Values of up to 200 mm s-’ are reported for some herbaceous plants (Zimmermann, 1983, p. 65) and, in young wheat plants, Passioura (1972) estimated a mean xylem-flow velocity of 250 mm s-I during the daytime in the basal part of the root. Excision of two of the three seminal roots from these wheat plants increased this velocity to some 800mm s-I in the remaining root. Peak flow rates would presumably have been even greater. A further consideration concerning solute movement is that during laminar flow, the pattern of velocities across the tube forms a parabola: the flow rate tends to zero at the periphery of the tube, while at the centre of the tube it tends to a peak velocity of twice the mean (Zimmermann, 1983). Thus, a compact pulse of solute added to one end of a tube will emerge from the other as an extended pulse, with a long tail. Interchange of fluid between the various vessels in a plant stem, which have a range of radii and correspondingly larger (squared) range of flow velocities, will further spread the solute elution profile, as could chemical interactions between the solute and the walls of the vessels. In view of such incoherent and unpredictable transport kinetics, it is difficult to envisage a root-shoot signalling system for close control (e.g. of whole-shoot stomatal aperture) which could operate efficiently using xylem-borne chemicals. Evidence from xylem anatomy and phyllotaxis, and from observation on the movement of tracers (Zimmermann and Brown, 1971; Jones and Lamboll, 1980; Zimmermann, 1983) suggests that any one region of the root (or shoot) may be preferentially coupled, by the xylem, to particular regions of the shoot. This could be problematic for root-shoot signalling by hydraulic dispersal. For example, signals for stomatal closure released from root tips in a partially dry soil (Davies and Zhang, 1991) might reach only certain leaves or parts of leaves, or they might reach some leaves long before others. The extent of this
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problem is unknown. In a number of herbaceous plants, localized treatments can induce systemic swelling of leaves (Fig. 4; section IIC) and the kinetics of this swelling are similar in most leaves throughout the shoot (see Fig. 7). This suggests that, if the xylem is separated into discrete sectors, there can be no significant barrier to pressure-driven flow between the sectors. During normal transpiration, however, the various parallel sectors could all be at similar pressure and there might then be virtually no interchange of water or solutes between them. Sectorial patterns might also develop or change with the age of the plant. This area merits closer study. Movement of chemicals with the transpiration stream will almost always be solely acropetal. Under some conditions, however, flow directions in the xylem can be disturbed and even transiently reversed. For example, reversal occurs if water is supplied artificially to the xylem at some point on the shoot (Bennett et al., 1984; Van de Pol and Marcelis, 1988). Transient reversal may also occur naturally, where the normal diurnal fluctuation in xylem water pressure meets tissues of large hydraulic capacitance and low transpiration rate, such as some fruit, or the heart leaves of lettuce (Malone, 1993b). Reversal of xylem flow also occurs on wounding (see below).
I.
Wound-induced hydraulic dispersal
A special situation develops around sites of localized wounding: hydraulic dispersal will occur rapidly and in all directions from such sites. This was first suggested by Ricca (1916) in connection with his experiments on Mimosa spegazzinii (Malone, 1994). This phenomenon occurs because water is released into the apoplast from damaged cells at the wound site, and it then becomes available at atmospheric pressure to the xylem. If the xylem is under tension, it will draw this water in and local xylem tension will be relieved. Local relief of xylem tension will spread rapidly, as an hydraulic pressure wave, throughout the xylem. Tissues over the entire plant tend toward a dynamic hydraulic equilibrium with the xylem (section IIC). Therefore, when xylem pressure suddenly rises systemically, after wounding, all the tissues of the shoot will begin to absorb water more rapidly from their nearest xylem. In “upstream” root tissues (those involved in water uptake from the soil), the cells will begin to lose water less rapidly to the xylem or, if the change in xylem pressure is sufficiently large, they may begin to absorb water from the xylem. Turgor pressure in all tissues will increase (Malone and StankoviC, 1991) and they will begin to swell (see Fig. 4; Malone, 1992; Boari and Malone, 1993). Swelling of the unwounded leaves requires a significant mass flow of water from the xylem (section IIC). This will tend to depress xylem pressure again, and cause more water to be drawn into the xylem at the wound site. Thus, mass flow will be pulled away from the wound site, through the xylem, and it will radiate towards all other parts of the plant. This wound-induced mass flow will continue until all the water released at the wound site is exhausted, or until tissues throughout the plant become saturated with water.
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The fluid released from damaged leaf cells is not pure water. It is mostly vacuolar sap, and contains considerable amounts of solutes including ions (chiefly K + but also Ca2+ and others; Malone and Tomos, 1992) as well as various organics. Mass flow from the wound site will convey all these solutes through the xylem. Given this situation, wound signalling requires merely that some soluble elicitor is present in the sap released from damaged cells, or is generated at the wound site from damaged cell constituents, or from interactions with the cell wall. Any such elicitor will be automatically conveyed, by the wound-induced mass flow (=hydraulic dispersal), from a wound site. Various such elicitors have been identified in tomato, including ‘‘systemin” (section IIIA). Systemic swelling can also occur by mechanisms other than wound-induced mass flow; for example, if transpiration were to decrease suddenly. However, this cannot explain the swelling that results from wounding because: 1 . Wound-induced swelling is faster than can be explained by water uptake across the root (Fig. 5 ) . This is because flows induced by wounding within the shoot do not have to traverse the large hydraulic resistance of the root endodermis. Thus, even if transpiration were to cease immediately and completely, the resultant swelling would not be as fast as that observed after wounding. 2. In shoots enclosed in polythene bags, and with their roots in mannitol solution, systemic wound-induced swelling can be induced as normal, even though transpiration rate remains negligible throughout (Malone and Alarcon, 1995). 3. Wound-induced swelling can occur even in excised shoots with no access to external water (Malone, 1992). Decreased transpiration could not account for actual swelling in this material; it could only slow the rate of shrinkage.
Excised plant organs placed in air normally contract rapidly as water is lost. However, internal redistribution of water within such organs can lead to transient localized expansion. This occurs because of internal pressure gradients generated by localized cell-wall relaxation and growth (Matyssek et al., 1991) or because of osmotic gradients generated by active redistribution of solutes, followed by passive redistribution of water (Weisz et al., 1989). In both these cases, the localized swelling is much slower (hours) than wound-induced swelling. The occurrence of rapid wound-induced swelling therefore provides a convenient diagnostic test for wound-induced hydraulic dispersal. One point needs to be emphasized: as described above, localized wounding destroys cell membranes and releases vacuolar water; it thus reverses local pressure gradients between tissue and xylem, and then within the xylem. It follows automatically that, basipetal to the wound site, the direction of xylem flow will be transiently reversed. Some workers have been reluctant to accept this point (e.g. Wildon etal., 1992) but it is supported by overwhelming evidence:
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seedling 1
Fig. 5 . Kinetics of wheat-leaf swelling after remote wounding and after rehydration of the root. Results from two seedlings run simultaneously are shown. A displacement transducer was placed on one leaf of each seedling. The steady-state leaf thickness obtained after substitution of the hydroponic root medium by 5-bar mannitol solution was first determined for each seedling (not shown). This serves as a reference point. The root medium was then changed back to water, and leaf thickness allowed to recover (not shown). Root rehydration (thinner lines): the roots were lifted clear of their hydroponic medium, and leaf thickness then began to decline because of water loss by transpiration. Severe dehydration of the roots was prevented by enclosing them in polythene bags. When leaf thickness had declined to the same level as previously reached in 5-bar mannitol, the roots were replaced in water (at the time indicated by the vertical line). The curves show recovery in leaf thickness with time after root rehydration. Wounding (thicker lines): the roots were returned to 5-bar mannitol solution (not shown). When leaf thickness had once again reached a steady level, a neighbouring leaf was wounded by scorching for 3 s with a flame (at the time indicated by the vertical line). These curves show the wound-induced swelling of leaves, with time. The thicker lines were taken subsequently from the same seedlings as the thinner lines. The transducers were not moved throughout, and the curves are therefore directly comparable. The absolute changes in leaf thickness were similar for both treatments, but the curves are normalized (to same total change over 0-7 min of treatment) to facilitate comparison of kinetics. The manipulations with mannitol ensured that both the wound-induced, and rehydration-induced swellings begin from approximately the same leaf water potential (thickness). It is evident that leaf swelling in response to wounding is faster than that in response t o rehydration of the root. This indicates that wound-induced leaf swelling is faster than can be accounted for by water influx across the root.
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1. Various tracers can be added to wound-induced mass flows, or to similar mass flows induced by submerged excision (see Fig. 9). These tracers are found to travel rapidly (c. 10mm s-I) through the plant, including in the direction basipetal to the wound site. Tracers that have been used in this context include heat (Houwink, 1939, and 14C-labelledcompounds such as Rhamnogalacturonan I (a pectic polysaccharide with PI-elicitor activity in tomato), adenine, sucrose and ABA (Malone et al., 1994a). In several cases the movement of these tracers has been shown to be unaffected by steam-girdling (Malone, 1993b). This demonstrates that they are xylemborne rather than phloem-borne. Further confirmation that basipetal xylem flow can occur comes from the classic experiment in which a pin is pushed into a transpiring herbaceous plant, through a droplet of dye on the surface of the stem: some of the dye is "sucked" almost instantaneously into the stem and it moves axially in both acropetal and basipetal directions (Simon etal., 1975, p. 189). The situation here involves local application of water at atmospheric pressure, to the xylem, and it is very much like that which occurs on wounding. 2. The rapid systemic increase in turgor pressure (Malone and StankoviC, 1991) and leaf thickness (Malone, 1992; Boari and Malone, 1993) that follows localized wounding, even in shoots with no access to water, can be explained only by mass flow of water from the wound site. Since the first stage of the flow is down the petiole of the wounded leaf, the initial mass flow, at least, must be mainly basipetal. 2. The kinetics of wound-induced h y d r d i c dispersal Velocity of transmission. The xylem itself presents a relatively low resistance to axial flow, and physiological pressure gradients can therefore induce very rapid flow in xylem. For example, if a pressure difference of 6 bar is applied to xylem vessels 25 pm diameter in a 40-mm petiole, the Hagen-Poiseuille equation predicts a mean flow velocity of about 300 mm s-' (Malone, 1994). Such rates will not normally be sustained after wounding in planta because radial flux of water from the xylem will quickly become limiting (but see section IID for examples of high flow rates sustained by transpiration). In the absence of data on xylem hydraulic capacity, it is difficult to model the kinetics of wound-induced flow. However, the velocity of this mass flow can be estimated experimentally. This was done in the petiole of wounded tomato leaves using three different methods (Malone, 1993b). For all three methods, the average flow velocity over the first 1 min after wounding was found to be about 8 m m s-I. The initial velocity could be much faster. Recently, the initial velocity of basipetal mass flow (close to a wound site) was measured directly using displacement transducers. In both tomato (see Fig. 10, section IIID) and Mimosa (see Fig. 13, section IIIB) this initial velocity exceeded the measurement resolution of 15-30 mm s-'.
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Extent of transmission. Leaf lamina are often only a few hundred micrometres thick. In absolute terms, therefore, the quantity of fluid released at wound sites on leaves is small (up to 0 . 3 ~ 1mm-2 wound area, for a leaf 300pm thick). However, the cross-sectional area of the xylem is also very small, and small volumes of water can thus displace xylem contents over substantial distances (see below). For example, consider a long, linear leaf such as that of maize. If we assume that the xylem in this leaf occupies 1% of the cross-sectional area of the leaf (excluding air space) then, assuming uniform flow across all the xylem, it follows that a heat-wound that kills a 1-mm wide strip across the leaf will release enough fluid to displace the xylem contents through a length of 100 mm of similar leaf tissue. This is not a particularly impressive distance, considering that a wound which killed an entire 1-mm strip of leaf was used. In the wounded plant, however, two additional factors operate that amplify the extent of this mass flow. First, the total area of the xylem in transverse section (TS) is virtually irrelevant for flow patterns: the r4 term in Hagen-Poiseuille dictates that the few largest vessels will carry the vast majority of any flow (Zimmermann, 1983). Thus, because most of the flow is confined to the few larger vessels, it must displace xylem contents through a much greater maximum distance for the same volume flux. Taking the above model leaf again, the few largest vessels might constitute only 20% of the total TS area of the xylem, giving a mass flow distance of up to 500 mm, rather than 100mm, for the same wound. The distribution of vessel radii in tomato petiole was measured using scanning electron microscopy (Fig. 6). In the example petiole described in Malone (1993b) the single largest vessel (of 26 in total), had a radius of 14pm, and carried over 13% of any flow through the petiole. This vessel occupied only about 0.06% of the total TS area of the petiole, and 8% of the total TS area of the xylem. A wound that damaged 1 mm2 on a subtending leaflet would therefore release sufficient fluid (0.3~1, above) to displace the contents of this vessel through 63mm ( = 63312pm = 13% x 0.3 x 109pm3/(?rx 142pm2)). The second source of amplification of wound-induced mass flow arises from the osmotic pressure of vacuolar sap. As discussed above, sap released from damaged cells enters the xylem and begins to flow away from the wound site. Healthy cells surrounding the xylem immediately basipetal to the wound site are therefore suddenly exposed to xylem sap of high osmotic pressure. These healthy cells therefore begin to lose water to the xylem, down the new osmotic gradient (despite the fact that xylem tension is reduced). This increases the volume of water available for mass flow and thus increases the extent of that mass flow. The extra water flowing from these healthy cells progressively dilutes the sap originating from the wound site, and so this effect diminishes with distance. This effect can be followed experimentally because it causes tissue close to the wound site to decrease in thickness (see Figs 10, 13), while more distant tissues increase. At a certain distance from the wound site the tissue initially increases in thickness, as xylem tension is released, but it then
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Fig. 6. Xylem vessel in a tomato petiole seen in transverse section under the scanning electron microscope. The section was from a position subtending the terminal leaflet of leaf three of a plant with four expanded leaves. The white vertical lines are part of a gauging tool, from which vessel diameter can be estimated. The distance between these lines in the example shown is 24.3 pm. The specimen was excised using a fresh razor blade, rinsed in water, then freeze-dried, shadowed with gold, and mounted.
changes sharply and begins to decrease in thickness (leaflet nearest the wound site in Fig. 7, marked *; Malone, 1993b). The onset of the decrease indicates the time at which solutes from the wound site arrive in that tissue and begin to exert their osmotic effect. It can thus be used to estimate the rate of solute flow from a wound site (Boari and Malone, 1993; see section IIIA). Because of these t w o amplifying mechanisms, even small wounds can generate sufficient fluid for hydraulic dispersal through considerable distances. In tomato leaves, for example, wounds imposed by the feeding of an individual Spodoptera larva can produce sufficient mass flow for hydraulic dispersal over a distance of 270 to 2700mm (Alarcon and Malone, 1994). As described above, hydraulic dispersal requires xylem tension. Its kinetics will therefore vary with the water status of the plant. This provides a useful diagnostic test for the involvement of hydraulic dispersal in physiological signalling: in the extreme case, when the plant is near-saturated with water there will be negligible xylem tension and no hydraulic dispersal (Malone and Alarcon, 1995). This situation obtains in a well-watered plant enclosed at high humidity, in which transpiration is minimal. Under these conditions, wound
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If4 If5
If3
Fig. 7. Systemic wound-induced increases in leaf thickness in tomato. The lines show simultaneous transducer recordings of leaf thickness, with time, in various leaves of an individual tomato plant. Leaf numbers are shown (right; the oldest leaf would be number 1). Two blank transducers (B), which did not contain leaves, were also run. At the time indicated by the long vertical line, one leaflet of leaf five was scorched for 3 s using a cigarette lighter. The transducer nearest to the wound site is marked with an asterisk. From Malone (1993b).
signalling in tomato can be eliminated (example in section IIIA). If signalling is not eliminated under these circumstances, then hydraulic mechanisms can normally be ruled out (but see section IIIB). Wound-induced mass flow is transient. It ceases when there is no further water available at the wound site. However, once soluble elicitors have been delivered into the xylem, they will continue to be carried by the transpiration stream long after the wound-induced mass flow has stopped. There will therefore be a second more prolonged and slower phase of hydraulic dispersal corresponding to flow with the transpiration stream. This will be in the acropetal direction only, and it will continue until all the solutes released from the wound site have been swept from the xylem into the leaves. Completion of this second phase may take up to an hour in tomato (see Fig. 10) and Mimosa (see Fig. 14; Malone, 1994). Most work on wound-induced hydraulic dispersal has involved localized
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wounding by heat. This procedure is very convenient for experimental use, and it releases large amounts of water at the treatment site, but it probably bears little relation to wounding in the natural environment. More realistic wounding can be achieved by mechanical damage or by insect feeding. These treatments also induce mass flows which, although small relative to those caused by scorching, are sufficient for long-distance hydraulic dispersal (Alarcon and Malone, 1994; Malone etal., 1994b). It is also conceivable that some nonwound stimuli generate limited basipetal hydraulic dispersal. This possibility is discussed in section IVB.
E. ELECTRICAL SIGNALS
It has been known for a century that brief electrical transients, usually termed “action potentials” (AP), precede or accompany rapid movement in the motor tissues of certain plants. Burdon-Sanderson, with encouragement from Charles Darwin (see Williams and Pickard, 1980), recorded electrical transients from the surface of the trap lobes of Dionaea muscipula (Venus flytrap) during rapid trap closure (Burdon-Sanderson, 1873; Burdon-Sanderson and Page, 1876). Such electrical events reflect transmembrane ion fluxes, and they can be monitored using either intracellular or extracellular electrodes. A close correspondence is usually found between the patterns with these two types of electrode (Frachisse-Stoilskovic and Julien, 1993; Due, 1993). Extracellular electrodes cost very little. For example, the six-electrode recording system described by Malone and StankoviC (1991) cost about f 150 to build, including power supplies, “Faraday” cage, and amplifiers, but excluding the PC and A-D convertor card. Extracellular electrodes are simple to use, but they are typically quite large and will measure a net response from many individual cells. Detailed studies of electrical events, including some intracellular measurements, have been carried out on several mechanically-stimulated sensitive plants, including Dionaea (Stuhlman and Darden, 1950; Benolken and Jacobsen, 1970; Sibaoka, 1980; Iijima and Hagiwara, 1987; Hodick and Severs, 1988), Aldrovanda (Iijima and Sibaoka, 1981, 1982), Drosera (Williams and Pickard, 1972a,b, 1980; Williams and Spanswick, 1976), and Mimosa (Sibaoka, 1962, 1980; Abe and Oda, 1976; Samejima and Sibaoka, 1983; Stoeckel and Takeda, 1993). It is widely believed that some electrical transients in sensitive plants are self-propagating action potentials akin to those of animal nervous systems, which permit rapid signalling and co-ordination over substantial distances (Sibaoka, 1966, 1969, 1991 ; Pickard, 1973; Roblin, 1979; Davies, 1987; Thain and Wildon, 1992). Transients recorded from sensitive plants often resemble A P from animal nerves, except that the plant A P is slower by a factor of about l o 3 (Burdon-Sanderson and Page, 1876; Sibaoka, 1991).
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Electrical activity is also found to spread rapidly from sites of localized wounding in most non-sensitive herbaceous plants, including laboratory favourites such as Phaseolus, Helianthus, Vicia, Pisum and Lycopersicon (Pickard, 1973; Van Sambeek and Pickard, 1976; Roblin and Bonnemain, 1985; Boari and Malone, 1993). Systemic electrical activity is especially pronounced following localized wounding by heat. However, the pattern of wound-induced electrical events in these herbaceous plants is more variable and prolonged than the single spikes or spike trains seen in mechanicallytriggered sensitive plants. An example is shown in Fig. 15. These patterns are considered to incorporate at least two distinct electrical phenomena (Pickard, 1973): (i) AP, as described above, and (ii) “variation potential” (VP, sometimes termed the “slow wave”), which is usually more prolonged than the AP . In herbaceous plants, A P are usually said to move with velocity of 1-20mms-’, while in sensitive plants, they move at up to 200mms-’ (Sibaoka, 1969). The pathway of AP is uncertain but intracellular recordings tend to locate most activity in the phloem parenchyma (Samejima and Sibaoka, 1983) or in the phloem sieve tubes (Sinyukhin, cited in Pickard, 1972; Eschrich et al., 1988; Wildon et al. 1995). The phloem cannot be the only location of AP because A P are also reported from the glandular tentacles of Drosera, which have no phloem (Williams, 1976). Many workers envisage a long-distance signalling role for A P in plants (Sibaoka, 1966, 1969, 1991; Pickard, 1973; Roblin, 1979; Davies, 1987; Thain and Wildon, 1992). By contrast, there is widespread agreement that the VP is not a self-propagating signal; rather, it is a local electrical response to the underlying passage of chemical substances (Houwink, 1935; Sibaoka, 1966, 1969; Pickard, 1973). These substances are released from the wound site and they move through the plant in the xylem (by hydraulic dispersal), eliciting electrical changes in any living cells which they contact, especially in those cells adjacent to the xylem. The nature of the chemical substance(s) that elicits the V P is not known. It may be a specific (hormonal) depolarizing substance, and various workers note parallels with Ricca’s factor in Mimosa (Houwink, 1935; Pickard, 1973; Sibaoka, 1991; Malone, 1994) and perhaps with “traumatin” (Zimmermann and Coudron, 1979) or “turgorins” (Schildknecht, 1983; Kallas et al., 1990). Alternatively, the VP may be a purely electrochemical reaction to the sudden appearance in the xylem and surrounding apoplast, of the cocktail of ions released from the wound site. For example, vacuolar sap from cells of the wheat-leaf epidermis contains about 200 mM K’, as well as some 3 0 m ~each of Ca2+ and C1- (Malone and Tomos, 1992). A solution containing these ions, even in diluted form, could well disturb the Nerstian component of the membrane potential in any cells that it contacts. It is often stated that the stimulating chemical(s) responsible for the variation potential moves in the xylem “with the transpiration stream” (Sibaoka, 1969; Hill and Findlay, 1981; Davies, 1987) and some workers have
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commented that the VP do not exhibit the slow unidirectional (acropetal) movement that this should imply (Hill and Findlay, 1981). In fact, of course, when the stimulating substance enters the xylem at a wound site, it will move by a process of hydraulic dispersal. As discussed above (section IIDl), woundinduced hydraulic dispersal can transport material in both acropetal and basipetal directions, and at rates considerably faster than the normal transpiration stream. The onset of the VP, at successive distances from the wound site, will therefore provide a further indication of the movement of solutes by wound-induced hydraulic dispersal. The topic of plant electrical signals is examined in more detail in section IV, and an alternative interpretation of some plant electrical phenomena is there discussed.
111. RAPID, LONG-DISTANCE SIGNALLING IN PLANTS: CASE STUDIES A. SYSTEMIC INDUCTION OF PI BY LOCALIZED WOUNDING IN TOMATO
During a biochemical study of endogenous PI in solanaceous plants, Ryan and coworkers noticed large and seemingly erratic variations in the levels of certain PI in tomato leaves. This led them to the discovery that damage to one leaf of a tomato plant can trigger accumulation of PI in other leaves (Green and Ryan, 1972, 1973). PI accumulation was later shown to involve woundinduced genes (Graham eta/., 1986). Because PI interfere with insects’ digestive enzymes, and are toxic to insects (Broadway eta/., 1986; Hilder eta/., 1987; Orozco-Cardenas eta/., 1993), wound-inducible PI is thought to be part of a systemic defence system against leaf-eating insects and possibly other pests (Bowles, 1992; Ryan, 1992). PI may also render the wounded tomato plant less attractive to insects (Edwards eta/., 1985). Similar systemic woundinducible PI occurs in potato shoots and in several other species (listed in section IC). The induction of PI in tomato and potato may not be truly systemic. For example, there is little or no induction in most root tissues (Stanford eta/., 1990; Narvaez-Vasquez eta/., 1993), nor within parts of the stem and petiole (Peiia-Cortes eta/., 1988; Keil eta/., 1989; Stanford et al., 1990). However, PI accumulation can occur throughout all the leaves (at least in young plants, see below) including those above and below the wound site (Peiia-Cortes eta/., 1988). Clearly, a signal must pass throughout the shoot from the wounded leaf. This signal is known as the proteinase-inhibitor inducing factor, or PIIF (Ryan, 1974). The velocity of this signal has been estimated by various means: Green and Ryan (1973), and Ryan (1974, 1977) excised the damaged leaf at various times after wounding, and found that the signal exited the wounded leaf with a half time of 40min or less. Wildon eta/. (1992) found that some
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of the wound signal exited from damaged cotyledons of tomato within 5 min. Increased PI activity can be detected in remote leaves within as little as 1 h after localized wounding in tomato (Malone et al., 1994b). Peiia-Cortes et al. (1988) showed that the mRNA for PI became detectable systemically within 20min of localized wounding in potato. The similar mRNA in tomato may become detectable in remote leaves within 15 min of wounding (StankoviC, pers. comm.) Even allowing zero time for signal generation, reception and transduction, these data indicate that the velocity of the wound signal can exceed 0.2 mm s-’. Each of the five mechanisms of signal transmission discussed in section I1 has been considered as a possible explanation for wound signalling in tomato: 1 . Volatile methyl jasmonate can be released from plant tissue, perhaps especially if wounded (Albrecht et al., 1993), and it can induce PI in tomato leaves (Farmer and Ryan, 1990, 1992; Bolter, 1993). Ethylene might also modulate PI induction (Weiss and Bevan, 1991). However, under most conditions involvement of these airborne signals in systemic wound induction can be discounted because wounding of a leaf on one plant does not induce PI in an adjacent plant, even if the two plants are held very close together (noted in Malone etal., 1994b); to be effective, the wound must be on the same plant. 2. Phloem transmission of elicitor chemicals is believed by many workers to be the mechanism of long-distance wound signalling in tomato (Ryan, 1992; Orozco-Cardenas etal., 1993). This is partly by default: many workers have imagined that the phloem is the only system to offer rapid bidirectional transport in plants (e.g. Weiss and Bevan, 1991; Rigby etal., 1994; but see section IIDl). However, transport of chemicals in the phloem would be consistent with the observed association of wound signals with the vascular system (Ryan, 1974; Keil etal., 1989; Stanford et al., 1990). Recent work has also demonstrated inhibition of systemic PI induction by p-chloromercuribenzene sulfonic acid (PCMBS), a known inhibitor of phloem translocation (Narvaez-Vasquez et al., 1994). Various endogenous chemicals are capable of inducing PI in unwounded tissue. These substances are known as PIIF (Ryan, 1992). There is evidence that the most active of these chemicals (“systemin”) can move in the phloem (Pearce et al., 1991; Narvaez-Vasquez et d.,1995). Some others are virtually immobile in phloem (Baydoun and Fry, 1985). Nelson et al. (1983) reported that steam girdling (which kills phloem) prevents passage of the wound signal in tomato. Conversely, other workers report that blockage of phloem transport by steam girdling (Fig. 8, Malone and Alarcon, 1995) or by chilling (Wildon etal., 1992) does not impair wound signalling in tomato. The phloem is notoriously sensitive to damage (Oparka, 1990) and, in the immediate vicinity of any wound site, it would presumably be disabled for a considerable period of time. The positive pressure of phloem
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sap would also tend to drive any flow towards the damaged sieve tubes at the wound site. This would not encourage rapid wound signalling through the phloem. 3. Hydraulic pressure signals are transmitted throughout the tomato plant from sites of localized wounding (evident as systemic leaf swelling in Fig. 7; Malone, 1993b). Systemic pressure signals can be mimicked without significant wounding, by excision through a petiole submerged in water (Fig. 9). However, this treatment does not induce PI in remote leaves and pressure changes have therefore been discounted as wound signals in tomato (Malone et al., 1994b). 4. Electrical events appear throughout the tomato plant soon after localized wounding and it has been proposed that they are travelling wound signals (Pickard, 1973; Wildon etal., 1989). Wildon e t a / . (1992) showed that neither electrical events nor the wound signal were stopped by a chilled region. However, there are two problems with the electrical theory. First, it is not clear whether the electrical events are self-perpetuating signals, or merely local responses to chemical signals (see section IVB). Second, there is no evidence that electrical events will induce PI; for example, electrical transients are also induced in tomato leaves by changes in lighting (Cheeseman and Pickard, 1977), but these do not induce PI. Much of the wound-induced electrical activity reported from wounded tomato plants looks more like variation potentials than action potentials (e.g. Fig. 1 in Wildon e t a / . , 1989; Fig. 15, and see section IVB). This is important because variation potentials are not self-propagating signals; they are local responses to chemicals travelling in the xylem (section HE). Variation potentials cannot therefore be long-distance wound signals in tomato. Wounded tomato plants often exhibit spike-like electrical events that resemble action potentials (Wildon et al., 1989; see Fig. 15) but again, it is questionable whether these events propagate through the plant (see section IVB). Finally, any involvement of self-propagating electrical signals in the tomato-PI system appears to be eliminated by the finding that wound signals readily traverse extensive heat-killed zones of the tomato petiole (Fig. 8; Malone and Alarcon, 1995). Electrical signals would not be able to traverse such zones because they require live membranes along which to propagate. 5 . The involvement of hydraulic dispersal in wound signalling in tomato was suggested by Malone and StankoviC (1991). Localized heat wounds cause rapid and systemic swelling of the tomato shoot (Fig. 7). This indicates that wounding triggers large and systemic mass flows from the damaged region in tomato. Quantitatively similar mass flows, induced by submerged excision, have been shown to be capable of conveying various solutes rapidly throughout the plant (Malone et al., 1994a). It follows that solutes present in the sap released from damaged cells will also be carried throughout the
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plant after localized wounding. Various chemicals with PIIF-activity, including “systemin” which is active at extremely low levels, are known to be among the solutes present in tomato cell sap (McGurl et al., 1992; Ryan, 1992). These will be carried rapidly away from wound sites along with the wound-induced mass flow. Various lines of evidence indicate that hydraulic dispersal occurs rapidly, and over long distances in the xylem of the tomato plant (section IID1). The fastest flow rates occur basipetal to the wound site, along the petiole of the wounded leaf. From the pattern of decreasing thickness in tissue close to the wound site (Fig. 10; see section IID2), solutes evidently traverse
n
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I
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10
I
I
I
15
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1
1
.-> CI
0
5 0.5 L
v
.-E
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time from wounding (h) Fig. 8. Wound signals pass through the heat-killed petiole of tomato. One day prior to the experiment, a zone of the petiole of leaf two was heat-killed (closed symbols), or not (open symbols). At time zero, the terminal leaflet of leaf two was wounded (triangles), or not (circles) by scorching for 3 s with a flame. At various times thereafter, the terminal leaflets of leaf three were harvested for PI assay. Each symbol shows mean &SE ( n = 10). These data show that passage of the wound signal is not blocked at heat-killed petioles. From Malone and Alarcon (1995).
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subrnerged excision 1
2,
scorch wound
Fig.9. Systemic swelling induced by wounding, and by submerged excision in tomato. Plants had three fully-expanded leaves. A displacement transducer was placed on the terminal leaflet of leaf three. The curves show change in leaf thickness with time, and each is a mean for six plants run simultaneously. At the time indicated by the arrow (upper trace only) one leaflet of leaf two was excised in air. At the time indicated by the vertical line, one leaflet of leaf two was wounded by scorching for 3 s with a match flame (lower curve) or was excised by a clean cut through its submerged petiole (upper curve). The two curves were taken successively from the same plants, without moving the transducers, and they are therefore directly comparable. Several hours were allowed for recovery between wounding treatments (not shown) and the curves are offset arbitrarily for clarity. From Malone etal. (1994b).
this stage at rates of at least 15-30mms-' in tomato, as in Mimosa (Malone, 1994; section IIIB, see Fig. 13). Eventually, after 30-60min, the returning transpiration stream sweeps any remaining solutes from the xylem of the wounded leaflet (Fig. 10). Wounding of less than 1% of a single leaflet has been estimated to generate sufficient mass flow for quasi-systemic signalling in tomato (Malone, 1993b; section IID2). This calculation is based on the volume of sap required to displace the contents of the major xylem vessels in the petiole of the wounded leaf, as far as their junction with the stem. From that point, even without further wound-induced mass flow, xylem-borne dispersal will occur with the normal transpiration stream, at least in the acropetal direction. Even wounds imposed by individual leaf-eating insects release sufficient sap to generate long-distance hydraulic dispersal in tomato (Alarcon and Malone, 1994). Significant hydraulic dispersal will occur only if there is a prevailing tension in the xylem of the shoot. If the plant is well-watered and enclosed in a polythene bag to minimize transpiration, there will be negligible xylem
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Fig. 10. Kinetics of change in leaf thickness close to a wound site in tomato. Four displacement transducers were spaced along the terminal leaflet of leaf four of an intact plant, at positions shown in the diagram (left). The plant had five expanded leaves in total, the oldest and lowest is designated leaf one. An area near the tip of the leaflet bearing the transducers (as indicated on the diagram) was wounded by scorching for 2 s with a flame, at the time indicated by the vertical line. The curves (centre and right) indicate changing leaf thickness at each transducer position, normalized so that the total change by 7 min from wounding is the same at each position. The two sets of curves (centre, right) are presented at different temporal resolutions (note separate time-scale markers). The central set shows the early wound-induced change at high resolution, while the right-hand set continues the data at a slower resolution. From the central set of curves it is evident that the onset of the decrease in thickness occurs almost simultaneously at all positions along the wounded leaf. This indicates that hydraulic dispersal of solutes (in cell sap) from the wound site occurs at rates of at least 15-30mms-I. The subsequent recoveries in leaf thickness shown in the right hand set of curves indicate the pattern of displacement of cell sap from the xylem underlying each transducer position, by “clean” water in the returning transpiration stream. From the onset of the steep part of these recoveries, at successive positions (indicated by vertical dashed arrows) the rate of this returning transpirational flow can be estimated as: 3. I mm min-’ (T4-+ T3); 3.3 mm min-’ (T3 + T2); 2.6 mm min-’ (T2 Tl). +
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tension and no hydraulic dispersal. Under these conditions, therefore, localized wounding should induce neither systemic swelling nor systemic PI. Both these predictions have been shown to hold true (Malone and Alarcon, 1995). This is very strong evidence in favour of wound signalling by hydraulic dispersal, as is the finding that wound signals pass freely through steam-killed regions of petiole (Fig. 8). Only the xylem remains functional within such regions. Two experiments that are supposedly inconsistent with hydraulic dispersal in the wounded tomato have been mentioned in the literature. First, Wildon et al. (1992) noted that transpiration continued from the wounded leaf, and they argued that this indicated no reversal of flow in xylem in the petiole of the wounded leaf. However, this argument is based on a misconception: transpiration from the leaf lamina can occur simultaneously with basipetal flow in the petiole, if the water for both processes comes from an intermediate position - that is, the wound site. Second, Thain and Wildon (1992) note that wound signalling can still occur in tomato shoots that have been excised and placed with their cut ends in water. They state that the xylem flow should be “very different” in this situation compared to that in the intact plant. The assumption here is that shoots with their cut ends in water should have no xylem tension, and therefore no wound-induced mass flow. Our experiments have confirmed that where xylem tension is eliminated (by enclosure in polythene bags) then wound signalling is also eliminated (Malone and Alarcon, 1995). However, we tested the assumption that shoots placed in water have no xylem tension, and found it to be false: such shoots show systemic swelling in response to wounding, exactly as in the intact plant (Fig. 11). This indicates that excised shoots can exhibit wound-induced hydraulic dispersal in the normal way. Evidently, xylem blockage occurs near the cut end of the excised shoot. Blockage may be associated with damage, active wound healing, or embolism. The block can be removed by excising a few millimetres from the base of cut stem. This causes transient swelling of the excised shoot (Fig. 11) exactly as with submerged excision through the petiole of an intact plant (Fig. 9) but the block soon regenerates. The hydraulic resistance of the (blocked) stem base is thus sufficient to ensure a substantial xylem tension (and therefore wound-induced hydraulic dispersal) in the excised shoot, as long as it is transpiring. Hydraulic dispersal is thus the most likely mechanism of systemic wound signalling in the tomato. The involvement of airborne- and hydraulic pressure signals can be discounted, and it remains to be established whether electrical events are signals in themselves or merely local responses to other travelling signals. The fact that wound signalling is unimpeded by an intervening heatkilled zone strongly favours xylem-borne signals over those requiring live cells - that is, electrical and phloem-borne signals. Note that heat-killing the
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excised shoots
intact plants
blank
1 30 min
Fig. 11. Wound-induced hydraulic signals in intact tomato plants and in excised shoots. Change in leaf thickness with time was measured using displacement transducers placed on the terminal leaflet of leaf three of intact tomato plants (lower traces) or of excised shoots with the base of the stem placed in water (upper traces). All six plants were at the three-leaf stage, and were run simultaneously. At the time indicated by the first vertical lines the soil was watered generously (lower traces, intact plants) or 3 mm was sliced, by a submerged cut, from the stem bases (upper curves, excised shoots). At the second vertical line, one leaflet of leaf two of each plant was wounded by scorching for 3 s with a match flame. Both the intact and the excised shoots showed marked wound-induced swelling.
petiole does not hinder xylem-borne flow, as indicated by the continued occurrence of wound-induced swelling in tissues on either side of such dead petioles (Malone et a(., 1994b). Given that hydraulic dispersal is involved, a general interaction between tomato shoot water relations and wound signalling can be predicted. This may account for the observed effects on PI induction of light, temperature, and C 0 2 (Green and Ryan, 1973; Ryan, 1977); each of these variables will affect transpiration rate (and thus shoot water status), and their effects may therefore be via hydraulic dispersal. The evidence from cold blocks and from steam girdling emphasizes that the
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phloem cannot play a primary role in wound signalling in tomato. Similarly, when the phloem could potentially function in isolation (in wounded plants held at high humidity to eliminate xylem tension) no wound signalling is observed (Malone and Alarcon, 1995). However, there remains a possibility that phloem transport could play some secondary or auxiliary role. For example, some reports indicate that exit of PIIF from wounded leaves can continue for over an hour (Green and Ryan, 1973). This is longer than would be expected for a mechanism involving only hydraulic dispersal. It is conceivable that the phloem can become secondarily involved in wound-signal transmission, by xylem-to-phloem transfer after there has been an initial ingress of PIIF into the xylem. Interchange between the xylem and phloem is reported in several systems (Van Bel, 1984; Minchin and McNaughton, 1987) and it is not inconceivable that it occurs with PIIF chemicals in tomato. This scenario would be compatible with the findings of Narvaez-Vasquez et al. (1995): 15 min after application to a wound site, ‘‘C-labelled systemin was located primarily in the xylem, but by 30min and thereafter, the label was located primarily in the phloem, and it appeared to spread progressively through the phloem over a period of 3 h or more. There are some question marks regarding the ecological significance of the wound-inducible PI system in tomato. Most work on this system has used young plants ( c 6 weeks old) which clearly show systemic induction of PI. In older plants, however, systemic wound induction of PI may be absent (Wolfson and Murdock, 1990). This is not because older leaves cannot accumulate PI; they can, but perhaps only in response to a nearby wound (Fig. 12). It is possible that the larger hydraulic capacity or more complicated hydraulic architecture of older plants dictates that hydraulic dispersal signals will extend only over a relatively limited range from wound sites in such plants. Local wound-induction could still confer a significant defence benefit to the plant.
B.
SIGNAL TRANSMISSION IN MIMOSA
Signalling in the “sensitive plant” Mimosa, especially M. pudica and M . spegazzinii, has attracted attention for centuries. The subject is summarized
Fig. 12. Local, but not systemic induction of proteinase inhibitor (PI) by wounding in large, mature tomato plants. Tomato plants were grown under natural lighting in a large outdoor polythene tunnel. By 42 days these plants were already larger than those normally used in our laboratory experiments, and by the end of the experiment (84 days old) they had over 15 leaves. Leaflets were harvested from the top, middle or bottom of the plant, and their PI level was assayed. Leaflets from the middle and bottom of the plants were harvested after wounding o f an apical leaflet (A “remote wound”). Leaflets from the top, middle and bottom of the plant were also harvested after wounding of an adjacent leaflet ( 0 “local wound”). Controls ( 0 ) were
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unwounded. In each case, 24 h elapsed between wounding and harvest. Leaves from all regions of the plant showed PI-induction in response to a local wound, but a remote (apical) wound did not induce PI in leaves on the middle and bottom of the plant. Such remote wounds induce substantial PI in young plants (Malone etal., 1994b).
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here only briefly. Further consideration can be found in Houwink (1939, Weintraub (1952), Sibaoka (1966), Pickard (1973), Schildknecht (1983), and Malone (1994). Mimosa shows rapid and remote leaf movements in response to wounding. Its responses to non-wound stimuli, such as touch, tend to be limited to the stimulated leaf. Both hydraulic and electrical theories of wound-signalling in Mimosa have been debated for many years (Ricca, 1926; Ball, 1927). For example, Bose (1914) states, rather loftily, that “The question of whether the transmitted effect in Mimosa is due to a hydro-mechanical or excitatory impulse is held to be of much interest in Plant Physiology”. The work of Ricca (1916), and others, provided strong evidence that the wound signal in Mimosa is a chemical messenger which moves in the xylem that is, in the terms used in this review, that wound signalling involves hydraulic dispersal. Ricca argued that the signal must be chemical rather than electrical since it could be isolated and stored in a dry form. The putative chemical has since been referred to as “Ricca’s factor”. Ricca also reported that the signal would pass through sections of stem that were steam-killed, or that had been removed and replaced by a water-filled glass tube. Malone (1994) re-examined Ricca’s ideas and demonstrated that hydraulic dispersal from wound sites in Mimosa would pass in both acropetal and basipetal directions. The initial rate of this hydraulic dispersal was shown to be 15-30 mm s-’ or more (Fig. 13) which is sufficiently rapid to explain signal transmission. The pattern of lag times for responses of leaves progressively further from the wound site is entirely consistent with an initial, brief but rapid bidirectional hydraulic dispersal, succeeded by a prolonged period of solely acropetal movement with the transpiration stream (Fig. 14; these two phases of hydraulic dispersal are described in section IID2). Mimosa shoots contain at least one tubular laticifer-like system. The fluid in this system is under considerable pressure, 15-20 bar or more, even in transpiring shoots (unpublished data); a precise determination of the pressure is difficult because the fluid coagulates spontaneously soon after puncture, and so it blocks up the tip of a pressure probe. This pressurized system ejects copious fluid at wound sites, and so permits extensive hydraulic dispersal even from wound sites which would be hydraulically negligible in plants like the tomato (Malone, 1994). I can find very little foundation for the view of various modern writers that Mimosa represents a classic model of electrical signalling in plants (e,g. Roblin, 1979; Satter, 1989; Tinz-Fiichtmeier and Gradmann, 1990; Thain and Wildon, 1992). Remote electrical events do occur in the wounded Mimosa plant, but they appear to be incidental to wound signalling. They may represent local responses to the underlying passage of Ricca’s factor (Malone, 1994). In the case of the slower signalling that occurs with touch stimuli in Mimosa, Ricca (1926) considered a form of stepwise hydraulic dispersal: when one pair of leaflets move (in response to a direct mechanical perturbation), the motor
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Fig. 13. Kinetics of hydraulic dispersal close t o a wound site in Mimosa. Four transducers (TO-T3, plus a blank) were arranged along one pinna-rachis and petiole of Mimosa in a similar manner t o the arrangement shown for tomato in Fig. 10. At the time indicated by the vertical line, the tip of the same leaf was scorched for 1 s with a flame. The numbers in brackets are the distance, in mm, of each transducer from the wound site. The vertical arrows shown on the upper curve indicate times of application of “blank” wounds. These involved holding a flame near to, but not touching, the leaf tip (From Malone, 1994).
cells in their pulvini collapse. This collapse involves ejection of a small amount of fluid which may be sufficient for hydraulic dispersal through the 1 mm or so of rachis to the next pair of leaflets, thus triggering their collapse with further ejection of fluid, and so on (see section IVB). Note that this mechanism could work even without xylem tension, because positive apoplastic pressures
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0
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time to leaf movement (s) Fig. 14. Response times for leaves above and below a wound site in Mimosa. One leaf in the centre of the plant was wounded by scorching for 3 s with a flame. The lag time to the motor reaction (leaf fall) in the main pulvini of successive leaves above and below the wound site is plotted against their distance from the wound site. There were leaves more than 15 cm below the wound site but they never responded. Data from three trials with the same plant are plotted, with a different symbol for each trial. The pattern indicates a brief initial phase of rapid flow in acropetal and (especially) basipetal directions, succeeded by a prolonged phase of purely acropetal movement (From Malone, 1994).
would be generated by the collapse of successive pulvini (Malone, 1994). Flow at positive pressure could travel through the intercellular space instead of, or as well as, through the xylem.
IV.
IMPLICATIONS AND DIRECTIONS FOR FURTHER RESEARCH
Various aspects of plant signalling that merit closer study have been identified in previous sections. These include the possible role of hydraulic dispersal in
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co-ordination of systemic acquired resistance (SAR, section lC), and the need for further information on sectorial patterns within the xylem (section IID). There is also a need for reappraisal of the nature of electrical signals in higher plants. In addition, recent information on hydraulic signalling may have wider implications, for example, for signalling of non-wound stimuli. These areas are discussed below.
A.
RE-ASSESSMENT OF ELECTRICAL SIGNALS IN THE HIGHER PLANT
The physiological role and even the existence of long-distance electrical signals in higher plants is a vexed question. The area is summarized briefly in section IIE. It is revisited here with some more speculative discussion on the nature of these electrical events. Physiologists have long been reluctant to accept the idea of long-distance electrical signalling in higher plants. Some interesting ideas on the origin of this reluctance have been advanced by advocates of plant electrical signals (Pickard, 1973; Simons, 1981; Davies, 1987; Thain and Wildon, 1992; Wayne, 1994). These ideas include: the desire of nineteenth-century botanists to emphasize differences between plants and animals; the emphasis on plant hormonal messengers that stems from influential work on auxins during the 1930s; doubts among some plant physiologists about whether a plasma membrane actually exists in plant cells and the unhelpful influence of contributions from the metaphysical fringe. Probably a more important reason is that the highly variable nature of some electrical phenomena in higher plants raises doubts about whether they could control anything. In addition, there is the long-standing suspicion that electrical events in plants represent local responses to the underlying passage of chemical signals (Ricca, 1916, 1926; Malone, 1994; Stahlberg and Cosgrove, 1995). Furthermore, the evidence for self-propagating electrical signals in most higher plants is slim and at best, correlative. As an example of the latter, consider the interesting and careful work of Williams and Pickard (1972a) on tentacles of Drosera: electrical events in the stalk were shown to follow stimulation of the head, and precede bending of the stalk. This is certainly a useful correlation. However, the conclusion made in the paper that . . the impulse has been identified with a train of AP" is premature because correlation alone will not necessarily indicate causal relationship; there remains the possibility that both the electrical activity and the bending are reflections of a local response induced by some other travelling signal. There seems no a priori reason why the tissues of higher plants should not be capable of generating, transmitting and responding to action potentials: the cells commonly maintain large transmembrane electric fields (Findlay and Hope, 1976) and the membrane permeability can change abruptly (as in Mimosa; Samejima and Sibaoka, 1982). The absence of nerves from plants
".
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is not a particular problem since various primitive animal tissues can propagate electrical events between ordinary cells (Thain and Wildon, 1982). Also, in many plant tissues, numerous plasmodesmata run between adjacent cells, and these could provide routes of low electrical resistance leading to strong electrical coupling between the cells (Spanswick, 1972; Cheeseman and Pickard, 1977; Iijima and Sibaoka, 1982; Lew, 1994). Moreover, nerve-like action potentials can be generated and transmitted in cells of Nitella and some other algae (Gradmann and Mummert, 1980; Sibaoka and Tabata, 1981; Beilby, 1989; Wayne, 1994). By analogy with the animal nervous system, the plant action potential should begin when a stimulus is perceived in particular receptor cells, it should propagate itself across the tissue without decrement, and it should initiate a response in distant cells (Thain and Wildon, 1992). The evidence for these stages is considered below; first in the sensitive carnivorous plants, then in non-sensitive plants.
I . Sensitive carnivorous plants The most convincing evidence for electrical signals in plants comes from the sensitive carnivores; Dionaea muscipula and Aldrovanda vesiculosa (Drosera to a lesser extent because its movements are rather slower). Utricularia might also fit into this category but the trigger cells are very close to the motor cells of the trapdoor, and there has been little study of its signal transmission (Sydenham and Findlay, 1975). The linkage in Utricularia might be purely mechanical (Lloyd, 1942). No further sensitive carnivores are known, although many other plants use static traps to catch insects (Darwin, 1875). There is evidence from Dionaeae and Aldrovanda of each of the three stages discussed above: Generation of A P. Membrane depolarization perhaps corresponding to a “receptor potential” has been observed during mechanical stimulation of cells in the base of the trigger hairs of Dionaea and Aldrovanda, and in the tentacle head of Drosera (Williams and Packard, 1980). The membranes can also be depolarized artificially by an injected current pulse. When this depolarization exceeds a critical value, a much larger depolarization is triggered: the AP. The AP is presumably caused by a massive transient increase in the permeability of the membrane toward particular ions: Na’ in the animal nerve, C1(and/or Ca2+) in the characeans (Gradmann and Mummert, 1980; Wayne, 1994). The electric currents associated with this strong depolarization may be sufficient to depolarize adjacent membranes beyond their critical threshold. If so, the AP will regenerate itself in neighbouring regions of membrane or in neighbouring cells, and thus become self-propagating at a rate independent of the initial depolarization. This is the basis of the all-or-nothing behaviour characteristic of AP in animal nerves. Propagation of AP. The propagation of AP can be assessed by comparing the
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timing of the electrical transient at successive distances from the site of stimulation. This approach has yielded propagation rates of 50-200 mm s-' across the trap lobes of Dionaea and Aldrovanda (Sibaoka, 1980; Iijima and Sibaoka, 1981), and about 20 mm s-' in the tentacle of Drosera (Williams and Pickard, 1972b).
Initiation of motor activity. There is a strong temporal correlation between occurrence of the A P and induction of movement in the motor tissue. The AP usually precedes movement, indicating that it may be the trigger for movement (Sibaoka, 1980, 1991). These sensitive carnivorous plants thus provide clear evidence of electrical signalling. Even in these, however, an alternative mechanism of signal transmission is conceivable (see section IVB). 2. Non-sensitive plants In non-sensitive plants, the situation is less clear. The most important classes of electrical events in these plants are the AP and the V P (introduced in section IIE). Other classes have been distinguished (Sibaoka, 1969; Bentrup, 1979) but these are probably unimportant in signalling. Obviously, when considering signalling of wound stimuli, it is critical to distinguish between the A P and the VP; the former is potentially a self-perpetuating signal, while the latter is merely a local response to passing solutes. In principal, the two phenomena are distinct (Pickard, 1973): AP should be brief, rapid, fullyreversible spiky events that conform to all-or-none characteristics. VP by contrast, will be (almost) any other electrical disturbance induced (usually) by wounding. In practice, however, it may be difficult to distinguish between these two phenomena. They often occur together and may be superimposed. They may also travel at roughly the same rate. Further entanglement may occur if the VP triggers new A P at sites distant from the original wound (Pickard, 1973). Moreover, V P are very variable and may incorporate rapid monophasic or biphasic changes in potential that resemble parts of an AP, and vice-versa (Van Sambeek and Pickard, 1976). Not surprisingly then, there are several published examples that are referred to as A P by the authors, but which could well be interpreted as VP by other workers (see e.g. Pickard (1973) discussion on Umrath (1959)). Pickard (1973) also notes problems of interpretation of potentials in the work of Kawano, and Lou; similarly Pickard (1972) queries some of the A P in the work of Sinyukhin. Further examples are mentioned in Findlay and Hope (1976). Unfortunately therefore, AP and VP can be far less distinct than some authors imply, and it becomes difficult to distinguish putative electrical signals (AP) from electrical responses
(W. One important distinguishing criterion should hinge on whether the electrical event can pass through dead regions of tissue (Houwink, 1935): V P can, but A P cannot because there are no live polarized membranes at which the
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AP can regenerate itself. This useful indicator has rarely been exploited. Even this may not be infallible because: (i) VP can sometimes peter out while crossing dead tissue (Pickard, 1973); (ii) in characeans at least, AP can sometimes jump across gaps of several millimeters if aided by salty media or by salt bridges (Ping etal., 1990; Osterhout and Hill, 1930); and (iii) an AP might appear to traverse a dead zone if it is retriggered in the living tissue at the other side by an accompanying VP (Pickard, 1973). If AP are to be viable as physiological signals they must propagate through the plant in a reasonably stable manner. There is evidence that they do so in the sensitive carnivores (above). In non-sensitive plants, the VP may spread in a logical fashion from wound sites (as expected since the mechanism involves hydraulic dispersal; section IIE and IID). However, the progressive spread of AP after wounding in such plants is much less clear. This is not only because of uncertainty about whether AP or VP are being measured, but also because in many published results too few, and sometimes only a single recording electrode (plus reference of course), have been employed. In some other cases, the recording and reference electrodes are placed rather close together on the plant, so that it may be difficult to distinguish whether the electrical event occurs at the position of the recording electrode, or at the reference electrode (e.g. Wildon etal., 1992; see legend to Fig. 17). A recording of surface electrical activity measured at multiple electrodes in a wounded tomato plant is shown in Fig. 15. Two major types of electrical feature are visible at all four electrodes along the stem below the wound site in this record: the first is the large early shoulder which recovers slowly over 3-4 min. From its asymmetry and long duration, this can be identified as the VP. It moves progressively from one electrode to the next at a mean rate of about 10 mm s-'. This reflects the underlying passage of xylem-borne solutes travelling by hydraulic-dispersal from the wound site. The second type of event in Fig. 15 is the spiky transient. These are symmetrical and brief, each lasting some 10s at '/2 peak height. Although these are considerably longer than AP in the carnivores, they can reasonably be identified as AP. However, it is evident from the figure that these spikes do not travel along the stem in any logical manner. Rather, the pattern at any one position appears to be virtually independent of those at preceding or succeeding positions on the same stem. Electrical recordings from wounded tomato plants are also shown by Van Sambeek and Pickard (1976) and by Wildon et al. (1989). The former show a pair of recordings very similar to those in Fig. 15, and suggesting a similar conclusion: the VP moves from one electrode to the next, but the AP do not. The results of Wildon etal. (1989) also show movement of the VP, but movement of AP cannot be judged from their data because spikes are shown only for a single electrode. Thus, although spiky electrical events occur in the wounded tomato plant, there is no convincing evidence that they propagate. A distinction must therefore be drawn between these events and the self-perpetuating AP of animal nerves.
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\
\
Fig. 15. Surface electrical patterns induced by wounding in tomato. Six electrodes
(EO-ES)were distributed along the stem and petiole of a tomato plant, at the positions arrowed in the diagram (left). A common reference electrode was pushed into the soil. The curves show change in surface electrical potential with time, at each electrode. At the time indicated by the vertical line, the terminal leaflet of leaf five (indicated by * on the diagram) was wounded by scorching for 3 s with a flame.
Single spikes and spike trains are common in electrical recordings from the wounded tomato plant. In our experience, however, they are atypical of herbaceous plants in general. In a survey of over a dozen species, we usually found only quite smooth V P after wounding, with few or no spikes (Boari and Malone, 1993). It may be that this type of spiky activity results from some morphological peculiarity. For example, spikes are found in tomato and some cucurbits; all of these have large glandular hairs at their surface which might generate local electrical activity, or which might alter the appearance of underlying activity, perhaps by offering low-resistance windows to the interior of the tissue. Such windows could have a large effect on recordings made with surface electrodes (Williams and Spanswick, 1976). It was concluded above that the spiky so-called “AP” do not travel in the wounded tomato plant. Spikes that do not appear to travel have also been reported from several other tissues (e.g. Pickard, 1972). In several reports on higher plants, however, workers claim to show the movement of AP. The beautiful recordings of Zawadzki and Trebacz (1985) using two recording
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A
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” Fig. 16. Basipetal (part A) and acropetal (B) travelling electrical events in the sunflower stem. At the times indicated by the vertical arrows, electrical stimuli were applied by passing current between the wires marked “ + n and ”-”. The positions of recording electrodes (1-3) are shown on the diagrams, together with that of the reference “Ref“. The curves show change in surface potential, with time, for each recording electrode. From Zawadzki et al. (1991) with permission.
electrodes on Lupinus, and of Zawadzki etal. (1991) using three recording electrodes on Helianthus (Fig. 16) serve as good examples: after localized stimuli, electrical phenomena are evident that clearly pass along the stem, affecting successive electrodes sequentially and in a logical manner. These are termed “AP” by the authors, and are referred to as good examples of propagating AP by several other workers (e.g. Thain and Wildon, 1992). However, I argue that these events are not AP; they are VP. This is because the duration of the electrical peak is rather long for an AP (c. 20s or more at ?hpeak height), and because the rate of travel (c. 8mms-’) is typical for the hydraulic dispersal associated with VP. Note that, although electrical stimulation, rather than wounding, was used in these studies, the applied voltage (4 V applied for 3 s) is very likely to have caused cell damage, or at least caused a change in membrane permeability, leading to release of solutes and water, and therefore to hydraulic dispersal, exactly as for wounding (see section IVB). This view is supported by the statement of Paszewski and
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Zawadzki (1976) that small heat wounds, involving “approaching or touching the stem with a hot glass rod” produce effects similar to those with electrical stimulation. The recurring dilemma of plant electrical “signals” is evident here: are these events AP or VP? That is, are they propagating electrical signals or merely local responses to a chemical signal travelling through the underlying tissue? The mechanism of the VP is known, at least in outline: cell sap is released at the wound site and is drawn along, via the xylem, by the pre-existing demand for water in all the tissues of the plant. It involves hydraulic dispersal (section IID). A number of predictions can therefore be made about VP: 1. The VP should not be blocked at dead regions. 2. (a) The propagation rate of hydraulic dispersal, and therefore VP, will depend on the prevailing water status of the plant; lower water status should tend to drive faster VP movement for a given wound; and (b) in the extreme case, where shoot water status is saturated (as with wellwatered plants enclosed for a few hours in polythene bags) xylem tension becomes negligible and VP should not travel at all. 3. The VP, because it is associated with rapid flows of water and solutes through the xylem, should also be associated with systemic changes in thickness of the tissue.
Higher plant AP are rather ill-defined in both character and mechanism. For example, Hill and Findlay (1981) stated that ”. . . it is quite disappointing to realise that a century after their discovery, the nature of propagated action potentials in higher plants is still poorly understood and we have no clear idea of their ionic basis”. It is therefore difficult to predict precisely how AP should behave in higher plants. Presumably, however, any self-propagating AP should be blocked at dead regions, should not be associated with large flows of water, and should be little affected by plant water status. Thus, AP ought to behave differently from VP under all three of the test conditions postulated above. These tests have not been reported from the species used by Zawadzki and colleagues. However, we have recorded similar electrical phenomena from various plants, including wheat seedlings, and we have tested the above predictions on these seedlings. Figure 17 shows that the wound-induced electrical event is accelerated when plant water status is depressed. Figure 18 shows that the electrical activity can pass across heat-killed tissue. Both of these observations are consistent with a mechanism involving hydraulic dispersal and VP, rather than one involving AP. It is also known that strong and systemic leaf swelling occurs in wheat seedlings and in other plants after wounding (see Fig. 4), and that this swelling is associated closely with wound-induced electrical events (Malone, 1992; Boari and Malone, 1993). Concerning prediction 2(b), we have found in wheat (not shown), as in tomato (Fig. 19), that enclosure of well-watered plants at high humidity will completely eliminate woundinduced electrical phenomena. Thus, according to each of the four criteria
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1 min
(El isolated) -
Fig. 17. Acceleration of wound-induced electrical events by reduced water status in wheat. Four electrodes were distributed along a wheat seedling (two-leaf stage) as shown in the diagram (A, right); three were placed along the blade of leaf two (E2-E4) and one was placed on the sheath of leaf one (El). A common reference electrode was placed in the hydroponic root medium. The curves show change in surface electrical potential with time, for each electrode. A. Roots were held in water. At the time indicated by the vertical line a small region near the tip of leaf one (vertical arrow) was wounded by scorching for 2 s with a match flame. B. The root medium was replaced by 5-bar mannitol solution and the seedling allowed to equilibrate for 4 h (not shown). Then, at the time indicated by the vertical line, a small region near the tip of leaf one (adjacent to that wounded in part A) was scorched as above. It is evident that, in the presence of mannitol (which reduces plant water status) electrical events are accelerated. The value recorded from each electrode is the difference in potential
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above, we can identify these wound-induced electrical events as VP rather than AP. The same conclusion is likely to hold for the (similar) recordings of Zawadzki and coworkers. I argue that most, possibly all, of the apparentlytravelling wound-induced electrical phenomona in higher plants are VP, reflecting hydraulic dispersal of solutes, rather than self-propagating AP. The tests described above should be applied in various other species, to assess whether their electrical activity is VP or AP. Tests involving enclosure in polythene (test 2 (b)), and transmission across dead tissue (test l), are particularly simple to apply. The spiky electrical phenomena from tomato cannot be explained as local responses to xylem-borne solutes, because these spikes do not appear to travel. However, the observation that spiky events in tomato are abolished if the plants are wounded at high humidity (Fig. 19) suggests that these too, may occur in response to hydraulic events. These spiky events normally occur during or shortly after the major wound-induced swelling of the tissue, and they may reflect pulses of ions emitted by cells during turgor- and/or volume regulation, as in Acetabularia (Wendler et al., 1983) and many animal cells (Sardaki and Parker, 1991). See section IVB, below. B. SIGNALLING OF NON-WOUND STIMULI - AN HYDRAULIC MECHANISM?
In addition to the electrical responses induced by wounding, there are many examples of electrical activity induced by non-wound stimuli, such as touch, cold water and small depolarizing currents. In these cases, it may seem that a mechanism based on hydraulic dispersal would be impossible because nonwound stimuli do not release the sap necessary to fuel hydraulic dispersal. However, non-wound stimuli can cause transient changes in membrane permeability (Jaffe, 1980). These would result in efflux of ions, followed passively by water, and they might release sufficient fluid to generate limited hydraulic dispersal. Rapid decreases in the axial electrical resistance of various (non-sensitive) plant tissues were recorded by Bose (1928) after gentle mechanical distortion, and by Jaffe (1980) after mild rubbing of the stem. This
between that electrode and the reference electrode in the root medium. In both A and B the three electrodes along leaf two (E2-E4) show an almost identical electrical pattern. These synchronous events are probably reflections of the electrical event at El (a variation potential) passing onward to register on the reference electrode (which is effectively at the root surface). Thus, they appear synchronously at all three positions and are inverted compared to that at E l . It follows that electrical events at the reference electrode will also be registered in electrode E l . The pattern at the reference electrode can be isolated approximately, as the inverted mean of the pattern in E2-E4. This mean reference pattern can then be subtracted from the trace El to yield the true pattern at E l (“El isolated”; dotted line). Applying the same reasoning, there is only negligible electrical activity at any of the positions E2-E4.
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E4
E3 E2
El
Fig. 18. Passage of electrical events across heat-killed regions of the wheat leaf. Four electrodes (El-E4) were arranged along a wheat seedling as shown in Fig. 17A. The four traces show change in surface potential with time, for each electrode. Four hours previous to this recording, the central region of the neighbouring leaf one was killed by scorching for 5 s with a match flame. By the start of this recording, the central part of leaf one was brown and shrivelled. At the time indicated by the vertical line, a small region near the tip of leaf one (distal to the previously killed zone), was wounded by scorching for 2 s. Wound-induced electrical events are evident at positions proximal to the dead zone, and they must therefore have traversed it. As argued in the legend to Fig. 17, much of the apparently synchronous electrical activity seen here at electrodes E2-E4 actually represents events occurring at the position of the reference electrode, which is also proximal to the dead zone.
probably indicates significant ion efflux from cells in the stimulated region. The rapid loss of ions postulated here would almost certainly be associated with water loss, and it could also depolarize the membrane potential, generating an electrical spike rather like an AP. Depolarization, the first portion of the AP, involves efflux of C1- in Charu, whereas repolarization to the resting potential involves efflux of K + (Wayne, 1994). The rrhimum ion flux necessary to cause electrical changes would be negligible in osmotic terms (Tinz-Fuchtmeier and Gradmann, 1990) but it appears that far more than the electrical-minimum flux often occurs in these cases. Thus, AP are associated with measurable water loss in Chara (Barry, 1970; Oda and Linstead, 1975; Wayne, 1994) and Nitella (Sandlin etul., 1968), and Wendler etal. (1983) discuss the role of AP in osmoregulation in Acetabularia. The latter authors show that the burst of C1- efflux associated with each periodic action potential imposes a significant stepwise decrease in cell turgor pressure and volume. Thus, an electrical spike, even if non-propagating and restricted to one region of the cell, will be associated with ion efflux which could influence the
SIGNAL TRANSMISSION IN HIGHER PLANTS
a. before mannitol addition
21 1
b. after mannitol addition
T1
Fig. 19. Elimination of wound-induced electrical activity in tomato plants at saturating water status. The shoot of a well-watered tomato plant (four-leaf stage) was enclosed in a polythene bag. Electrodes were placed on the base of the petiole of leaf three (El) and on the stem between leaf two and leaf three (E2). A displacement transducer was placed on the terminal leaflet of leaf two. A. The plant was allowed to equilibrate for 4 h inside the polythene bag then, at the time indicated by the first vertical line, half of the terminal leaflet of leaf three was wounded, through the polythene bag, by placing a hot (c. 90°C) metal block on it. B. The soil was irrigated to run-off with mannitol solution (10 bar) and the plant allowed to equilibrate for 4 h (not shown). At the time indicated by the vertical line, the remaining half of the terminal leaflet of leaf three was wounded, through the bag, as in part A. Parts A and B were conducted on successive days on the same individual plant. The transducer and electrodes remained in the same positions throughout and the two parts are therefore directly comparable. It is evident that, at saturating water status, wounding induces neither remote hydraulic nor electrical responses. Both types of response are restored if the plant's water status is reduced (still inside the bag) by irrigation with mannitol. Note especially that the spiky electrical events, as well as the slower variation potential, are eliminated at saturating water status.
remainder of the cell by causing a transient depression of turgor. A local water current could also be established transiently across the membrane, with efflux at the depolarized patch of membrane, where apoplastic solutes are most concentrated, and influx across the remainder of the cell where the solute gradients are unchanged. In the higher plant, where turgid, elastic cells are packed together and surrounded by an apoplast of negative pressure and small volume, a modest release of water and ions (causing depolarization) at one cell could provide enough sap to fuel hydraulic dispersal to neighbouring cells. Therefore, even without any electrical propagation or plasmodesmatal connections, events associated with depolarization at one cell could be signalled to neighbouring cells. If the membranes of these neighbouring cells are sensitive to a sudden change in turgor or apoplastic ion composition, they might also depolarize in
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response to this signal, with further loss of fluid. Thus, an hydraulic-dispersal signal, induced without wounding, could propagate across plant tissue. Electrically, such a signal might appear very much like a propagating AP, because successive ranks of cells would depolarize as the signal passed. An extreme example of this process, involving loss of ions from stimulated but unwounded cells, may happen in the “motor tissue” of some sensitive plants (Findlay, 1978). In Mimosa, for example, rapid movement of the leaf is driven by collapse of turgor pressure and volume in cells of the pulvinar motor cortex (Hill and Findlay, 1981; Sibaoka, 1991). This collapse is caused by a sudden and massive loss of solutes from the cells. K + and C1- are the major components, but other ions are probably also involved (Samejima and Sibaoka, 1982; Kumon and Suda, 1984, 1985). Because the cell walls are elastic, when turgor pressure coIlapses, cell volume must also collapse and water will be ejected from the collapsing cell. The water efflux could follow as a response to the efflux of ions, by diffusional flow down the new osmotic/ hydrostatic gradient. Alternatively, some workers argue that rapid efflux of sap from the motor cells of Mimosa resembles bulk flow rather than diffusional flow (Hill and Findlay, 1981; Iijima and Sibaoka, 1983; Kumon and Suda, 1984; Sibaoka, 1991); in this case, cell sap, including all the solutes it contains, will be squirted into the surrounding apoplast. If rapid flow in the Mimosa pulvinus is indeed a bulk flow, the membranes of this tissue might contain some very unusual transmembrane proteins, perhaps giant versions of the “aquaporins” reported from other tissues (Chrispeels and Maurel, 1994). Regardless of the mechanism of efflux, the fluid released to the apoplast will flow away from the cell which ejected it, because of its positive pressure, and because of apoplastic water tension. Such fluid will quickly reach neighbouring cells. If one of the ions or other constituents expelled from a collapsing motor cell, such as Ca2+ or Ricca’s factor, can stimulate opening of membrane channels, it will trigger a sudden change in membrane permeability in neighbouring cells leading to their collapse with further release of sap containing this active substance. In this way a cascade would occur such that the entire motor cortex will react, with each cell triggered by hydraulic dispersal of active substance from its collapsing neighbours. Furthermore, the catastrophic ion efflux from collapsing cells will generate an electrical transient, and this will appear to propagate across the tissue, as successive ranks of cells collapse. The initial ion efflux (and thus the associated electrical transient) could appear significantly in advance of actual leaf movement. This is because leaf movement might not begin until a large proportion of cells in the motor cortex had completed their collapse, since the few remaining turgid cells might support the leaf for a time. The lag of about 0.5 s observed between electrical transient and leaf movement in Mimosa (Sibaoka, 1966; Samejima and Sibaoka, 1980), might therefore be consistent with an hydraulic-dispersal mechanism of signal propagation, as well as with an electrical theory. Thus, even where “AP” appear to propagate rapidly across non-wounded tissue immediately prior to
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a motor response, hydraulic-, rather than electrical-, transmission could be involved. This possibility should be a priority for further study. At the molecular level, the electrical and hydraulic-dispersal mechanisms might not be totally distinct since, if the signal carrier is an ion, it will move according to local gradients of both pressure and electrical potential. One fundamental difference is that the major signal molecule could be neutral according to the hydraulic mechanism, but it would have to be electrically charged to be involved in a propagating AP. Note that, where many cells are collapsing catastrophically, as in the pulvini of Mimosa, positive apoplastic pressures could be generated transiently. Bose (1928) demonstrated by clever use of dyes, that sap was forcibly ejected from the primary pulvinus of Mimosa during its motor reaction. This probably also explains why the tertiary pulvini of Mimosa darken visibly for a moment as they collapse - the forcible displacement of intercellular air by flooding will change the refractive index of the tissue. In this case, hydraulic dispersal could occur over limited distances without the normal requirement for xylem tension. From the foregoing, it is concluded that non-wound stimuli, as well as wound stimuli (section IIDl), may be signalled by hydraulic dispersal in both nonsensitive plants and in the sensitive non-carnivores like Mimosa. The question then arises as to whether the motor reactions in the sensitive carnivores (principally Dionaea and Aldrovanda) might also be co-ordinated hydraulically, with the AP following the front of the motor reaction, and as a reflection of it rather than a trigger of it. This view would overturn a century-old conception of signals in these plants, but it is difficult to find other than anecdotal evidence pertaining to it in the literature. Examples of such evidence include: 1 . The trap of Dionaea closes much faster in light than in dark (Jaffe, 1973). This is suggestive of an hydraulic mechanism because hydraulic dispersal should also be faster under light (transpiring) conditions, when xylem tension is greater. 2. Wounding of a small number of cells, particularly in the motor region of the trap, can induce rapid closure in both Dionaea and Aldrovanda (Darwin, 1875, p. 292; DiPalma et al., 1966; Sibaoka, 1966; Williams, 1976; Iijima and Sibaoka, 1982). This is consistent with a mechanism involving hydraulic dispersal because the wounded cells would release sap, including any putative active substance, to the apoplast. Thus the normal receptor process would be circumvented and the signal cascade would begin from the wound site. 3. The trigger hairs are the structures that normally perceive mechanical stimuli from insects in the trap lobes of Dionaea and Aldrovanda. One particular cell layer in Dionaea has indented cell walls and, since these indentations will focus any bending stresses in the hair, these cells are believed to be the major receptors (Bentrup, 1979). They have been identified as such
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from their electrical behaviour (Benolken and Jacobsen, 1970). These cells are markedly larger than their neighbours (Haberlandt, 1914; Bentrup, 1979; Williams and Mozingo, 1971; Mozingo etul., 1970). One might predict that receptor cells at the start of an hydraulic signalling chain would be of large volume so that they can release a strong pulse of fluid to get the signal underway. 4. The organization of the xylem in Dionueu appears compatible with hydraulic transmission across the lobes. There is a massive midrib with vascular bundles branching off at right angles to it into the trap lobes. Signal transmission is fastest parallel to these vascular bundles (Darwin, 1875; Sibaoka, 1966). In Droseru, each tentacle contains central xylem elements, which extend into the glandular head (Williams, 1976; Darwin, 1875, p. 5). These might provide a route for hydraulic dispersal. Histology of the trap lobes of Aldrovundu is discussed briefly by Iijima and Sibaoka (1982) but they make no mention of vasculature. A strongly suberized or cutinized “endodermoid layer” is noted by Williams (1 976) in the mechanoreceptor tissues of various sensitive carnivorous plants. Williams uses this layer to trace evolutionary relationships between the genera, but he assigns no function to it and presumes it is vestigial in some species. The hydraulic viewpoint would predict the presence of such a layer to permit accumulation of aqueous secretions at the trap surface (against the prevailing hydrostatic gradients), and to prevent their escape into the apoplast and xylem. Accumulation of such secretions is necessary to trap insects in Droseru and to facilitate their digestion in both Droseru and Dionueu (Darwin, 1875). One major obstacle for speculations concerning the nature of signal transmission in traps of the sensitive carnivores is that the cellular water relations of trap closure are not understood. Almost all rapid plant movements involve rapid, reversible turgor loss and cell collapse, as in the pulvinus of Mimosa. Contrary to popular belief (Williams, 1976), however, there are no hinge-type pulvinar zones in the traps of Dionueu or Aldrovandu (or in the tentacles of Droseru), and their closure involves a different mechanism. Several workers have concluded that trap closure in these plants involves rapid and irreversible expansion of the entire outer surface of the trap lobe (Brown, 1916; Williams and Pickard, 1980; Williams and Bennett, 1982), while other workers favour the involvement of elastic changes instead or as well (Darwin, 1875; Stuhlman, cited in Hill and Findlay, 1981; Ashida, cited in Iijima and Sibaoka, 1981; Hodick and Severs, 1989). Williams and Bennett (1982) concluded that during trap closure in Dionueu, the outer epidermis undergoes irreversible cell growth at rates of up to 10% per second (dV/V), and they proposed the involvement of an acid-growth mechanism. But cell-wall loosening and cell growth at these unprecedented rates seems unlikely. Also, there would have to be a net water influx into the entire outer epidermis to support this growth, and water flows into leaves usually have half-times of minutes rather than seconds (see Figs 3,
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4, 9). An alternative view is that the basic mechanism involves rapid decrease
in turgor of epidermal cells over the outer or inner trap lobes, and that preexisting tissue tensions within the lobe then cause it to distort into the closed position (Iijima and Sibaoka, 1983; Hodick and Severs, 1989). This view seems inconsistent with the careful observations of Brown (1916), which revealed that the entire trap became larger with each cycle of closure and opening. These fascinating trap systems warrant re-examination with modern techniques for cellular water relations, such as the pressure probe. If their closure really does involve cell growth at fantastic rates, then hydraulic transmission of the impulse can be ruled out: signalling by hydraulic dispersal would not be possible if motor cells absorb water, rather than release it, when triggered. In this case, signalling by a neuroid, self-propagating action potential would appear to be the only available mechanism capable of delivering the observed high transmission rate. It is not inconceivable that the sensitive carnivores use AP in a manner fundamentally different from all other plants. If cells of these traps can grow at 10% per second, their cell walls must have some unique properties. A biochemical analysis of cell-wall constituents, including proteins, in the trap lobes might be very interesting. Finally, some tests which might be used to distinguish between hydraulic and electrical signalling in the sensitive carnivorous plants can be considered: 1. If the signal is a hydraulic dispersal, then many cells of the motor zone
must contain the putative “active substance”. An aqueous extract of the motor zone should therefore be capable of inducing closure when applied to the trap via the xylem. The putative active factor might then be purified using trap closure as a bioassay. Aqueous leaf extract can trigger movement in Mimosa (Ricca, 1916) and Berberis (Sibaoka, 1969). Large scorch wounds applied t o one trap should also trigger long-distance hydraulic dispersal of the active substance, leading to closure in other traps on the same plant. 2. As discussed earlier, transmission across dead zones of tissue provides a method of distinguishing between hydraulic and electrical transmission. As far as I am aware, this has not been tried with the sensitive carnivores, although Darwin (1875) noted that transmission could pass around transverse cuts in the trap Iobe of Diunaea. One approach might be to use a hot metal cylinder to apply a circular heat wound to one trap lobe of Dionaea. The wound should encircle at least one trigger hair. Then, after the leaf had recovered and reopened, the trigger hair could be brushed or the enclosed leaf surface stimulated by wounding. If the entire trap can still be induced t o close under these circumstances, then the involvement of selfpropagating electrical signals can probably be ruled out. 3. Similarly, the enclosure of well-watered plants at saturating humidity should eliminate hydraulic signals and should inhibit or at least retard leaf
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closure, even if a positive-pressure hydraulic signal is involved (as suggested for the Mimosa leaf). Afdrovanda is aquatic and its traps capture aquatic insects, but I am not clear whether the plant normally grows in an entirely submerged position. Iijima and Sibaoka (1981) state that Aldrovanda “floats just below the water surface” whereas Le Maout and Decaisne (1876, p. 407) state that it “floats on stagnant waters in S. France, N. Italy, and Bengal”. Even if a part of the plant normally protrudes above the water surface (and thus generates transpiration and xylem tension) Iijima and Sibaoka (1981) state that isolated traps of Aldrovanda can be triggered when completely submerged. It would be interesting to determine whether the rate of closure is reduced under fully submerged cf. partially submerged conditions. Dionaea is indigenous to damp boggy habitats in N. Carolina, but it is not habitually submerged, neither are the Drosera spp. Investigations of these types should be carried out to resolve the mechanism of rapid signalling in the sensitive carnivores.
V.
CONCLUSION
Higher plants exhibit various forms of rapid communication. Hydraulic pressure signals, phloem-borne- and airborne chemical signals can travel long distances in plants. Each offers a mechanism for long-distance co-ordination within the plant. Electrical “signals” in plants are more enigmatic. Many electrical events could represent local responses t o the passage of other signals. The most convincing evidence of self-propagating electrical signals comes from the leaf traps of the sensitive carnivores (Dionaea and Afdrovanda).Further analysis of signalling in these trap structures will require fundamental information on the water relations of trap closure. Transmission of chemical signals by hydraulic dispersal in the xylem could be the major mechanism of wound signalling in plants, and it may be of widespread ecological importance in the co-ordination of plant defences against leaf-eating insects and some lesion-inducing pathogens. Sucking insects which precisely target the phloem (or xylem), such as aphids, cause little cell damage and probably trigger negligible hydraulic signals. Plants may not be able rapidly to mobilize defences against these pests. Hydraulic dispersal may also play a role in signalling of non-wound stimuli.
ACKNOWLEDGEMENTS The author is funded by the Biotechnology and Biological Sciences Research Council (BBSRC, UK). Helpful comments on this chapter were provided by Professor Lyn Jones and Dr David Gray of HRI Wellesbourne. Professor E. Davies (University of Nebraska) kindly granted permission to reproduce
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Fig. 16. Grateful t h a n k s are due t o Jim Justice of HRI Wellesbourne f o r help with electron microscopy, and t o m y colleagues, Drs Bratislav Stankovii-, Francesca Boari, G i n a P a l u m b o and Juan Alarcon.
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Wingate, V. P. M. and Ryan, C. A. (1991). Uniquely regulated proteinase-inhibitor-1 gene in a wild tomato species. Plant Physiology 97, 496-501. Wolf, O., Jeschke, W. D. and Hartung, W. (1990). Long-distance transport of ABA in NaC1-treated intact plants of Lupinus albus. Journal of Experimental Botany 41, 593-600. Wolfson, J. L. and Murdock, L. L. (1990). Growth of Manduca sexta on wounded tomato plants: role of induced proteinase inhibitors. Entornologia Experirnentalis et Applicate 54, 257-264. Woods, F. W. (1960). Biological antagonisms due to phytotoxic root exudates. Botanical Review 26, 546-569. Zawadzki, T. and Trebacz, K. (1985). Extra- and intracellular measurements of action potentials in the liverwort Conocephalurn conicurn. Physiologia Plantarurn 64, 477-481. Zawadzki, T., Davies, E., Dziubinska, H. and Trebacz, K. (1991). Characteristics of action potentials in Helianthus annuus. Physiologia Plantarurn 83, 601-604. Zhu, G. L. and Boyer, J. S. (1992). Enlargement in Chara studied with a turgor clamp: growth rate is not determined by turgor. Plant Physiology 100, 2071-2080. Zimmermann, D. C. and Coudron, C. A. (1979). Identification of traumatin, a wound hormone, as 12-0x0-trans-10 dodecenoic acid. Plant Physiology 63, 536-541. Zimmermann, M. H. (1983). “Xylem Structure and the Ascent of Sap”. Springer Verlag, Berlin. Zimmermann, M. H. and Brown, C. L. (1971). “Trees: Structure and Function”. Springer Verlag, New York. Zimmermann, U. (1977). Cell turgor pressure-mediated transport processes. In “Integration of Activity in the Higher Plant” (D. H. Jennings, ed.), pp. 117-154. Cambridge University Press, Cambridge. Zimmermann, U., Haase, A., Langbein, D. and Meinzer, F. (1993). Mechanisms of long-distance water transport in plants: a re-examination of some paradigms in the light of new evidence. Philosophical Transactions of the Royal Society (London) B341, 19-31.
Keeping in Touch: Responses of the Whole Plant to Deficits in Water and Nitrogen Supply
A . J . S. McDONALD' 2nd W . J . DAVIES'
'Department of Plant and Soil Science. Aberdeen University. Cruick Shank Building. St . Machar Drive. Aberdeen. AB9 2UD. UK. 'Division of Biological Sciences. I.E.B.S., Lancaster University. Bailrigg. Lancaster. LA1 4YQ. UK
...........................................................................
230
.............................................
231
I.
Introduction
11.
Manipulating Water and N Supply
I11.
Acclimation of C and N Uptake ................................................ A . Framework for Analysing Limitations to CO, Uptake ............. B . Stomata1 Responses ........................................................... C . Changes in the Mesophyll .................................................. D . Acclimation of NO; Uptake ..............................................
235 235 238 240 243
IV .
Acclimation of Extension Growth .............................................. A . Framework for the Analysis of Extension Growth .................. B . Growth of Roots and Shoots when Water Supply is Restricted . C . Growth of Roots and Shoots when N Supply is Restricted .......
246 246 252 258
v.
Implications ........................................................................... A . Sink Strength ................................................................... B . Regulation of N Balance .................................................... C . Regulation of Water Use Efficiency .....................................
263 263 264 266
VI *
Information Transfer ............................................................... A . Responses t o Soil Drying ................................................... B . Responses t o N Limitation .................................................
267 267 273
VII
What is in the Xylem Sap and How Can Changes in Water and N Availability Change the Xylem Sap Contents? .............................. A . Collection of Xylem Sap ....................................................
275 275
Advances in Botanical Research Vol . 22 incorporating Advances in Plant Pathology
ISBN 0-12-005922-3
Copyright 0 1996 Academic Press Limited All rights of reproduction in any form reserved
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Soil Drying and N Deprivation and the Effects on Xylem Contents ......................................................................... 276 C. Interaction and the Concept of Sensitivity Variation ............... 280 B.
VIII. Conclusions: An Integrated Stress Response System for the Plant? References .............................................................................
I.
..
286 289
INTRODUCTION
At the crop level, the framework of analysis provided by Montieth’s light interception model shows that when water or nutrient supply is limited, dry matter accumulation can be restricted by two broad classes of effects (Fig. 1) (Jarvis, 1985). These are direct effects of environmental perturbation on the processes that lead to the interception of solar radiation by plant parts and also possible effects on the efficiency with which intercepted light energy is converted into chemical energy. Although this model provides a very useful summary statement of how total biomass production may be influenced by restricted water or nitrogen (N) supply, it does not (and was not intended to) tell us anything about the underlying growth phenomena and physiology. For a more mechanistic appreciation of whole-plant response to perturbations in water and N supply, the extension growth of leaves and roots, in conjunction with the acclimation of specific uptake capacities for carbon (C) and N in leaves and roots, respectively, must be studied. Regulation at the level of single plant organs is, of course, only part of the response picture and it is important to note that effects of stress on root and leaf demography can assume progressively greater importance with prolonged drought and N deprivation. However, here we emphasize the more rapid responses of limitations to C and N uptake and the physiological mechanisms underlying the differential extension of roots and leaves at low water potentials and low N supply. First, we consider how water and N supplies can be manipulated around plant roots. These are important considerations, because the techniques employed can have a very great bearing on the results and conclusions reached. We then consider the acclimation of C and N uptake systems to deficits in water and N supply. We consider a framework for analysing extension growth and review the information on single leaf and root responses. We comment upon possible implications of response to whole plant functioning at limited availability of water and N. Finally, we review the literature on information transfer in response to water and N deficits and emphasize the importance of considering how the sensitivity of stomata1 and growth responses to chemical signals in the transpiration stream may be modulated by other variables.
PLANT RESPONSES TO WATER AND NITROGEN SUPPLY
23 1
/
/ /
c /
/
/
0
lo00 Photosynthetically active radiation intercepted (MJ m-2season")
Fig. 1. Dry matter production as a function of photosynthetically active radiation for C4 and C3 crops with adequate water, nutrients and temperature. Changes in leaf area decrease light interception and move production from A B. Impaired metabolism caused by stress decreases production at a given light interception (A --* D or B C). Stresses which decrease both area and efficiency decrease production via A + B C. From Jarvis (1985). -+
+
-+
11.
MANIPULATING WATER AND N SUPPLY
Our main consideration is the regulation of growth and uptake systems under conditions where the supply of water and N are growth-limiting. This can be termed supply-limitation and implies that uptake is insufficient to accommodate maximum growth in a given environment. Before we consider growth responses, it is appropriate to comment on the approaches used in manipulating water and N supply. In general, a useful approach to understanding the nature of any system, is t o perturb a relevant input variable and follow the time-series of response in output variables. Where soil drying and N deprivation are input variables, there are at least two aspects of perturbation that should be considered in designing any experiment, namely the magnitude of change and the rapidity with which it is effected. Because both of these aspects may affect conclusions with regard to growth response and regulatory
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mechanisms, it is important that the time-course of soil drying or N input should be very clearly documented. Much early literature shows that if plants are dehydrated rapidly, then stomata1 and other responses can be very different from those shown by plants that experience rather more gradual soil drying. In what follows, we cite many examples of what can result when the rate of soil drying is varied. One of the reasons for these differences can be the limited extent of solute regulation that can occur when rapid dehydration occurs. We argue later that plant growth regulators can mediate the responses of many plants to drought but Hartung and Slovik (1991) pointed out that synthesis of extra abscisic acid (ABA) is a relatively slow process and that rapid stress responses must involve redistribution of the hormone. Both of the above points must be borne in mind when, for example, investigating the molecular biology of drought stress. Here, detached shoot tissue is sometimes rapidly dehydrated on the laboratory bench, which is just about the most unrealistic droughting treatment imaginable. One common technique for studying the effects of limited water supply on plant growth has been to manipulate the water potential of plants by growing them in hydroponics and adding osmotica of different strengths. This technique has been used successfully on many occasions and has the merit that a relatively consistent degree of drought stress can be imposed. When soil is allowed to dry, the availability of water is restricted more severely as more and more water is lost and the analysis of the response can be complicated. One problem with osmotic treatments to simulate soil drying is that plant growth regulators generated in the roots may be leached from the roots, significantly altering the response of the whole plant to the drought stress treatment. There may also be other effects of solution culture on roots, one of which can be the structure of roots themselves. The production of root hairs is often inhibited in plants grown in solution culture. We will see later that low water potential treatments can have very different effects on cell wall properties and on turgor maintenance of maize primary roots, depending on whether they are imposed in vermiculite or in solution culture. One clever manipulation to overcome problems that may arise as a result of hydroponic treatments is to grow plants in soil inside large-diameter dialysis tubing. The tubes are sealed at the bottom and placed in large containers with osmotic solutions of the desired strength. In this way, plant roots are in contact with soil but the water potential around the roots is kept relatively constant. One important consideration in drought stress experiments is that, as a soil dries, both water and N supply may be restricted (Turner, 1986). If we are to distinguish between the effects of these two variables, we may have to use bathing solutions of different osmotic strength, rather than withholding water from a solid substrate. The literature contains a great many descriptions of techniques devised to restrict water supply to whole root systems or to parts of the root. Most recently it has become clear that to understand how plants respond to soil
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233
drying it is necessary to break the link between the soil drying process and the supply of water to the shoots. Davies and coworkers (Blackman and Davies, 1985; Neales e t a / . , 1989) used a split root system to try to ensure an unrestricted supply of water to the shoots of plants with some roots in contact with dry soil. These manipulations suggest that plants are sensitive to soil drying around the roots, even when water supply is not limited. Passioura and Munns (1984) describe a whole-plant pressure chamber that can be used to keep plants effectively fully turgid even when roots are in contact with very dry soil (Fig. 2). As the soil dries, pressure on the root soil system is increased so that the shoot water relations of unwatered plants are comparable to those of plants that have been watered regularly. Gollan eta/. (1986) showed that where this was achieved, stomata1 conductance declined, and both pressurized and non-pressurized plants showed essentially the same relationship between conductance and soil moisture status, indicating that much of the plant’s response to soil drying was not necessarily related to restricted water supply. The nature of the solid medium around the roots can also have an important effect on the response of plants in a soil-drying experiment. As soil dries, mechanical impedance of the soil (soil strength) can increase significantly and it is well known that this change will limit plant growth (Passioura, 1988). The morphology of the plant can also be changed. For example, roots growing in soil with a high mechanical impedance often show increases in thickness (see Scott Russell, 1977). Part of the response of roots to soil drying can involve a response to increased soil strength. We shall see later that roots grown in vermiculite (where soil strength is negligible) show restricted radial growth at low water potentials. Such techniques may be necessary to disentangle the separate effects of increased soil strength and restricted water supply as the soil dries. Passioura (1988) also used the whole-plant pressure chamber to address this question. In a great many studies of N uptake where supply limitation has been assumed, it would appear that insufficient thought has been given to the choice of N variable. Two emphases in manipulating N supply can be identified. In the vast majority of cases, the concentration of N in solution about the root has been varied. However, it has been argued that, where this is practised, interpretation of the growth response can be difficult (cf. Ingestad, 1982; Ingestad and Lund, 1986; Ingestad and h r e n , 1992). This is because plant growth can be maximized at very low concentrations of N in the bathing solution (cf. Olsen, 1950). Therefore, assuming that the external concentration is actually maintained, no effect of concentration on plant growth is to be expected over the wide range of N concentrations often investigated. In practice, treatment differences are often found but we assume these may be attributed to a lack of control over the N concentration in solution. If N concentrations are not closely monitored and maintained, then, as the plants become larger, N deficiency occurs. Typically, this will first be observed in the low-N treatments. With this approach, the plant will experience a whole range of N availability
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\
light
1
p .C r
U
1
I
Fig. 2. A Passioura-type pressure chamber to control the hydrostatic pressure of the xylem sap to atmospheric pressure. Plants are grown in soil in pots that can be enclosed in a pressure chamber, and xylem water potential is increased by applying pneumatic pressure. The pressure in the chamber is controlled by an electronic device that includes a light sensor, which is connected to the xylem of the plant via a waterfilled tube. From Gollan et al. (1986).
from excess consumption to extreme deprivation as it grows larger. It becomes extremely difficult to relate the growth response at any one time to a defined level of N deprivation. More recently, the flux of N to the root has been varied to control the growth of the plant (e.g. Ingestad and Lund, 1979). With this approach, the amount of N added to solution over a given period of time is manipulated. The N-flux approach can result in precise control of plant growth and is therefore most suitable for studies of growth regulation with respect t o supply limitation. Most studies with this approach have involved the addition of
PLANT RESPONSES TO WATER AND NITROGEN SUPPLY
235
variable, exponentially increasing amounts of N to plants grown in solution. This results in a range of plant relative growth rate (RGR) and plant N concentration which is maintained throughout the exponential phase of growth, and the plants are said to be at steady-state nutrition. We believe that the potential of this approach in preparing plant material for physiological studies in a growth context has yet to be fully realized. We are not saying that external concentration per se is unimportant to N uptake. However, for the most part, the range of external N concentration investigated has been much too high for supply limitation to take place. Recently, Macduff etal. (1993) varied the flux of nitrate such that a range of steady-state nutrition was achieved. These workers found that, for each value of flux and its associated plant growth, there was a unique, equilibrium value of external nitrate concentration. The crucial piece of information is that, in a separate experiment, when this range of equilibrium values of nitrate concentration was chosen as the experimental variable, plant growth and the values of all physiological and morphological variables investigated were identical to those where nitrate flux had been manipulated. In fact, in maintaining different external concentrations over the range investigated, Macduff et al. (1993) would necessarily have manipulated nitrate flux in the growth-limiting range. The point is that these were extremely low nitrate concentrations much lower than have generally been used in studies where supply-limitation has often been assumed.
111.
ACCLIMATION OF C AND N UPTAKE
A. FRAMEWORK FOR ANALYSING LIMITATIONS TO C02 UPTAKE
It has been standard practice to assess the stomatal and non-stomata1 contributions to the overall control of photosynthesis and consider these as quite separate influences. There is, however, considerable evidence that shows that there are significant interactions between the two levels of control. Historically, stomatal and non-stornatal limitations to photosynthesis have been assessed using electrical analogues to describe the resistances to carbon dioxide (CO,) uptake provided by the gas phase and the liquid phase of the transport pathway between the ambient air and the sites of fixation, Such calculations have several uncertainties (cf. Jones, 1992). A useful graphical device for investigating the relative contribution of gas and liquid phase resistances was first introduced by Jones (1973) and further developed by Farquhar and Sharkey (1982) and is illustrated in Fig. 3. By varying ambient CO, concentration and plotting the photosynthesis rate obtained against the calculated intercellular C 0 2 concentration (ci), a demand function for photosynthesis is obtained. As the photosynthetic rate increases, ci will fall below c, (the atmospheric CO, concentration) and the extent to which this occurs will
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A. J. S. McDONALD and W. J. DAVIES
t
A
cc
r
~ , - r
--
-
c, c, +-
Normal atmospheric concentration (c,) cO, concentration (c,)
Fig. 3. (a) Response curve or demand function relation A (assimilation) to ci (intercellular CO, concentration). (b) Two supply functions relating A to ci for different gas phase resistances with the slopes of the lines equal to leaf conductance. (c) Calculation of photosynthetic limitation (see text for details). The response curve represents the demand function for a hypothetical leaf. The CO, concentration drop and that across the gas phase by across the mesophyll is represented by (ci (c, - ci). Farquhar and Sharkey’s definition of the gas phase limitation is given by a/ ( a b). The recommended definition is ( rg/ r g + r* ) . From Jones (1992).
r)
+
depend upon the resistance provided by the gas phase (essentially the stomata). High resistances can result in substantial limitation in ci. Figure 3 shows the relationship between the photosynthetic rate and ci for two different values of gas phase resistance; the straight lines represent the photosynthetic supply functions. The actual values of ci and the photosynthetic rate under any particular set of conditions is obtained from the intersection of the supply and demand functions (Fig. 3).
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237
Farquhar and Sharkey (1982) suggested that the analysis described above can be used to calculate the gas-phase limitation to photosynthesis (I,). From the relationship shown in Fig. 3 we can see that a comparison of the photosynthetic rate at the normal operating point and the photosynthetic rate at a ci equal to c, (assumed to be an infinite gas phase conductance) can be used t o calculate lg ( a / ( a b ) in Fig. 3). Jones (1992) suggested that it would be more realistic to define the gas phase limitation as the relative sensitivity of photosynthesis to a small change in the gas phase resistance (I, = rg/(rg I-*), where r* is the slope dci/dP at the operating point (see Fig. 3). A critical assumption in analysis of this kind is that the stomata are equally open over the whole area of the leaf whose gas exchange is being measured. If this assumption does not hold, as for example where the stomata close in patches, the photosynthetic rate, gas phase resistance and the calculated value of ci only represent average values. This can give very misleading indications of the photosynthetic response to the environment. The relationship between photosynthetic rate and ci is non-linear, with changes in ci having little effect on photosynthesis when stomata are nearly fully open (Fig. 3). We can compare what happens if (i) a healthy leaf closes all its stomata by 50%, or (ii) if half the stomata in discrete patches close completely. In the first case, stomatal conductance (g) falls to g/2, assimilation falls according to the response curve (>A/2) and the calculated ci will decrease significantly, implying increased stomatal control of photosynthesis. In the second case, conductance will fall to g/2 but photosynthesis will now decrease to A/2, as only half the area is now photosynthesizing. Because conductance and photosynthetic rate have changed to a similar extent ci is apparently unchanged. Calculations of this kind made on an area basis where homogeneity of stomatal response is assumed can lead to the conclusion that photosynthetic capacity is directly reduced by the treatment (as shown by the apparent shift in the photosynthetic demand function in Fig. 3), even under circumstances where there may be no real shift in mesophyll properties. There is much analysis of this kind in the literature (e.g. Lange, 1988) but in the absence of a demonstration of a homogeneous stomatal response, interpretation of the results must be open to question. As early as 1983, Laisk proposed that inhomogeneities in stomatal response could cause problems for the analysis of gas exchange responses and since then he and others (e.g. Downton etal., 1988; Terashima etal., 1988) have shown a patchy stomatal response to a number of stomatal closing agents, particularly the application of the plant hormone ABA. Others have reported a patchy photosynthetic response which may be due to a collapse of parts of the mesophyll due to loss of turgor, with a resulting reduction in lateral C 0 2 diffusion capacity (Daley etal., 1989). Wise et al. (1992) note that patchy photosynthetic behaviour in severely stressed leaves cannot always be reversed by high C 0 2 concentrations, which would be the case if it was attributable to a patchy response of the stomata.
+
+
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More recent reports have shown that patchy photosynthetic responses do not always occur (Gunasekera and Berkowitz, 1992; Ort etal. 1994) but more detailed analysis of patchiness is probably required before we can unequivocally quantify the effect of soil drying or N deprivation on rnesophyll properties. It may be that patchy responses occur mainly when perturbation in the input variable is rapid. This is often the case, for example, when dehydration is rapid in experiments with drying soils and potted plants (Comic, 1994).
B. STOMATAL RESPONSES
1. Soil drying Stomata are sensitive to changes in the availability of soil water but the mechanism of this response is currently the subject of some debate (see e.g. Davies and Zhang, 1991). Stomata1 movements depend on changes in turgor pressure inside the guard cells and in adjacent epidermal cells, and anything that causes an increase in the relative turgor of the guard cells will promote opening of the stomatal pores. If soil drying restricts the supply of water to the leaves, stomatal apertures are generally reduced. This observation tells us that stomatal responses to soil drying result from active, energy-requiring processes, rather than simple hydraulic responses. This must be the case because epidermal cells have a mechanical advantage over guard cells, such that equal increases in pressure in both, which might result from increased water availability, will cause some stomatal closure. Most stomatal movements, including those in response to soil drying, involve active changes in the osmotic status of the guard cells. A range of opening stimuli will increase the potassium content of guard cells, with uptake from surrounding cells being driven by ATP-powered primary proton extrusion at the guard cell plasmalemma. The proton gradient between the outside and the inside of the guard cell will drive cation entry. This may be accompanied by chloride uptake and/or by stimulation of malate synthesis from storage carbohydrate in the cytoplasm. There are many published relationships between stomatal conductance and leaf water relationships. If soil drying occurs relatively slowIy, closure of stomata will occur over a wide range of leaf water potentials. Rapid drying can result in a threshold response. Relationships can also be modified by previous exposure of plants to stress and hardening can greatly decrease the sensitivity of guard cells to decreasing leaf water potential. Although it is clear that stomata can respond to local changes in leaf water status, there is accumulating evidence that stomatal response to soil drying may often be controlled by additional factors. Turner et al. (1985) describe some experiments with sunflower and Nerium, where leaf water relations are manipulated by varying leaf-air vapour pressure difference. The relationship between stomatal conductance and leaf water potential depended on the leaf air vapour pressure
239
PLANT RESPONSES TO WATER AND NITROGEN SUPPLY
-2.0
-1.5
-1.0
-0.5
Leaf water potential (MPa)
0 0
20
40
60
80
Extractable soil water
100 (%)
Fig. 4. Relationship between leaf conductance and (left) leaf water potential and (right) extractable soil water in a single leaf in a temperature- and humidity-controlled gas exchange cuvette at A wi= 10 Pa kPa-', while the remainder of the plant in a growth cabinet was at A w , = 10Pa kPa-' (low transpiration, 0 ) or at Aw, = 30 Pa kPa-' (high transpiration, 0).A w, leaf to air vapour pressure difference. From Turner e t a f . (1985).
deficit (Fig. 4). There was a single relationship, however, when all of these data were plotted as a function of soil water status, suggesting that the stomata were actually responding to some function of soil water availability. Some data of Jones (1985) collected with Bramley apple trees growing in the field, suggested that leaf water status can often be controlled by stomatal behaviour rather than the converse. Here, stomatal closure in unwatered trees resulted in an increase in shoot water potential. The fact that stomata did not respond to this rehydration suggests that some interaction between roots and drying soil generated a non-hydraulic influence that kept the stomata closed. This possibility is supported by the manipulations of soil drying and water supply that are described in section 11.
2. N deprivation Stomata can also be sensitive to changes in N supply. Where N is withheld from roots of plants grown in solution, stomatal conductances are often reduced compared with values shown by plants supplied with more N (e.g. Chapin et al., 1988a,b). In other instances, stomatal response to N deprivation seems to be lacking. For example Palmer eta/. (1996) grew Helianthus plants according to the N-flux approach described above. When the N supply to these plants was abruptly reduced from 25% per day to 12% per day, no stomatal reaction was detected. Where it does occur, partial stomatal closure following
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A. J . S. McDONALD and W. J. DAVIES
N deprivation is not necessarily associated with a change in leaf water potential (Chapin et al., 1988a,b). Other studies have shown that decreases in leaf water potential can occur following N deprivation (Radin and Boyer, 1982). There is little information on how the rate of decrease in N supply or how previous exposure to fluctuation in N affect the reponse. However, there is evidence that the extent of the decrease in leaf water potential and stomatal conductance following N deprivation can be influenced by root temperature (Radin, 1990).
C. CHANGES IN THE MESOPHYLL
1. Soil drying
In the estimation of photosynthetic limitation, problems of stomatal heterogeneity can be overcome with the use of several non-destructive techniques developed in the last few years. For example, the leaf disc oxygen electrode can be used to assess photosynthetic capacity even when the stomata are completely closed (see e.g. Kaiser, 1987). Figure 5 from Cornic (1994) shows variation in photosynthetic capacity, measured in an oxygen electrode, as a function of leaf water deficit. Leaf material was dehydrated either rapidly by excising leaves or slowly by withholding water from the plants over a period of 3 weeks. Measurements were made at CO, concentrations up to 17%, since Kaiser (1987) has shown that even at 5 % CO,, diffusion resistance of some plants is not completely overcome. For all of the plants measured, it was not until the leaf water deficit reached around 30%, that the leaf biochemistry began to be affected. Net photosynthetic rate is negligible at this water deficit and a comparison of the data suggests that stomatal limitation explains most of the observed decrease in photosynthesis in this range of water deficit. Rather surprisingly, the sensitivity of photosynthetic mechanisms is very similar across a wide range of plant material. We are also forced to conclude that ABA has little significant effect on the biochemistry of photosynthesis, since dehydration to this extent would be expected to result in massive accumulation of this plant hormone. Several reports seem to support this view (e.g. Downton etal., 1988). Maximum apparent quantum yield of photosynthesis measured at high CO, on three different plants did not vary much over a 40% range of leaf water deficit, suggesting that whole chain electron transport and related processes are also very resistant to leaf water deficit (Cornic etal., 1992). There are suggestions that the high CO, concentrations necessary for these measurements can themselves inhibit mesophyll activity but Cornic (1 994) argues that this is not a significant effect, at least in C3 plants. Several workers have now shown that maximum photosystem I1 (PSII) photochemical efficiency, estimated as the ratio of variable chlorophyll fluorescence to maximum fluorescence, is not significantly affected in the range of water deficit up to 30-40% (e.g. Bjorkman and Powles, 1984; Ben etal.,
PLANT RESPONSES TO WATER AND NITROGEN SUPPLY
1
24 1
\ 2
0
20 40 60 w ) 1 0 0 Water deficit (%)
Fig. 5 . (a) Relationship between photosynthetic capacity (C02 evolution, expressed 070 of maximum value) and leaf water deficit (LWD) for six different plant species: 1, Elatostema repens (understorey plant of the rain forest); 2,3 and 6, French bean, spinach and sunflower (cultivated mesophytes); 4, Arbutus unedo (xerophyte); 5 , Impatiens valeriana (hygrophyte). The relationship between leaf net C 0 2 uptake at 340ppm C 0 2 (9’0 maximum value, V) and LWD is also shown in the case of the French bean. (b) Relationship between apparent quantum yield of O2 evolution measured at high C 0 2 concentration and LWD. The numbers refer to the same plant species as specified in (a). From Cornic (1994). as
1987). The measurement of chlorophyll fluorescence from intact leaves using non-actinic modulated light means that, with an appropriate model, we can conveniently investigate photosynthetic whole chain electron transport and the regulation of light utilization by PSII (Weis and Berry, 1987; Genty etal., 1989). Again the conclusions from this work are that the regulation of PSII activity is the same whether or not the leaf is modified by changing leaf water status. These conclusions conflict with early work, largely involving isolation of organelles from wilted leaves, which seemed to suggest that photosynthetic mechanisms of higher plants were very sensitive to desiccation. We can now
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suggest that these responses described in early reports were either artefacts due to isolation procedures or the result of photoinhibition. Although the data described above do seem t o suggest that the photosynthetic metabolism of most plants is relatively robust as long as the water deficit is not too severe, there are data in the literature that suggest a down-regulation of photosynthetic processes as water potentials fall (see review by Ort eta/., 1994). One intriguing observation is that measured COz concentrations within leaves can increase as water deficit increases (Lauer and Boyer, 1992). It may be that this observation is consistent with the observations of Cornic, Genty and coworkers, if water deficit increases the resistance to diffusion of COz into the chloroplast more than the diffusion of COz into the leaf. Under these circumstances, ci could increase even under circumstances where the fixation of C 0 2 is not limited by photosynthetic biochemistry. One implication of the relative insensitivity of the photosynthetic apparatus to partial dehydration of the leaf is that the C 0 2 concentration in the chloroplasts of an illuminated, dehydrated leaf will be very low. This means that there will be light energy that cannot be utilized through CO, reduction. If photoinhibition is not to occur, this energy must be dissipated in other ways. Possibilities include leaf movements, non-radiative dissipation of energy and enhanced photosynthetic reduction of oxygen (see e.g. Cornic, 1994). If leaf water deficit is severe enough, the photosynthetic apparatus will eventually be damaged such that after rehydration photosynthetic activity will not be fully resumed. The nature of this damage is not well understood. 2. N deprivation Leaf N accounts for a large portion of the total-plant N, particularly in early vegetative growth. The majority of this N is in the enzymes of the photosynthetic carbon reduction (PCR) cycle and in the thylakoid proteins (Evans and Seeman, 1989). About haIf of this protein is involved in the light reactions of photosynthesis, including thylakoid membrane-bound proteins associated with light harvesting, electron transport and photophosphorylation. The other half is in soluble proteins involved in the dark reaction of the PCR cycle. These latter proteins include those involved in COz assimilation, photorespiration, ribulose bisphosphate (RuBP) regeneration, as well as sucrose and starch synthesis (Evans and Seeman, 1989). Where N supply and retranslocation are insufficient to support the growth rate of the plant, leaf N amounts decrease below values that are associated with more optimal supplies of N. In the short term, this may affect both the carboxylation efficiency and the C0,-saturated value of photosynthesis. With decreasing leaf N (per unit leaf area), the proportion of the total leaf N in the thylakoids remains about the same, the proportion in soluble proteins (such as Rubisco) decreases and the proportion in other nitrogenous compounds tends to increase (Evans, 1989). N-deficient plants may therefore tend to be more limited by Rubisco (decreased slope of an A/ci curve, Fig. 3) than by RuBP regeneration (i.e. the plateau). However, the Rubisco activity and
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243
the electron transport capacity seem generally to be closely coupled (Evans, 1989). Rubisco and RuBP regeneration therefore often continue to co-limit photosynthesis even under N deprivation and a linear dependence of several photosynthetic variables upon leaf-N concentration has been reported (Fig. 6; Sage et al., 1990; Harley et al., 1992). Lawlor et al. (1989) reported similar proportions of chloroplast components (less than 20% variation) over a wide range (10-fold) of leaf N content in wheat. There are reports that indicate that longer-term acclimation to supplylimitation in N can be associated with a constancy in photosynthetic rates (Waring et al., 1985; McDonald et al., 1986b, 1992; Pettersson et al., 1993) and in the variables obtained from A/ci analysis (Pettersson and McDonald, 1994). This would indicate that any down-regulation or inhibition of photosynthesis in response t o variability in N supply can be of a transient nature. It was only at more extreme N deprivation that A/ci analysis revealed changes in photosynthetic characteristics (cf. Fig. 7). In addition to assimilation of COz, electrons are used to reduce nitrate and sulphate, resulting in the formation of amino acids (Oakes, 1986; cf. Lawlor, 1994). Nitrate (NO;) is reduced in the cytosol by nitrate reductase and, in the chloroplast stroma, nitrite is further reduced by nitrite reductase to ammonia. The reduction of CO, and NO; d o not occur in strict stoichiometry. Under conditions of N deprivation (a larger ratio of CO, to NO; supply), larger fractions of the products of the light reactions and electron transport are consumed in CO, assimilation, and more carbohydrates tend to accumulate than at higher availability of NO;. Where nitrogen supply is less limiting to growth (a smaller ratio of COz to NO; supply), amino acids tend to accumulate.
D. ACCLIMATION OF NO;
UPTAKE
There are many reports that V,,, for NO; uptake in roots, which have been pregrown with a plentiful supply of NO;, increases when NO; is withheld (Clarkson, 1986; Jackson et al., 1986). This is apparent from significantly higher uptake rates in these roots when subsequently exposed to higher concentrations of nitrate, than in roots that have been maintained at the higher concentration for longer periods of time. Evidence that this can relate to the N demand of the whole plant is provided by results from split-root experiments where it has been reported that NO; uptake by roots fed with full nutrient solution may be stimulated by NO; starvation of other parts of the root system (cf. Touraine et al., 1994). Where NO; is freely available at the root surface and potentially nonlimiting to growth, uptake of NO; is considered to be demand-limited and it is apparent that there is a feedback of NO; usage in plant growth upon subsequent rates of NO; uptake (cf. Ismande and Touraine, 1994; Touraine et al., 1994). The evidence indicates that signals of N-demand probably d o not
244
A. J. S. McDONALD and W. J. DAVIES 150 V,
-
(35)= -9.6 + 60.0 N (P = 0.90)
125 100 75
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/4
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Leaf nitrogen (g m '1
Fig. 6. The relationship between key model parameters describing the A/ci response and total leaf N in cotton grown in ambient and elevated COz. Estimates of V,,,, (maximum rate of carboxylation), J,,, (maximum rate of electron transport) and TPU (triose phosphate utilization), obtained by non-linear least squares regression, plotted as a function of leaf N. Estimates are obtained from recently fully-expanded and from leaves up to 18 d after full expansion (0).For V,,,,. indepenleaves (0) dent linear regressions were obtained for leaves grown in ambient and elevated COz . Filled symbols denote elevated C 0 2 grown plants. Regressions of J,,, and TPU data on leaf N content are based on combined ambient and elevated COz grown plants. From Harley etal. (1992).
PLANT RESPONSES TO WATER AND NITROGEN SUPPLY
-
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Internal GO, (pmol mol.') Fig. 7. Leaf photosynthetic COz uptake (assimilation) as a function of internal CO, concentration (q) for small birch plants grown at optimal nitrogen supply (0) or at two suboptimal nitro en supply rates (0 = 0.07 mol N mol N-' d-' and 0 = 0.15 mol N mol N-' d - ) and grown at present or elevated CO,. Filled symbols denote elevated COz . Each response curve is based on data from 2 to 4 plants. From Pettersson and McDonald (1994).
7
arise in (or are not confined to) roots themselves but are emitted in the shoot and transported to the root (Edwards and Barber, 1976; Simpson et al., 1982; Burns, 1991). Larsson (1994) recently reviewed studies of N deprivation at supply limitation where NO; flux has been varied. Two types of response of specificuptake capacity (V,,,,,) to nitrate supply have been reported. Oscarson et al. (1989) found proportionately higher values of V,, at higher supply rates of NO;. In other studies, V,, was found to increase with increasing supply over the lower range of supply rates (phase I) but was found to decrease at higher rates of supply (phase 11) (Fig. 8; Mattsson et al., 1991). An apparent discrepancy exists between the acclimated response of V,,, in the phase I studies reported by Larsson and the findings of other workers. The findings of Larsson and his colleagues show that V,,, can be lower in NO;-deficient roots. Presumably the discrepancy between the dependency of V,,, on NO; supply in phase I and that more generally reported in depletion studies, may be accounted for by differences in the acclimation period and the sustained nature of N deprivation at extremely low fluxes and concentrations of NO; in the studies of Larsson and his colleagues.
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A . J. S. McDONALD and W. J . DAVIES
>I
"0
0.10
0.20
RA (d-1)
Fig. 8. Relative growth rate (RGR) and V,,, for net nitrate uptake, in vegetative barley maintained at different relative addition rates of nitrate (RA). Data from Mattsson etuf. (1991). Phases of uptake responses (see text) indicated by Roman numerals. From Larsson (1994).
IV. ACCLIMATION OF EXTENSION GROWTH A.
FRAMEWORK FOR THE ANALYSIS OF EXTENSION GROWTH
An understanding of plant response to environmental perturbations, assumes the measurement of relevant growth and physiology. However, recent advances indicate that accepted dogma on growth phenomena and traditional methods of measurement may be inadequate. In this section we provide support for this statement and advocate the use of a more mechanistic model of growth and more focused methods of measurement. During the past 30 years, it has been common practice to consider the expansive growth of plant organs in terms of a model originally formulated to describe the expansion of single cells (Lockhart, 1965). A driving force for growth, the cell turgor, is assumed to stretch the cell wall irreversibly at a rate determined by its yielding properties. In this model, the relative growth rate of the cell (r) is related to turgor ( P ) by two yielding properties of cell walls, the extensibility (m)and the yield threshold (Y). We can write the equation r = m(P - Y), where the amount of turgor that drives growth is the difference between P and Y . Lockhart's model tells us nothing about the nature of wall yielding or how wall properties can change in response to a change in the environment. For many years, Y and m were viewed as parameters that
PLANT RESPONSES TO WATER AND NITROGEN SUPPLY
247
were properties of cell-wall structure and which only varied over an extended time-scale. More recently, however, it has become clear that wall-yielding properties can be highly variable over very short time-scales (e.g. Frensch and Hsiao, 1994) and that this variation can act to maintain a relatively constant elongation rate. While there have been many reservations over the use of this equation to describe the growth of whole organs and even over its validity as a model for single cells (Ray etal. 1972; Zhu and Boyer, 1992), it continues to be a commonly used framework for analysing growth (Cosgrove, 1993). Is it really relevant? One of the main criticisms of Lockhart’s model has been the lack of evidence in support of turgor as a regulatory variable. In later sections, we discuss with respect to water and nitrogen supply, instances where differences in growth rate within the extension zone of plant organs were apparently associated with constancy in turgor. In these cases, it may be assumed that metabolism associated with cell-wall growth was regulating the rate of expansion in single cells. In some cases, turgor has been manipulated without any apparent effect on growth. For example, from their study of irreversible extension as a function of manipulated turgor in single giant algal cells of Chara coralha, Zhu and Boyer (1992) concluded that the crucial requirement with regard to turgor was that it should exceed the yield threshold value. Above this value (and assuming turgor did not attain damagingly high values) these authors found no regulatory role for turgor in cell extension. Their data indicate that the turgor-driven component of cell expansion ( P- Y ) in the Lockhart equation may be conceptually inadequate and that cell expansion may respond to a turgor switch: either ON when P - Y > 0, or OFF when P - Y I0. The regulatory mechanisms would then relate to growth processes in the cell wall. We consider this to be a definitive observation where turgor did not regulate growth. However, its relevance to cell growth in the tissues of higher plants has yet to be ascertained. For many years, it was only possible t o estimate turgor in tissues (not single cells) of higher plants by measuring water potential and osmotic potential and calculating the difference. This was commonly done on large plant parts using the pressure chamber and an osmometer. Water relations of small samples of plant tissue can be measured using the psychrometer but this is problematical with growing tissue because cells continue to grow in the psychrometer and, with a restricted water supply, cell turgors decrease with time to the yield threshold (Cosgrove, 1985). This can be avoided by using particular precautions (Michelena and Boyer, 1982) and other techniques (Matsuda and Riazi, 1981). Interpretation of earlier results from studies with dicot. leaves, where turgor has been varied by manipulating the transpiration rate and calculated from measurements on tissue samples, indicates that growth rate in cells of higher plant organs may be dependent upon P - Y . For example, Taylor and Davies (1986) made stepwise decreases in calculated turgor and measured corresponding decreases in the expansion of whole dicot. leaves (Fig. 9). However, interpretation of such data is problematical for two reasons. First, extension is measured for the whole organ and, second, turgor is calculated on tissue
248
A. J. S. McDONALD and W.J . DAVIES
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Fig. 9. Relationship between growth and turgor for illuminated (0)and darkened ( 0 ) leaves of Betulu pendula. Regression lines: illuminated, Y = 0.46~-0.031, r = 0.726; darkened Y = 0.13~-0.009.r = 0.750. From Taylor and Davies (1986). samples without taking into account any spatial variation in developmental stage of different cell populations. One intepretation of the Taylor and Davies data is that cell expansion was proportional to P - Y . This would be consistent with a turgor-driven plastic deformation of the cell wall, relating to the mechanical properties of the wall as measured, for example, by an Instron apparatus (Van Volkenburgh et al., 1983). The mechanical properties presumably cannot persist indefinitely in the absence of further metabolic loosening and incorporation of new wall material. Moreover, the extent to which irreversible deformation can occur without damaging wall structure and reducing its capacity for further growth is uncertain. Zhu and Boyer (1992) found evidence of damage to the extension process after turgor had been artificially increased to a higher value. For the several-hour period in which they investigated the response, these authors found no recovery of extension growth and it may well be that the wall was irreversibly damaged. It is uncertain whether a longer delay would have shown an eventual recovery to the metabolically driven rate. It has been common practice to measure the plastic extensibility of tissues and discuss the findings in terms of m in the Lockhart equation (cf. Pritchard and Tomos, 1993; Taylor etal., 1993, 1994). However, apart from the problems of measurement (Cosgrove, 1993) and the possible irrelevance of the forces applied (potentially excessive) to those normally associated with living cell walls, the approach can be criticized on other grounds. For example, increase in cell-wall size is dependent upon loosening and synthetic processes
PLANT RESPONSES TO WATER AND NITROGEN SUPPLY
249
of which wall plasticity is a result. The crucial point here is that the rates of these processes may change, affecting the rate at which the wall grows, without necessarily affecting the plasticity of the end product. It is even conceivable that, where P - Y I0, wall growth processes may continue, without the cell expanding. It is to be expected that Y might decrease (cf. Frensch and Hsiao, 1994), but, until this results in a resumption of cell expansion, it is possible that wall plasticity might increase. We are proposing a possible negative feedback of cell expansion on rn which might therefore be inversely related to current expansion. On recovery of P - Y > 0, we might expect an expansive spurt (a type of stored growth) until the metabolically limited rate is resumed. We do not dispute that plastic extensibility is less in older, more slowly growing tissues (e.g. Taylor et al., 1993) but we doubt the relevance of this property to the regulation of cell expansion. Moreover, reliable measurements of turgor in conjunction with relevant growth data are sufficient to assess the likely importance of wall phenomena to the regulation of growth. We have argued that plasticity measurements on cell walls are potentially incorrect and misleading. They are also probably unnecessary since biochemical studies have now provided us with fundamental models of wallloosening phenomena (see below). For all of these reasons, we now find it hard to motivate the measurement of plasticity in studies of the regulation of plant growth. The observed dependence of leaf extension upon manipulated turgor in Fig. 9 may not necessarily have been attributable to turgor-driven plastic deformation. If there were populations of cells within the leaf at different developmental stages, with predictably higher yield thresholds (Y) in older cells than in younger cells, an alternative explanation may be proposed. With decreasing turgor, the pressure in an increasingly large number of cells would fall below the yield threshold value. If a turgor switch was operating (similar to that apparent in the Zhu and Boyer study) there would have been zero growth where P - Y 5 0. Cell expansion would occur at a rate limited by cellwall metabolism where P - Y > 0 without any further regulatory significance being necessarily attached to turgor. If this latter interpretation is valid, then a decrease in leaf growth, such as that in Fig. 9 caused by increasing transpiration rate, would be attributable to a decrease in the number of cells rnomentarily contributing to leaf expansion. We can postulate that the expansion of an organ might be manipulated by varying the pressure in its constituent cells even though turgor pressure may have no regulatory role beyond a discrete ON/OFF switching. This argument illustrates the possible inadequacy of discussing growth regulation at the cell level from measurements of extension growth on whole organs and from turgor pressure calculated from tissue samples (cf. Spollen etal., 1993; section IVB). Such a n approach does not provide information for estimating parameters and variables in models of regulation at the single-cell level. We have discussed two possible ways in which a step-decrease in humidity
250
A. J . S . McDONALD and W. J . DAVIES
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Time (h) Fig. 10. Time-series of dependence of leaf expansion rate upon humidity. Note that the rate (slope) is less at lower humidity. A direct measure of turgor would confirm that this is because of a reduction in cell turgor. Wall loosening has been unaffected by the humidity (turgor) change and leaf size at the end of the run is the same as would have been attained had the humidity been maintained at 68% throughout. Unpublished data of the authors for Betula pendula.
about a dicot. shoot might cause a temporary reduction in the extension growth of leaves. If such a reduction is followed by a step-increasein humidity to its original value, a recovery in extension growth is observed (Fig. 10). Initially, the rate of extension is in excess of the original value but, after a delay, the original rate of extension is resumed. Apparently, total extension over the period investigated is unaffected by perturbations in turgor. (Although not discussed by Zhu and Boyer, this phenomenon is also apparent from their data.) Irrespective of the manner in which turgor affects growth (ON/OFF switching or plastic deformation), it is apparent that the longer-term rate of extension growth in Fig. 10 was dependent upon something other than turgor. Presumably, this related to cell-wall metabolism. What then of Lockhart? In our view, the most convincing data currently available to help us understand the regulation of cell growth are those reported by Zhu and Boyer (1992) where turgor did not have a regulatory role beyond a switching function. We know of no data where cell turgor and growth measurements are of sufficiently high spatial resolution that we can argue for a turgor-dependent regulation of cell growth in organs of higher plants. Thus we find no reason to pursue the Lockhart equation as a framework for analysing growth response to environmental perturbations. However, it is possible
PLANT RESPONSES TO WATER AND NITROGEN SUPPLY
25 1
that a degree of turgor-dependent plastic deformation can occur in the short term but not predictably in the longer term. A possible scenario is thus one where the long-term metabolically controlled rate of irreversible expansion may be punctuated by irreversible deformations associated with short-term variation in turgor pressure. It remains one of the more immediate challenges in plant science to demonstrate whether or not turgor pressure has a regulatory role in the expansion of cells in higher plant organs. What are the alternatives to Lockhart? It has become clear that to make progress in understanding the factors regulating plant growth requires increased understanding of the biochemistry of the cell wall, coupled with the development of a model of yielding properties at the molecular level (Fry, 1989; Passioura and Fry, 1992; Passioura, 1994). The main postulates of Passioura’s latest model are (i) that there are two functionally disparate populations of hemicellulose molecules in the wall that tie cellulose microfibrils together: those that are taut and load-bearing and those that are slack and not load-bearing; and (ii) that there are enzymes that cleave or loosen the loadbearing molecules. Passioura suggests that as the wall stretches during growth, slack molecules become taut, are recruited to load bearing and thereby stiffen the wall. Stiffening can be undone by enzymes that cut or loosen load-bearing tethers. The rate of expansion of the cell wall can depend on the frequency with which load-bearing molecules are loosened, a function of the activity of wall-loosening enzymes which may be a highly dynamic variable, and presumably also a function of tension which can also unzip hydrogen bonds between molecules. Expansion rate will also depend on the number of loadbearing molecules that are recruited per unit increase in wall length. This latter property may depend on the composition of the wall (e.g. the ratio of cellulose to hemicellulose) and could be modulated on a time-scale pertaining to synthesis of new wall material. Our current understanding of the biochemical basis of wall stiffening and loosening during growth is described below. Data are now accumulating t o allow us to analyse the effects of different environmental variables on the growth of plant parts in terms of variation in the properties described above. In some cases we have information on specific wall biochemistries in conjunction with improved measurements of turgor in cells of the growing zone. A great deal of progress has been made in recent years with the development of the cell pressure microprobe (see e.g. Steudle, 1993). Using this instrument, it is possible to measure directly the turgors of individual growing cells and even to investigate the spatial distribution of turgor within the elongation zone of an organ. Indeed, in view of the advances being made in single cell measurements, it is unlikely that measurements at the tissue level comprising cells of different developmental stages can any longer be motivated in growth studies where the regulating model pertains to cell turgor and the wall phenomena of single cells. It remains a challenge to adapt the use of the pressure probe to a wider variety of plant cells and measurement environments than have so far been investigated.
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A. J. S. McDONALD and W. J. DAVIES
With the combined awareness of how wall chemistry may relate to loosening and yielding phenomena, and with an improved resolution of turgor measurement, it has become possible in recent years to make progress in understanding the nature of growth response to perturbations in variables such as water and nitrogen supply and make more definitive statements about regulatory mechanisms than was previously possible. These mechanisms are now discussed in the following sections.
B. GROWTH OF ROOTS AND SHOOTS WHEN WATER SUPPLY IS RESTRICTED
When plants experience a reduction in water availability from the soil, shoot growth is often more inhibited than root growth (Sharp et al., 1988). The total growth rate of the plant might be expected to decrease but, in some cases, the absolute biomass of roots has been shown to increase relative to the biomass of well-watered controls (Sharp and Davies, 1979; Malik etal., 1979; Blum etal., 1983). Absolute increases in root growth probably only occur under rather specialized circumstances where carbohydrate supply would otherwise be limiting root growth. Where shoot growth is restricted but photosynthesis is able to continue, increased carbohydrate supply from the shoot to the root might be expected. It is generally accepted that this situation can arise during soil drying because the effects on stomatal behaviour and photosynthesis are not manifest until shoot growth is very significantly restricted (Hsiao, 1973). It is important to understand why shoot growth is generally more sensitive to drought than stomatal behaviour and also ask why root cells are able to continue to expand under circumstances where shoot-cell growth is restricted. There is no dispute that, under many circumstances, reduced water availability in the soil will result in reduced leaf turgor and a reduction of growth. However, it is difficult to know the extent to which the limitation in growth can be attributed to reduction in turgor. Indeed, from our previous discussion (section IVA), it is apparent that a causal dependence of growth on turgor is not necessarily to be expected. It is interesting to note that, in a range of grass species, restricted growth has been found in low water potential treatments, despite the complete maintenance of turgor in the growing cells as a result of osmotic adjustment (e.g. Michelena and Boyer, 1982; Nonami and Boyer, 1989). This result indicates that cell walls in the leaves become less yielding under reduced water availability. Results of Nonami and Boyer (1990a,b) support this view. Appreciation of this point has considerable significance for those interested in developing genotypes for drought-prone environments. It has been suggested that selection of plants showing effective osmotic adjustment in aerial plant parts can result in yield advantage because turgor maintenance can be equated with growth maintenance at low water potential. However, if growth at low water potentials is actually restricted by wall yielding, then we might expect to find solutes accumulating in shoots that have stopped growing or where growth rates have been slowed (see Munns, 1988).
PLANT RESPONSES TO WATER AND NITROGEN SUPPLY
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Relative reduction in leaf elongation rate Fig. 1 1 . Relationship between leaf osmotic adjustment and the relative reduction in leaf elongation rate in wheat over 7 days after withholding water from the soil during which the roots were ( ) or were not (0) pressurized to maintain a high leaf qW. From Kuang et al. (1990).
In fact, this is the case, and growth sensitivity to water deficit can commonly be related to the degree of osmotic adjustment in a genotype, with those genotypes showing the greatest solute accumulation being those in which growth is most significantly reduced by water deficit (Fig. 11; Kuang etal., 1990). This is not to say that selection for osmotic adjustment in aerial plant parts cannot result in yield advantage for other reasons, such as the postponement of cell death or the avoidance of desiccation damage to reproductive plant parts. The results described above indicate that, if we are t o understand how low water potential treatments restrict growth of above-ground plant parts, we must understand how the yielding properties of cell walls are regulated. The same is also true for roots. Here, several workers have shown that, following immersion of roots in solutions of low water potential, root elongation recovered more rapidly than root-tip turgor, indicating that the low water potential treatment actually increased the yielding properties of cell walls, maintaining growth at low water potential (Hsiao and Jing, 1987; Itoh etal., 1987; Kuzmanoff and Evans, 1981). It seems extremely important to understand why the wall properties of leaf and root cells respond so differently to a given treatment.
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A . J. S. McDONALD and W. J . DAVIES
A series of well-controlled studies has now confirmed early suggestions (Weaver, 1926) and shown that root growth is intrinsically less sensitive to low water potential than is the growth of the shoot (Westgate and Boyer, 1985; Spollen et af., 1993). In these experiments, small seedlings were grown in moist vermiculite under non-transpiring conditions so that water potentials of roots and shoots were in equilibrium with the water potential of the vermiculite. Under these conditions, growth and water relations of roots and shoots can be monitored with precision. In the maize primary root (the best-studied root system in investigations of the effects of low water potential on growth), longitudinal expansion rates are maintained preferentially towards the apex of the root, even when the water potential is reduced to - 1.6 MPa (Sharp et af., 1988). This results in a shorter elongation zone than at high water potential (Fig. 12). Pressure probe measurements show that cell turgor is essentially constant throughout the elongation zone, suggesting spatial variation in wall-yielding properties over only a few millimetres. Turgor at -1.6 MPa is 50% less than at -0.02 MPa, showing that, while osmotic adjustment was not complete, it was sufficient to maintain growth close to the apex (Fig. 12). Sharp et al. (1990) and Voetberg and Sharp (1991) have investigated osmotic adjustment in the growing regions of maize primary roots at low water potential, They found that concentrations of the amino acid proline were particularly high towards the apex of the root where elongation rates have been shown t o be completely maintained over a wide range of water potentials. Proline accumulation accounted for almost half of the osmotic adjustment in the apical millimetre of roots growing at -1.6 MPa, indicating that proline deposition plays an important role in the maintenance of P - Y > 0 and root growth at low water potential. Ober and Sharp (1994) have recently shown that application of the carotenoid synthesis inhibitor fluridone to root tips held at low water potential will decrease proline accumulation throughout the root. This suggests that ABA may play a regulatory role in maintaining the osmotic status of roots at low water potential. This is particularly interesting because Saab et af. (1990, 1992) have shown that ABA plays important roles in both the maintenance of root growth and the inhibition of shoot growth in maize seedlings at low water potential. Again, fluridone has been used to reduce the accumulation of endogenous ABA at low water potential and the effect of this treatment is to reduce the growth rate of roots and increase the growth rate of shoots at a water potential - 1.6 MPa. These authors have described spatial variation in growth and ABA distribution through the root and mesocotyl. In both cases, low water potential treatment reduces the length of the growing zone. In roots, fluridone treatment reduces this further. In the mesocotyl, fluridone treatment restores the growth rate almost to that of the well-watered control. Comparison of the ABA distribution and the ABA profile suggests a gradient in sensitivity of the growing cells to the ABA treatment. Young cells are relatively insensitive, compared to more mature cells. It should also be noted
PLANT RESPONSES TO WATER AND NITROGEN SUPPLY
255
0.7 0.6
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Fig. 12. (a) Spatial distribution of growth through the growing zone of primary roots of maize seedlings in vermiculite at the water potentials shown. (b) Turgor pressure of cells in the growing zone of the maize roots described in (a). From Spollen and Sharp (1991).
that Saab et al. (1990) report a relative insentivity to ABA of growth processes of plants maintained at high water potential. Later in the chapter, we discuss the importance of sensitivity variation in the hormonal response as a more general phenomenon. In section IVA, we discussed the necessity of making relevant measurements of growth and physiology in understanding growth regulation in plant organs. The work of Sharp and colleagues on the regulation of root growth at low
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A. J. S. McDONALD and W. J. DAVIES
water potential shows the importance of being able to assess spatial variation of cell properties in this kind of study. Spollen eta/. (1993) noted that one of the results of treating a growing zone as a homogeneous mass and plotting overall root elongation rate versus root-tip turgor can be the conclusion that elongation rate is reduced as a function of a reduction in turgor when in fact the reduced elongation rate of the whole root is simply a result of a reduction in the size of the growing zone. If we are to explain the maintenance of cell growth rate close to the tip of the maize primary root, even when the turgor is reduced by around 50%. it is necessary to argue that wall yielding is increased in this region. Passioura’s molecular model of cell-wall properties allows us to think of such changes in terms of an enzyme cleaving tethers between adjacent cellulose molecules. One enzyme that has recently received much attention in this context is xyloglucan endotransglycosylase (XET) (Fry et al., 1992). This enzyme has the property of both cutting and rejoining xyloglucan polymers which bind strongly to cellulose and are usually long enough to link adjacent microfibrils (Fry, 1989). Spollen et al. (1993) and Wu eta /. (1994) report on the spatial distribution of XET activity in the apical few millimetres of maize primary roots growing at two water potentials (Fig. 13). At low water potential, activity was significantly enhanced immediately behind the apex, where wall yielding is thought to increase, In contrast to this result. Pritchard and Tomos (1993) report that maize roots growing in mannitol at -1.OMPa showed no change in XET activity compared to roots growing at higher water potential. In their study, however, root-tip turgor was completely maintained and therefore this result may not be entirely inconsistent with the study discussed above. Wu etal. (1994) show that treatment of maize roots at low water potential with fluridone delayed the increase in XET activity at low water potential. This effect was largely overridden when internal ABA concentrations were restored by external application. The loss of activity associated with decreased ABA was associated with inhibition of the deposition of activity. This calculation allows the determination of the deposition rate profile that must occur to maintain the density of XET in the face of tissue expansion and displacement from the root apex. The calculation is necessary to show that the effects of ABA are due to variation in XET synthesis and/or activity and not to accompanying changes in root morphology. To demonstrate that the reported variation in XET activity is important for the maintenance of root growth at low water potential it will be necessary to alter XET activity in vivo. From other systems, there is some evidence against XET as a wall-loosening enzyme supporting wall extension (McQueen-Mason et al., 1993). Long-term extension activity of the isolated cucumber cell wall was not promoted by this enzyme but was dependent on a novel class of proteins named expansins (McQueen-Mason etal. , 1992). Wu et at. (1994) report that preliminary experiments have also shown that expansin-like activity is also increased in the maize root apical region by low water potential treatment.
PLANT RESPONSES TO WATER AND NITROGEN SUPPLY
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d 2
4
6
8 1 0 1 2
Distance from apex (mm)
Fig. 13. Spatial distribution of xyloglucan endotransglycosylase (XET) activity in the apical 10 mm of maize primary roots at high \k, ( , 0; -0.03 MPa) or low \k, (A; - 1.6 MPa, 48 h after transplanting). High 'Ir, measurements were made either 20 h ( ; development control) or 48 h (0; temporal control) after transplanting. XET activities are expressed o n the basis of fresh weight (FW; a), total soluble protein (b), and cell wall dry weight (DW; c). Data are means f SE (n = 3 [high *,I, n = 4 [low +,I. From Wu etal. (1994).
25 8
A. J. S. McDONALD and W. J . DAVIES
Passioura (1994) has suggested that the effects of changing water status on expansion rate may be mediated by changes in the hydration of the cell wall. Shrinkage at low water potential may inactivate enzyme molecules by restricting their freedom of movement. Some changes in cell-wall components have also been reported in roots held at low water potential. Net synthesis of cellulose and other cell-wall polysaccharides may be inhibited in both shoots and roots (e.g. Zhong and Lauchli, 1988) as water potential falls but the amount of particular proteins in the cell wall can increase (Bozarth et al., 1987; Surowy and Boyer, 1991). In soybean hypocotyls, genes for both a 28- and a 31-kDa protein were expressed at high water potential, while the production of the 28-kDa protein was up-regulated by water deficit. In the roots, the mRNA for the 31-kDa protein accumulated as water potential fell. The cDNAs for these messages were homologous to those of vegetative storage proteins. The authors suggest that they may also have a dual function as growth proteins. Surowy and Boyer also report on increased expression of an H+-ATPase mRNA in roots of soybeans at low water potential. Such an enzyme could play a role in the acidification of the apoplast that may be necessary for the maintenance of growth. Creelmann and Mullet (1991) reported differential expression of a proline-rich cell-wall protein in shoot and root elongation zones of soybean. In roots, low water potential reduces the expression of this gene, while the converse is true in the elongation zone of the hypocotyl.
C. GROWTH OF ROOTS AND SHOOTS WHEN N SUPPLY IS RESTRICTED
When plants experience a reduction in N supply, the growth of shoots is reduced more than that of roots and, in terms of dry matter increment, the growth rate of the whole plant decreases (e.g. Ingestad and Lund, 1979). This is not a general phenomenon with regard to all nutrient stresses. Deprivation of other mineral nutrients (e.g. K, Mg) can be associated with little or no shift in favour of root growth and can even result in higher shoot to root ratios than are found in control plants (Ericsson and Kahr, 1993; Cakmak etal., 1994). However, with respect to N deprivation, the observation is similar to that previously made with respect to reduction in soil-water availability (section IVB). Apparently, for a given degree of N deprivation, the extent to which a shift in dry matter partitioning occurs can depend upon the type of plant (cf. Aerts, 1994). This may possibly relate to inherent differences in modes of phloem loading (Van Be1 and Visser, 1994). In studies where plant growth has been manipulated by varying the flux of Mg, the allocation of dry matter between roots and shoots has been affected by the amount of N absorbed (T. Ericsson, pers. comm.). Where excess uptake of N was decreased, allocation of dry matter to the root increased while plant growth rate was still being limited by Mg supply. This may indicate a central
PLANT RESPONSES TO WATER AND NITROGEN SUPPLY
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role for N in determining the allocation of dry matter between shoots and roots, even where other nutrients are limiting the growth rate of the plant. Total dry matter increment of the plant seems to be a function of the total N availability to the roots and it may be unimportant that N deprivation occurs about one part of the root if N supply is increased by the same amount around another part. In split-root studies, Samuelson et al. (1992) showed that the growth rate of the plant (Hordeurn vulgare) was proportional to the total amount of N absorbed, irrespective of how it was supplied t o two parts of the root. The two parts of the root system, however, grew at quite different rates, in response to the amount of N that they were absorbing. With time, this resulted in a stable root to shoot ratio at the whole-plant level (Fig. 14). It was also noted that the frequency of lateral roots and their ultimate length was unaffected by reduced supply of N. However, laterals took much longer to develop and attain final length at lower N supply than at higher supply. Following N deprivation, an inhibition of expansion growth can proceed more rapidly than the shift in dry matter partitioning (Fig. 15, McDonald et al., 1986a). Because it was concurrent with accumulation of starch in leaves, this rapid inhibition of shoot expansion in Betula pendula was not attributed to a carbohydrate limitation of expansive growth. However, it has yet to be definitively demonstrated that, following N deprivation, carbohydrate supply is in fact non-limiting to the growth of cells in individual, expanding leaves. Presumably, carbohydrate metabolism and transport phenomena associated with, for example, the availability of xyloglucan in the growing cell wall could be crucial. It has been observed that N deprivation leads to rapid reduction in the expansion of single leaves of Helianthus annuus (Fig. 16, Palmer et a f . , 1996). Here, reduction in leaf expansion was attributed to reduced cell growth in older expanding leaves but may also have partly been attributable to a reduced rate of cell production in younger leaves. Reduction in final leaf size was accounted for by a decrease in the size of epidermal cells in Salix viminalis (McDonald, 1989). However, reduced cell numbers were more important in accounting for decreased blade extension in N-limited Festuca sp. (MacAdam etal., 1992). We conclude that cell division and cell expansion, can both be reduced following N deprivation and that reduced numbers and activity of meristems may assume increasing importance with time in inhibiting the development of shoot area (cf. Dale and Milthorpe, 1983). Here, we confine our thoughts to the regulation of cell expansion following N deprivation. At the outset, it can be stated that the quality of physiological and growth information available in the context of N deprivation is lacking compared with that for soil drying. We do have a good understanding of morphological development following N deprivation (cf. Clarkson and Touraine, 1994; Rooney, 1994) but it is apparent that critical studies of growth, physiology and biochemical gradients within the growth zones of leaves and roots have
260
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--
J. DAVIES
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0, .a) 5 c
0
P
40
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z
20 Yo
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5
10
15
20
Days of N addition
Fig. 14. (a) Relative growth rate (RGR) of whole plant, root and shoot as a function of relative addition rate of nitrate (RA) in standard cultures. Dashed line: RGR = RA. (b) Weight proportions of subroots of split-root cultured plants growing at RA 0.09d- , with the nitrate addition divided at a ratio of 20:80 between the subroots, as a function of time. Also shown are data from a culture with perturbed nitrate additions. From Samuelson et al. (1992).
to be carried out before definitive statements on the regulation of cell expansion can be made. Radin and Boyer (1982) addressed the question of possible mechanisms of growth reduction in leaves of H. annuus when plants were grown in solutions of lower-N concentration compared with leaves on plants at higher-N
PLANT RESPONSES TO WATER AND NITROGEN SUPPLY
26 1
0.4
0.3 0.2 0.1 h
a,
0.0
cn
a .-K
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a,
0.2
c
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a
5 0.0
a
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10 20 Time (d)
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40
Fig. 15. Time-series of relative rates of increase (d-') in (a) leaf nitrogen ( 0 ) . root nitrogen (0)and total plant nitrogen (+); (b) leaf starch (a), root starch (0) and plant starch (+); (c) in root dry weight (O), plant dry weight (+) and in leaf area ( ). On Day 0, the relative rate of increase in nutrient supply decreased from 0.20 d - ' to 0.05 d-I. The dashed vertical lines denote four stages of the experiment. From McDonald et al. (1986a).
concentration. (The problems with this approach to studying the effects of N deprivation are dicussed in section 11.) They found that the hydraulic conductivity of the root was decreased at lower N concentration and that, during periods of high transpiration, the water potential of growing leaves was reduced in plants at lower N compared to leaves of plants at higher N. They concluded that the reduction in calculated turgor may have accounted for the observed reduction in leaf expansion at lower N. In the light of the discussion
262
A. J. S . McDONALD and W. J. DAVIES
A
B
m
E
a
ij
F
c'
0
3
6
9
12
1 5 0
3
6 ; 9 12
15
t step
Time (Days in unit) Fig. 16. Time-series of leaf area and turgor in leaf epidermal cells of sunflower plants grown at (A) 26g N N-' d'-' or grown at (B) 26g N gN-' d'-' and then transferred to 0.04 g N g N-'d-'. The dashed vertical line shows the occasion, tstepr of decrease in nitrate supply. Data are for the first leaf pair (0)and for the second leaf pair (V). From Palmer et al. (1996).
in section IVA, we might conclude that this may have been attributable to either a reduced turgor-driven rate of plastic deformation or to expansion ceasing in a number of cells where P - Y I0. More recently, Palmer etal. (1996) measured turgor with a pressure probe in epidermal cells of growing leaves of H . annuus following a rapid depletion of N about the roots. Palmer did not find a significant difference in turgor between cells measured either before or after N deprivation or between cells in similar leaves of control and N-deprived plants (Fig. 16). Here, it would seem likely that the rapid reduction in leaf expansion was attributable to changes in cell-wall properties. At present, we do not know the reason for the discrepancy between the data of Radin and Boyer and those of Palmer but suppose that it may relate either to the
PLANT RESPONSES TO WATER AND NITROGEN SUPPLY
263
measurement methods, the nature of N deprivation or to some unidentified interaction. Other attempts have been made to identify mechanisms regulating leaf expansion following N deprivation (Taylor et al., 1993; Stadenberg et al., 1994) but interpretation of the data is difficult. This is because growth has been measured at the organ level (without targeting specific growth zones) and mechanisms a t the cell level have been inferred from measurements of water relations and mechanical properties made at the tissue and organ level. We conclude that priority must be given to direct measurements of turgor and relevant wall biochemistry in well-defined regions of growth in plants where N availability has been varied. This is necessary if definitive statements about the regulation of cell expansion, consistent with current models of cell-wall growth, are t o be made with respect to N deprivation. It is possible that effects of N supply on cell-wall growth relate directly to N substrate limitations to the synthesis of wall enzymes such as XET and other wall proteins in the expansin class (see section IVB). It is also possible that N deprivation may be more indirect, affecting the activity of XET and expansins in the cell wall at the time of N decrease. Hormonal balance (ABA and cytokinins) in the plant can be affected by perturbation in N supply (cf. Clarkson and Touraine, 1994) and increased ABA concentrations in plants with decreased availability of N or all nutrients have been reported (Goldbach etal., 1975; Mizrahi and Richmond, 1972; Radin e t a f . , 1982; Chapin etal., 1988a,b; Thorsteinsson etal., 1990; Palmer et al., 1996). The causal significance, however, of ABA involvement in regulating growth processes in the cell wall following N deprivation, has yet to be established. An interesting possibility is that N deficiency may accelerate the process of cross-linking wall components, thus making the wall less yielding. MacAdam et al. (1992) provided evidence of a link between growth deceleration and a dramatic increase in the activity of peroxidase enzymes in the meristems of tall fescue leaves. Chaloupkova and Smart (1994) recently reported the ABA stimulated induction of a novel peroxidase in Spirodela. Apparently, induction is antagonized by high cytokinin concentrations.
V.
IMPLICATIONS A. SINK STRENGTH
Because the initial events in cell expansion are associated with loosening in the cell wall and, because there is no apparent mechanistic basis for assuming a carbohydrate limitation to the activities of wall-loosening enzymes and proteins discussed above, it may be more logical to think of shifts in carbohydrate partitioning between shoot and root growth as a consequence of differential wall-loosening activities rather than a cause of such. Following a decrease in
264
A. J. S. McDONALD and W . J. DAVIES
nitrate supply, the proportional decrease in average-cell length was found to be less in the root cortex than in epidermal cells of the leaf in sunflower (unpublished data of the authors). This was associated with a decrease in plant-growth rate and a shift in dry-matter partitioning in favour of root growth. If the wall-loosening events produce important components of sink strength by, for example, creating sites for the subsequent incorporation of xyloglucan, then differentials in loosening could create differentials in sink strength, ultimately affecting the directional fluxes of sucrose. With time, other components of sink strength such as secondary wall thickening and the differential activity of root and shoot meristems will presumably contribute to the fate of sucrose in the phloem. However, we propose that part of the shift in dry matter partitioning in favour of root growth may be a direct result of differences in sink strength induced by differences in the extent to which the expansion of root and leaf cells is inhibited by N deprivation or a restriction in water supply.
B. REGULATION OF N BALANCE
It is apparent from longer-term studies of growth response to N availability that acclimation tends to confer a stability of C : N ratio in the whole plant. Under conditions of demand limitation, Touraine eta/. (1994) cite case studies with root temperature (Clarkson et a/., 1986), sodium-chloride salinity (Helal and Mengel, 1979; Luque and Bingham, 1981; Touraine and Ammar, 1985), aluminium toxicity (Tan and Keltjens, 1990) and soil pH (Schubert e t a / . , 1990). The common finding to all of these studies is that growth rate was limited by a variable in the root environment but N concentration in the plant was unaffected. Under conditions of demand-control, enhancement of N uptake by some roots can be obtained by withdrawing N from the solution surrounding other roots (cf. Touraine e t a / . , 1994). The general conclusion is that some internal regulation ensures that N0;uptake matches the N requirements of the plant in growth. A similar conclusion might be drawn from the data of Ingestad and McDonald (1989) where birch plants were grown over a wide range of photon flux density. Here, different growth rates were achieved but very similar plant N concentrations were found where N was made freely available in solution to plants which had different C assimilation rates (cf. McDonald etal., 1992). However, it should be noted that there are examples where growth limitations caused by one mineral nutrient do not always result in conservation of N concentration. For example, Goransson (1994a,b) reported significantly higher N concentrations in birch at growthlimiting supplies of Mn or Fe, where N was made freely available in solution. Stability of plant N concentration has also been reported as an acclimated response to sustained N deprivation (e.g. Ingestad and Lund, 1979; Ingestad and McDonald, 1989). The split-root studies of Samuelson e t a / . (1992)
PLANT RESPONSES TO WATER AND NITROGEN SUPPLY
265
indicate that, at supply limitation, it is the total N absorbed, irrespective of how it is made available to different parts of the root system, that confers stability to the prevailing C : N ratio of the whole plant. The actual value and stability of plant-N concentration may best be considered as a consequence of regulatory activity in the C and N uptake systems, without necessarily conferring any importance to the maintenance of plant N concentration as a set point. Lawlor (1994) makes this point with regard to relating mechanisms of CO, and N0;assimilation to C :N ratios in the whole plant. Rooney (1994) comes to a similar conclusion with regard t o plant development, Although there is much evidence that indicates that the external C and N supply influences development, there is little to support the importance of C : N ratio per se in the regulation of morphological changes and stages through which a plant progresses. Presumably, there are concurrent feedbacks of the end products of C and N metabolism in shoots on both the subsequent uptake of C in leaves and N0;in roots but there may be no mechanistic reason for assuming a complete matching of these fluxes. Larsson (1 994) discussed the combined significance of acclimation of V,,, for N0;uptake and changes in root extension to the further acquisition of N at supply limitation. In the type of hydroponic culture system used by Larsson and his colleagues, the shift in favour of root extension following N deprivation may be of little consequence to the further uptake of N. However, if what is being observed reflects an adaptation to reduced NO; availability, then the findings are presumably of significance to the uptake of nitrate from a soil, where root extension allows new soil volumes t o be exploited. An interesting calculation has been made by Larsson (1994) of relative V,,,, in which he relates the uptake capacity to the amount of N in the plant. This allows a discussion of uptake capacity with respect to the instantaneous rate of NO; uptake which is required to maintain a given plant RGR (Fig. 17). The conclusion from the data of Mattsson etal. (1991) is that relative V,,, is far in excess of the uptake requirement at low supply rates associated with low plant RGR but approaches the actual uptake requirement at highest supply rates associated with highest plant RGR. Because these are steady-state responses of the uptake system and root extension, it may be concluded that, on NO;-deficient sites, the plant can maintain a high scavenging capacity for available NO;. Interestingly, the relative V,,, at the lowest supply rate is sufficient to accommodate an NO; uptake which would shift the plant from a low RGR to a maximum RGR, should a flush of sufficient NO; occur (McDonald and Stadenberg, 1994: calculated from the data of Oscarson et al., 1989). Down-regulation of uptake capacity at higher supply rates (Mattsson eial., 1991) resulting in a relative V,,, which approximates the uptake requirement for steady-state growth might be interpreted as an economy of energy investment in the uptake system. The data of Oscarson etal. (1989), where V,, was maintained at a high value, indicate that down-regulation at high supply rates may not always be the case. The extent to which there may
266
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2.5
-k c
2.0
increased carrier density _ increased R.T increased tissue N conc
-
increased metab reg
[I
U
-*
1.5
I
-
0
I
0.10
II
>
)n
0.20
RA (d.') Fig. 17. The relative V,,, for net nitrate uptake in vegetative barley cultures grown at different relative addition rates of nitrate (RA). Relative growth rate (RGR) values included for comparison. Directions of physiological responses (observed and putative) of significance for the relative V,,, are shown in the top of the figure. From Mattson etal. (1991) and Larsson (1994).
be a species difference in the trade-off between regulation of V,,, and root extension in the maintenance of high uptake capacity for the whole plant, is predictably an interesting phenomenon contributing to the competitive fitness of individual plants and species on sites of variable N supply. Over a very wide range of N supply, the acclimated response can be such that plant growth rate, in terms of biomass increase per plant N and unit time (plantN productivity), tends to be conserved (e.g. Ingestad, 1979). This is in the same range of N supplies for which a tendency towards the conservation of photosynthetic rate (leaf area basis) has also been found and means that the regulation of leaf extension by N supply can be associated with a conservation of photosynthetic rate and plant-N productivity. Apparently, this acclimation can coincide with that for NO; uptake such that a capacity sufficient to accommodate the N requirement for sudden, rapid growth can be maintained. C.
REGULATION OF WATER USE EFFICIENCY
Implicit in our discussion of the regulation of growth and C gain in response to soil drying is the assumption that plant water status is maintained within
PLANT RESPONSES TO WATER AND NITROGEN SUPPLY
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a range favourable to growth. We understand that below some critical water status threshold, the plant may be damaged or at least growth and gas exchange will be limited. In a competitive situation, however, there is little to be gained by conserving water which will be used by competitors although, once captured, efficient use of water can convey some advantage. The concept of water-use efficiency is relevant here (see Davies and Pereira, 1992). Much has been written about the instantaneous water-use efficiency of different species and we know that closure of stomata can increase instantaneous water-use efficiency under certain conditions (see e.g. Jones, 1993). The early writings of Jones (1976) and Cowan and Farquhar (1977) show clearly that maximizing water-use efficiency on a day-to-day basis does not necessarily mean that the instantaneous ratio between transpiration and C gain must always be maximized. Further analysis suggests that over the longer term, plants may regulate growth and stomatal behaviour in an optimal fashion with respect to the amount of water available in the soil and, to some extent, the uncertainties in the future environment (Cowan, 1982). Regulation of this kind requires that the plant has some means of estimating the amount of water available in the soil. Such mechanisms are discussed in the following section. Jones and Sutherland (1991) and Jones (1993) suggested an alternative kind of regulation. This involves a trade-off between open stomata for high assimilation and closing stomata to prevent damage to the water conducting system (Tyree and Sperry, 1989). The model proposes that high productivity could be achieved by closure of stomata only when a proportion of xylem vessels are embolized. Beyond this point, catastrophic xylem failure is avoided by stomatal closure. Since such catastrophic failure rarely if ever occurs in natural environments, Jones (1993) suggests that a major function of stomata may be to avoid this phenomenon. It is interesting that the model proposed by Jones and Sutherland was able to explain many of the well-known features of stomatal response to soil drying, including stomatal regulation by soil water status and atmospheric humidity rather than by leaf water potential.
VI.
INFORMATION TRANSFER A.
RESPONSES TO SOIL DRYING
When the soil dries, water uptake by the roots declines and this reduction in water supply will eventually result in the development of a water deficit in the shoot. The extent of this water deficit will depend not only on the extent to which water supply is restricted but also on the rate at which water is lost from the shoot. This is determined by the evaporative demand of the atmosphere and the diffusion resistance provided by the Ieaf. We have described above some experiments where stomatal behaviour is linked to the water supply from the soil rather than to the water status of the shoot and in these plants, the
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A. J . S. McDONALD and W. J. DAVIES
water status of the shoot is often controlled by the stomata, such that shoot water potential does not vary as the soil dries. Plants that exhibit this type of behaviour can be described as isohydric, while those that show reduced water potential as soil dries are anisohydric. At least part of the stomatal control shown by anisohydric plants in drying soil can be a response to the changing water status of the shoot. Because of the influence of both edaphic and climatic variables on plant water status, it is difficult to see how the plant can assess the water supply from the soil from the shoot water status. During the day, this can vary significantly over a very short time-scale and cannot therefore be a suitable developmental regulator. Predawn shoot water status is much more stable and may therefore be a more suitable regulator but in very dry soil this variable may come into equilibrium for only a short time before the end of the dark period. Quite recently, several authors (see e.g. Davies and Zhang, 1991) have suggested that interaction between the plant and drying soil may generate a chemical signal which can move from the root to the shoot to provide a measure of soil water status and/or soil water availability. There are many possible candidates for such chemical signals (see section VIIB) but most attention has been given to the possibility that the plant growth regulator ABA can act as a signal in this context. We know that ABA is synthesized in increased quantities in root cells as they dehydrate (e.g. Cornish and Zeevaart, 1985) and that ABA will move in the transpiration stream to the shoot to regulate both stomatal behaviour and growth. For potted maize plants, Zhang and Davies (1989) have shown a relationship between soil water content and the ABA content of roots in contact with this soil, while Tardieu etal. (1992a) have shown a clear relationship between xylem ABA concentration and predawn leaf water potential for plants growing in the field. When xylem ABA concentrations are plotted against soil water availability for the field crop, there is a clear relationship for non-transpiring plants (sampled at night). For plants sampled during the day, the ABA signal seems to depend upon soil properties as well as the water status of the soil, as different relationships are obtained for plants in well-mixed and compacted soil (Fig. 18). This is presumably a function of different water fluxes through the roots of plants in the two types of soil. Clumped roots in compacted soil might be expected to dry the soil locally and generate stronger ABA signals as roots dehydrate further and water fluxes are reduced. These data indicate that the ABA signal cannot provide the shoot with an absolute measure of the amount of water available in the soil. Rather, the signal seems to indicate the access that plant roots have to soil water, a property that depends both on soil water status and on the distribution of roots in relation to the water. Figure 18 also indicates that the ABA signalling system is relatively insensitive to soil drying, which we would expect if stomata are not to be partially closed most of the time. It has been suggested (Passioura, 1988) that root signals may be generated as a result of an interaction between
PLANT RESPONSES TO WATER AND NITROGEN SUPPLY
500
.
400 '
300' 200 '
.
Predawn
1
0
269
Day time
40 80 120 Transpirable soil water (mm)
Fig. 18. Relationship between abscisic acid (ABA) concentration in the xylem and transpirable soil water beneath a crop of maize plants growing in the field in France. Data was collected before dawn o r during the day. and 0 represent plants grown in soil with low mechanical impedence and A and A represent plants growing in soil with a compacted plough layer. From Tardieu et al. (1992a).
the root and the changing physical properties of the soil. This is clearly not the case in the system described in Fig. 18, as the root signalling pattern during the day is different from that generated at night, suggesting a response to root dehydration. We would not necessarily expect the physicaI influence of the soil to vary between the day and the night. Although there are many papers that report relationships between xylem ABA concentration and stomatal behaviour of droughted plants (e.g. Loveys 1984; Zhang and Davies, 1990; Khalil and Grace, 1992, 1993), the many feedbacks in the control system mean that perturbations in the system do not always produce responses that are easily predicted. Tardieu and Davies (1993) modelled the control of stomatal behaviour by ABA signals and described a system that is comprised of five simple equations (Fig. 19). ABA is generated in the roots by dehydration of the root cells as the soil/root interface resistance
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- J, = (w, - VJ 1R, [ABA] = a y, (J, + b)
Fig. 19. Representation of variabIes and equations of control in an interactive model describing water flux through maize plants [model described by Tardieu and Davies (1993)l. Input variables: a,,, net radiation; T, and T,, air and dew point temperatures; *s, soil water potential. R, and R,, are the plant and the soil-plant resistances to water flux. Unknowns: g, , stomatal conductance; and \k,, root and water flux; [ABA], concentration of abscisic acid in the leaf water potentials; .Iw, xylem. Other symbols are constants (see Tardieu and Davies, 1993). Arrows symbolize transfers of water and/or ABA.
is increased. The concentration of ABA in the xylem is determined by the extent of root dehydration and the flux of water though the plant. This latter variable is important because we might expect that on hot dry days high rates of water loss will dilute the signal. Evidence for this is provided by our observation of a relatively stable xylem ABA concentration as the day progresses (Tardieu et al., 1992b). The effects of increasing root dehydration on ABA concentration in the xylem are apparently offset by increasing water flux as evaporative demand increases through the day. In the field, stomatal conductance is commonly restricted late in the day and it is not easy to reconcile this observation with the relative stability of
PLANT RESPONSES TO WATER AND NITROGEN SUPPLY
27 1
ABA concentration in the xylem. It appears that this can be done if the sensitivity of the stomata to the ABA signal is made to vary with time of day, as is apparently the case in the field (Tardieu and Davies, 1992). We have shown that this variation may be a function of varying shoot water potential (see section VIIC) and the model therefore calculates stomatal conductance as a function of ABA concentration in the xylem with a variable sensitivity depending on shoot water status. We can therefore explain limitation in conductance late in the afternoon as a function of increased stomatal sensitivity to an ABA signal as the leaf water potential falls. Output from the model (Fig. 20) of Tardieu and Davies (1993) shows control of stomata on a diurnal basis t o be largely a function of sensitivity variation, with increased restriction in conductance on a daily basis as the soil dries and the baseline concentration of ABA in the plant increases. Interestingly, shoot water potential is also controlled by the system. A crucial role for sensitivity variation is highlighted when the model is run with chemical control alone (no interaction with water status to increase sensitivity). This version of the model fails to control plant water potential and fails to restrict stomatal conductance to any appreciable extent (Tardieu, 1993). Our analysis seems to suggest that plants growing in the field can regulate stomatal behaviour as a function of the access that plant roots have to water in the soil. The extent to which the stomata respond to the root signal will depend upon a n interaction with leaf physiology and the microclimate around the leaf and may be viewed as a sensitivity modulation. The nature and extent of this type of interaction is discussed further in section VIIC. Control of stomata can be important in the regulation of gas exchange, particularly in rough, relatively uncoupled canopies. The system of regulation that we have described above will also be important in as much as the ABA concentrations in the plant and the water status of the shoot will also be regulated. This can have important consequences for development of plants growing in situations where water supply is restricted. There has been considerable interest in the possibility that chemical or genetic modification of the plant’s ABA balance in the field will modify its response to soil drying and possibly modify its drought resistance (e.g. Quarrie, 1991). Despite much early promise, it is not clear that even quite substantial genetic variation in the capacity for ABA accumulation has a significant influence on plant growth and development. This may be because of the many feedbacks between the water and the ABA relations of the plant (e.g. Tardieu, 1993) which may mean that a capacity to accumulate large amounts of ABA will not always be realized. Recent work on Mediterranean forages by Puliga etal. (1996) has highlighted one character that may be important for the control of leaf growth in the field. It seems that summer-active species - that is, those that continue to produce leaves even when soil drying is quite severe - may show a restricted capacity to produce ABA when expressed as a function of soil water status, even though the ABA production per unit of
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a
8
12 Time (h)
b
16
20
8
12
16
20
Time (h)
Fig. 20. Simulations of the daily pattern of stomata1 conductance (gs),water flux in the soil-plant atmosphere continuum (J,,,), leaf and root potentials (9, and qr), abscisic acid concentration in the xylem sap ([ABA]) and ABA flux to the leaf (JAB*). Soil water potential (9) is -0.3 MPa, meteorological conditions (net radiation, R,, and air vapour pressure deficit, VPD) are those measured on 26 July 1990. Soil characteristics: silty clay loam. (a) Calculations with values of soil-root resistance to water transfer (Rsp) simulating a root system in favourable conditions. (b) Calculations with a value of R,, multiplied by 20, simulating unfavourable characteristics of the root system for water uptake (such as root clumping). From Tardieu and Davies ( I 993).
plant water deficit is not unusual. These data seem to provide further evidence that plants may regulate their growth and development in the field as a function of a measure of soil water availability and that there may be some genetic variation here that may be of interest.
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B. RESPONSES TO N LIMITATION
1. Stomata1 conductance and leaf growth We consider two possible types of signal to N deprivation. The first is a possible role for ABA, either on its own or in conjunction with cytokinins, and the second relates to the possible importance of hydraulic signals in the plant. There is evidence that these may not be independent variables. Apparently, an external supply of ABA can increase the hydraulic conductivity (Lp) of root membranes (Karmoker and Van Steveninck, 1978; Pitman and Wellfare, 1978; Glinka, 1980). It might be supposed that a synthesis of ABA in response to N deficit might therefore oppose the apparent decrease in hydraulic conductivity but, as Clarkson and Touraine (1994) point out, this apparent paradox may presumably be resolved by distinguishing between hydraulic conductivity of root cell membranes at the site of ABA synthesis in the root tips and the hydraulic conductance of other (the vast majority) of root cells effectively isolated from this region. It is also possible that shifts in Lp of roots may cause changes in the water potential of the leaf, triggering synthesis of ABA in the leaf (cf. Clarkson and Touraine, 1994) or causing shifts in the compartmentation of leaf ABA. The combination of simultaneous increase in ABA and decrease in cytokinins might constitute a strong signal in response to N deprivation. Jackson (1993) recently reviewed the occurrence of decreased cytokinin synthesis in roots following mineral nutrient shortage. A number of studies were cited in support of the earlier findings of Kulaeva (1962) that cytokinins had a central role in delaying leaf senescence and that, following N deprivation, cytokinin synthesis in roots can be reduced. In two separate studies with Solanurn tuberosum, Sattelmacher and Marschner (1978) and Krauss (1978) showed that NO; deficiency induced decreases in cytokinin-like activity and increases in ABA in the xylem, respectively (cf. Clarkson and Touraine, 1994). Importantly, Radin etal. (1982) found that increased application of cytokinin decreased the sensitivity of stomata to ABA in response to water stress. Thus, there is evidence that the ABA :cytokinin ratio may constitute a strong signal but one that is not necessarily specific to N deprivation.
2. Nitrate uptake There is no evidence for effects of ABA on the NO;-uptake system. Chapin et al. (1988a,b) applied ABA externally and found no effect and, even where small changes in the ABA concentration of roots were measured after N deprivation, these were not in phase with changes in NO; transport. In mutants that are defective in ABA synthesis, the NO; transporter activity increased in ways similar to wild-type tomato. Thus, Clarkson and Touraine (1994) make the important point that the NO; transport responses appear to be controlled separately from the morphological responses. At demand limitation, the evidence is in support of a shoot-sourced signal producing feedback
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inhibition of N uptake. A consequence of NO; assimilation in the shoot is a stoichiometric synthesis of amino acids and organic acids. Both these components are among the main nitrogenous components of phloem sap (Hall etal., 1971; Richardson etal., 1982) and thus are potentially important as regulatory signals of NO; uptake (Touraine et al., 1994). Touraine et al. (1994) discuss the stimulation of NO; uptake in the root by carboxylates transported in the phloem from the shoot. Details of the mechanism by which carboxylate (mainly malate), imported into the roots, stimulates NO; uptake are still not clear. Anions are believed to enter root cells via transporters with protons or via antiports with hydroxyl ions or bicarbonate ions at the plasma membrane (Clarkson, 1986; Glass, 1988). The overall control loop in the whole plant relates to the models of Dijkshoorn et al. (1968) and Ben Zioni et al. (1971) in which a cycling loop of K ions supplies the accompanying cations for NO; in the xylem and carboxylate ions in the phioem. Decarboxylation of malate in the root leads to the formation of bicarbonate ions which are released into the external solution. The tight stoichiometry between NO; absorbed and hydroxyl released provides a regulatory possibility. There is good evidence that amino acids in the phloem can play a regulatory role in the uptake of NO;. Touraine etal. (1994) make the important point that the relative isolation of the cycling pool of amino-N might make its composition sufficiently specific to convey information. Where protein synthesis is reduced in the shoot, both qualitative and quantitative changes in the aminoacid composition of the phloem may be expected. The idea is that an increase in one or more amino acids transported from the shoot will cause a downregulation of NO; uptake. The mechanism of this down-regulation is uncertain but it is thought to relate to transcriptional events rather than any direct effects of amino acids on the NO; transporter (see review by Touraine et al., 1994). It may be concluded that, although some details of mechanism remain obscure, a good working model for NO; uptake and its regulation in the whole plant at demand limitation can be assumed, in which the key components are a stimulation of uptake by phloem-sourced carboxylate and an inhibition of uptake by phloem-sourced amino acids (Fig. 21). Larsson (1994) suggests that the mechanisms behind the change in V,,, in phase I1 (Fig. 8) may be the same as those involved during N starvation of plants pregrown with a plentiful supply of NO;. He considers the possibility of a central role for the cytosolic NO; pool in control of NO; uptake. This possibility exists because the cytosolic concentrations are extremely low in phase I but rise sharply in phase 11. It also seems feasible that regulation at demand limitation, based on the phloem content of amino acids and carboxylate, might be relevant to that at supply limitation. A sudden decrease in NO; supply might initially stimulate the uptake system as a result of a decrease in the amount of amino acids and
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Fig. 21. A model for the control of nitrate uptake by leaf-generated signals during (a) rapid vegetative growth and (b) rapid pod fill. During vegetative growth (a), nitrate ions are rapidly absorbed by the root and transported via the xylem to the leaf. In the leaf, nitrate reduction produces organic acids (OA) and amino acids (AA). Most of the newly formed OA are translocated to the root where a carboxyl group is released in exchange for a nitrate ion, whereas the newly assimilated N is incorporated primarily into leaf N compounds. During rapid pod fill (b), leaf proteolysis occurs and much of the amino N in the leaf is exported to the filling pods. Consequently, the phloem is enriched with amino compounds, which repress nitrate uptake and consequently diminish the rate of nitrate reduction. From Ismande and Touraine (1994). a n increase in the amount of carboxylate cycling via the phloem. This would be consistent with the commonly reported increase in V,,, following NO; deficiency. However, with time, a decrease in carbon assimilation by the shoot a n d changes in C and N usage in growth will occur which might result in changes in the composition of the amino acid pool. It is possible that longerterm changes in the composition of amino acids in the phloem, associated with retranslocation a n d cycling of N (cf. Mattsson etal., 1991), might then downregulate the uptake system, giving the type of acclimated response in phase I discussed by Larsson (1994).
VII. WHAT IS IN THE XYLEM SAP AND HOW CAN CHANGES IN WATER AND N AVAILABILITY CHANGE THE XYLEM SAP CONTENTS? A.
COLLECTION OF XYLEM SAP
One substantial difficulty in assessing the effects of any root perturbation o n the concentrations of the different substances in the xylem stream is to take
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samples for analysis without disrupting contents or concentration (see e.g. Jackson, 1993). Xylem sap is commonly expressed from cut leaves or cut stumps under pressure (e.g. Zhang and Davies, 1990) or under partial vacuum (Pate et al., 1994) and may also be collected from bleeding stumps exhibiting root pressure (e.g. Loveys, 1984). All of these techniques may be criticized on the grounds that plants are severed before sampling so that the transpiration stream is no longer moving through the plant part sampled. It is argued that this can result in the concentration of substances in the xylem and Else et al. (1994) demonstrated that this may be a problem when sap is sampled from relatively small roots of tomato. These authors also demonstrated that contamination of xylem sap samples with sap from damaged cells at the cut surface can also lead to a substantial overestimate of concentrations of hormones in the xylem stream. Tardieu and others sampled xylem sap from large maize leaves from plants grown in the field (e.g. Tardieu etal., 1992a). These experiments revealed large effects of transpiration flux on the concentration of ABA in the transpiration stream and these effects have been elucidated using a destructive sampling technique. These authors argued that xylem sap samples from such large leaves can be taken from sap which was present in the xylem before the leaf was sampled. Of course the amount of redistribution of xylem contents between xylem conduits and xylem parenchyma is unknown. It is generally accepted that problems of the concentration of xylem sap as a result of sampling non-transpiring plants can be avoided if the whole plant pressure chamber shown in Fig. 2. is used to sample sap (see e.g. Munns, 1989; Jeschke and Pate, 1991). Here, a small overpressure can be applied to roots to force sap from the cut tip of a transpiring leaf. Of course, the pressure chamber cannot be used with field-grown plants but it probably is a good idea to validate particular sap collection methods against the pressure chamber to check for overestimations of concentration.
B. SOIL DRYING AND N DEPRIVATION AND EFFECTS ON XYLEM CONTENTS
I . Plant hormones As we have discussed above, considerable attention has been given to the possibility that plant hormones can move in the xylem stream to act as signals to the shoot of the availability of soil water. Many authors have reported that soil drying can increase xylem sap ABA concentrations by several orders of magnitude (see e.g. Wartinger et al., 1990; Davies and Zhang, 1991; Khalil and Grace, 1992, 1993; Jackson et al., 1995; Correira and Pereira, 1995). Munns (1989) suggested that the high concentrations of ABA detected by these authors may be artefactual due to the techniques used for sap collection but similar or even greater concentrations were detected by Schurr et al. (1992) in sunflower sap sampled with the whole plant pressure chamber. In all of the studies cited above and in many others, generally good correlations were
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reported between xylem sap ABA concentration and stomatal conductance. If we are to argue for a regulatory role for ABA, it is important to determine that this is something more than a correlation and that it is the ABA that is influencing the stomata and not the converse. In their model of chemical control of stomatal behaviour described above, Tardieu and Davies (1993) show that there can be a relationship between stomatal conductance and the concentration of chemical constituents in the xylem, even when these constituents have no effect on conductance. This relationship arises simply as a result of the concentration of constituents as stomata close and transpiration is restricted. The activity of xylem sap constituents can be tested by comparing concentration :response relationships produced by droughting the plants with those generated by applying the constituent of interest externally. Zhang and Davies (1990) have done this for ABA in maize plants and found an excellent correlation between relationships generated by the two methods. Further confirmation that the constituent in question is active and indeed is the only active component can be obtained by removing it from the xylem sap sample and testing the remaining sap for activity in an appropriate bioassay. Munns and King (1988) have done this with xylem sap extracted from wheat plants. ABA was removed from sap samples with a immunoaffinity column but when sap was tested in a transpiration bioassay, much antitranspirant activity still remained (Fig. 22). Similar results were obtained by Trejo (1994) working with Phaseolus. Subsequent experiments by Munns (1992) suggest that there is similar non-ABA growth-inhibiting activity in the xylem stream of wheat and Chandler etal. (1993) report on the enhanced expression of dehydrins as a result of treatment with sap extracted from droughted plants but with ABA removed. Although there has been some confusion over whether this non-ABA activity can be found in newly collected xylem sap (Munns et al., 1993), it has recently been suggested that it may be some kind of ABA-adduct that could release free ABA in the leaf under certain conditions (Munns and Sharp, 1994). Netting et al. (1992) reported on the existence of such a compound but unequivocal identification has not yet been achieved. Zhang and Davies (1 991) failed to find any non-ABA-like antitranspirant activity in the xylem sap of maize plants (Fig. 22) but they did filter their sap to remove large molecular-weight compounds which blocked up the xylem and caused wilting and it is therefore possible that they also removed any ABA-adduct by this process. Interestingly, Trejo (1994) used an identical filtering procedure in their experiments but still detected non-ABA antitranspirant activity. In the experiments reported above, xylem sap collected from well-watered and droughted plants of a variety of species contained ABA at concentrations which are between micromolar and nanomolar. In most experiments, micromolar ABA will close stomata but this is not always the case and Trejo et al. (1993) have shown that this is because of the activity of the mesophyll cells
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-2 -
100
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c
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80
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0
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Q)
60
_-
c
9 40 --
20
--
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3
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::::u+)
100
:
::4 : : : J
1000
ABA concentration (nM)
Fig. 22. Transpiration of detached wheat leaves as a function of concentration of abscisic acid (ABA) in assay solutions. The solutions were: synthetic ABA in artificial xylem sap (O), xylem sap from well-watered maize plants (m), xylem sap from unwatered maize plants (A),and xylem sap from unwatered maize plants but with ABA removed by immunoaffinity colum (A). The dotted line shows the dose response of detached wheat leaves to synthetic ABA solutions. The crossed point is a typical result for xylem sap extracted from unwatered wheat plants and fed to detached wheat leaves. Data are expressed as percentages of the rate of water loss from control leaves fed with artificial xylem sap only (no ABA) and are means of five observations. The bars are double standard deviations divided by the mean transpiration rate of the control leaves. From Davies and Zhang (1991).
in metabolizing and compartmentalizing ABA. If the mesophyll tissue is removed and ABA is applied to epidermal tissue alone, stomata will respond to ABA concentrations as low as lo-'' M (Trejo etal., 1995). These results suggest that there is enough ABA in xylem sap of well-watered plants to close stomata, assuming that the sap reaches the guard cells without modification. Clearly, since stomata are not permanently closed, this cannot be the case. Hartung and coworkers (see e.g. Hartung and Slovik, 1991) have worked hard to elucidate the factors influencing compartmentation of ABA in the leaf. We know comparatively little, however, about ABA metabolism. It seems likely that the regulation of these processes will have an important controlling influence on stomata1 behaviour by determining what proportion of ABA in the transpiration stream gets through to the guard cells. We need more information on how rates of metabolism and compartmentation might be
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influenced by the daily pattern of variation of climate and by soil drying. When one considers the massive flux of ABA into a leaf on a daily basis, it is clear that the removal of ABA from sites of action in the leaf must be extremely effective. Nevertheless, Gowing et af. (1993) show that stomatal behaviour of cherry leaves is influenced by the amount as well as the concentration of ABA arriving in the leaf. Trejo et af. (1995) report a similar phenomenon for Commefina epidermis with the mesophyll removed. When whole leaves received different fluxes of different ABA concentrations, stomatal responses could be interpreted mostly as a function of a concentration response. This indicates that we are justified in using concentration as a variable in the analysis of control of gas exchange. This is not to say that we should not estimate flux in an attempt to demonstrate unequivocally that signalling is taking place (see Jackson, 1993). Thus far, we have concentrated on the role for ABA, a growth inhibitor, in the process of signalling between roots and shoots. The possibility that root to shoot signalling may also involve the modification of the transport of promoters has also received considerable attention. Indeed, much of the early work in this field concerned the possibility that cytokinin transport from roots to shoots could be reduced by soil drying. Cytokinins are required for the normal functioning of shoots and may be required for the opening of stomata. Incoll and Jewer (1987) considered the possible importance of cytokinins in the drought responses of stomata and made a strong case for their involvement. The effects of augmenting the supply of cytokinins on stomatal behaviour can be seen particularly in older leaves, where cytokinin contents may be declining (Blackman and Davies, 1984) and there is some suggestion that modification in cytokinin supply may modify the responses of stomata to C 0 2 . Blackman and Davies (1983) showed that high cytokinin concentrations could reduce stomatal responses to COz and suggest that the observation that soil drying enhances the C 0 2 response of stomata may be explained by a soil dryinginduced reduction in the supply of ABA to leaves. We have noted above that relatively mild soil drying may enhance the concentration of ABA in the xylem stream by perhaps two orders of magnitude, making this molecule a powerful candidate for a signal molecule in this context. It seems unlikely that even quite severe soil drying can do more than reduce cytokinin transport by 50 or even 75%. We can perhaps argue that along with many other components of the xylem stream, this molecule has a role in providing the shoot with a general indication of root functioning. The potency of these compounds as growth regulators perhaps make them particularly important in this regard. There has been very little attempt to address the importance of cytokinins in the control of stomatal behaviour in field-grown plants. Fussader et af. (1992) suggest that these compounds may be important. 2. Ions, amino acids and pH Although many authors have addressed the possibility that part of the influence
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of soil drying on the physiology of droughted plants may be a function of reduced uptake of ions (e.g. Turner, 1986), it is only since the development of the whole plant pressure chamber that it has been possible to quantify reliably the concentrations and fluxes of different ionic components of the xylem sap as plants are exposed to increasing degrees of soil drought. Gollan et al. (1992) sampled sap from turgid, transpiring sunflower plants and found that concentrations of NO; and orthophosphate decreased with soil water content, whereas concentrations of the other anions remained unaltered (Fig. 23). Calcium concentrations also decreased, presumably as a function of the relative immobility of this ion in the soil as the soil water content is reduced. A similar argument can be made for orthophosphate (Nye and Tinker, 1977). K, Mn, Mg and Na concentrations in the xylem sap were not affected by soil drying. The pH, the buffering capacity at a pH below 5 and the catiodanion ratio increased after soil water content fell beyond a certain point. Amino acid concentration of the xylem sap increased as the soil dried, although the concentration of amino acids transported in the xylem sap of sunflower is negligible compared to the NO; concentration. Jeschke and Pate (1991) used the whole plant pressure chamber to collect xylem sap from transpiring castor bean seedlings. NO; concentrations and concentrations of reduced N were measured at various levels up the shoot in xylem exudates and in phloem sap. An experimentally based modelling technique allowed some quantification of N fluxes into different leaves and it was apparent that younger leaves were heavily dependent on xylem import for their N requirements. In the N-flux experiments performed by Palmer eta/. (1996) and described earlier, an abrupt decrease in N supply to roots caused a very abrupt decrease in NO; concentration in the xylem and therefore in NO; flux into leaves. It seems possible, therefore, that reduced NO; flux in the xylem could act as a chemical signal to the shoots of NO; deprivation around the roots.
C.
INTERACTION AND THE CONCEPT OF SENSITIVITY VARIATION
In most studies of the effects of environmental perturbation on the physiology, growth and development of plants, the effects of individual environmental variabIes are considered in isolation. As we focus in on the mechanisms of response it is necessary to become more and more reductionist in our approach. It has, however, become increasingly clear that many of the plant responses to stress are whole-plant phenomena and that by focusing on cellular and even molecular responses in isolation, we risk being unable to elucidate the whole story. Work with whole plants presents problems but these are issues that we must address if we are to understand how plants respond to perturbations in the natural environment. In the context of the present chapter, a central issue is the extent to which responses of plants to soil drying can be
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28 1
Fig. 23. Relationship between the concentrations of the cations: potassium, magnesium, calcium and sodium, and the anions: nitrate, phosphate, sulphate and chloride, and the soil water content in the xylem sap samples of eight plants. Values of significance P < 0.001 are indicated. From GolIan eta/. (1992).
understood simply in hydraulic terms and the extent to which we must consider the importance of the various chemical effects that are produced by soil drying. We have made a case here and in other writings for a central role for ABA in the regulation of functioning of droughted plants. Nevertheless, as we have discovered more and more about the operation of the hormonal control
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system, it has become necessary to introduce concepts such as sensitivity of response t o explain what we see. For example, the responses of root cells to ABA, reported by Saab el al. (1992) seem to show clear increases in sensitivity as cells age. It seems important to examine further the idea of sensitivity variation and determine the extent to which this idea will allow us to account for whole-plant responses to deprivation in water and N supply. In our discussions above, we have noted that increases in endogenous ABA concentration occur both in response to soil drying and in response to a reduction in N supply. We examine in turn interactions involving ABA which are important in the context of the regulating physiology, growth and development of plants deprived of water and/or N. We also examine the possibility that N may act in its own right as a plant hormone. I . Interactions between the effects of ABA and plant water relations In their work on the behaviour of maize stomata in the field, Tardieu and Davies (1992) noted that stomata were apparently more sensitive to endogenously produced ABA during the afternoon hours than during the morning. Burschka etaf. (1983) made a similar observation in their experiments where ABA was applied to field plants at different times throughout the day. Working in the laboratory, Tardieu and Davies (1992) were able to show that the basis of this response was an apparent interaction between ABA and the water status of the plant, such that stomata became more sensitive to ABA as leaf water potential declined. These experiments were performed with isolated epidermal strips held in polyethylene glycol solutions at different water potentials. Trejo and Davies (1994) confirmed these results with experiments performed with whole leaves. Here, water potentials were reduced by adding a fine capillary to the end of a petiole of detached Phaseolus leaves. A reduction in water potential of only a few tenths of a megapascal brought about a significant increase in sensitivity of the stomata to ABA. This appeared to be a relatively dynamic response, however, as closure of stomata caused rehydration of the leaves and the difference of stomatal sensitivity to the ABA dose decreased. There are many reports of variation in stomatal sensitivity to ABA following drought stress. A general response seems to be that drought stress or ABA treatment reduces stomatal sensitivity to ABA (see e.g. Dorffling et al., 1977) but this is not always the case (e.g. Peng and Weyers, 1994). Work by MacRobbie (1990) on ion fluxes through guard cell membranes shows that following an initial challenge with ABA, a repeat application after as long as 28 min has no effect on ion flux. This work and the work of Tardieu and Davies (1992) suggests a fundamental variation in sensitivity of guard cell membrane processes to ABA application. This may have to d o with binding of the hormone on the guard cell but we have little information on binding sites for this hormone and therefore, speculation in this area is unrewarding. One further complication is current uncertainty over the loca-
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tion of ABA-binding sites on guard cells. The work of Hartung (1983) seemed to show conclusively that ABA binding was restricted to the outside of the plasma membrane of the guard cell. Anderson et al. (1994) seemed to confirm this conclusion, but work by Allan et al. (1994) suggested that there may be some binding on the inside of the membrane. Much ABA can be sequestered in the guard cell and if this pool of the hormone is important for guard cell functioning, then we need to reassess our whole concept of stomatal functioning in stressed plants. Many reports of apparent variation in stomatal sensitivity to ABA dose may simply be a function of variation in the amount of ABA reaching the guard cells (see e.g. Trejo et al., 1993). We have discussed above how compartmentation and metabolism can influence apparent sensitivity and it is clearly important to distinguish between these two types of sensitivity variation. We know that soil drying will result in an increase in pH of the xylem sap (Hartung et al., 1988) and we would expect this to release ABA from sites of sequestration in the leaf (e.g. mesophyll cell chloroplasts) to increase the availability of the hormone t o the sites of action on the guard cell. It may be then that water deficit and other stresses can influence both fundamental and apparent sensitivity of guard cells to ABA. One other very important effect of plant water status on the plant’s sensitivity to ABA is shown by Saab etal. (1990) who reported that ABA will act to sustain root growth at low water potential but has little effect at high water potential. Saab and coworkers offer no explanation for this phenomenon but it seems likely that cellular water status has caused a fundamental change in cellular properties such that the binding of the hormone or the signal transduction chain have been influenced. In fact, the situation may be even more complicated than this.
2. Interactions between the effects of ABA and other chemical components of the xylem sap We have noted above that experiments with the whole plant pressure chamber have revealed substantial effects of soil drying on the ionic status of xylem sap. Osonubi etal. (1988) have shown similar changes for spruce trees growing in the field. It is well known that stomatal functioning depends upon perturbation of ion fluxes across guard cell membranes and that changes in the mineral nutrition of plants (e.g. K nutrition and Ca nutrition) can be shown to influence stomatal behaviour (Atkinson et af., 1990). Nevertheless, it seems likely that changes in ion composition seen as a result of soil drying may not be large enough to function on their own as modifiers of stomatal behaviour. The possibility of interaction with ABA effects have been considered, however, and Radin etal. (1982) have shown that both N and P deficiency can enhance stomatal sensitivity to an ABA signal. These possibilities have been addressed in greater detail by Schurr etal. (1992) who note that for their
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J. DAVIES
\
Fig. 24. Stornatal conductance of individual plants versus abscisic acid (ABA) concentration in their xylem sap during the first day with significant reduction of leaf conductance due to soil water depletion. From Schurr et al. (1992).
sunflower plants exposed to a soil drying treatment, a single relationship between ABA in the xylem sap and leaf conductance could not be shown. Rather, a series of relationships was found for individual plants (Fig. 24). These differences were explained by variation in the ionic status of xylem sap. Both the concentration of NO; and Ca in the xylem sap were positively correlated with sensitivity to an ABA signal and pH was negatively correlated with sensitivity. These changes are of a very substantial magnitude and whiIe we have some mechanistic understanding of the interaction between the effects on stomata of ABA and Ca (De Silva etal., 1985; McAinsh etal., 1990), we have little understanding of how ABA and NO; can interact to influence stomata unless this is via effects of N metabolism on changes in the pH relations of the leaf (Raven and Smith, 1976). If this is the case, this will be an apparent change in stomata1 sensitivity, while the Ca interaction may be a more fundamental effect on guard cell ion fluxes. Trewavas (1981) raised the possibility that NO; should be classified as a plant growth substance in the sense that this term is used to describe auxins, gibberellins and the other major classes of growth substances. Of these substances, we have discussed signalling by ABA and cytokinins. Implicit in our discussion of the communication of information around the plant is that the source of the substance conveying the information is localized in one part of the plant and transport of the substance is to an area which is relatively defi-
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cient in that substance. These criteria can apply to both ABA (see e.g. Davies and Zhang, 1991) and cytokinin (see e.g. Incoll and Jewer, 1987) although even this is a controversial area (Trewavas, 1981). The criteria may not necessarily apply to N, where there is considerable internal circulation (Pate et al., 1979) but there is evidence that variation in NO; supply to leaves as a result of soil drying can regulate NO; reductase activity in maize leaves (Shaner and Boyer, 1976). These authors differentiated between an effect of NO; already in the leaves of these plants and that arriving in the transpiration stream. In other words, the NO; is acting as a signal in this system, rather than a substrate. We have no evidence that this is the case in the regulation of leaf growth as a result of soil drying but this possibility should be considered. Leaves generally have a very high N content compared to stems and roots, probably because of a substantial protein requirement for construction of the photosynthetic apparatus. Despite this, the apical transport of N in a wellwatered plant may exceed N use by only 20% or so (Schulze, 1991). We have seen above that when the soil dries substantially, the N supply to leaves can be very substantially restricted by a combination of a reduced concentration in the xylem stream and a reduced transpiration flux. It may be then that a link between a soil-drying induced reduction in N supply and a reduction in leaf growth can be postulated, with N acting as both a signal and a substrate. This will depend to some extent on the amount of N that can be remobilized from stores to make up for the shortfall in supply from the roots. In plants with a relatively small vacuolar store of N, this can be drawn down rapidly in response to inadequate external supply (e.g. Chapin et al., 1988a,b).
3. Interactions between the effects of ABA and temperature We consider here the interaction between ABA and temperature because of the link between high temperature and soil drying. This link may be particularly important in the control of leaf growth of grasses where the meristem is commonly at or even below the surface of the soil. As high insolation drives evapotranspiration, the soil and the leaf meristem will warm. Dodd and Davies (1994) showed that ABA supplied to the leaf through the transpiration stream is more effective as leaf temperature increases. Patterns of leaf growth of plants in drying field soil can be interpreted in terms of this interaction (see e.g. Gallagher and Biscoe, 1979). These authors reported that soil drying has little effect on leaf extension rate early in the morning when temperatures are low, while the soil-drying induced reduction in leaf growth is increased later in the day as leaf temperature increases (Fig. 25). These differences in apparent sensitivity may be explained by differences in the accumulation of ABA in the meristem of the leaf and/or the result of a fundamental variation in sensitivity of the growing cells to the ABA signal. A similar interaction between ABA and temperature was reported in a study of stomata1 responses of maize plants
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/ /
2.1
/
8
5
10
11
I
15
Temperature (“C)
Fig. 25. Two-hourly mean values of leaf extension rate (0-O), turgor potential (U-
0). and water potential ( A - A ) plotted as functions of meristem temperature on 16 May 1975. The numbers on the lines refer to the time at the start of the 2 h period. The dashed line AB represents the expected response of extension rate of temperature in the absence of water stress. From Gallagher and Biscoe (1979).
(Rodriguez and Davies, 1982) and Allan el al. (1994) recently noted that changing the growth temperature for plants may alter the responses of the guard cells to the hormone by changing the signal transduction chain.
VIII. CONCLUSIONS: AN INTEGRATED STRESS RESPONSE SYSTEM FOR THE PLANT? In this chapter, we have argued for the regulation of stomata1 behaviour and leaf growth of plants in drying soil by root-derived chemical signals. In our view, ABA plays a central role in this signalling system. There is now good evidence for enhanced ABA synthesis in roots and transport to leaves following soil drying. Increasing evidence suggests that some of this extra ABA may
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result from reduced N availability. Clearly, ABA does not operate in isolation. There is evidence that synthesis and transport of other growth regulators, some as yet unidentified, are affected by soil drying but we need much more information in this area. We need to quantify both concentrations and fluxes of regulators in plants subjected to soil drying and N deprivation. It appears that altered fluxes of some other xylem components (e.g. cytokinin, N and Ca) will influence stornatal behaviour and leaf growth in their own right but changes in the concentrations and fluxes of these substances may not be large enough to explain the sensitive responses of leaf growth and stornatal behaviour that are sometimes seen. We propose here that soil-drying induced variation in the xylem transport of regulators such as cytokinin, N and Ca will interact with a modified supply of ABA to regulate leaf growth and functioning. Chapin (1989, 1991) proposed a similar hypothesis. We extend his ideas by arguing a modulating role for leaf water status and for several climatic variables as well as for N. Figure 26 shows an outline of the control system. The production of ABA and cytokinin will reflect the extent of soil drying but the supply of these regulators to leaves will be highly variable depending on the transpiration rate of the plant (see Tardieu and Davies, 1993) and the penetration of the transpiration stream to the sites of action within the leaf (see e.g. Trejo etal., 1993). The sites of action for stomata are either on the inside or the outside of the plasmalemma, while there is some uncertainty over sites of action for the regulation of leaf growth. The degree of penetration of the hormones to the sites of action will depend on metabolism and compartmentation of the hormones. Variation in the sensitivity of response to what hormone does reach the site of action is another crucial variable in our system but we have little information on the mechanistic basis of this variation. Our integrated stress response system also contains several feedback responses which are discussed in detail above. Hormonal accumulation in roots will influence root growth and development which will affect uptake of both water and N. We also show feedback effects of the modification of growth and development and gas exchange on water and N availability and balance. There may also be feedback effects of growth on N uptake capacity. The variation in the sensitivity of stomata to an ABA signal reported by Schurr et al. (1992) can be of a very significant magnitude and so it is clear that the processes determining this are at least as important in our control system as variation in the supply of the major substrates for growth and variation in the supply of our main hormonal regulator, ABA. In the extreme, even the ABA content of the xylem sap of well-watered plants will close stomata and may well reduce leaf growth. To understand why this does not occur and how sensitivity is nearly always damped down is a major challenge for the years ahead. In recent years, the subject of root to shoot signalling has received a considerable amount of attention. Some authors, in focusing on these
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phenomena, have tended to forget that stomata and leaf growth can and probau1y
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between hydraulic and chemical control should be carefully considered. A shoot response to a modified chemical signal arriving from the roots and providing information on edaphic conditions is a simple but attractive concept. Nevertheless, this idea has never been able to explain the more dynamic minute by minute or even hour by hour responses of stomata, particularly where these are occurring in tall trees where a signal may take 10 days to arrive from the roots. The idea of sensitivity variation provides a more dynamic link between the root message and the climatic environment. In our revised formulation, the root signal in response to soil drying or N deprivation may provide a stable long-term regulation of development, while interaction with temperature, leaf water status etc. will determine the extent of short-term, dynamic response to the signal.
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AUTHOR INDEX
Numbers in italics refer to pages on which full references are listed at the end of each chapter
A Abbott, L.K., 12, 13, 14, 17, 18, 19, 20, 21, 35, 43 Abe, T., 186, 217 Adams, W. W., 143, 152 Adamse, P., 142, 149 Adir, N., 125, 126, 157 Aerts, R., 258, 289 Agren, G. I., 233, 293 Ahmad, M., 105, 149 Akizuki, M., 126, 130, 159 Alarcon, J. J., 164, 177, 180, 182, 184, 186, 189, 190, 191, 192, 194, 196, 21 7, 222, 223
Albrecht, T., 168, 189, 217, 224 Alexander, T., 31, 35 Alexandre, J., 77, 78, 88 Allan, A. C., 50, 56, 57, 88, 283, 286, 289
Allan, W. T. G., 57, 58, 59, 60, 68, 86, 94
Allen, E. B., 23, 24, 40 Allen, G. J., 79, 81, 82, 88 Allen, L. H., Jr, 122, 127, 161 Allsopp, N., 26, 35 Almeida, J. M., 130, 158 Ambach, W., 100, 150 Amijee, F., 3, 18, 19, 35 Ammar, 264, 299 Amos, W . B., 55, 65, 96 Amundson, J., 85, 88 Amundson, R. G., 114, 116, 156 Anderson, A. J., 3, 35 Anderson, B. E., 283, 289 Anderson, J.G., 98, 100, 151 Anderson, J. M., 101, 103, 105, 111, 113, 122, 123, 124, 125, 126, 127, 128, 129, 130, 134, 135, 141, 142, 152, 154, 155, 159, 170, 171, 217 Anderson, B., 125, 150
Annamalainathan, K., 148, 157 Anraku, Y.,5 1 , 60, 92 Anthony, R., 107, 111, 113, 114, 126, 129, 132, 134, 135, 136, 140, 154, 155
Aono, M., 120, 149 Appenroth, K. J., 130, 149 Armstong, G. A., 137, 138, 149, 161 Armstrong, J., 167, 217 Armstrong, W., 167, 217 Arwidsson, E., 243, 300 Ashford, A.E., 14, 40 Ashley, C. C., 56, 65, 90, 96 Assmann, S. M., 85, 88, 93 Atkinson, C. J., 283, 289 Augner, M., 166, 217 Augsten, H., 130, 149 Auh, C.K., 109, 156 Austin, J., 98, 149 Ausubel, F.M., 111, 136, 137, 153, 155, 159
Ayling, S. M., 50, 52, 56, 58, 88, 89 Azcon, R., 17, 28, 35
B Backster, C., 167, 217 Bajer, A., 51, 59, 92 Baker, A. J., 121, 131, 155 Baker, D. A., 170, 217, 221, 274, 292, 297
Baker, N. R., 241, 292 Balakumar, T., 143, 144, 149 Baldwin, I.T., 167, 217 Ball, J. T., 237, 291 Ball, N. G., 196, 217 Ballart, C. L., 106, 149, 167, 217 Barber, J., 125, 150 Barber, S. A., 245, 291 Barker, R. F.,188, 221
302
AUTHOR INDEX
Barkowitz, G. A., 238, 292 Barnes, J., 116, 117, 120, I50 Barnes, P.W., 101, 102, 103, 106, 122, 149, 150 Barry, P. H., 210, 217 Barry, W., 210, 225 Basiouny, F.M., 122, 150 Batschauer, A., 106, 107, 118, 150, 153 Battey, N. H., 86, 88 Baughan, R., 166, 225 Baydoun, E. A. H., 171, 189, 217 Baylis, G. T. S., 13, 28, 36 Baylor, S. M., 56, 92 Beale, M. H., 50, 56, 57, 88, 283, 286, 289 BCcard, G., 14, 31, 36 Becker-AndrC, 137, 158 Beech, S. M., 14, 36 Beeson, R.C., Jr, 127, 162 Beever, R. E., 6, 36 Beilby, M. J., 202, 217 Ben, G. Y., 241, 289 Ben Zioni, A., 274, 289 Bennet, A. B., 214, 227 Bennett, A. B., 179, 217 Benolken, R. M., 186, 214, 217 Bentrup, F. W., 164, 203, 209, 210, 213, 214, 217, 227 Beraud, J., 170, 217 Berger, F., 51, 58, 88 Bergounioux, C., 133, 159 Berridge, M. J., 47, 49, 56, 69, 74, 75, 76, 78, 79, 80, 83, 85, 86, 88, 91 Berry, J., 241, 300 Berry, J. A,, 237, 291 Berta, G., 9, 38 Bertl, A., 80, 89 Besford, R.T., 121, 141, 150, 161 Bethenod, O., 270, 299 Bethke, P . C . , 81, 89 Bethlenfalvay, G. J., 19, 36 Bevan, M., 170, 189, 227 Bevan, M. W., 170, 188, 189, 226 Bhartia, P. K., 98, 99, 153 Biel, M., 48, 92 Biggs, R. H., 122, 150 Bingham, F. T., 264, 294 Bird, G.St.J., 82, 83, 94 Biscoe, P. V., 285, 286, 291 Biyaseheva, A. E., 50, 59, 89 Bjorkman, O., 240, 289 Bjorn, L.O., 101, 144, 147, 157, I60
Black, R. L. B., 17, 21, 41 Blackbourn, H. D., 86, 88 Blackman, P. F., 233, 289 Blackman, P. G . , 279, 289 Blakeley, S. D., 107, I50 Blatt, M. R., 56, 57, 77, 84, 89, 95 Bleecker, A. B., 84, 89 Block, A., 137, 138, 148, 156, I 6 1 Bloom, J., 14, 37 Blum, A., 252, 289 Blumthaler, M., 100, 150 Blumwald, E., 80, 81, 82, 89, 91, 94 Boari, F., 165, 166, 173, 174, 179, 182, 184, 186, 187, 189, 190, 192, 195, 198, 205, 207, 218, 223 Bohnert, H. J., 106, 153 Bol, J.F., 148, 151 Bolter, C. J., 189, 218 Bonfante-Fasolo, P., 2, 5 , 13, 14, 30, 33, 36 Bonnemain, J. L., 170, 187, 225 Bono, J. J., 59, 91 Bootman, M., 86, 89 Bornman, J. F., 101, 103, 112, 113, 121, 122, 132, 133, 142, 146, 147, 150, 152, I57, 159 Borowicz, V., 23, 43 Bosac, C., 248, 299 Bose, J.C., 165, 166, 196, 209, 213, 218 Bossen, M . E . , 107, 108, 150, 15I Boudet, A. M., 77, 94 Boullard, B., 12, 14, 34, 36 Boulter, D., 188, 221 Bowes, G., 145, I51 Bowler, C., 46, 47, 85, 89, 108, 109, 120, 151, 157, 161 Bowles, D. J., 166, 171, 180, 188, 189, 190, 194, 204, 218, 227 Boyer, J. S., 143, 151, 171, 172, 173, 175, 176, 180, 188, 218, 223, 228, 240, 242, 247, 248, 250, 252, 254, 258, 260, 285, 289, 294, 295, 296, 297, 289, 298, 300 Boyer, N., 165, 220 Boynton, A. L., 75, 92 Bozarth, C. S . , 258, 289 Bradshaw, H. D., 165, 224 Brain, P., 31, 43 Brandle, J. R., 124, 151 Braun, J., 115, 160 Braunberger, P. G . , 19, 36
AUTHOR INDEX
Bray, C. H., 119, 159 Bream, J., 165, 218 Brearley, C.A., 76, 94, 108, I57 Brederode, F.Th, 148, 151 Brent Reeves, F . , 15, 41 Briantais, J . M . , 240, 241, 290, 292 Briggs, W. R., 105, 106, 109, 136, 138, 155, 158, 159, 167, 223 Brinck, R . W., 56, 92 Brini, M., 66, 94 Britikov, E. A., 165, 226 Britt, A. B., 119, 151, 152 Britz, S . J . , 142, 149 Broadbent, P., 116, 117, 120, IS0 Broadway, R. M., 188, 218 Brosnan, J . M . , 47, 77, 78, 80, 81, 89, 92 Brown, C.L., 172, 178, 228 Brown, M. F., 13, 37, 39 Brown, M . S . , 19, 36 Brown, S . B., 126, 151 Brown, W. H . , 214, 215, 218 Brownlee, C., 50, 51, 52, 56, 57, 58, 64, 73, 81, 86, 88, 89, 93, 94, 284, 294 Bruce, A., 19, 36 Brun, A., 170, 217 Brundrett, M. C., 1, 2, 9, 12, 13, 15, 26, 28, 30, 36, 37 Brune, W . H . , 98, 100, 151 Bruno, E., 106, 107, 150 Bruns, B., 106, I51 Buchanan, B. B., 116, 156 Burdon-Sanderson, J., 165, 171, 186, 218 Burg, E.A., 168, 218 Burg, S. P . , 168, 218 Burns, D. J . W., 6, 36 Burns, I . G., 245, 289 Burschka, C., 282, 289 Busa, W. B., 75, 78, 79, 85, 90 Bush, D. S . , 46, 47, 50, 51, 59, 89 Butchart, N., 98, 149 Butcher, R. W., 47, 94 Butler, W. L., 124, 153 Buwalda, J. G., 18, 37 C Cabrini, L., 121, 158 Cadenas, E., 127, I51 Cakmak, I., 258, 289
303
Caldwell, C. R., 146, 151 Caldwell, M.M., 101, 103, 104, 112, 113, 122, 124, 132, 142, 150, 151, 152, 153, 157, 158, 160, I61 Callaham, D. A., 51, 52, 56, 57, 58, 60, 86, 89, 93, 96 Camp, L. B., 103, 151 Campbell, A. K., 49, 50, 51, 66, 67, 68, 92 Campbell, W.F., 124, 151 Campos, J.L., 121, 150 Canaani, O., 124, 125, 126, 153 Canney, M. J . , 164, 171, 172, 175, 177, 218 Canut, H., 72, 77, 89, 95 Carling, D.E., 13, 37 Carpentier, J. L., 80, 95 Carrasco, A., 77, 89, 94 Cashmore, A . R., 105, 149 Catford, J.G., 31, 40 Catt, J. W., 130, 156 Cen, Y.P., 142, 152 Chae, Q., 50, 59, 89 Chaloupkova, K., 263, 289 Chandler, P. M . , 277, 289 Chang, C., 84, 89 Chapin, F. S., 26, 37 Chapin 111, F . S., 239, 240, 263, 273, 285, 287, 289, 290 Chappell, J., 137, 152 Chazen, O., 164, 176, 218 Cheeseman, J. M . , 190, 202, 218 Chen, J . J . , 119, 151, 152 Chew, F. S . , 30, 38 Chilvers, G. A., 14, 40 Chino, M., 170, 221 Chory, J., 110, 111, 141, I59 Choudhary, A. D., 138, 156 Chow, W. S., 101, 103, 105, 111, 113, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 134, 135, 141, 142, 152, 154, 155, 159 Chrispeels, M. J., 212, 218 Chua, N. H., 46, 47, 85, 89, 108, 109, 151, 157 Clapham, D., 85, 88 Clark, A., 80, 95 Clarkson, D. T., 6, 31, 37, 43, 50, 52, 56, 58, 88, 89, 239, 240, 243, 259, 263, 264, 273, 274, 285, 290, 299 Clay, K., 22, 37
304
AUTHOR INDEX
Cleland, R.E., 247, 297 Clepham, A. L., 277, 278, 279, 299 Close, T. J., 277, 295 Cobbold, P. H., 60, 65, 89, 90 Cody, S. H., 59, 91, 96 Collins, J . H., 56, 90 Coohill, T. P., 103, 152 Cooke, M.A., 15, 37 Cooper, K.M., 13, 14, 18, 37 Cordero, M. J., 165, 218 Cork, R. J., 59, 90 Cornic, G., 238, 240, 241, 242, 290 Cornish, K., 268, 290 Coronado, R., 80, 89 Correira, M. J., 276, 290 Corzo, A., 72, 73, 93 Cosgrove, D. J., 71, 73, 90, 166, 176, 201, 166, 219, 247, 248, 256, 290, 295 Costa e Silva, de, O., 137, 138, 161 Cote, G. G., 76, 77, 90 Coudron, C. A., 187, 228 Cowan, I.R., 171, 175, 176, 177, 219, 267, 290 cox, G., 4, 5, 37 Cox, H., 188, 220 Cox, R. C., 284, 291 Crain, R. C . , 76, 77, 90 Cramer, G. R., 164, 219 Creelmann, R. A., 258, 290 Criessen, G., 116, 117, 120, 150 Crush, J. R., 19, 37
D Dacey, J. W. H., 168, 219 Dale, J.E., 164, 219, 259, 290 Daley, P. F., 237, 291 Dalling, M. J., 245, 298 Dangl, J.L., 137, 156, 158 Daniels-Hetrick, B. A., 14, 37 Darden, E. B., 186, 226 Darlington, A. B., 171, 219 Darwin, C., 164, 165, 168, 202, 213, 214, 215, 219 Dassen, H . H . A . , 107, 150 Davies, E., 206, 228 Davies, E. C., 124, 142, 152 Davies, E. D., 166, 186, 187, 201, 219 Davies, H., 142, 152 Davies, W. J., 164, 177, 178, 219, 220, 233, 238, 239, 248, 252, 259, 262,
263, 267, 268, 269, 270, 271, 272, 276, 277, 278, 279, 280, 282, 283, 285, 286, 287, 289, 291, 295, 296, 297, 298, 299, 300 Davis, J. M., 170, 219 Davis, R. W., 165, 218 Davy, A., 21, 43 Day, T. A., 112, 152 De Wit, C.T., 274, 291 De Wit, P. J.G.M., 165, 219 Dean, N. M., 75, 92 Dean, R. A., 165, 219 Decaisne, dand, J., 216, 222 Deckmyn, G., 126, 152 DeLucia, E. H., 112, 152 Demmig-Adams, B., 143, 152 Depka, B., 124, 125, 154 DeRocher, E. J., 106, 153 Desbiez, M.O., 166, 219 DeSilva, D. L. R., 284, 291 Dexheimer, J., 6, 8, 10, 15, 40, 42 Dhankar, J. S., 252, 295 Dicke, M., 166, 219 Dickson, S., 5 , 34, 42 Digby, J., 166, 220 Dijkman, H., 166, 219 Dijkshoorn, W., 274, 291 Dilley, R. A., 143, 157 Ding, J. P . , 74, 90 DiPalma, J . R., 213, 219 DiPalrna, M., 213, 219 Dixon, M.A., 171, 219 Dixon, R.A., 138, 156 Dodd, I. C., 285, 291 Doherty, H.M., 166, 171, 180, 188, 189, 190, 194, 204, 227 Dohler, G., 130, 149 Dorffling, K., 282, 291 Dormer, K. J., 182, 226 Doubrava, N. S., 165, 219 Dove, S . K . , 77, 90 Downton, W. J. S., 237, 240, 291 Drobak, B.K., 76, 77, 90, 91 Drouillard, D. L., 29, 37 DubC, S.L., 146, 150 Duc, G., 9, 38 Duckett, J. G., 14, 22, 37, 41 Due, G., 164, 186, 220 Duffey, S. S . , 188, 218 Dumas-Gaudot, E., 9, 38 Dumbroff, E. B., 121, 158 Dunsmuir, P., 120, 160
AUTHOR INDEX
Dupont, G., 56, 69, 75, 78, 79, 86, 88, 91 Durachko, D. M., 256, 295 Durall, D.M., 26, 43 Dziubinska, H., 206, 228
E Eagles, G., 190, 204, 227 Easton, W. R., 23, 24, 40 Ebel, J . , 148, 154 Eckelkamp, C., 165, 220 Eckert, H. J., 124, 125, 157 Eckey-Kaltenbach, H., 110, 153 Edelman, M., 124, 125, 126, 153, 154 Edwards, J . H., 245, 291 Edwards, P. J . , 188, 220 Ehmann, B., 106, 110, 153, 165, 220 Ehrlich, B.E., 75, 90 Eilam, Y., 59, 91 Eisenberg, R., 80, 89 Eissenstat, D. M . , 3, 20, 23, 24, 29, 32, 37, 38, 40 Eliasson, L., 259, 260, 264, 298 Elliott, D. C., 52, 59, 90 Elliott, G., 26, 39, 100, 158 Else, M. A., 276, 291 Endrigkeit, A., 13, 37 Enstone, D.E., 28, 41 Ericsson, A., 243, 259, 261, 295, 300 Ericsson, T., 243, 258, 263, 291, 295, 299, 300 Erixon, K., 124, 153 Ernst, D., 110, 153 Esau, K., 177, 220 Eschrich, W., 187, 220 Etter, E. F., 56, 90 Evans, D.E., 83, 90 Evans, D. W., 17, 41 Evans, J . R., 243, 291 Evans, M. L., 59, 92, 253, 294 Evert, R.F., 187, 220 Ewers, F. W., 172, 226
F Fahey, D. W., 98, 100, 151 Falkenstein, E., 168, 220 Farham, J . C . , 98, 153 Farmer, E.E., 120, 153, 168, 189, 220 Farquhar, G. D., 235, 237, 267, 290, 291, 299
305
Fasolato, C., 69, 76, 82, 83, 90 Fautz, E., 148, 154 Fawcett, T. W.,129, 156 Fay, F.S., 56, 90 Feder, W . A . , 133, 146, 153 Fein, A., 75, 94 Feinbaum, R. L., 137, 153 Feiser, G., 115, 160 Feray, A., 170, 217 Ferguson, I. B., 77, 90 Ferris, R., 248, 299 Fewtrell, C., 69, 78, 79, 90 Feyerherm, S. M., 14, 37 Finch, S., 167, 220 Findlay, G., 80, 89 Findlay, G. P . , 73, 95, 165, 187, 188, 201, 202, 203, 207, 212, 214, 220, 221, 226 Firn, R.D., 164, 166, 220 Fitter, A. H., 22, 26, 31, 37 Flint, S. D., 101, 103, 104, 112, 122, 132, 142, 150, 151, 152, 153 Flockerzi, V., 48, 92 Flynn, L., 98, 99, 153 Fontana, A., 13, 36 Fontin, J . A . , 14, 31, 36 Fordham, M., 5 5 , 65, 96 Fosset, M., 71, 91 Fotinou, C., 120, 155 Fourcy, A., 13, 42 Fowler, S. V., 167, 220 Frachisse-Stoilskovic, J . M., 186, 220 Franceschi, V. R., 171, 188, 189, 196, 223 Francois, J. M., 56, 90 Frankland, J.C., 13, 15, 37 Franklin, F.C. H., 51, 56, 90 Franklin-Tong, V. E., 51, 56, 90 Freer-Smith, P. H., 248, 248, 263, 299 Frensch, J . , 172, 220, 247, 249, 291 Frey, B., 23, 37 Fricker, M. D., 50, 51, 52, 56, 57, 86, 88, 91, 92, 95, 283, 286, 289 Frohnmeyer, H., 106, 109, 110, 137, 153, 156 Fromm, J., 187, 220 Fromme, R., 124, 125, 157 Fry, S. C., 171, 189, 217, 251, 256, 257, 291, 295, 296, 300 Fujii, S., 75, 93 Fujino, I., 75, 93 Fujiwara, T., 170, 221
306
AUTHOR INDEX
Furuichi, T., 75, 93 Furuya, M., 105, 106, 110, 153 Fussader, A., 279, 291
G Gaba, V., 124, 125, 126, 153 Galaud, J. P., 165, 220 Galione, A., 49, 69, 75, 78, 79, 85, 90 Gallacher, D. V . , 85, 95 Gallagher, J . N., 285, 286, 291 Gallaud, I., 2, 9, 10, 11, 13, 37 Garcia-Ferris, C., 129, 130, 153 Gardiner, B. G., 98, 153 Gardner, S. D. L., 248, 299 Garrard, L . A . , 122, 127, 161 Garrec, J. P., 13, 42 Gaspar, T., 165, 166, 220, 224 Gasteiger, L., 167, 221 Gatehouse, A. M. R., 188, 221 Gehring, C . A . , 50, 59, 84, 91, 92, 96 Gelli, A., 80, 81, 82, 91, 94 Gemma, J . N., 3, 31, 39 Genty, B., 240, 241, 290, 292 Gerasimenko, O., 78, 79, 95 Gerats, T., 138, 156 Gerdemann, J. W . , 4, 13, 14, 30, 38, 39, 41 Ghanotakis, D., 120, 155 Ghashghaie, J., 240, 290 Gianinazzi, S., 6, 7, 8, 9, 15, 19, 31, 32, 38, 40 Gianinazzi-Pearson, V., 2, 6, 7, 8, 9, 13, 14, 15, 19, 23, 29, 30, 31, 32, 38, 39, 40, 42 Gies, H. P., 100, 158 Gillot, I., 51, 56, 58, 75, 78, 79, 85, 90, 94 Gilman, G . A . , 108, 153 Gilroy, S., 46, 47, 50, 51, 52, 56, 57, 59, 60, 64, 65, 77, 86, 91, 92, 164, 220 Giovannetti, M., 2, 3, 9, 30, 38 Givan, C. V., 131, 154 Glass, A. D. M., 274, 292 Gleason, J. F., 98, 99, I53 Glenn, M. G., 30, 38 Glinka, Z., 273, 292 Goh, K. M., 18, 37 Gold, W.G., 101, 150 Goldbach, E., 263, 292 Goldbach, H. W., 263, 292
Goldbeter, A., 56, 91 Goldsmith, M. H . M, 166, 220 Gollan, R., 276, 284, 287, 298 Gollan, T., 233, 234, 238, 239, 280, 28 1, 292, 299 Gollotte, A., 9, 38 Goodchild, D. J., 105, 152 Goransson, A,, 264, 292 Gordon, M. P., 165, 170, 219, 224 Gosselin, A., 127, 162 Goth, R. W . , 147, 158 Goudman, H.M., 136, 155 Could, C. L., 167, 220 Gourret, J. P., 13, 42 Gowing, D. J . G., 178, 220, 279, 292 Gozlan, G., 252, 289 Graber, P., 124, 125, 157 Grace, J., 269, 273, 276, 293 Gradman, D., 80, 89 Gradmann, D., 198, 202, 210, 220, 226 Graham, J . H., 3, 20, 28, 29, 32, 37, 38 Graham, J. S., 165, 188, 221 Grange, R. I., 177, 221 Grant, W. J . R., 237, 240, 291 Grayson, R. L . , 171, 175, 227 Graziana, A., 59, 71, 72, 73, 87, 91, 95 Green, P. B., 247, 297 Green, T. R., 165, 188, 195, 196, 221 Greenall, J. M., 14, 38 Greenberg, B. M., 115, 124, 125, 126, 153, 161 Greppin, H., 166, 224 Griffiths, A., 50, 94 Grime, J . P., 23, 24, 38 Grolig, F., 50, 51, 52, 59, 94 Gross, D. J . , 52, 56, 86, 93 Groth, B., 168, 220 Grove, T. S., 23, 38 Gruissem, W., 135, I55 Grynkiewicz, G., 5 5 , 91 Gubb, I . R., 166, 171, 180, 188, 189, 190, 194, 204, 227 Guillemin, J.P., 9, 38 Guinn, G., 263, 273, 283, 297 Gunasekera, D., 238, 292 Gundlach, H., 120, 154
H Haase, A., 175, 228 Haberlandt, G., 177, 214, 221
AUTHOR INDEX
Haeder, D. P., 109, 160 Hagiwara, S., 50, 57, 71, 81, 95, 186, 221 Hahlbrock, K., 106, 109, 110, 114, 115, 137, 138, 139, 148, 149, 151, 152, 153, 154, 155, 156. 158, 161 Hahm, S. H., 50, 51, 59, 91 Hahne, G., 133, 154 Halachmi, D., 59, 91 Hall, G., 165, 188, 221 Hall, I. R., 9, 19, 38 Hall, J. L., 107, 150 Hall, K. C., 276, 291 Hall, M. A., 170, 221 Hall, S. M., 274, 292 Hamanaka, Y., 75, 93 Hamm, H.E., 109, 161 Hammerschmidt, R., 170, 224 Hani Babu Vincent, V., 143, 144, 149 Harkins, A. B., 56, 92 Harley, J. L., 10, 12, 14, 21, 25, 38 Harley, P. C., 243, 244, 292 Harris, D., 26, 38 Harris, P. J . , 59, 96 Harrison, A. F., 13, 15, 37 Harrison, M . A . , 124, 125, 156 Harrison, M. J . , 138, 156 Harter, K., 106, 110, 153 Hartoonian, A. T., 60, 96 Hartshorne, J. N., 182, 226 Hartung, W., 170, 228, 232, 276, 278, 279, 282, 283, 289, 291, 292, 300 Harvey, H. J., 72, 91 Harwood, J. L., 142, 152 Haugland, R.P., 54, 59, 91 Hayashi, H., 170, 221 Hayman, D.S., 13, 17, 38 Hays, J. B., 118, 142, 146, 157 Haystead, A., 23, 38 He, J., 101, 111, 113, 127, 128, 129, 130, 141, 154, 155 Heath, I . B., 60, 92 Hedrich, R., 71, 73, 90 Heilmeier, H., 276, 279, 283, 291, 296, 300 Helal, H. M., 264, 292 Heller, W., 110, 153 Hendrix, D. L., 283, 292 Hendrix, J. W., 19, 27, 28, 39, 40 Hendry, G. A. F., 126, I51 Hengeler, C., 258, 289
307
Henkow, L., 129, 134, 162 Henson, I.E., 253, 294 Hepler, P. K., 5 1 , 52, 56, 57, 58, 59, 60, 86, 89, 93, 96 Hepper, C . M., 31, 39, 40 Herman, J. R., 98, 99, 153 Herrmann, A., 137, 161 Hetherington, A. M., 50, 51, 56, 57, 58, 64, 71, 72, 73, 76, 77, 81, 86, 87, 91, 93, 95, 256, 257, 284, 291, 294, 300 Hetrick, B. A. D., 28, 39 Hilder, V.A., 188, 221 Hill, B. S., 165, 187, 188, 207, 212, 214, 221 Hill, S. E., 204, 224 Hill, T. D., 75, 92 Hille, B., 80, 89 Hillier, S . H., 23, 24, 38 Hirano, H., 170, 221 Hirrel, M. C., 30, 39 Ho, L. C., 177, 221, 274, 297 Hodge, S. K., 256, 291 Hodick, D., 51, 52, 56, 92, 186, 214, 215, 221 Hoeijmakers, J. H. J., 118, I54 Hofmann, F., 48, 92, 133, 154 Hohm-Veit, S., 124, 125, 157 Holley, J. E., 13, 39 Hollingworth, S., 56, 92 Holman, S. R., 102, 150 Honda, C., 170, 221 Hong, S. D., 50, 59, 89 Hope, A. B., 201, 203, 220 Hopper, M. J., 264, 290 Horowitz, K. A., 167, 221 Horvath, I., 131, 161 Houghton, J . D . , 126, I51 Hourmant, A., 170, 217 Houwink, A. L., 165, 166, 171, 182, 187, 196, 203, 221 Howarth, M.J.,13, 22, 41 Hsaio, T. C. , 247, 249, 252, 253, 254, 291, 292, 298 Hu, X . , 129, 134, 162 Huang, L. K., 101, 113, 130, 141, 154 Huffaker, R. C., 127, 129, 130, 154, 157 Hughes, W. A., 52, 59, 60, 65, 91 Hunt, S., 248, 300 Hurley, T. W . , 56, 92 Hutchinson, F., 117, 154
308
AUTHOR INDEX
I Iida, H., 51, 60, 92 Iijima, T., 186, 202, 203, 212, 213, 214, 215, 216, 221 Impens, I., 126, 152 Incoll, L., 279, 285, 292 Ingemarsson, B., 265, 296 Ingestad, T., 233, 234, 258, 264, 266, 292, 293, 295 Innocenti, B., 69, 76, 82, 83, 90 Inze, D., 120, 161 Irvine, J., 276, 293 Irvine, R. F., 74, 88 Irving, H. R., 50, 59, 84, 91, 92 Ishiwatari, Y., 170, 221 Ismande, J., 243, 275, 293 Itoh, H., 108, 155 Itoh, K., 253, 293 Ivorra, I., 75, 94 Iwanzik, W., 127, 131, I60
J Jackson, G. E., 276, 293 Jackson, J. F., 117, 132, 154 Jackson, M.B., 170, 171, 178, 221, 273, 276, 291, 293 Jackson, S . L . , 60,92 Jackson W.A., 243, 293 Jacobsen, S . L., 186, 214, 217 Jacquelinet-Jeanrnougin,S., 13, 14, 23, 39 Jaffe, M. J., 165, 209, 213, 221 Jahnen, W., 115, 158 Jakobsen, I., 21, 41 James, P., 107, 111, 113, 114, 126, 129, 134, 135, 136, 140, 155 James, R.A., 277, 295 Janos, D.P., 17, 21, 22, 23, 24, 25, 27, 39 Janse, J. M., 12, 14, 25, 39 Jansen, M.A.K., 124, 125, 154 Jarvis, P. G . , 230, 231, 293 Jarvis, S . C . , 235, 294 Jechke, W. D., 276, 296 Jerstrom, P., 80, 95 Jeschke, W. D., 170, 228, 276, 280, 293 Jewer, P. C . , 279, 285, 292 Jiang, N., 118, 154 Jing, J., 253, 292 Johannes. E., 47, 77, 80, 81, 92 Johansson, E., 245, 265, 266, 275, 295
John, T.R., 1 1 5 , 161 Johnson, E. L. V., 30, 42 Johnson, J. D., 59, 92 Johnson, S., 165, 171, 189, 224 Johnston, E. L. V., 17, 28, 40 Jones, A. M., 84, 92 Jones, F. M., 165, 221 Jones, H. G . , 165, 167, 178, 186, 189, 190, 192, 195, 198, 220, 221, 223, 235, 236, 237, 239, 267, 279, 292, 293 Jones, K., 28, 39 Jones, L. H. P., 264, 290 Jones, M. D., 26, 43 Jones, R. L., 50, 51, 59, 81, 89, 91, 98, 100, 151 Jones, W. J., 279, 292 Jordan, B. R . , 102, 103, 105, 106, 107, 111, 113, 114, 119, 121, 124, 126, 127, 128, 129, 130, 131, 132, 134, 135, 136, 140, 141, 142, 152, 154, 155, 159, 162 Jordan, E. T., 167, 217 Jordan, P. W., 101, 150 Julien, J. L., 186, 220
K Kado, R.T., 77, 78, 88 Kahr, M., 258, 291 Kaila, K., 58, 92 Kaiser, W. M., 240, 293 Kallas, P., 187, 222 Kamprath, E. J., 243, 293 Kariya, N., 14, 39 Karlin-Neumann, G. A., 106, 155 Karmoker, J. L., 273, 293 Kasai, H., 8 5 , 92 Kasterberger, G . , 164, 226 Katerji, N., 270, 299 Katou, K., 164, 223 Kaufman, L.S., 109, 161 Kawa, S. R., 98, 100, 151 Kawashima, I., 170, 221 Kawata, H., 253, 293 Kaziro, Y., 108, 155 Kehlen, A., 168, 189, 217 Keil, M., 170, 188, 189, 222 Keith, C. H., 51, 59, 92 Keltjens, W. G . , 264, 298 Kendrick, B., 12, 13, 14, 15, 26, 30, 36
AUTHOR INDEX
Kendrick, R.E., 106, 107, 108, 149, 150, 151 Kern, R., 108, 157 Kerr, R. A., 98, 99, 155 Kessler, K. J., 13, 39 Khalil, A. A.M., 269, 276, 293 Kickert, R. N., 146, I55 Kinden, D.A., 13, 39 King, R. W., 277, 295 Kiss, H. G . , 59, 92 Klaff, P., 135, 155 Klein, P., 214, 223 Kleinkopf, G. E.. 129, 157 Knight, A. E., 169, 222 Knight, H., 57, 58, 59, 60, 68, 86, 94 Knight, M.R., 49, 50, 51, 57, 58, 59, 60, 66, 67, 68, 73, 86, 92, 94 Knofel, H. D., 168, 189, 217 Koes, R.E., 107, 155 Kohler, K., 80, 89 Koide, R. T., 26, 27, 28, 29, 30, 39, 41 Kolb, H.A., 80, 89 Kombrink, E., 139, 148, 155 Komhyr, W.D., 98, 99, 153 Kondo, N., 120, 126, 130, 149, 159 Konishi, M., 56, 92 Kontturi, M., 243, 294 Korner, C., 27, 39 Koske, R.E., 3, 31, 39 Kotzabasis, K., 120, 155 Kozasa, T., 108, 155 Kramer, G. F., 120, 131, 143, 155 Krapp, A., 130, 141, 155 Krause, J . H., 80, 95 Krauss, A., 273, 294 Krebs, C. J., 166, 222 Kretsch, T., 106, 110, 153 Krischer, J., 80, 95 Krizek, D.T., 120, 131, 143, 155 Krupa, S. V., 146, 155 Kruse, W., 282, 291 Ku, H.S., 168, 222 Kuang, J. B., 253, 294 Kuba, Y., 56, 93 Kubasek, W. L., 136, 155 Kubo, A., 120, 149 Kuc, J., 165, 219, 222 Kuhlemeier, C., 135, 155 Kuhn, M.A., 56, 90 Kiihn, K. D., 13, 39 Kulaeva, 0. N., 273, 294 Kulandaivelu, G., 101, 148, 157, 160
309
Kumon, K., 212, 222 Kutchan, T. M., 120, 154 Kutschera, U., 176, 222 Kuzmanoff, K. M., 253, 294 Kwok, S.F., 84, 89 L Labow, G., 98, 99, 153 Laisk, A., 237, 294 Lamb, C. J., 120, 138, 148, 154, 156, 161 Lambers, H., 26, 27, 39, 41, 245, 298 Lamboll, D., 178, 221 Langbein, D., 175, 228 Lange, 0. L., 237, 294 Langebartels, C., 120, 161 Larkow, D., 98, 99, I53 Larsson, C. M., 235, 245, 246, 259, 260, 264, 265, 266, 274, 275, 294, 295, 296, 298 Larsson, M., 245, 265, 266, 275, 295 Larsson, S., 243, 300 Larthwell, D. J., 274, 291 Lassalles, J. P., 77, 78, 88 Last, R. L., 114, 116, 156 Lauchli, A., 50, 59, 93, 258, 300 Laudi, L., 121, 158 Lauer, M. J., 242, 294 Lawlor, D. W., 243, 265, 294 Lawrie, A. M., 85, 95 Lawton, J. H., 167, 220 Lawton, K. A., 165, 222 Lawton, M. A., 120, 161 Layzell, D. B., 285, 296 Lazdunski, M., 71, 91 Leake, J. R., 25, 40 Lee, J. A. C., 65, 89 Le Fay, J.M., 124, 142, 152 Lei, J., 31, 40 Leigh, R.A., 72, 73, 93 Lenaz, G., 121, 158 Lenton, J. R., 239, 240, 263, 273, 285, 290 Leonard, R.T., 3, 38 Leopold, A. C . , 175, 226 Lerman, L., 210, 225 Lery, S., 75, 94 Letham, L. S . , 166, 223 Leun, van der, J.C., 100, 157 Lew, D. P., 80, 95 Lew, R. R., 202, 222
310
AUTHOR INDEX
Lewis, D.C., 167, 221 Lewis, D. H., 22, 40 Lewis, E. R., 214, 223 Lherminier, J., 9, 38 Li, J., 114, 116, 156 Li, W. W., 85, 93 Li, Y.X.,85, 92 Ligrone, R., 14, 22, 37, 40 Linderman, R. C . , 20, 38 Ling-Lee, M., 14, 40 Linstead, P. J., 164, 210, 223 Linthorst, H. J. M., 148, I51 Lips, S. H., 274, 289 Lloyd, C. W., 77, 90 Loake, G.L., 138, 156 Lockhart, J. A., 176, 222, 246, 294 Logi, C., 3, 30, 38 Lohammar, T., 243, 259, 261, 264, 295, 300 Lois, R., 113, 116, 148, 156 Lomax, T. L., 164, 227 Loreto, F., 166, 222 Lorimer, G.H., 127, 156 Los, D.A., 131, 161 Louis, I., 12, 40 Loveys, B. R., 237, 240, 269, 276, 291, 294 Lozoya, E., 148, 156 Luan, S., 85, 93 Lumsden, P., 105, 107, 156 Lund, A. B., 233, 234, 258, 264, 293 Lundborg, T., 245, 265, 266, 275, 295 Luque, A. A., 264, 294 Lydon, J., 144, 160 Lynch, J., 50, 59, 93
M MacAdam, J. W., 259, 263, 294 McAinsh, M. R., 50, 51, 56, 57, 58, 64, 73, 81, 86, 87, 93, 95, 284, 294 McCoy, A., 102, 162 McCray, J.A., 5 5 , 93 McDonald, A. J. S., 239, 243, 248, 249, 259, 261, 262, 263, 264, 265, 280, 293, 294, 295, 296, 297, 298. 299, 300 McDougall, A., 75, 78, 79, 85, 90 MacDougall, A. J., 170, 189, 225 Macduff, J. H., 235, 294 McGee, P.A., 23, 40
McGurl, B., 188, 189, 191, 223 McKenna, D.S., 98, 100, 151 Mackey, J. M. L., 23, 24, 38 McKenzie, R. L., 100, 158 McKillop, A., 136, 155 McLaughlin, C., 116, 117, 120, 150 McLennan, E. I., 13, 25, 40 McMichael, R., 213, 219 McNabb, R. F. R., 13, 36 McNaughton, G.S., 171, 196, 223 McNeil, D. L., 285, 296 McPeters, R., 98, 99, 153 McQueen-Mason, S., 256, 295 MacRobbie, E., 80, 89 MacRobbie, E. A. C., 171, 222, 282, 294 Maggard, S., 102, 150 Maimon, E., 166, 223 Makus, D., 171, 189, 223 Malajczuk, N., 23, 38 Malho, R., 51, 52, 56, 57, 58, 59, 60, 68, 86, 93, 94 Malik, R. S., 252, 295 Malkin, S., 124, 125, 126, 153 Malone, M., 164, 165, 166, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 179, 180, 182, 183, 184, 185, 186, 187, 189, 190, 191, 192, 194, 195, 196, 198, 200, 201, 205, 207, 21 7, 218, 222, 223 Mandoli, D. F., 167, 223 Mansfield, T. A., 283, 284, 289, 291 Maout, Le, E., 216, 222 Marcelis, L. F. M., 179, 226 Marchner, H., 273, 298 Markham, K. R., 114, 156 Marschner, H., 258, 289 Marshall, J., 72, 73, 93 Martens, C., 126, 152 Martin, C., 138, 156 Martin, D. J., 256, 291 Martin, G.,112, 152 Martinez del Rio, C., 17, 21, 42 Marx, C., 6 , 8, 15, 40 Masia, A., 233, 295 Matsuda, K., 247, 295 Matthews, K. J., 256, 291 Matthews, M. A., 176, 225 Mattner, A., 130, 149 Mattoo, A.K., 124, 125, 126, 129, 153, 156 Mattsson, M., 245, 265, 266, 275, 295
AUTHOR INDEX
Matyssek, R., 180, 223 Maurel, C., 212, 218 Mavandad, M., 138, 156 Maxfield, F. R., 51, 59, 92 Mayer, J., 252, 289 Mazars, C., 71, 72, 91 Mehravaran, H., 30, 39 Mehta, R. A., 129, 156 Meidner, H., 58, 96 Meier, R., 31, 35 Meier-Augenstein. W., 187, 222 Meinzer, F., 175, 228 Meinzer, F.C., 171, 175, 227 Meissner, G., 80, 89 Melis, A., 124, 125, 156 Meloh, K.A., 14, 40 Menge, J . A . , 3, 6, 17, 20, 28, 30, 38, 40, 42 Mengel, K., 264, 292, 298 Merkle, T., 137, 156 Messiaen, J., 51, 52, 93 Meyerowitz, E. M . , 84, 89 Michael, G., 263, 292 Michelena, V. A., 247, 252, 295 Michikawa, T., 7 5 , 93 Mikoshiba, K., 75, 93 Milborrow, B. V . , 277, 295 Milburn, J.A., 170, 217, 274, 292 Millard, P., 130, 156 Miller, A. J., 98, 99, 153 Miller, C., 80, 89, 105, 152 Miller, D. D., 52, 56, 86, 93 Miller, H. M., 19, 36 Miller, R. M., 18, 20, 40 Miller, S. L., 23, 24, 40 Millner, P., 56, 95 Milthorpe, F. L., 259, 290 Mimura, T., 204, 224 Minchin, P. E . H.. 166, 171, 180, 188, 189, 190, 194, 196, 204, 223, 227 Mirecki, R.M., 120, 131, 142, 143, 155, 156 Mitchell, D., 119, I51 Mitchell, D. L., 119, 152 Mithofer, A., 168, 220 Miyashita, Y., 85, 92 Miyawaki, A., 75, 93 Miyazaki, S., 79. 93 Miziorko, H. M., 127, 156 Mizrahi, Y., 263, 295 Modjo, H. S., 19, 27, 40 Mol, J. N. M., 107, 155
31 1
Moll, R. H., 243, 293 Molotkovskii, Y.G., 50, 59, 89 Mornonov, L.K., 50, 59, 89 Money, N. P., 58, 93 Monteleone, M., 165, 186, 189, 190, 192, 195, 198, 223 Montgomery, L., 87, 95 Moore, R., 166, 223 Moorman, T. B., 20, 40 Moran, N., 71, 95 Moreno, J., 129, 130, 153 Mori, S., 170, 221 Morris, C., 5 , 34, 42 Morrison, T. M., 13, 36 Morse, S. R., 171, 223 Morton, J. B., 14, 40 Mosse, B., 13, 17, 18, 31, 40 Mozingo, H. N., 214, 223, 227 Muir, S . R., 79, 88 Muller, B., 243, 264, 274, 299 Muller, M. J., 120, 154 Mullet, J. E., 135, 156, 258, 289, 290 Mullineaux, P., 116, 117, 120, 150 Mummert, H., 202, 220 Mums, R., 177, 223, 233, 234, 252, 276, 277, 289, 292, 295, 296 Murali, N. S., 101, 145, 156 Murata, N., 131, 161 Murdock, L. L., 165, 196, 225, 228 Murphy, T.M., 109, 156 Murray, B. J., 166, 223 Murrman, M., 172, 224 Muxfeldt, B., 282, 291 N Nagatani, A., 106, 110, 253 Nakade, S., 75, 93 Nakafuko, M., 108, 155 Nakagawa, T., 75, 93 Nakahori, K., 164, 223 Nakamura, S., 170, 221 Nakamura, Y., 253, 293 Narvaez-Vasquez, J., 171, 188, 189, 196, 223 Natori, T., 120, 149 Neales, T. F., 233, 295 Nedunchezhian, N., 148, 157 Needs, P. W . , 170, 189, 225 Negash, L., 144, 157 Neher,, E., 80, 89 Neljubow, D., 168, 171, 223
312
AUTHOR INDEX
Nelsen, C.E.,22, 40, 171, 189, 223 Nelson, C. J . , 259, 263, 294 Nemson, J . A . , 124, 125, 156 Netting, A. G., 277, 295 Neuhaus, G., 85, 89, 108, 109, 151, 157 Neumann, P.M., 164, 176, 218 Newman, E . I., 23, 24, 40 Newman, P., 98, 99, 153 Nicolson, T. H., 12, 41 Nikiforov, E. L., 50, 51, 59, 96 Niklas, K. J . , 172, 223 Nobel, P.S., 171, 172, 178, 223 Nobiling, R., 59, 93 Nonami, H., 252, 296 Nooden, L. D., 166, 223 Norman, H . A . , 120, 131, 155 Northcote, D. H., 170, 188, 189, 226 Nye, P. H . , 280, 296
0 Oakes, A . , 243, 296 O’Bannon, J . H., 17, 41 Ober, E . S., 254, 296 Obermeyer, G., 51, 52, 93 Ocampo, J . A., 17, 28, 35 Oda, K., 164, 186, 210, 217, 223 O’Donnell, P. J . , 166, 171, 180, 188, 189, 190, 194, 204, 227 Ohad, I., 125, 126, 157 O’Halloran, I., 15, 37 Ohnishi, T., 116, I59 Ohta, E., 253, 293 Okamoto, H., 164, 223 Okano, H., 75, 93 Olsen, C.,233, 296 O’Neill, S. D., 132, 157 Oparka, K. J . , 189, 223 Oren, R., 283, 296 Orozco-Cardenas, M., 188, 189, 191, 223 Orozco-Cardenas, M. L., 171, 189, 196, 223 Ort, D. R., 237, 238, 242, 296, 300 Orth, A . B . , 147, 157 Ortiz-Lopez, A., 237, 300 Osborne, D. J., 168, 224 Oscarson, P., 235, 265, 294, 296 Osmond, C.B., 237, 241, 289, 299 Osonubi, O., 283, 296 Osterhout, W . J . V., 204, 224
Ou-Lee, T. M., 114, 116, 156 Oxborough, K., 238, 242, 296
P Pacovsky, R. S., 19, 36 Page, J . F. M., 186, 218 Pais, M . S., 51, 52, 56, 93 Palade, P., 80, 89 Paliwal, K., 143, 144, 149 Palmer, S., 263, 270, 298, 299 Palmer, S . J . , 239, 259, 262, 263, 280, 296 Palmerley, S. M., 17, 21, 41 Palta, J . A . , 171, 224 Palumbo, L., 165, 182, 186, 189, 190, 192, 195, 198, 223 Pan, R.S., 143, 157 Panagopoulos, I., 147, 157 Pang, Q., 118, 142, 146, 157 Pann, W . L., 243, 293 Pantoja, O., 82, 94 Pardossi, A., 164, 177, 224 Parekh, A. B., 82, 94 Parish, R. W., 50, 59, 84, 91, 92, 96 Park, H. J . , 50, 59, 89 Parker, I . , 75, 94 Parker, J . C.,209, 225 Parker, L. L., 263, 273, 283, 297 Parmar, P. N., 76, 94, 108, 157 Parsons, T. J., 165, 224 Parthier, B., 120, 158 Partis, M. D., 105, 106, 142, 152, 155, 157 Pasquati, P., 121, 158 Passioura, J . B., 177, 178, 224, 233, 234, 251, 258, 268, 277, 292, 295, 296 Paszweski, A., 206, 224 Pate, J . S., 276, 280, 285, 293, 296 Paul, E . A., 26, 38 Paul, N., 116, 117, 120, I50 Payne, R., 75, 94 Peaden, R. N., 17, 41 Peak, M. J., 100, 157 Pearce, G., 165, 171, 188, 189. 191, 196, 218, 221, 223, 224 Pearce, R. W . , 243, 297 Pearson, J . N . , 21, 41 Penel, C., 166, 224 Peng, 2.Y., 282, 297 Penot, M., 170, 217
313
AUTHOR INDEX
Peiia-Cortes, H., 165, 168, 170, 188, 189, 224 Percy, K., 116, 117, 120, 150 Pereira, J. S., 267, 268, 276, 290, 291 Perotto, S., 2, 36 Perumalla, C. J., 28, 41 Petersen, 0. H . , 78, 79, 85, 95 Peterson, C. A., 28, 41, 172, 224 Peterson, L. W . , 129, 157 Peterson, R. C., 9, 13, 37 Peterson, R. L., 13, 19, 22, 36, 39, 41 Petkoff, H . F., 52, 59, 90 Pettersson, R., 243, 245, 297 Peuss, H., 28, 41 Pfiindel, E.E., 143, 157 Piche, Y., 9, 13, 14, 31, 36, 37, 40 Pickard, B. G., 74, 90, 166, 168, 170, 186, 187, 190, 201, 202, 203, 205, 214, 218, 224, 226, 227 Picken, A. J., 177, 221 Pineros, M., 72, 73, 94 Ping, Z., 204, 224 Pitman, M. G., 273, 297 Planet, W., 98, 99, 153 Platt, R. G., 17, 28, 40 Pocock, K., 22, 41 Poenie, M., 51, 52, 5 5 , 60, 91, 95 Poff, K.L., 115, 161 Polito, V. S., 50, 59, 93 Poole, L. R., 98, 100, 151 Poole, R. J . , 80, 94 Poorter, H . , 26. 27, 39 Poovaiah, B. W . , 46, 47, 67, 94, 164, 224 Porath, D., 129, 156 Porra, R. J . , 114, 126, 159 Potter, B. V . L., 78, 79, 90 Potter, J. D., 56, 90 Potter, S. L., 165, 222 Powles, S. B., 240, 289 Pozzan, T., 66, 69, 76, 82, 83, 90, 94 Prasil, O., 125, 126, 157 Prat, H. K., 168, 222 Prat, S., 168, 224 Pratt, L.H., 105, 157 Price, A. H . , 50, 94 Pritchard, J . , 164, 177, 224, 248, 254, 255, 256, 282, 283, 297 Puliga, S., 271, 297 Pulsford, A. L., 52, 56, 58, 89 Purves, R. D., 57, 94 Purwin, C., 148, 154
Putney, J. W., 82, 83, 94
Q
Qian, Y.C., 109, 156 Quail, P. H . , 105, 157 Quaite, F.E., 104, 118, 157 Quarrie, S. A., 271, 297 Quick, P. W . , 130, 141, 155
R Raba, R., 114, 116, 156 Radin, J. W . , 240, 260, 263, 273, 283, 292, 297 Ramos, M. I. R. F., 23, 24, 40 Ranasinghe, S., 248, 299 Randall, H. C., 180, 227 Randriamampita, C., 82, 83, 94 Ranjeva, R., 59, 71, 72, 73, 77, 87, 89, 91, 94, 95 Rappaport, L., 168, 222 Raschke, K., 175, 224, 237, 291 Rasenick, M. M., 109, 161 Rasmussen, J. B., 170, 224 Ratan, R., 51, 59, 92 Raven, J. A., 284, 297 Raventos, D., 165, 218 Ray, P. M . , 247, 297 Rayner, J. H . , 18, 42 Rea, P. A., 80, 94 Read, D. J., 1, 23, 24, 38, 41 Read, N. D., 49, 50, 5 1 , 52, 56, 57, 5 8 , 59, 60, 64, 66, 68, 77, 86, 90,91, 92, 93, 94, 95, 108, 158 Reddy, A. S . N., 46, 47, 67, 94, 164, 224 Redhead, J.F., 13, 41 Reilly, A. J . , 166, 171, 180, 188, 189, 190, 194, 204, 227 Reinsvold, R. J., 15, 41 Reiss, H. D., 52, 59, 60, 93, 95 Renger, G., 124, 125, 157 Renwick, K. F., 87, 95, 256, 291 Reupold-Popp, P., 120, 161 Reynolds, J. F., 243, 244, 292 Rhodes, J. D., 187, 227 Rhodes, L. H . , 4, 41 Riazi, A., 247, 295 Ricca, U., 179, 196, 198, 201, 215, 225 Richardson, C. M . , 121, 150 Richardson, P. T . , 274, 297
314
AUTHOR INDEX
Richmond, A. E., 263, 295 Ride, J. P., 51, 56, 90 Ries, S., 166, 225 Rigby, N.M., 170, 189, 225 Rink, T. J., 55, 60, 90, 96 Ripley, S. J., 50, 94 Rizzuto, R., 66, 94 Robberecht, R., 112, 151, 157 Roberts, D. R., 121, 158 Roberts, S. K., 51, 56, 58, 94 Robertson, M., 277, 289 Robinson, G. A., 47, 94 Robinson, K. R., 51, 94 Roblin, G., 170, 186, 187, 198, 225 Robson, A. D., 13, 17, 18, 19, 20, 21, 35, 43 Rocha-Sosa, M., 165, 170, 188, 189, 224 Rocholl, M., 106, 110, 153 Rodriguez, J. L., 286, 297 Rooney, J. M., 259, 265, 297 Rosamond, J., 119, 159 Rossignol, M., 77, 89 Roubelakis-Angelakis, K. A., 120, 155 Roux, S. J., 51. 52, 60,95, 107, 158 Roy, C.R., 100, 158 Ruiz, L. M.P., 277, 283, 287, 299 Russ, U., 50, 51, 52, 59, 94 Russell, A., 57, 58, 59, 60, 68, 86, 94 Ryals, J., 165, 222 Ryan, C. A., 120, 153, 165, 168, 170, 171, 188, 189, 191, 195, 196, 218, 220, 221, 223, 224, 225, 227, 228 Ryan, M. P., 56, 92 Ryo, Y., 75, 93 Ryser, P., 27, 28, 41 S
Saab, I. N., 249, 254, 255, 256, 282, 283, 297, 298 Sachs, T., 166, 225 Sage, R.F., 145, 158, 243, 297 Saif, S.R., 12, 13, 41 St John, T. V., 28, 42 Saji, H., 120, 149 Sakai, W.S., 22, 42 Sakata, M., 253, 293 Salema, R., 130, 158 Samejima, M., 186, 187, 202, 212, 225 SamueIson, M.E., 259, 260, 264, 298 San Segundo, B., 165, 218
Sancer, A., 117, 118, 158 Sancer, G.B., 117, 118, 158 Sanchez-Serrano, J. J., 165, 170, 188, 189, 222, 224 Sandermann, Jr, H., 110, 120, 153, 161 Sanders, D., 47, 72, 73, 77, 78, 79, 80, 81, 82, 88, 89, 92, 93 Sanders, F. E., 17, 21, 41 Sanderson, J., 171, 225 Sandlin, R., 210, 225 Santos, I., 130, 158 Sardaki, B., 209, 225 Satoh, T., 108, 155 Sattelmacher, B., 273, 298 Satter, R. L., 71, 95, 198, 225 Sattin, M., 164, 219 Saunders, M. J., 50, 51, 59, 91 Savithiry, S., 166, 225 Sbrana, C., 3, 30, 38 Scannerini, S., 30, 33, 36 Schfer, E., 106, 107, 109, 110, 137, 148, 150, 151, 153, 154, 156 Scheel, D., 114, 148, 154, 156 Schel, J. H.N., 52, 60, 95 Schenck, N. C., 17, 43 Schenck, N. S., 17, 41 Scheuerlein, R., 51, 52, 60, 95, 109, 160 Schildknecht, H., 187, 196, 222, 225 Schlapfer, B., 28, 41 Schmelzer, E., 115, 158 Schmidt, K., 51, 52, 60, 95 Schmidt, S.K.,20, 40 Schopfer, P., 165, 220 Schram, A. W., 50, 96 Schreiber, S. L., 85, 93 Schreiner, R. P., 29, 30. 41 Schroeder, J. I., 50, 57, 71, 72, 73, 80, 81, 82, 87, 95,96, 283, 289 Schubert, E., 264, 298 Schubert, S., 264, 298 Schiiepp, H., 23, 37 Schultz, J. C.,167, 217 Schultz, R. C., 13, 16, 43 Schulz, W., 137, 158 Schulze, E. D.,238, 239, 276, 279, 280, 281, 283, 284, 285, 287, 291, 292, 296, 298, 299, 300 Schulze-Lefert, P., 137, 156, 158, I61 Schumaker, K. S., 77, 95 Schurr, U., 276, 280, 281, 284, 287, 292, 298
AUTHOR INDEX
Schuster, A., 166, 219 Schwab, S. M., 6, 30, 42 Scopel, A . L., 167, 217 Scott Russell, R., 233, 298 Searles, P. S . , 103, 104, 142, 152 Sechi, A . M . , 121, 158 Sechmeyer, G., 100, 158 Seeman, J. R., 242, 291 Seemann, J. R., 145, 158 Seftor, C., 98, 99, 153 Selvendran, R. R., 170, 189, 225 Sembder, G., 120, 158 Sembdner, G., 168, 189, 217 Semeniuk, P . , 147, 158 Serpe, M.D., 176, 225 Serrigny, J., 10, 42 Setlow, R. B., 118, 158 Shacklock, P . S . , 50, 51, 57, 58, 59, 60, 68, 86, 94, 95, 108, 158 Shaner, D. L., 285, 298 Shanklin, J. D., 98, 153 Sharkey, T.D., 145, 158, 166, 222, 235, 237, 241, 243, 289, 291, 297 Sharp, R.E., 249, 252, 254, 255, 256, 259, 263, 277, 282, 283, 294, 295, 296, 297, 298, 300 Sharp, W.E., 255, 256, 257, 298, 300 Sheen, J., 141, 158 Sheerman, S. E., 188, 221 Shelanski, M. L . , 51, 59, 92 Shibata, K., 13, 42 Shimizu, H., 126, 130, 159 Shine, K. P . , 98, 149 Shirakawa, H., 79, 93 Shirley, B. W., 136, 155 Short, T. W., 105, 106, 109, 158 Shotton, D., 5 5 , 65, 95 Shrier, R., 133, 146, 153 Shukle, R. H., 165, 225 Sibaoka, T., 164, 167, 186, 187, 196, 202, 203, 212, 213, 214, 215, 216, 221, 225, 226 Severs, A . , 186, 214, 215, 221 Silk, W. K., 252, 254, 298 Simon, E. W., 182, 226 Simons, P. J., 201, 226 Simpson, A . W. M . , 66, 94 Simpson, R. J., 245, 298 Sinclair, T. R., 180, 227 Sinyukhin, A. M . , 165, 226 Sisler, H. D., 147, 157 Sisson, W. B., 122, 124, 151, 158
315
Skipper, Y. D., 166, 171, 180, 188, 189, 190, 194, 204, 227 Slovik, S., 232, 278, 292 Smart, E. C . , 263, 289 Smit, B . A . , 170, 219 Smith, F . A . , 2, 5, 6, 7, 8, 15, 19, 23, 24, 29, 30, 31, 32, 33, 34, 38, 42, 43, 284, 297 Smith, G. S., 17, 41 Smith, K. C . , 118, 158 Smith, P . M . , 85, 95 Smith, R. C . , 256, 291 Smith, S.E., 2, 5 , 6, 7, 8, 12, 15, 18, 19, 21, 23, 24, 25, 29, 30, 31, 32, 33, 34, 36, 38, 42, 43, 49, 50, 51, 66, 67, 73, 92 Soberon, M. J., 17, 21, 42 Sokolovski, S. G., 50, 51, 59, 96 Son, C. L., 18, 19, 42 Spanswick, R.M., 179, 186, 202, 205, 217, 226, 227 Sparling, G. P., 19, 42 Spelt, C.E., 107, 155 Sperry, J. S., 267, 299 Spollen, W. G., 249, 254, 255, 256, 257, 298, 300 Spreitzer, R. J., 127, 158 Stacchiarini-Seraphim, E., 164, 219 Stadenberg, I., 243, 248, 249, 263, 265, 294, 297, 298, 299 Stahl, K., 168, 189, 217 Stahl, M., 14, 42 Stahlberg, R., 166, 176, 201, 226 Staiger, C. J., 77, 90 Stanford, A. C . , 170, 188, 189, 226 Stankovic, B., 166, 179, 182, 186, 190, 222 Stapleton, A. E., 103, 116, 117, 118, 159 Stasz, T. E., 22, 42 Staves, M. P., 175, 226 Staxen, I., 132, 133, 159 Steinmiiller, D., 116, 117, 159, 160 Stendahl, O . , 80, 95 Step, E., 169, 222 Steudle, E., 172, 220, 224, 251, 298 Stewart, G. R., 276, 296 Stitt, M . , 130, 141, I55 Stock, W. D., 26, 35 Stoeckel, H., 186, 226 Stolarski, R. S . , 98, 99, 153 Strain, B. R., 243, 244, 232, 278, 282, 292
316
AUTHOR INDEX
Streich, J., 282, 291 Stribley, D. P., 3, 18, 19, 35, 42 Strid, A, 103, 107, 111, 113, 114, 122, 123, 124, 125, 126, 127, 129, 134, 135, 136, 138, 140, 141, 152, 155, 159, 162 Strong, M. E., 22, 43 Strullu, D.G., 13, 42 Strydon, D., 171, 189, 224 Stuhlman, O., 186, 226 Stuhmer, W., 82, 94 Suda, S., 212, 222 Sugahara, K., 126, 130, 159 Suge, H., 168, 222 Sullivan, J. H., 101, 102, 144, 145, 159, 160, 162 Sun, L., 106, 155 Surowy, T. K., 258, 298 Susek, R., 110, 111, 141, I59 Sutherland, B. M., 104, 118, 157 Sutherland, E. W., 47, 94 Sutherland, J.C., 104, 118, 157 Sutherland, R. A., 267, 293 Sweger, B. L., 179, 217 Sydenham, P. H . , 202, 226 Sylvertsen, J. P., 29, 37 Syvertsen, J. P., 28, 38 Sze, H., 77, 95 Sztein, A. E., 101, 160
T Tabata, T., 202, 226 Taber, R. A., 22, 43 Takakashi, A., 116, 159 Takeda, K., 116, 159, 186, 226 Takeshita, S., 56, 93 Takeuchi, Y., 126, 130, I59 Tan, K., 264, 298 Tanaka, K., 119, 120, 149, 159 Tang, A. C., 180, 223 Tardieu, F., 268, 269, 270, 271, 272, 276, 277, 282, 287, 299 Tasaki, I., 210, 225 Taylor, A., 50, 94 Taylor, G., 247, 248, 249, 263, 299 Taylor, J. E., 51, 86, 87, 93, 95 Taylor, J. S., 118, 154 Taylor, L. P., 138, 259 Taylor, R. M., 119, 159 Taylor, W. C., 110, 141, 159 Tazawa, M., 204, 224
Teller, S., 130, 149 Tenhunen, J. D., 282, 289 Tepperman, J.M., 120, 160 Teramura, A. H., 101, 102, 113, 142, 143, 144, 145, 147, 150, 151, 156, 157, 159, 160, 162 Terashima, I., 237, 299 Terlau, H., 82, 94 Terry, B. R., 73, 95 Tester, M., 19, 36, 72, 73, 94 Tester, M. A., 6, 29, 30, 43 Tevini, M., 101, 103, 113, 115, 116, 117, 127, 131, 132, 143, 145, 146, 151, 159, I60 Thain, J.F., 166, 171, 180, 186, 187, 188, 189, 190, 194, 198, 201, 202, 204, 206, 226, 227 Theibert, J. L., 56, 90 Theler, J. M., 80, 95 Thellier, M., 166, 219 Thiel, G., 56, 57, 77, 84, 89, 95 Thimann, K . V . , 166, 225 Thoma, U., 127, 131, 160 Thomas, B., 105, 106, 107, 132, 150, 155, 157, 160 Thomas, R. B., 243, 244, 292 Thompson, D. S., 239, 259, 262, 263, 280, 296 Thompson, J.E., 121, 158 Thompson, W.F., 106, 135, 160 Thomson, B. D., 18, 20, 21, 31, 43 Thomson, C.M., 130, 156 Thorn, P., 78, 79, 85, 95 Thornham, K . T., 171, 175, 227 Thorsteinsson, B., 263, 299 Thuleau, P., 71, 72, 73, 87, 91, 95 Tiburcio, A. F., 121, I50 Tillberg, E., 263, 299 Timmers, A. C . J., 52, 60, 95 Tinker, P. B., 3, 4, 5 , 6, 17, 18, 19, 21, 26,35,37, 41, 42, 43, 280, 296 Tinz-Fuchtmeier, A., 198, 210, 226 Tirlapur, U., 109, 160 Tobin, A. K., 119, 159 Tobin, E.M., 106, 155 Tomos, A. D., 164, 171, 175, 177, 180, 187, 222, 224, 248, 256, 297 Toohey, D. W., 98, 100, I51 Tort, M., 166, 219 Toth, R., 14, 31, 35, 39 Touraine, B., 243, 259, 263, 264, 273, 274, 275, 290, 293, 299
AUTHOR INDEX
Trappe, J. M., 1, 22, 30, 43 Trebacz, K., 206, 228 Trebst, A., 124, 125, 154 Trejo, C. L., 178, 220, 277, 278, 279, 282, 283, 287, 299 Trentham, D. R., 5 5 , 57, 77, 89, 93 Trewavas, A. J., 46, 47, 49, 50, 51, 52, 56, 57, 58, 59, 60, 64,65, 66, 67, 68, 72, 73, 77, 86, 88, 90, 91, 92, 93, 94, 95, 108, 158, 283, 284, 285, 286, 289, 299 Trewavas, T., 164, 220 Trouvelot, A., 9, 38 Trudel, M. J., 127, 162 Tsien, R. W., 48, 69, 71, 73, 74, 78, 80, 83, 85, 96 Tsien, R.Y., 48, 5 5 , 59, 60, 69, 71, 73, 74, 78, 80, 82, 83, 85, 91, 94, 95, 96 Tsong, T.Y., 60, 96 Tsunoda, Y., 47, 8 5 , 96 Tumlinson, J . H . , 166, 226 Turlings, T. C. J., 166, 226 Turner, M., 78, 79, 90 Turner, N . C . , 232, 238, 239, 252, 253, 280, 294, 295, 299 Tyerman, S. D., 73, 95 Tyree, M. T., 172, 226, 267, 299
U Uknes, S . , 165, 222 Umrath, K., 164, 226
V Vaadia, Y . , 274, 289 Valenta, R., 77, 90 Van, T . K . , 122, 150 Van Baarlen, P., 166, 219 Van Bel, A. J. E., 171, 196, 226, 258, 299 Van Camp, W., 120, 161 Van Cutsem, P., 51, 52, 93 Van de Pol, P. A., 179, 226 Vander Plank, J . E., 21, 43 Van Duijn, B., 50, 96 Van Montagu, M . , 120, 161 Van Nuffelen, M., 17, 43 Van Oosten, J . J., 141, 161 Van Sambeek, J . W., 166, 170, 187, 203, 204, 226
317
Van Steveninck, R. F. M., 273, 293 Van Volkenburgh, E., 248, 300 Vazzana, C., 271, 297 Venis, M. A., 72, 91 Vergara, B.S., 102, 150 Verhey, S. D., 164, 227 Verhoeven, C., 109, 156 Vierstra, R. D., 115, 161, 167, 217 Vigh, L., 131, 161 Vince-Prue, D., 107, I50 Visser, A. J., 258, 299 Voetberg, G. S., 254, 255, 283, 297, 300 Vogelmann, T. C., 112, 152, 161 Voipio, J . , 5 8 , 92 Volgelmann, T. C., 112, 142, 150 Volker, M., 124, 125, 157 Volotovski, I.D., 50, 51, 59, 96 Vredenberg, W. J . , 107, 108, 150, I51 Vu, C.V., 122, 127, 161
W Wagner, G., 50, 51, 52, 59, 94 Wagner, 0. E., 167, 227 Walbot, V., 116, 159 Walker, C., 2, 43 Walker-Simmonds, M., 171, 189, 223 Walker-Simmons, M., 165, 227 Walter, C. H . S., 239, 240, 263, 273, 285, 290 Walz, B., 75, 94 Wang, M., 50, 96 Ward, J . L., 50, 56, 57, 72, 80, 81, 82, 87, 88, 95, 96, 283, 286, 289 Ward, J. M . , 283, 289 Waring, R. H . , 243, 300 Warner, C. W., 103, 142, 151, 161 Warpeha, K . M . F., 109, 161 Wartinger, A., 276, 279, 291, 300 Waters, J . R., 23, 43 Watkins, P. A. C., 77, 90 Watras, J., 75, 90 Watson, S. P., 78, 79, 90 Wayne, R., 164, 175, 201, 202, 210, 226, 227 Weaver, J.E., 254, 300 Webb, A. A. R., 51, 86, 87, 93, 95 Weber, H . C., 13, 31, 35, 39 Wedders, I., 166, 225 Weiler, E. W., 168, 189, 217, 220, 224 Weintraub, M., 196, 227
318
AUTHOR INDEX
Weis, E., 241, 300 Weisenseel, M. H . , 51, 52, 93 Weiss, C., 170, 189, 227 Weisshaar, B., 137, 138, 149, 161 Weisz, P. R., 180, 227 Welbaum, G. E., 171, 175, 227 Wellburn, A., 116, 117, 120, 150 Wellemeyer, C., 98, 99, 153 Wellfare, D., 273, 297 Wellman, E., 106, 161 Wendler, U . , 209, 210, 227 Werk, K. S., 283, 296 Wert, V., 166, 225 Wessels, R., 166, 219 Westgate, M. E., 254, 300 Weyers, J . , 58, 96 Weyers, J. D. B., 282, 297 Whitaker, M., 75, 78, 79, 85, 90 White, A., 78, 79, 90 White, I . R., 56, 95 White, J.G., 5 5 , 65, 96 White, M. J., 106, 135, 160 Whitecross, M. I . , 101, 113, 130, 141, 154 Whitford, P. N., 276, 291 Whittier, D. P., 13, 22, 41 Widden, P., 15, 37 Wildon, D.C., 166, 171, 180, 186, 187, 188, 189, 190, 194, 198, 201, 202, 204, 206, 226, 227 Wilimitzer, L., 170, 188, 189, 222 Wilkins, D., 141, 161 Willekens, H., 120, 161 Williams, D. A., 59, 91, 96 Williams, P. H., 30, 38 Williams, S. E., 165, 168, 186, 187, 201, 202, 203, 205, 213, 214, 227 Williamson, R. E., 65, 96 Willmer. C . M., 74, 96 Willmitzer, L . , 165, 168, 170, 188, 189,
224 Willmott, N., 75, 78, 79, 85, 90 Willows, R. D., 277, 295 Wilson, M. I . , 115, 161 Wingate, V. P. M., 120, 161, 165, 228 Winston, G. W., 119, 162 Winter, A . G . , 14, 43 Wiren, A., 243, 300 Wise, R. R . , 237, 238, 242, 296, 300
Wolf, O . , 170, 228 Wolfson, J. L., 196, 228 Wong, S. C . , 237, 299 Wood, J. W., 51, 86, 89 Wood, R . D . , 119, 159 Woodall, G., 276, 296 Woods, F. W., 166, 228 Woolhouse, H. W., 6, 43 Wratten, S. D., 188, 220 Wu, Y . , 249, 254, 256, 257, 298, 300 Wykoff, D., 119, 151 Wyn Jones, R.G., 171, 224
Y Yagawa, Y., 51, 60,92 Yallop, A., 21, 43 Yamada, N., 75, 93 Yamada, T., 253, 293 Yamagata, H . , 85, 89, 109, 15f Yawney, W. J., 13, 16, 43 Yelle, S., 127, 162 Yoshikawa, S., 75, 93 Young, A. T . , 243, 294 2 Zannoni, D., 51, 52, 96 Zawadzki, T., 205, 206, 224, 228 Zeevaart, J . A. D., 268, 290 Zeevi, Y . , 214, 223 Zenk, M . H . , 120, 154 Zhang, D. H., 51, 52, 56, 96 Zhang, J., 129, 134, 162, 164, 177, 178, 219, 233, 238, 268, 269, 270, 276, 277, 278, 285, 291, 295, 299, 300 Zhong, H . , 258, 300 Zhu, G. L . , 176, 228, 247, 248, 250,
300 Zimmermann, D. C., 187, 228 Zimmermann, M. H . , 172, 178, 183, 288 Zimmermann, U., 175, 209, 210, 227, 228 Zinchenko, V. P., 50, 51, 59, 96 Ziska, L. H., 101, 102, 145, 160, 162 Zook, M. N., 170, 224 Zottini, M., 51, 52, 96 Zuroske, G . , 171, 189, 223
SUBJECT INDEX
A Abscisic acid calcium ions, 69, 71, 81, 84, 86 signal transmission, 170, 182 U V radiation, 137 water and nitrogen supply, 232, 237, 240, 254-6, 263, 268-88 Acaulospora, 14 Acer, 6-7, 13 pseudoplatamus, 15 saccharum, 15, 16 Aceraceae, 13 Acetabularia, 209, 210 Acetoxymethyl (AM) esters, 59 Acetylcholine, 85 Achlorophyllous plants, 25, 34 Acid loading, dye, 56, 58-9 Action potentials (AP), 186-8, 201, 202-9, 212-3 Adenine, 182 Adenosine diphosphate, 47, 49 Adenosine 5 ‘-diphosphoribose (cADPR), 74, 78-80, 88 Adenosine monophosphate, 46, 47, 81, 85, 107 Adenosine triphosphate, 122 Aequorin, 49-60, 65-8, 87 Aeguorin victoria, 65 Airborne signals, 167-70, 216 Aldrovandu, 164, 186, 213-6 vesiculosa, 202-3 Alfalfa, 118, 165 Allelochemicals. 165 Allium, 10, 13, 28, 32 cepa, 4-5, 6-9, 15, 17, 28 porrum, 9, 28 Aluminium toxicity, 264 Amino acids, 214-5, 279-80 Ammonia, 4, 169, 243 a-Amylase, 83, 85 Anaeorobiosis, signal transmission, 164 Anemone, 13 nemorosa, 12
Angiopteris, 13 Anisohydric plants, 268 Antheraxanthin, 143 Anthocyanin, 108, 109, 110, 114, 116, 138, 144 Antioxidants, 119-21, 143 Antirrhinum, 166 Apoaequorin, 66-8 Apple, 239 Appressoria, 30 Aquaporins, 212 Arabidopsis, 105, 111, 116, 118-9, 126, 129, 135, 136, 140, 146 Araceae, 13 Araliaceae, 12, 34 ‘Arbuscular’ mycorrhizal symbiosis, 1-35 cheating by myco-heterotrophic plants, 25 costs and benefits, 26-9 development and control of, 29-33 transfer of solutes, 3-9 variations in nutritional efficiency, 17-25 variations in structure, 9-17 Arbutus unedo, 241 Arisaema, 13 Aristolochiaceae, 13 Arum, 13 maculatum, 8, 9-17, 19-20, 22, 28, 31-2, 34 Asarum, 13 Ascorbate, 119 ATPase calcium, 49, 80, 81, 82, 83 mycorrhizal symbiosis, 4, 6-9, 10, 15, 20, 32, 34 UV radiation, 129, 135 Atriplex confertifolia, 20 Attractants, 166 Autofluorescence, dyes, 64 Auxin, 69, 84, 166, 201, 284 Avena sativa, 12
320
SUBJECT INDEX
B Barium, 72, 107 Barley, 59, 60, 116, 127, 131 Bean, 116, 127, 131 Bepridil, 71, 72, 73 Berberis, 165, 215 Betula pendula, 259 Bidens, 166 Bignonia, 165 Biomass assimilation, UV radiation, 141, 142 Birch, 264 Blue/UV-a photoreceptor, 105-6, 107, 110 Brassica napus, 1 15 Brassicas, 29-30 BTC dye, 53, 55 Bud break, 166 Burmanniaceae, 13
C Cadmium, 72, 146 Caesium, 72 Caffeine, 75, 78 Calcium Crimson dye, 53 Green dye, 53, 54, 5 5 , 56 induced calcium release, 79-82 influx factor (CIF),82-3 ions, intracellular second messengers, 45-88 homeostatic apparatus, 69-83 measurement of stimulus-induced changes in calcium, 68-9 problem of specificity, 84-7 Orange dye, 53 release activated calcium channel (CRAC), 83 signal transmission, 177, 180, 187 ‘signature’, 85-7 UV radiation, 107, 108-9 water and nitrogen supply, 280, 283-4, 287 Calmodulin, 81, 83, 87, 108-9 Calvin cycle, UV radiation, 141 Capacitative calcium entry, 82-3 Capsicum, 174 Carambola, 165 Carbohydrates mutualism and parasitism, 8, 9, 24, 31 UV radiation, 121, 130, 132, 141
water and nitrogen supply, 238, 243, 252, 259, 263-4 Carbon see Mycorrhizal symbiosis, Water and Nitrogen supply Carbon dioxide signal transmission, 171 UV radiation, 102, 142-3, 145-6 water and nitrogen supply, 235-8, 240-5 Carboxylate, 274-5 Carnivorous plants, 202-3, 204, 213-6 see also Aldrovanda, Dionaea, Drosera Carotenoids, 114 Castor bean, 280 Catalase, 144 Cauliflower, 66 Cell-cycle, UV radiation, 132, 133-4 Cellulose, 251, 258 Centaurea cyanus, 116 Centaurium erythraea, 23 Cercospora beticola, 147 Chalcone isomerase (CHI), 136 Chalcone synthase (CHS), 136, 137 Chara, 210 corallina, 247 Charge Coupled Device (CCD), 61, 62-3 Chemical control, 268, 288 Chemical defences, 166 Chemical signals, 167-71 see also Hydraulic dispersal Chenopodium, 174 album, 112 guinoa, 147 Chenopods, 29-30 Chloride, stomata1 response, 238 Chlorine, 187, 202, 210, 212 Chlorofluorocarbon (CFC), 99 Chloromercuribenzene sulfonicacid (PCMBS), 189 Chlorophyll UV radiation, 119 water and nitrogen supply, 240-1 see also Photosynthesis Chlorophyllides, 126 Chloroplast proteins, 108, 110-1, 134-5, 136, 140 Chlorotetracycline, 109 Cholecystokinin, 85 Cholodny-Went Theory, 166 Cinnamic acid, 114 Citrus, 28
SUBJECT INDEX
Clover, 165 Cornrnelina, 279 ocrnrnunis, 76 Common plant regulatory factors, 138 Confocal scanning laser microscopy (CSLM), 55, 60, 64-5 Copper, 72 Copro-porphyrins, 126 Corn, 127, 131 Cornaceae, 12, 14, 34 Cosrnospora robusta, 9 Crassulacean acid metabolism (CAM), 171 Cucumber signal transmission, 165 UV radiation, 101, 116, 130, 143,
144, 146, 147 water and nitrogen supply, 256
Cucurnis, 174 sativus, 147 Cucurbitaccae, 13 Cucurbito, 13 Cyclobutane pyrimidine dimer (CPD),
116, 117, 118-9 Cytochrome, 109, 134 Cytokines, 166,263,273,279,284-5,287 Cytoskeleton, UV radiation, 133-4
D DAG, 74, 85, 88 Datura strarnonium, 165 Daucus carota, 60 Daylength, 166 Defence genes, UV radiation, 135-8 Defence reactions, 165 Dehydrins, 277 Development, UV radiation, 102, 139 Diacylglycerol, 46, 47, 48-9 4' -6-Diamidino-2-phenylindoledihydrochloride monohydrate,
109 Diethylstilbestrol (DES), 7 Digalactosyldiacylglycerol (DGDG), 13 1 Digitonin permeabilization, dyes, 56, 60 Dihydroflavonol reductase (DFR), 136 1,4-Dihydropyridines, 71, 73, 78 Diltiazem, 72 Dimethyl sulphate (DMS), 137 Dionaea, 164, 165, 213-6 rnuscipula, 186, 202-3 Dioscoraceae, 13
321
Diphenylbutylpiperidines, 71 DNA recombination technology, 66-8 UV radiation, 103-4, 113, 117-9, 132, 133, 139 Drosera signal transmission, 164, 168-9, 186, 187 further research, 201, 202, 203, 214, 216 filiforrnis, 169 rotundvolia, 169
E Ectomycorrhizas, 5 Elatosterna repens, 241 Electrical signalling, 167, 171, 186-8 case studies, 190, 194, 196, 198 further research, 201-16 Electropermeabilization, dyes, 56, 59-60 Electroporation, dyes, 56, 59-60 Endodermoid layer, 214 Endomycorrhizas, 9 Epoxidation, 143 Ericales, 5, 10, 15 Erythronium, 13 Escheriehia coli, 1 18, 120 Ester loading, dye, 56, 59 Ethanol, 75 Ethylene, 168, 189 Euglena gracilis, 109 Evolution, mycorrhizal symbiosis, 24-5,
27 Exodermes, 28 Expansins, 256 Extended ISIS-M camera. 61
F Fern, 60 Festuca, 259 ovina, 23 Flavin, 105 Flavones, 110, 114 Flavonoids, UV radiation, 106, 112 effects on cellular processes, 122 effects on gene expression, 135-6, 138 interactions with other stresses, 143,
144, 146. 147 protective mechanisms against, 110,
114-7, 120
322
SUBJECT INDEX
Flavonols , 114 Flavoprotein, 105 Flowering, U V radiation, 132 Fluo-3 dye, 53, 54, 55, 59, 60 Fluorescence ratio imaging, 61-4 Fluorescence ratio photometry, 61 Fluorescent dyes, calcium, 49-56, 68 Fluphenazine, 109 Fluridone, 254, 256 Fluspirilene, 71 Free oxygen radicals, 119 Fructose, UV radiation, 130 Fucus, 86 serratus, 58 Fura-2 dye, 53, 54, 55, 57 Fura-BSA dye, 56 Fura-Cle dye, 56 Fura Red dye, 53, 55 Furanocoumarin, 110, 147 G Gadolinium, 72, 73, 74, 81 Galactolipids, 13 1 Genes, U V radiation, 110-1, 134-41 Gentiana, 13, 14 Gentianaceae, 13, 23, 25 Geotropism, 166 Germination, UV radiation, 132-3 Gibberellic acid (GA), 83, 132 Gibberellins, 284 Gigaspora calospora, 20 margarita, 14, 31 Gingko, 13 Gingkoacaeae, 13 Gliocladium deliquescens, 67 Glomalean fungi, 2, 10, 14, 25 Glomus, 32 etunicatum, 16 fasciculatus, 9, 20 macrocarpum, 27-8 mosseae, 4-5, 6-9, 31 Glucose, UV radiation, 130, 141 Glutamate synthase, 131 Glutamine synthetase, 131 Glutathione (GSH), 109, 119 Glutathione reductase (GR). 119, 120, 138
Glycerophosphatidylinositol, 76 Glycerophosphatidylinositol bisphosphate, 76
Glycerophosphatidylinositol phosphate, 76
Glycine, 13, 174 Glycogen metabolism, 47 G protein calcium ions, 48, 74, 83, 84 UV radiation, 107, 108, 109-10 Gramineae, 12, 13 Grape, 165 Grass, 252 Greenhouse effect, 146-7 Griselinia, 14 Growth, signal transmission, 176-7 see also Water and Nitrogen supply Guanosine diphosphate (GDP), 74, 108 Guanosine monophosphate (GMP), 47, 79, 85, 88, 109
Guanosine triphosphate (GTP), 74, 108, 109
H Hagen-Poiseuille ‘law’, 172, 182, 183 Heat shock proteins, 148 Helianthus, 174, 187, 206, 239 annuus, 259, 260-3 Hemicellulose, 251 Heparin, 75, 77, 81 Hevea, 177 Hexadecanoic acid, 131 Hibiscus, 133 Homeostasis, calcium, 46, 69-83, 87 Homeotic genes, U V radiation, 132 Hordeum vulgare, 259 Hormones signals, 168-9, 171, 201 water and nitrogen supply, 276-9 see also Abscisic acid Hydraulic conductivity, 273 Hydraulic dispersal signals, 167, 177-86, 187, 188,
case studies, 190-6, 197, 198-200 further research, 200-1, 204, 206-7, 209- 16
Hydraulic pressure signals, 167, 171-7, 190, 194, 216
Hydrogen peroxide, 109 Hydropassive effects, stomata, 175-6 Hydroxycinnamic acids, 120 Hyoscyamus niger, 132 Hyphae coils, mycorrhiza, 2, 10, 14, 15-17, 32, 34
see also Paris quadrifolia
SUBJECT INDEX
I Impatiens valeriana, 241 Incarvillea, 165 Indo-1 dye, 53, 54,5 5 Inositol, 130 Inositol phosphate, UV radiation, 107 Inositol phosphate receptor (IPR), 75-6 Inositol (1,3,4,5)-tetrakisphosphate,70,
75-6 Inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3), 47,48,70,74-82,
85, 108 Instron apparatus, 248 ‘Internal winds’, 167-8 Intracellular transduction, 164 Ion channel, mycorrhizal symbiosis, 6 ‘Ion trapping’, 59 Ions, water and nitrogen supply,
279-80,283 Iontophoretic injection, 57-8 Iron, 264 Irritability, 163-4 Isoflavonoids, I14 Isohydric plants, 268 Isoprene, 166
K Kaempferol, 116 Khaya, 13
323
Lupinus, 206 Lycopersicon, 174, 187 Lyso lipids, 46
M Magnesium Green dye, 56 UV radiation, 107,129 water and nitrogen supply, 259-60,
280 Magnoliacae, 13 Mahonia, 165 Maize calcium ions, 59, 72 mutualism and parasitism, 14-15 signal transmission, 165, 175, 183 UV radiation, 116, 138, 146 water and nitrogen supply, 232,
254-6,268,276,282,285-6 Malate, 238,274 Malus, 174 Manganese, 264,280 Marattiaceae, 13 Mechanical impedance, soil, 233 Meliaceae, 13 Mesophyll, water and nitrogen supply,
240-5 Messengers see Calcium Metals, 146 N-Methyl-D-aspartate (NMDA), 73 Methyl jasmonate (MeJ), 120, 168, 170,
L Lanthanum, 72,73,74,82, 107, 109 Leguminoseae, 13, 177 Lettuce, 179 Light mutualism and parasitism, 17-18,32 signal transmission, 167 UV radiation, 103, 110, 132, 139,
141-3 Liliaceae, 13 Linolenic acid, 131 Lipids, UV radiation, 104, 116, 119,
131, 141, 147 Lipoxygenase, 120 Liquidambar, 14 Liriodendron, 13, 14 Lithium, UV radiation, 108 Lockhart’s equation, 176,246-52 Lotus corniculatus, 23 Luffa, 13
189 Microinjection, dye, 56-8,66,71 Microtubules, UV radiation, 133 Mimics, 21 Mimosa, signal transmission 165, 182,
185, 186, 187,202 case studies, 192, 196-200,202 further research, 212-3,214,215-6 pudica, 167, 177, 196 spegazzinii, 179, 196 Mimulus, 165 Mitosis, UV radiation, 133 Molinia, 13 Molybdate, 7 Monoclonal anticalmodulin, 109 Monogalactosyldiacylglycerol (MGDG),
131 Motor activity, 203,212 Mutualism see ‘Arbuscular’ mycorrhizal symbiosis
324
SUBJECT INDEX
Mycorrhizal symbiosis see ‘Arbuscular’ mycorrhizal symbiosis
N Neodymium, 72 Neomycin, 108 Nerium, 238 Nickel, 72 Nicotiana, 133, 174 Nicotinamide adenine dinucleotide (pNAD), 78-9 Nifedipine, 71, 72, 81, 82, 109 Nitella, 202, 210 Nitrate, 243-5, 264, 265-6, 273-5, 280, 284-5 Nitrate reductase, 130-1, 244 Nitrendipine, 7 1 Nitrite, 243 Nitrite reductase, 244 Nitrogen mutualism and parasitism, 8, 23 UV radiation, 130-1 see also Water and Nitrogen supply NMR, 87 Non-radiometric dyes, calcium, 49-56, 68 NuCaGreen dye, 56 Nucleic acids, UV radiation, 104 see also DNA, RNA Nuphar lutea, 168 Nymphoides peltata, 168, 170 0 Oats, 59, 121, 146 Oligosaccharides, 17I Onion see Allium Orchids, 5 , 10, 15 Osmoregulation, 210 Osmotica, signal transmission, 164 Osmosis see Water and Nitrogen supply Ovaries, 132 Oxalic acid, 165 Oxalis, 165 Oxidative stress, 113, 129, 130, 141 Ozone, 98-101, 110, 120, 132, 146
P Paraquat, 120 Parasitism see ‘Arbuscular’ mycorrhizal symbiosis
Parasitoids, 165 Paris, 13 quadrifolia, 8, 9-17, 19-20, 22, 25, 31-2, 34 Parnassia, 13 Parsley, 110, 114, 137, 147-8 Passioura-type pressure chamber, 233, 234 Patch-clamp studies, 57, 71, 72, 73-4, 84, 87 Pathogens mutualism and parasitism, 22 signal transmission, 166, 216 UV radiation, 102, 110, 139, 147-8 Pea calcium ions, 59 UV radiation, 101, 113 effects on cellular processes, 122-3, 126, 127-9 effects on gene expression, 135, 140, 141 protective mechanisms against, 119 Peroxidase, 263 Peroxidation, 131 Peroxides, 119 Petunia, 107 Petunia hybrida, 132, 133 PH calcium, 46, 56, 58-9, 84 water and nitrogen supply, 264, 279-80, 283 Phaeoceros, 14 Phaseolus, 174, 187, 277, 282 Phenolic compounds, 113 Phenylalanine, 127 Phenylalanine ammonia lyase (PAL), 120, 136, 148 Phenylalkylamines, 71, 72 Phenylpropanoids, 102, 114, 135-6, 138, 140, 147, 148 Pheromones, 165-6 Phloem translocation, 167, 170-1, 216 Phorbol 12-myristate, 108 Phosphate, 280 see also Mycorrhizal symbiosis Phosphatidylcholine, 131 Phosphatidylglycerol, 131 Phosphatidylinositol (PtdIns), 74,76, 108 Phosphatidylinositol(4,5)bisphosphate (PTDIns(4,5)P2), 48, 74-8 Phosphatidylinositol 4-phosphate (PTdIns(4)P), 74, 76
325
SUBJECT INDEX
Phosphoinositide cycle, 48 Phosphosynthate, 3, 23-4 Phospholipase. 121 Phospholipase C (PLC), 48, 74, 77, 108 Phospholipids, 131 Phosphorus, 144 Photoinhibition, 241 Photolyase, 105, 118 Photomorphogenesis, 105-6 Photoperiodism, 105, 132 Photophosphorylation, 121, 122, 123, 129 Photoproteins see Aequorin Photoreceptors, UV radiation, 104-7 Photosynthesis mutualism and parasitism, 17 see also Ultraviolet-B radiation, Water and Nitrogen supply Photosynthetic carbon reduction (PCR) cycle, 242 Photosynthetically active radiation (PAR), 102, 104, 112, 141-3, 145 effects of cellular process, 121 effects of gene expression, 134, 139 protective mechanisms, 120 Photosystem I1 (PSII), 240-1 Phototropism, 105, 166 Phyllotaxis, 178 Phytochrome, 105, 106, 107, 110 Phytotoxic root exudates, 166 Picea pungens, 1 12 Pigmentation, UV radiation, 102, 103, 104, 112, 113-4 effects on cellular processes, 122, 123, 124, 132 effects on gene expression, 138, 139, 140 interactions with other stresses, 141, 142, 143, 144, 147 protective mechanisms against, 114-7 Pinus, 102 Pisurn, 9, 174, 187 Plasmodesmata, 202 Plasticity, cell wall, 248-51 Plastoquinone, 125-6, 143 Pluronic F-127, 59 Podocarpaceae, 13 Podocarpus, 13 Pollen, 132-3 Polyamines, 120-1, 143 Polychromatic action spectrum, 103
Polysomes, 166 Poplar, 165 Potassium calcium ions, 72, 73, 77, 81 mutualism and parasitism, 4 signal transmission, 180, 187, 210, 212 UV radiation, 107, 109 water and nitrogen supply, 238, 258-9, 274, 280, 283 Potato, 165, 188, 189 Potato virus, 147 Predators, 165 Pressure chambers, 233, 234, 247, 276 Pressure injection, 57-8, 66 Pressure signals, 175-9 Proline, 144, 254, 258 Protein biosynthesis, UV radiation, 132 Protein kinase C, 49, 108 Proteinase, 165 Proteinase inhibitor, 188-96, 197 Proteinase-inhibitor inducing factor (PIIF), 188, 191, 196 Proteins, UV radiation, 104 Protoplasts, UV radiation, 107-8 Protoporphyrin, 126 Psilotaceae, 13 Psiloturn, 13 Psychrometer, 247 Pteridaceae, 13 Pteridiurn, 13 Pyrimidine dimer, 104, 116, 140 Pyrimidine-(6-4 ’)-pyrimidone photoproducts, 117, 118-9 Pyrophosphatase, 80, 8 1
Q Quin-2 dye, 53, 59 Quinones, UV radiation, 104
R Radiometric dyes, calcium, 49-56, 68 Radish, 126, 130, 143-4 Ranunculaceae, 13 Ranunculus, 13 Receptor-operated channels, 48, 69, 7 1, 73 Recombinant aequorin, 66-8 Recombinant DNA technology, calcium, 66-8
326
SUBJECT INDEX
Regnellidium diphyllum, 169 Relative growth rate (RGR), 235, 260, 265-6 Reproductive biology, UV radiation, 131-3 Rhamnogalacturonan I, 182 Rhod-2 AM dye, 53, 56 Ribulose bisphosphate (RuBP), 146, 242-3 Ribulose 1,5-bisphosphate carboxylase (Rubisco) UV radiation, 109, 146 effect on cellular processes, 121, 122, 126-9, 130 effect on gene expression, 135, 137, 141 water and nitrogen supply, 242-3 Ricca’s factor, 198, 212 Rice, 101-2, 113, 145-6 RNA, 111, 113, 115, 117, 134, 136 Root, signal transmission, 164 Rubisco see Ribulose 1,5-bisphosphate carboxylase Rumex acetosa, 23 Ruscus, 13 Ryanodine, 78-82 Rye, 146 S Salicylic acid, 170 Salix viminalis, 259 Salt, signal transmission, 164 Saxifragaceae, 13 Scutellospora calospora, 20- 1 Second messenger operated channels, 48, 69, 71, 74, 75 see also Calcium ions Secretory cells, 177 Sensitivity variation, 280-6, 288 Sequoia, 13 Shoot response, 164 Signal transmission rapid, long-distance, 163-216 case studies, 188-200 further research, 200-16 mechanisms of, 167-88 UV radiation, 107-11, 140-1 water and nitrogen supply, 267-75, 276, 287-8 Signal-to-noise ratio, dyes, 64 Sinapate esters, 114
Sink strength, 263-4 Slow vacuolar (SV) channel, 80 Smilacina, 13 stellata, 113 Sodium, 202, 264, 280, 287 Solanum tuberosum, 273 Soybean, 101, 127, 142, 144-5, 166, 258 Sparmania, I65 Specificity, 21-2, 46, 84-7 Spinacia, 174 Spirodela, 263 polyrhiza, 130 Spodoptera, 184 Sporangioles, 12 Squash, 165 Starch, 130, 141, 242 Stimulus-induced changes in calcium, 68-9 Stomata signal transmission, 164, 166, 175-6, 178-9 UV radiation, 141, 143-4 see also Water and Nitrogen supply Stornatal response, 238-40 carbon dioxide uptake, 235-8 Strawberry, 165 Stress see Water and Nitrogen supply Stretch activated channels (SAC), 73-4 Stunt disease, 27-8 Stylidium, 165 Sucrose, 130, 141, 182, 242, 264 Sugar, 6 Sugar beet, 147 Sugar maple, 15, 16 Sulphate, 243 Sulpholipid, 131 Sulphoquinovosyldiacylglycerol, 13I Sundew see Drosera Sunflower, 146, 238, 264, 280 Superoxide dismutase, 119, 120, 138, 144 Supply-limitation, 23 1 Sycamore see Acer Symbiosis, 1-2 Systemic acquired resistance (SAR), 165, 200-1 Systemin, 170, 180, 189, 191
T Tamus, 13 Taxaceae, 13
327
SUBJECT INDEX
Taxodiaceae, 13 T a m , 13 Temperature mutualism and parasitism, 17-18, 32 signal transmission, 164 UV radiation, 146-7 water and nitrogen supply, 264, 285-6 Texas red dye, 53, 54, 55 Thismia, 13 Thylakoid membrane, 119, 120-1, 123-6, 131, 142, 143, 242 Tobacco, 27-8, 117, 120, 165 Tomato signal transmission, 165, 166, 180, 183, 184-5 case studies, 188-96, 197, 198 future research, 204, 205, 207, 209, 21 1 mechanisms of, 180, 182, 183, 185-6 UV radiation, 108, 127 water and nitrogen supply, 276 Total ozone mapping spectrometer (TOMS), 98 Tradescantia, 22, 166 Trap systems, 214-6 ‘Traumatin’, 187 Trifolium, 13, 31 subterraneum, 14, 20-1 Trillium, 13 Triticum, 174 Tryptophan, 127 Tubulin, 133 Tulip tree, 13, 14 Turgor signal transmission, 166, 172, 176-7, 179, 182, 211-3 water and nitrogen supply, 237, 238, 246-63 ‘Turgorin’, 187 Tyrosine, 127 Tyrosine kinase, 74
U Ultraviolet-B radiation, 97-149 effect on cellular processes, 121-34 effect on gene expression, 134-41 interaction with other stresses, 141-8 perception of, 103-14 protective mechanisms against, 114-21 Uro-porphyrins, 126 Utricularia, 164, 202
V Vanadate, 7 Variation potential (VP), 187-8, 203-9 Venus flytrap, 186, 202-3 Verapamil, 71-3, 82, 107 Vesicular-arbuscular mycorrhizas see ‘Arbuscular’ mycorrhizal symbiosis Vicia, 13, 174, 187 faba, 73, 81 Vigna sinensis, 148 Viola, 13 Violacacae, 13 Violaxanthin, 143 Vitis, 14 Voltage-independent Ca sensitive K influx channel (VK), 80-1 Voltage operated calcium channels (VOC), 48, 69-73 Water and Nitrogen supply, 230-88 acclimation of extension growth, 246-63 acclimation of uptake, 235-45 implications, 263-7 information transfer, 267-75 manipulating supply, 23 1-5 xylem sap, 275-86
W Water stress, UV radiation, 102, 143-5 Water vapour, 167 Water-use efficiency, 267 Wax, 102, 112, 116-7 Wheat calcium ions, 60 signal transmission, 173, 175, 178, 181, 187, 207, 208 UV radiation, 116, 145 water and nitrogen supply, 277 Wild tomato, 165 Woody rose, 166 Wounds, signal transmission, 165-6 hydraulic dispersal, 179-86, 188, 206-9, 213
x Xanthophyll, 143 X-ray crystallography, 87
328
SUBJECT INDEX
Xylem signal transmission, 171-86 see also Water and Nitrogen supply Xyloglucan, 259, 264 Xyloglucan endotransglycosylase (XET), 256-8, 263 Y Yeast, 60
Yield threshold, cell, 246 2
Zea, 13 mays, 72, 122 Zeaxanthin, 143 Zinc, 4, 72, 81 Zingiberaceae, 22
